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PMC516040

Background ========== Prostate cancer is the most common non-skin malignancy and the second leading cause of male death in the United States \[[@B1]\]. The incidence of prostate cancer increases most rapidly with age, and multiple genetic and epigenetic factors have been implicated in the initiation, progression, and metastasis of prostate cancer. Nevertheless, scientific knowledge of the molecular mechanisms underlying the disease is still limited. The problem often faced with the clinical management of prostate cancer is derived not only from the fact that no single gene or molecule can serve as a reliable marker \[[@B2],[@B3]\], but also that there is still no effective therapeutic regimen available without serious, sometimes fatal side effects. Unfortunately, at the time of clinical diagnosis, human prostate cancers mostly present themselves as heterogeneous entities -- hormone-dependent and hormone-independent, and proliferating and non-proliferating. The tumor re-growth that occurs after post-treatment remission is largely due to progression of initially androgen-dependent to androgen-independent cancer cell \[[@B4]\] and/or non-proliferating to proliferating tumor cells. Therefore chemotherapeutic strategies should focus on eradicating all cancer cells irrespective of state of growth or sensitivity to hormone. This calls for a search for drug combination treatment that works through different mechanism of action. The facts that prostate cancer cells retain the inherent apoptotic machinery potentially subject them to an appropriate efficacious chemotherapeutic intervention. The molecular mechanism(s) and intracellular mediators of both spontaneous-and treatment-induced apoptosis are not fully elucidated. However, evidence from several research investigations seem to indicate that a variety of stimuli, including physiological, pathologic, environmental or cytotoxic, can trigger the process of apoptosis in many mammalian cells \[[@B5],[@B6]\], and that both apoptosis and necrosis may share some upstream events in the molecular pathways that lead to induction of apoptosis \[[@B7]-[@B11]\]. An emerging strategy for cancer chemotherapy is the choice of drugs that induce apoptosis and/or disruption of angiogenesis with eventual elimination of the cancer. It is suggested that blocking of caspase activation in an apoptotic process may divert apoptotic cell death to a necrotic demise \[[@B10]\]; implying that apoptosis and necrosis may share some upstream events in the molecular pathways of apoptosis induction. Among the dietary phytochemicals of potential therapeutic significance, are genistein isoflavone and β-lapachone, both of which induce apoptosis and also inhibit angiogenesis (genistein) in an array of cancer cells \[[@B6],[@B12],[@B13]\]. Genistein isoflavone \[4\',5\',\]-trihydroxyisoflavone) is a metabolite of soy \[[@B14]\] and has a heterocyclic, diphenolic structure similar to estrogen \[[@B14]\]. The phytochemical isoflavonoid family to which genistein belongs is a group of plant chemicals that resemble steroid estrogens and mimic their biological reactions \[[@B15],[@B6],[@B16]\]. Several clinical studies indicate that genistein has some chemoprotective and chemotherapeutic potential against many tumors, including prostate, breast, and colon cancers through several mechanisms of action including: apoptosis induction; modulation of cell cycle activity by arresting cell cycle at the G~2~-M stage \[[@B17]\]; inhibition of DNA topoisomerase-II and tyrosine protein kinase \[[@B18]\]; competitive inhibitor of ATP binding to the catalytic domain of tyrosine kinase \[[@B14],[@B18]\]; stimulating the production of sex hormone-binding globulin (SHBG), which may lower the risk of hormone related cancers by decreasing the amount of free and active hormones in the blood \[[@B19],[@B20]\]. The other phytochemical of potential therapeutic significance is β-lapachone \[3, 4-dihydro-2, 2-dimethyl-2H-naphtol (1,2-b) pyran-5,6-dione\], a simple plant product with a chemical structure different from currently used anti-cancer drugs. It has been previously demonstrated that the primary mode of cytotoxicity of β-Lapachone is through the induction of apoptosis \[[@B21],[@B22]\]. Structural similarities between β-lapachone and other members of the naphthoquinone family, such as menadione, suggest that the enzyme, NAD(P)H:Quinone oxidoreductase enzyme (NQO1) may be involved in the activation or detoxification of β-lapachone \[[@B23]-[@B25]\]. While a number of *in vitro*effects of β-lapachone and genistein have been described, knowledge of the key intracellular targets of β-lapachone and genistein is limited. Recent reports have suggested that by β-lapachone-induced apoptosis is non-caspase mediated in breast \[[@B26]\] and prostate cancer cells \[[@B21],[@B34]\], and that the cytotocxicity of this compound is dependent on the activity of NAD(P)H:Quinone oxidoreductase enzyme (NQO1/xip3) \[[@B26],[@B27]\]. B-Lapachone has been shown to be an inhibitor of DNA repair that sensitizes cells to DNA-damaging agents \[[@B28],[@B29]\]. It directly inhibits DNA topoisomerase I and II \[[@B30]-[@B32]\] and induces a cell-cycle delay in G~1~and/or S phase followed by apoptotic and/or necrotic cell death \[[@B13]\]. The apoptosis induced by β-lapachone is p53 independent \[[@B21]\], and has been associated with upregulation of Bak as well as cleavage of caspase-7 \[[@B33]\] and caspase 3 in a variety of mammalian cells \[[@B13],[@B33]\]. The objective of this study was to determine the potential chemosensitivity of human prostate adenocarcinoma, PC3 cells to β-lapachone and Genistein and the role of caspase 3 (CPP32) and NAD(P)H:Quinone oxidoreductase enzyme (NQO1) in the signaling of β-Lapachone and genistein-induced apoptosis in PC3 cells. The hypothesis is that combination treatment with the two phytochemicals will be strongly preventive and/or interceptive against prostate cancer by modulating epigenetic events (apoptosis) associated with the progression of active and latent cancer cells to clinical malignancy. Results ======= Genistein and β-lapachone inhibit growth and proliferation of human prostate carcinoma cells, PC3 ------------------------------------------------------------------------------------------------- Human prostate carcinoma cells PC3, was used to determine the chemosensitivity of prostate cancer to genistein isoflavone and β-lapachone *in vitro*using Trypan blue exclusion, LDH and MTS bioassays. In single and combination treatments, both genistein and β-lapachone inhibited cell growth and decreased cell survival through induction of cell death \[Figures. [1](#F1){ref-type="fig"},[2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"}\]. The data indicated that PC3 sensitivity to both single and combination treatment is dose-dependent, and that PC3 was significantly more sensitive (P \< 0.05) to the combination treatment than to the single treatment; indicating a potential synergism between genistein and β-lapachone \[Figures [2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"}\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### β-lapachone-induced growth inhibition in PC3. PC3 cells (1 × 10^4^cells/well) were cultured in 24-well plates for 48 hr to allow 85--90% confluence; treated with varyingconcentrations of bLap and assessed for post-treatment viabilitywith the MTS assay. Note the dose-dependent growth inhibitionin PC3. Data points represent means ± SEM of three independentexperiments performed in triplicates :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Genistein (Gn)/β-Lapachone combination treatment of PC3. Cells were treated as described in the methods and subjected to post-treatment viability with MTS colorimetric assay. Data points represent the means ± SEM of three independent experiments performed in triplicates. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Single and combination of PC3 cells with genistein (Gn) and β-lapachone (bLap) βLap. Briefly, PC3 cells were seeded at 1 × 10^4^cells/well in 48-well MTP and co-cultured with Gn~0-70~with/without bLap (1.2 μM); followed by determination of treatment-induced cytotoxicity as described in the methods. Data points represent means ± SEM of three experiments performed in triplicates :::  ::: Genistein and β-lapachone induce apoptosis in human prostate cancer cells ------------------------------------------------------------------------- Extensive cell death was observed in proliferating human prostate cancer cells after treatment with β-lapachone and genistein isoflavone. To determine if the treatment-induced cell occurred through cytotoxic necrosis and/or apoptosis, cells were harvested and assayed for apoptosis induction with Annexin V-FITC and TUNEL apoptosis assays to detect early and late apoptosis respectively. Aliquots of cells were also stained with acridine orange/ethidium bromide nuclear stain to distinguish between apoptotic and necrotic cells. The results revealed that in both single and combination treatments, cell death was mostly through apoptosis in a dose-dependent manner \[Figures [4](#F4){ref-type="fig"},[5](#F5){ref-type="fig"}\]. With increasing concentration of the agents, cell death through necrosis increased correspondingly. Furthermore, combination treatment induced significantly more apoptosis in PC3 (p \<0.01) than individual treatment with either agent. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### βLap)-induced cell death in PC3 cells. PC3 cells were co-cultured with varying concentrations of bLap; and and the degree of treatment-induced apoptosis and/or necrosis assessed with the Annexin V-FITC assay, as described in the methods. Data points are means ± SEM of three independent experiments performed in triplicates. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Combination treatment-induced cell death in PC3 cells. PC3 cells were co-cultured with varying concentrations of Gn (Gn~0-70~) with or without bLap (1.2 μM), and apoptotic/necrotic cell death assessed with theAnnexin V-FITC assay as described in the methods. Data points are means ± SEM of three independent experiments performed in triplicates :::  ::: Dicoumarol enhanced the survival of human prostate cancer cells (PC3) following single treatments with β-lapachone (bLap) and Gn/bLap combination but not in PC3 cells treated with genistein alone --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- To determine the potential role of the enzyme NAD(P)H:quinone oxidoreductase (NQO1) in β-lapachone (bLap)-and genistein (Gn)-induced apoptosis in PC3, the cells were exposed to Gn and bLap in the presence or absence of dicoumarol in single and combination treatments; and then assayed for apoptosis by the Annexin V-FITC and TUNEL assays. Dicoumarol is a specific inhibitor of NQO1. The results revealed that blocking NQO1 activity with dicoumarol (50 μM) significantly reduced bLap-induced apoptosis \[Figure [6](#F6){ref-type="fig"}\]; indicating that bLap-induced apoptosis requires involvement of NQO1 target. However, dicoumarol did not appear to have significant effect on Gn-induced apoptosis \[Fig [7](#F7){ref-type="fig"}\]; indicating that NQO1 did not play significant role in Gn-induced apoptosis. \[Figures [6](#F6){ref-type="fig"},[7](#F7){ref-type="fig"}\]. The degree of apoptosis induction was highest in the Gn-bLap combination treatment without inhibiting NQO1 activity with dicoumarol \[Figure [7](#F7){ref-type="fig"}\]; implying that a possible synergy between Gn and bLap may be due to NQO1 activity. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Role of NQO 1 in β-Lapaphone-mediated apoptosis in PC3 cells. PC3 cells were treated with bLap alone or in combination with 50 μM dicoumarol (NQO 1 inhibitor) as described in the methods; and TUNEL assays performed to monitor apoptosis. Data points represent means + SEM of three independent experiments performed in triplicates. :::  ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### NQO1 is the main target in bLap/Gn-induced apoptosis in PC3 cells. Cells were treated with genistein (Gn) and Gn/bLap combination with or without 50 μM dicoumarol as described in the methods; and TUNEL assays performed to monitor apoptosis. Data points represent means ± SEM of three independent experiments performed in triplicates. :::  ::: Activation of CPP32 in genistein-induced apoptosis in PC3 but not in β-lapachone-induced apoptosis in PC3 --------------------------------------------------------------------------------------------------------- To determine if apoptosis induced by β-lapachone and/or genistein involved activation of caspase 3 protease (CPP32), the PC3 cells, were subjected to treatments with Gn and/or bLap co-administered with or without CPP32 inhibitor (DEVD-fmk), and then cultured as previously described. Post-treatment apoptosis was determined as previously described. As shown in Figures [8](#F8){ref-type="fig"} and [9](#F9){ref-type="fig"}, blocking the release of caspase 3 significantly decreased genistein induced apoptosis but not bLap-induced apoptosis; indicating the significant role of CPP32 in the molecular pathway of Gn-induced apoptosis; and minor involvement of CPP32 in bLap-induced apoptosis in PC3. Furthermore, blocking CPP32 activity did not significantly affect combination treatment-induced apoptosis (Figure not shown). ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Caspase-3 (CPP32) activity in genistein-induced apoptosis in PC3 cells. PC3 cells (2.5 × 10^3^cells/well) were cultured; then treated with/without 100 μM caspase inhibitor (zVAD-fmk) for 2 hr; and then with 10--70 μg/mL genistein for 4 hr as described in the methods. Cells were thenanalyzed for caspase (CPP32) activity and corresponding apoptosis in the cells. Data points were the means ± SEM of two independent experiments performed in triplicates. :::  ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### CPP32 is the major pathway in genistein-induced apoptosis in PC3 cells. PC3 cells (2.5 × 10^3^cells/well) were cultured in 48-well culture plates; treated with/without 100 μM caspase inhibitor (zVAD-fmk) for 2 hr; then with 1--8 μM β-Lapachone (bLap) for 4 hr as described in the methods. Cells were then analyzed for caspase (CPP32) activity and corresponding apoptosis. Data pointsare the means ± SEM of two independent experiments performed in triplicates :::  ::: Discussion and Conclusions ========================== In this study, we determined the role of caspase 3 (CPP32) and the enzyme NAD(P)H:quinone oxidoreductase (NQO1) in the signaling of β-lapachone (bLap)-and genistein (Gn)-induced apoptosis in human prostate adenocarcinoma, PC3 cells. Data from this study demonstrate significant inhibition of cell growth and proliferation in PC3 cells, with significant difference in chemosensitivity of PC3 to genistein and β-lapachone (P \< 0.01). Furthermore, growth inhibition of PC3 cells strongly correlated with the MTS and LDH assay results. The pattern of response and percent post-treatment live cells was consistent with previous results \[[@B6],[@B37]-[@B39]\]. The genistein-and bLap-induced morphological changes observed in the cells were identical in pattern but differed in severity at a given exposure time; indicative of differences in chemosensitivity of PC3 to genistein and β-lapachone. These observations were consistent with previous results \[[@B5],[@B6]\]. Furthermore, previous studies have shown that β-lap \[[@B22]\] induces morphologic changes indicative of apoptosis in human breast cancer cells. Similar alterations in morphology including cell shrinkage and chromatin condensation in the PC3 cells following single and combination treatment with β-lapachone and genistein isoflavone. The present data also implicates caspase-3 protease, CPP32, in the molecular pathway of genistein-induced apoptosis in prostate PC3 cancer cells, consistent with previous investigations \[[@B10],[@B11],[@B39]\]. Using the caspase inhibitor DEVD-fmk, caspase activity was arrested concurrent with significant decrease in genistein-induced apoptosis in PC3 cells. However, it is noteworthy that inhibition of caspase did not confer 100% protection against genistein-induced apoptosis; implying alternative death pathways, which suggests that some death receptor signals do not utilize the caspase CPP32 death pathways in PC3. We have previously demonstrated the significant role of caspase-3 protease in the genistein-induced apoptosis pathway in both testes and prostate (PC3) cancer cells \[[@B38],[@B39]\]. The present data indicate a possible alternate CPP32 pathway in bLap-induced apoptosis in PC3. However, unlike the observation in genistein-induced apoptosis, blocking the CPP32 activity with the specific caspase inhibitor, DEVD-fmk, did not significantly change the percentage of bLap-induced apoptosis in PC3 cells; indicating that CPP32 many not be the main death pathway of bLap-induced apoptosis in PC3 cells. Activation of the caspase 3 in bLap-induced apoptosis has been reported in previous studies \[[@B10]\]. The potential role of NAD(P)H:quinone oxydoreductase (NQO1) activity in genistein-and bLap-induced apoptosis in PC3 was investigated. Co-culture of PC3 cells with dicoumarol, a specific inhibitor of NQO1 activity, significantly reduced the cytotoxicity of β-Lapachone in PC3 cells, as reflected in the significant reduction in the percentage of treatment-induced apoptosis. Dicoumarol increased cell survival. These results implicate NQO1 as the main target in bLap-induced apoptosis in PC3, consistent with previous observations \[[@B26],[@B34],[@B40]\]. However, the fact that blocking of NQO1 did not confer 100% protection against induction of apoptosis indicates a possible alternate pathway in bLap-induced apoptosis. The present data indicate some involvement of caspase protease CPP32, though not with the same significance as NQO1. The activation of cysteine protease has been observed after bLap treatment \[[@B26]\]. Pink et al \[[@B26]\] reported activation of the cysteine protease in MCF-7 and T4D breast cancer cells in bLap-induced apoptosis. Contrary to the observation in bLap-induced apoptosis, blocking NQO1 activity did not significantly influence genistein-induced apoptosis in PC3 cells; implying NQO1 may not be the major target in genistein-induced apoptosis in PC3 cells. However, the overall data indicate a synergistic effect of genistein-bLap combination treatment of PC3 and, that the major target in the combination treatment-induced apoptosis in PC3 cells is NQO1. Investigation into genistein/bLap synergism in a number human cancer cells is on-going. Conclusion ---------- It is concluded from the data obtained that: i) both genistein and bLap exert growth inhibition effects in PC3 cells, with significant differences in chemosensitivity of PC3 to the two agents; ii) there is a manifestation of synergism between genistein-bLap combination treatment; iii) both genistein and bLap induce apoptosis in PC3 cells; iv) the major target in genistein-induced apoptosis in PC3 cells seems to be CPP32; v) the major molecular target in bLap-induced apoptosis in PC3 cells appear to be NQO1; vi) the major target in the genistein-bLap combination treatment-induced apoptosis appears to be NQO1; and vii) combination treatment appears significantly more efficacious than single treatments. More extensive studies are ongoing to delineate and clarify the molecular mechanisms underlying the combination effects. Materials and Methods ===================== Chemicals --------- Genistein isoflavone (Indoline Chemical Co. Summerville, NJ, USA) was constituted in DMSO (dimethylsulfoxide) solvent as 10, 20, 30, 50 and 70 μg/ml solutions (G~10-70~) and frozen at -37°C until used. β-lapachone (Sigma Scientific (St. Louis, MO, USA) was constituted in DMSO solvent as 1, 2, 3, 5 and 8 μM/ml solutions (bL~1-8~) and stored at -37°C. Dicoumarol (Sigma Scientific St. Louis, MO, USA) was constituted in DMSO as 50 μM/ml and frozen at -37°C until used. The caspase 3 inhibitor, DEVD-fmk and substrate DEVD-afc were purchased from Biovision (Palo Alto, CA). Culture media (RPMI 1640), antibiotics, trypsin-EDTA, and other reagents were purchased from Sigma scientific (St. Louis, MO, USA). Cell lines ---------- Human prostate adenocarcinoma (PC3) was a generous donation from Rambaugh-Goodwin Cancer Research Institute, Plantation FL. Cell Culture ------------ To assess the chemosensitivity of human prostate cancer cells to single and combination treatment with genistein (Gn) and β-lapachone (bLap), cells were sub-cultured under 5% CO~2~at 37°C for 48 hrs to reach 80--90% confluence. All cells were grown and maintained as monolayers in 25 m^2^tissue culture flasks (Sigma Scientific, St. louis, MO, USA) in RPMI 1640 containing 15 mM HEPES, and supplemented with 0.45% glucose (w/w), 5.0% FBS and 100 U^.^mL^-1^penicillin + 100 mg^.^ml^-1^streptomycin. The cells were then harvested by gentle scraping with a cell scraper. The cell suspensions were then grown at a density of 2.5 × 10^3^cells/well in 24-well microtiter plates (MTP) for 36 hr to allow adherence. The supernatants were discarded and the agents (Gn or bLap) were added over a range of 5 cytotoxic concentrations in single and combination treatments. In preliminary studies with bL~1-8~, the IC~50~ranged between 1.8--3 μM for a number of cells. Therefore in the present studies, 2.0 μM (bL~2~) was used in the combination studies with varying concentrations of genistein. All treatments were in triplicates. Dicoumarol was added to the cells and incubated for 4 hr before treatment with either genistein or β-lapachone alone or in combination. All plates were then incubated at 37°C in a humidified atmosphere of 5% CO~2~in air for a maximum of 72 hr. At 12, 24 and 36 hr of incubation, 100 μl of the supernatant from each well was gently aspirated and replenished with 100 μl of fresh media. The supernatants were stored at -37°C until assayed for lactate dehydrogenase (LDH) enzyme activity. At 36 hr incubation, the cells were harvested by trypsinization with trypsin-EDTA, and processed for post-treatment metabolic activity using cell viability and apoptotic assays as described. A. Cell Viability Assays ------------------------ ### A1.1 MTS assay MTS assay depends on mitochondrial enzyme reduction of MTS solution to detect and determine cell viability. The MTS cell proliferation assay is a colorimetric method for determining the number of viable cells in proliferation. It is composed of solutions of a tetrazolium compound \[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS\] and an electron coupling reagent (phenazine ethosulfate; PES). MTS is bioreduced by the cells into formazan product that is soluble in cell culture medium. Following cell culture as described above, 100 μL of cells were harvested from each treatment group and added to a 96 MTP followed by addition of 20 μl of MTS (2.5 mg/mL: Sigma Chemical Co) stock solution to each well. After 2 hr incubation under standard conditions of 5% CO~2~and 37°C, the purple formazan product (indicative of reduction of MTS) was visible. The absorbance was read on Multiskan biochromatic automated microplate reader (Multiskan, DC) at 490 nm. The signal generated (color intensity) is directly proportional to the number of viable (metabolically active) cells in the wells. Relative cell numbers can therefore be determined based on the optical absorbance (optical density, OD) of the sample. The blank values were subtracted from each well of the treated cells and controls; and the mean and standard error for each treatment (singles and combination) were calculated relative to the control:  where A~C~= absorbance of the control (mean value): A~T~= absorbance of the treated cells (mean value) A~B~= absorbance of the blank (mean value) ### A1.2 Trypan Blue exclusion assay For the Trypan blue exclusion test, cells were treated and cultured as described. They were harvested and Trypan blue dye solution was added to the cell suspensions. Total cell counts and viable cell number (survival rate) were determined by a standard hemocytometer procedure. Live-viable cells were seen as colorless (impermeable to the dye due to intact cell membrane) and dead cells were seen as blue (permeable to dye due to disruption of cell membrane):  A2. LDH assay ------------- Lactase dehydrogenase activity was measured by a non-radioactive protocol using the LDH cytotox kit (Cat. \#. 1644 793: Boehringer-Mannheim GmbG, Bochemica). The previously frozen supernatants were thawed for LDH determination. Briefly, 100 μL/well of each cell-free supernatant was transferred in triplicate into wells in a 96-well microtiter plate (MTP) and 100 μL of LDH-assay reaction mixture (Kit: dye-catalyst mixture) added to each well. After 90 min incubation under standard conditions the absorbance of the color generated was read on Multiskan biochromatic automatic microplate reader at 490 nm. The mean absorbance/optical density (OD) for each treatment group was calculated. The blank values were subtracted from each well and the mean percent treatment-induced cytotoxicity for each cell line and treatment type (single and combination) was calculated as:  Where: ABS~expt~= mean absorbance from the treated cells: ABS~low~= mean absorbance from controls (untreated cells)(spontaneous release of LDH) ABS~hi~= mean absorbance from Triton X-100 treated cells (standard/maximum LDH release)(positive control). B. Detection of Treatment-induced Apoptosis ------------------------------------------- Treatment-induced apoptosis was assessed by two independent assays, Annexin V-FITC assay and the DNA Fragmentation (TUNEL) assay. PC3 cells were treated and co-cultured with the test agents as previously described in this study; and the subjected to the apoptosis determination assays as below: B1.1 Annexin V-FITC assay ------------------------- Apoptosis-associated translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane in GC27 and K833 cells was assessed with the use of FITC-labeled Annexin V, a calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylserine; using AnnexinV-FITC Staining Kit (Boehringer Mannheim). Briefly, 100 μl aliquots of the previously prepared cell suspensions were centrifuged, and the cell pellets re-suspended in Annexin binding buffer, incubated with AnnexinV-FITC substrate; then cells were smeared onto microscope slides and either evaluated immediately with fluorescence microscope, or smears were fixed with 4% depolymerized paraformaldehyde and stored at -40°C for later examination as previously described \[[@B35]\]. Percentage of apoptosis in the cells was quantified based on morphological and fluorescence characteristics of apoptotic cells as previously described \[[@B5],[@B35],[@B36]\]. All tests were run in triplicates. B1.2 DNA Fragmentation (TUNEL) assay ------------------------------------ The presence of apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), using the ApopTag^R^kit (Boehringer Mannheim Co, Indianapolis, IN) as previously described \[[@B37]\]. The kit reagents detect apoptotic cells *in situ*by specific end labeling and detection of DNA fragments produced by the apoptotic process. To perform the TUNEL assay, slides of the PBS suspended cells were fixed with 4% paraformaldehyde for 30 minutes. The cells (slides) were then permeabilized with Triton X-100 at 4°C for 2 min; then flooded with TdT enzyme and digoxigenin-dUTP reaction buffer (TUNEL) reagent for 60 min in a humidity chamber at 37°C, washed with distilled water, incubated for 10 minutes with streptavidin-horseradish peroxidase complex. The stained mounted cells were examined at 100×, 200× and 400× magnification of the microscope (Olympus BH-2). Cell death was quantified by counting 150 cells in 5--7 separate fields of view per slide, and noting the percentage of apoptotic cells based on morphological appearance, as previously described \[[@B5],[@B36]\]. C Potential mechanism(s) of action ---------------------------------- The potential involvement of caspase-3 protease (CPP32) and/or the enzyme NQO1 \[NAD(P)H:quinone oxidoreductase\] in the molecular pathways of β-lapachone-and/or genistein-induced growth inhibition and apoptosis in PC3 cells were determined, after treatment of the cells as already described. C1.1 Caspase-3 expression/activity in treatment-induced apoptosis ----------------------------------------------------------------- In order to determine the potential role of caspase-3 proteases (CPP32) in the common pathways of β-lapachone and genistein-induced growth inhibition and apoptosis, human prostate cancer cell lines were treated as previously described above. The activity of caspase 3 was determined using a the fluorometric substrate DEVD-afc and caspase 3 inhibitor DEVD-fmk according to the protocol of the Caspase Activity Assay kit. Briefly, PC3 cells were treated and incubated as previously described. At 24, 48 and 72 hr cells were scrapped into suspension and centrifuged at 10,000 rpm for 10 min. The pellet was resuspended in 100 μl of lysis buffer and incubated at 4°C for 10 min, followed by centrifugation at 10,000 rpm for 10 min. Fifty μl aliquots of the supernatants were removed and placed in a 96-well microtiter plate (MTP) containing reaction buffer. The DEVD-afc substrate was added and the MTP was incubated at 37°C for 30 min. Activity was monitored with the linear cleavage and release of the afc side chain; and compared with a linear standard curve generated by the controls on the same MTP. C1.2 NQO1 Activity in treatment-induced apoptosis ------------------------------------------------- In order to determine the potential role of enzyme NQO1 \[NAD(P)H:quinone oxidoreductase\] in the molecular pathways of β-lapachone-and genistein-induced growth inhibition and apoptosis in human prostate cancer, PC3 cell lines were treated as previously described. Dicoumarol (3-3\'-methylene-bis (4-hydroxycou-marin) is a commonly used inhibitor of NQO1, which competes with NADH or NADPH for binding to the oxidized form of NQO1. Dicoumarol thereby prevents reduction and activation of various target quinines like β-lapachone. The cells, cultured as previously described, were treated concomitantly in single and combination treatments of varying concentrations β-lapachone (bLap), genistein (gen), and bLap-Gn combination with and without 50 μM dicoumarol as previously described. The treated cells were harvested and tested for treatment-induced apoptosis by the methods previously described in this study. Authors\' contributions ======================= JKD contributed 50% in all aspects of the research. All other authors contributed equally-50% All authors read and approved the final manuscript. Acknowledgments =============== The authors acknowledge the generous offer of cell lines by the Rambaugh-Goodwin Cancer Research Institute at Plantation FL. and thank Florida Atlantic University for partial support of this research.

PubMed Central

2024-06-05T03:55:47.470866

2004-8-17

PMC516041

Background ========== Successful implantation depends on the ability of the embryo to degrade the basement membrane of the uterine epithelium and to invade the uterine stroma. Cytotrophoblastic cells (CTB) are derived from trophoectodermal cells of the blastocyst. CTB ensue to become either the villous cytotrophoblastic cells which will proliferate and differentiate by fusion to form the syncytiotrophoblast, or they will stream out of the syncytiotrophoblast to form mononuclear multilayered invasive extravillous cytotrophoblastic cells. The temporal and spatial regulation of trophoblast invasion is mediated in an auto-and paracrinic way by trophoblastic and uterine factors \[[@B1]\]. Several factors have been studied, including hormones, cytokines and growth factors \[[@B2],[@B3]\]. Trophoblast invasion is facilitated by degradation of the extracellular matrix of the endometrium/decidua by various proteinases, among them, the matrix metalloproteinases (MMPs). The tissue inhibitors of matrix metalloproteinases (TIMPs) inhibit the activity of the MMPs by binding to the highly conserved zinc-binding site of active MMP \[[@B4]\]. Successful implantation and trophoblast invasion are closely linked to the expression of MMPs, which are able to degrade basement membranes. The gelatinases (gelatinase A: MMP-2: 72-kDa and gelatinase B: MMP-9: 92-kDa) which degrade collagen IV, the main component of the basement membrane, are expressed by trophoblast cells and are therefore regarded as key enzymes in the invasion process \[[@B5]\]. Several studies have shown that MMP-2 and MMP-9 synthesis and activation are required for trophoblast invasion \[[@B1],[@B5]-[@B9]\]. Some studies have found either MMP-9 \[[@B5],[@B7],[@B8]\], or MMP-2 \[[@B9]-[@B11]\] to be more pronounced during the first stage of trophoblast invasion. However, the exact changes in protease expression during the first trimester are still not clear. Xu et al \[[@B11]\] found differential expression of MMP-2 and -9 in first trimester trophoblast cells, with MMP-2 being the main gelatinase secreted until 9 w and hereafter MMP-9. In this study, trophoblast cells from first trimester were therefore divided into two groups according to their MMP secretion profile. The aim of this study was to examine the expression and importance of MMP-2 and -9 in human trophoblast invasion, and to test the similarity between primary trophoblast cell culture and the JAR choriocarcinoma cell-line. The JAR cell-line serves as a widely used model for 1^st^trimester trophoblasts \[[@B12]-[@B14]\]. The limited availability of 1^st^trimester trophoblast tissue often requires the use of such a model, and therefore a comparison study between JAR cell-line and 1^st^trimester trophoblasts is of significant importance to ensure similarity. This study shows a differential, dynamic importance of each gelatinase in trophoblast invasion during the 1^st^trimester. Methods ======= Cell culture ------------ The JAR (Jar, HTB 144) human choriocarcinoma line was established from a trophoblast tumor of the placenta (1988 American Type Culture Collection Catalogue). The JAR cells were a generous gift from Prof. Hochberg A Department of Biological Chemistry, Hebrew University, Jerusalem, Israel. JAR cells (1 × 10^4^cells/well) were cultured in M-199 medium (Beit-Ha\'Emek, Israel) containing 10% Fetal Calf Serum (FCS, Beit-Ha\'Emek, Israel) and penicillin/streptomycin (Beit-Ha\'Emek, Israel). Cell culture was maintained in a humidified atmosphere containing 5% CO~2~at 37°C. After 24 hours of culture to facilitate cell attachment, the medium was removed, and M-199 medium with 1.5% serum supplemented with antibiotics was added. The cells were cultured with various concentrations of: a) Forskolin 1--100 μM, b) Epidermal Growth Factor (EGF), 0.8--80 ng/ml (Sigma). Control consisted of M-199 with 1.5% serum alone. Cells were cultured for an additional 48 hours, and media were removed for analysis of MMP secretion and stored at -20°C until use. Cell count was performed with XTT in order to normalize MMP secretion to cell number. Isolation and cultivation of human cytotrophoblast cells -------------------------------------------------------- Human trophoblast cells were obtained from legal abortions (6 to 12 weeks of gestational age), with the approval of the local ethical committee (in compliance with the Helsinki Declaration) and the consent of the participating patients. Trophoblast cells were isolated as described previously in detail elsewhere \[[@B8],[@B9],[@B15],[@B16]\] with modifications. Briefly, tissues were digested by 0.25% trypsin (Sigma) and DNase I (Sigma) at 37°C, then trophoblast cells were separated from blood cells and decidua on a discontinuous Percoll gradient (Sigma) and immunopurified with magnetic antibody CD45RB (DAKO, Denmark). The cells were plated at 1--2 × 10^5^cells/well in 96-well plates or in Transwell plates (Corning) with M-199 medium supplemented with 1.5% FCS and 1% penicillin/streptomycin and kept in 5% CO2 at 37°C. Inducers (10 μM forskolin or 8 ng/ml EGF (chosen as working concentrations after a dose-response study in JAR cell-line) were added to medium, and after 48--72 h media were collected for analysis of MMP secretion and cell count was performed. This method supplies a 95--98% purity of trophoblastic cells, including all trophoblastic sub-groups. We verified the purity of trophoblast cells by using immunohistochemistry with specific antibodies to cytokeratin 7 (positive) and vimentin (negative), commonly used for indication of trophoblast purity \[[@B7],[@B8],[@B15]\]. Figure [1](#F1){ref-type="fig"} shows representative pictures of this analysis. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Representative immunohistochemical analysis of isolated placental cells (6--8 w) after 24 h in culture. (A) Cells stain positive for anti-human cytokeratin 7 (1:100, Biogenics). (B) Cells stain negative for anti-human vimentin (1:200, Zymed). Magnification: ×100. :::  ::: Cell count assay ---------------- Evaluation of cell proliferation was performed with XTT Reagent kit (XTT, cell proliferation kit, Beit-Ha\'Emek, Israel) according to manufactures protocol. This is based on the activity of mitochondria enzymes in live cells, reducing tetrazolium salts, XTT, into colored formazan compounds, which can be detected colorimetric with a spectrophotometer at 450 nm (ELISA reader). Dye absorbance is proportional to the number of cells in each well. Substrate-gel-electrophoresis (zymography) ------------------------------------------ In order to detect proteolytic activity in conditioned media (CM) collected after 48--72 h culture, substrate-gel-electrophoresis (Zymography) on gels containing gelatin as the substrate were used as was described by our previous manuscript \[[@B4]\]. Briefly, CM, was diluted in sample buffer (5% sodium dodecyl sulphate (SDS), 20% glycerol in 0.4 mol/l Tris, pH 6.8 containing 0.02% Bromophenol Blue without 2-mercaptoethanol) and electrophoresed, through a 10% polyacrylamid gel containing 0.5% gelatin (50 mg/ml). Afterwards gels were washed twice in 2.5% Triton X-100 for 15 min. and incubated for 24 h at 37°C in 0.2 mol/l NaCl, 5 mmol/l CaCl~2~, 0.2% Brij 35 and 50 mmol/l Tris, pH 7.5. The buffer was decanted and the gels stained with Coomassie Blue G in 30% methanol and 10% acetic acid for 10 min at room temperature on a rotary shaker. Stain was washed out with water until clear bands were seen. Areas where proteolytic activity degraded the gelatin were seen as absence of staining. Identification of each gelatinase band was done in accordance to their molecular weight and commercial standards (gelatinize A and B, 7 μl; Oncogene Science, Cambridge, MA, data not shown). These bands (proMMP) were quantified using the BioImaging gel documentation system (Dinco & Renum, Jerusalem, Israel) endowed with TINA software (Raytest, Staubenhardt, Germany). MMP secretion was expressed as percent of control. Matrigel invasion assay ----------------------- Matrigel invasion assay was prepared in our laboratory with modifications as described in detail elsewhere \[[@B17],[@B18]\]. Briefly, diluted 1:10 Matrigel (1 mg/ml) (BD Biosciences, Beit-Ha\'Emek, Israel) in serum free cell culture media was added to upper chamber of 24-well transwell plate, and incubated at 37°C 3--4 h for gelling. JAR Cells were harvested from tissue culture flasks by Trypsin/EDTA, washed and resuspended in 1.5% FCS in M-199 medium and added to upper wells at a density of 10^5^cells/well in 200 μl medium, while 500 μl medium was added to lower well. 1^st^trimester trophoblast were cultured in upper wells at a density of 2 × 10^5^cells/well in 100 μl medium. The same density of cells, in the absence or presence of activators, was seeded in a well without transwell and counted at time of the invasion assay, as reference of total cells. Preliminary studies found no significant matrigel-mediated changes in multiplication rates between 6--8 w and 9--12 w trophoblasts, whether seeded on matrigel or at plastic bottom of well (data not shown). Activators (10 μM Forskolin or 8 ng/ml EGF) and inhibiting MMP-2 or MMP-9 antibodies (Oncogene Cat. IM33L, Cat. IM09L; concentration as recommended by manufacture) were added to medium in upper and lower wells. Plates were incubated at 37°C for 36--48 hours, and then non-invaded cells on top of the transwell were scraped off with a cotton swab. The amount of invaded cells in the lower well as a percent of total seeded cells was evaluated with XTT Reagent kit. The percent of invasion was calculated as:  Invasion was expressed as Invasion Index (Percent of control). Statistical methods ------------------- Results are expressed as mean ± SEM of 5--10 independent experiments, each treatment performed in duplicates. Statistical analysis was performed using the SPSS statistical software. Student\'s t-test and \"one way analysis of variance\" (ANOVA) were used when appropriate. P \< 0.05 was considered significant. Immunohistochemistry -------------------- Immunohistochemistry was performed as previously described \[[@B19]\] using the Histostain-Plus kit (Zymed laboratories Inc., USA). Briefly, cultured cells were fixed with cytospray for 20 min and quenched with 3% hydrogen peroxidase in methanol to eliminate endogenous peroxidase activity. The slides were washed, blocked and incubated at room temperature with primary antibodies (mouse anti-human cytokeratin-7 (1:100, clone OVTL12/30, Biogenics) and mouse anti-human vimentin (1:200, clone V9, Zymed laboratories Inc., USA). Secondary antibody used: Histostain-Plus broad-spectrum biotinolated second antibody (Zymed laboratories Inc., USA). Slides were then developed with a substrate-chromagen solution of aminoethyl carbazole (Zymed laboratories Inc., USA). Results ======= Relative secretion profile of proMMP-2 and proMMP-9 in 6--8 W, 9--12 W trophoblasts and in JAR cells without treatment ---------------------------------------------------------------------------------------------------------------------- JAR cells (1 × 10^4^cells/well), 1^st^trimester trophoblast cells 6--8 w and 9--12 w (1--2 × 10^5^cells/well) were incubated for 48 hours, then media collected and gelatinase secretion analyzed by zymography. Figure [2](#F2){ref-type="fig"} summarizes the results. In 6--8 w trophoblast, proMMP-2 secretion was only slightly dominant (statistically not significant) compared to proMMP-9, 53.2% vs. 46.8% respectively (SEM ± 4.3). In 9--12 w trophoblasts the picture was different, with proMMP-9 being dominant (P \< 0.05) over proMMP-2, 61.7% vs.38.3% respectively (SEM ± 4.6). In JAR cells proMMP-2 was dominant (P \< 0.05), whereas proMMP-9 only had a small contribution to the gelatinase secretion, 90.2% vs.9.8% respectively (SEM ± 1.4). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **(A)**Representative secretion pattern of proMMP-2 (72 kD) and proMMP-9 (92 kD) in 6--8 w, 9--12 w trophoblasts and in JAR cells without treatment as examined with zymography. **(B)**Bar graph describing the relative percentage of gelatinases secretion, representing mean ± SEM from 5 independent experiments. Black bars represents proMMP-2 and white bars represent proMMP-9, \*P \< 0.05. :::  ::: Dose-dependent effect of forskolin and EGF on the proMMP-2 and -9 secretion by JAR cells ---------------------------------------------------------------------------------------- JAR cells (1 × 10^4^cells/well) were incubated 48 hours in the absence or presence of forskolin (1 μM, 10 μM or 100 μM) or EGF (0.8 ng/ml, 8 ng/ml or 80 ng/ml) and media was analyzed by zymography for gelatinase secretion. Fig [3](#F3){ref-type="fig"} summarizes the results. 10 μM forskolin significantly enhanced secretion of proMMP-2 compared to control (128.5% ± 12.0, P \< 0.05). Forskolin (1 μM and 10 μM) significantly enhanced proMMP-9 secretion compared to control (131.3% ± 11.1, and 183.2% ± 27.9, P \< 0.05, respectively) (Fig. [3A](#F3){ref-type="fig"}). 8 ng/ml EGF significantly enhanced secretion of proMMP-2 (133.5% ± 15.0, P \< 0.05) and of proMMP-9 (223.9% ± 30.4, P \< 0.05) compared to control. 80 ng/ml EGF, on the contrary, decreased proMMP-2 and proMMP-9 secretion compared to control (65.1% ± 18.3, and 66.6% ± 3.7, P \< 0.05) (Fig. [3B](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Dose-dependent effect of Forskolin and EGF on gelatinase secretion in JAR cells. JAR cell (1 × 10^4^/well) were incubated 48 hours with or without Forskolin (1 μM, 10 μM or 100 μM) or EGF (0.8 ng/ml, 8 ng/ml or 80 ng/ml) and media collected for measurement of Gelatinase secretion. (**A)**Bar graph, representing mean ± SEM of 10 independent experiments, of cells treated with forskolin. (**B)**Bar graph, representing mean ± SEM of 10 independent experiments, of cells treated with EGF. Black bars represent proMMP-2 and gray bars represent proMMP-9. \*P \< 0.05 vs. control. :::  ::: Effect of forskolin on proMMP-2 and proMMP-9 secretion by JAR cells, 1^st^trimester trophoblast cells 6--8 w and 9--12 w of gestation ------------------------------------------------------------------------------------------------------------------------------------- JAR cells (1 × 10^4^cells/well), 1^st^trimester trophoblast cells 6--8 w and 9--12 w (1--2 × 10^5^cells/well) were incubated for 48 hours in the absence or presence of forskolin 10 μM. Figure [4A](#F4){ref-type="fig"} shows representative zymography gels. Figure [4B](#F4){ref-type="fig"} and [4C](#F4){ref-type="fig"} summarizes the results. Gelatinase secretion was enhanced by forskolin in all cell groups: Forskolin significantly increased proMMP-2 secretion in JAR cells (144.3% ± 8.8, P \< 0.05), in 6--8 w trophoblast (125.9% ± 6.3, P \< 0.05) and in 9--12 w trophoblast (169.7% ± 20.3, P \< 0.05) as compared to control (Fig. [4B](#F4){ref-type="fig"}). Forskolin also significantly increased proMMP-9 secretion in JAR cells (226.6% ± 50.7, P \< 0.05), in 6--8 w trophoblast (128.4% ± 6.4, P \< 0.05), and in 9--12 w trophoblast (120.3% ± 4.5, P \< 0.05) as compared to control (Fig. [4C](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Secretion of proMMP-2 and proMMP-9 after 48 hours incubation of JAR cells, 1^st^trimester trophoblast cells 6--8 week or 1^st^trimester trophoblast cells 9--12 week in medium in absence or presence of 10 μM Forskolin. (**A)**Representative zymography gels. (**B)**Bar graph, representing mean ± SEM from 10 independent experiments detecting proMMP-2 (72 kD). (**C**) Bar graph, representing mean ± SEM from 10 independent experiments detecting proMMP-9 (92 kD). White bars represent control medium of cells without treatment. Black bars represent medium from cells with forskolin treatment. \*P \< 0.05 vs. control. :::  ::: Effect of EGF on proMMP-2 and proMMP-9 secretion by JAR cells, 1^st^trimester trophoblast cells 6--8 w and 9--12 w of gestation ------------------------------------------------------------------------------------------------------------------------------- JAR cells (1 × 10^4^cells/well), 1^st^trimester trophoblast cells 6--8 w and 9--12 w (1--2 × 10^5^cells/well) were incubated for 48 hours in the absence or presence of EGF 8 ng/ml. Figure [5A](#F5){ref-type="fig"} shows representative zymography gels. Figure [5B](#F5){ref-type="fig"} and [5C](#F5){ref-type="fig"} summarizes the results. EGF significantly increased proMMP-2 secretion in JAR cells (130.4% ± 13.1, p \< 0.05), in 6--8 w trophoblast (141.22% ± 14.8, P \< 0.05) and also in 9--12 w trophoblast (168.3% ± 18.2, P \< 0.05) as compared to control (Fig. [5B](#F5){ref-type="fig"}). EGF significantly increased proMMP-9 secretion in JAR cells (187.8% ± 27.3, P \< 0.05), in 6--8 w trophoblast (138.8% ± 10.3, P \< 0.005), and in 9--12 w trophoblast (117.3% ± 3.8, P \< 0.05) as compared to control (Fig. [5C](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Secretion of proMMP-2 and proMMP-9 after 48 hours incubation of JAR cells, 1^st^trimester trophoblast cells 6--8 week or 1^st^trimester trophoblast cells 9--12 week in medium in absence or presence of 8 ng/ml EGF. (**A**) Representative zymography gels. (**B)**Bar graph, representing mean ± SEM from 10 independent experiments detecting proMMP-2 (72 kD). (**C**) Bar graph, representing mean ± SEM from 10 independent experiments detecting proMMP-9 (92 kD). White bars represent control medium of cells without treatment, black bars represent medium from cells with EGF treatment. \*P \< 0.05 vs. control. :::  ::: Effect of forskolin on cell invasion properties in JAR cells, 1^st^trimester trophoblast cells 6--8 w and 9--12 w of gestation ------------------------------------------------------------------------------------------------------------------------------ JAR cells (10^5^cells/well), 1^st^trimester trophoblast cells 6--8 w and 9--12 w (2 × 10^5^cells/well) were incubated for 36--48 hours in the absence or presence of forskolin 10 μM on top of Transwell wells containing a transwell membrane coated with matrigel. Forskolin (10 μM) significantly enhanced trophoblast invasion in all cell groups. Forskolin increased cell invasion in JAR cells (110.6% ± 3.4, P \< 0.05) (Fig. [6A](#F6){ref-type="fig"}), in 6--8 w 1^st^trimester trophoblast (189.7% ± 14.2, P \< 0.05) (Fig. [6B](#F6){ref-type="fig"}) and in 9--12 w (302.4% ± 56.0, P \< 0.05) as compared to control (Fig. [6C](#F6){ref-type="fig"}). The addition of inhibitory MMP-2 antibody significantly decreased invasion of control cells of JAR cells (86.5% ± 3.6, P \< 0.05) and of 6--8 w (73.8 ± 10.6, P \< 0.05) but did not affect 9--12 w trophoblasts. In forskolin-induced cells the presence of inhibitory MMP-2 antibody caused a significant decrease in invasion of JAR cells compared with induced cells alone (96.6% ± 1.5 versus 110.6% ± 3.4, respectively P \< 0.05), of 6--8 w trophoblasts compared with induced cells alone (114.1% ± 24.6 versus 189.7% ± 14.2, respectively, P \< 0.05) and of 9--12 w trophoblasts compared with induced cells alone (188.8% ± 18.4 versus 302.4% ± 56.0, respectively, P \< 0.05) (Figure. [6A,6B](#F6){ref-type="fig"} and [6C](#F6){ref-type="fig"}) ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Cell invasion ability of JAR cells, 1^st^trimester trophoblast cells 6--8 week or 1^st^trimester trophoblast cells 9--12 week tested with Transwell Invasion Assay. (**A)**represents JAR cells, (**B**) 1^st^trimester trophoblast cells 6--8 week and (**C**) 1^st^trimester trophoblast cells 9--12 week. Cells were treated with 10 μM Forskolin and incubated 36 hours on Matrigel coated membrane, with or without MMP-2 or MMP-9 inhibitory antibodies. Cells that invaded the membrane to lower well were counted with XTT. Results represent mean ± SEM from 10 independent experiments. Black bars represent control (cells without treatment or cells treated with Forskolin) with no antibodies. Gray bars represent cells (without treatment or treated with Forskolin) with addition of MMP-2 inhibitory antibody. White bars represent cells (without treatment or treated with Forskolin) with addition of MMP-9 inhibitory antibody. ANOVA for all groups results in p \< 0.05, post test confirmed the t test results. \*P \< 0.05 :::  ::: The addition of inhibitory MMP-9 antibody surprisingly increased invasion of control cells of 6--8 w and 9--12 w trophoblast (132.0 ± 8.3 and 134.9 ± 15.2, respectively, P \< 0.05) as compared to control, and only in 9--12 w forskolin-induced cells caused a significant decreased invasion compared with induced cells alone (201.5% ± 6.3 versus 302.4% ± 56.0, respectively, P \< 0.05). ANOVA post test confirmed the t test results (Fig. [6A,6B](#F6){ref-type="fig"} and [6C](#F6){ref-type="fig"}). Effect of EGF on cell invasion properties in JAR cells, 1^st^trimester trophoblast cells 6--8 w and 9--12 w of gestation ------------------------------------------------------------------------------------------------------------------------ JAR cells (10^5^cells/well), 1^st^trimester trophoblast cells 6--8 w and 9--12 w (2 × 10^5^cells/well) were incubated for 36--48 hours in the absence or presence of EGF 8 ng/ml on top of Transwell wells containing a transwell membrane coated with matrigel. The results of EGF resembled those of forskolin. EGF enhanced trophoblast invasion in all cell groups. EGF increased cell invasion in JAR cells (112.6% ± 2.9, P \< 0.05) (Fig. [7A](#F7){ref-type="fig"}), in 6--8 w 1^st^trimester trophoblast (157.9% ± 10.4, P \< 0.05) (Fig. [7B](#F7){ref-type="fig"}) and in 9--12 w (192.4% ± 10.5, P \< 0.05) compared to control (Fig. [7C](#F7){ref-type="fig"}). In EGF-induced cells the presence of inhibitory MMP-2 antibody caused a significant decrease in invasion of JAR cells (100.2% ± 0.8 versus 112.6 ± 2.9, P \< 0.05), of 6--8 w trophoblasts (129.7% ± 8.0 versus 157.9 ± 10.4, P \< 0.05) and of 9--12 w trophoblasts (161.1% ± 22.0 versus 192.4 ± 10.5, P \< 0.05) compared with induced cells alone (Figure. [7A,7B](#F7){ref-type="fig"} and [7C](#F7){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Cell invasion ability of JAR cells, 1^st^trimester trophoblast cells 6--8 week or 1^st^trimester trophoblast cells 9--12 week tested with Transwell Invasion Assay. (A) represents JAR cells, (B) 1^st^trimester trophoblast cells 6--8 week and (C) 1^st^trimester trophoblast cells 9--12 week. Cells were treated with 8 ng/ml EGF and incubated 36 hours on Matrigel coated membrane, with or without MMP-2 or MMP-9 inhibitory antibodies. Cells that invaded the membrane to lower well were counted with XTT. Results represent mean +SEM from 10 independent experiments. Black bars represent control (cells without treatment or cells treated with EGF) with no antibodies. Gray bars represent cells (without treatment or EGF treated) with addition of MMP-2 inhibitory antibody. White bars represent cells (without treatment or EGF treated) with addition of MMP-9 inhibitory antibody. ANOVA for all groups results in p \< 0.05, post test confirmed the t test results. \* P \< 0.05. :::  ::: The addition of inhibitory MMP-9 antibody affected 9--12 w EGF--induced cells and caused a significant decreased invasion compared with induced cells alone (169.7% ± 18.1 versus 192.4 ± 10.5, respectively, P \< 0.05), ANOVA post test confirmed the t test results (Figure. [7A,7B](#F7){ref-type="fig"} and [7C](#F7){ref-type="fig"}). The inhibitory affect of MMP-2/-9 antibody on control cells was described in the previous section. Discussion ========== Trophoblastic invasion of the endometrium is highly regulated by interrelated reactions between invasion-promoting factors, such as cytokines, growth factors, MMP-2 and MMP-9, and invasion-inhibiting factors such as TIMPs. In the current study, the secretion and activity of MMP-2 and MMP-9 in human cytotrophoblastic cells from different weeks of gestation was measured and compared with a choriocarcinoma cell-line. A differential secretion profile of proMMP-2 and -9 was found between 6--8 w and 9--12 w as expressed by a shift in the relative proportion of each gelatinase. Elevated proMMP-9 secretion in 9--12 w was observed, compared with both 6--8 w and JAR cells. These results are consistent with those of Xu et al \[[@B11]\], who found MMP-2 production to be dominant between 6--8 weeks of gestation and then declining, whereas MMP-9 production significantly increased from 8 to 11 weeks, with a shift of dominant gelatinase from MMP-2 to MMP-9 from 9 week of gestation. MMP-2 is predominant in human preimplantation embryos \[[@B20],[@B21]\], whereas MMP-9 is dominant in the third trimester \[[@B6]\]. Niu et al \[[@B22]\] reported a dominance of MMP-2 secretion over MMP-9 from 1^st^trimester villous tissue, and a dramatically decrease in MMP-2 levels in the second trimester. These results support our findings of a dynamic gelatinase secretion profile during the 1^st^trimester. In our study, MMP secretion was induced by two separate signal pathways: PKA and PTK. Several factors with importance in embryo implantation act via the cAMP-protein kinase A (PKA) signal transduction pathway, including human chorion gonadotropin (hCG), the primary signal of an implanting pregnancy \[[@B23]\]. Forskolin is a prototypical stimulator of the cAMP pathway by direct activation of adenylate cyclase \[[@B24]\]. In this study, forskolin was found to significantly enhance proMMP-2 secretion in JAR choriocarcinoma cell-line, 6--8 w and 9--12 w trophoblasts. Forskolin also enhanced proMMP-9 secretion, in choriocarcinoma cells and in 1^st^trimester trophoblast (from both groups). This indicates that forskolin might influence trophoblast cells invasiveness by enhancing the secretion of gelatinases. Zymography measures all forms of MMP (active and inactive) and therefore does not reliably represent the true physiological activity, which is influenced by the presence of MMP inhibitors and activators. In order to examine the role of the gelatinases in the implantation process, we examined cell invasion after PKA stimulation in JAR cells, 6--8 and 9--12 w trophoblasts. Our results showed a significant increase in invasive ability in JAR cells and in early and late 1^st^trimester trophoblasts, after stimulation with forskolin. In order to detect the contribution of each gelatinase to this invasive process, inhibitory antibodies to MMP-2 and MMP-9 were added to cell culture and cell invasive ability examined. MMP-2 inhibitory antibody caused a significant decrease in cell invasion in JAR cells, 6--8 w and 9--12 w trophoblasts treated with forskolin, whereas MMP-9 inhibitory antibody only caused a decrease in 9--12 w trophoblasts. This indicates, that most probably MMP-2 and not MMP-9 is the key-enzyme in the invasion process of JAR cells and early 1^st^trimester trophoblasts stimulated by forskolin, whereas MMP-9 together with MMP-2 plays a role in late 1^st^trimester trophoblasts. Only a few published data describe the relationship between forskolin and trophoblast invasion. Human chorionic gonadotropin acts via cAMP, and is considered a sign of differentiation of trophoblasts to syncytiotrophoblast. The hCG receptor was shown to be expressed on invasive trophoblast and in choriocarcinoma cells, and hCG was found to increase *in vitro*invasion and migration of a trophoblastic cell line, an effect that was also mimicked by forskolin \[[@B23]\]. Several studies demonstrated a positive correlation between hCG level and successful implantation \[[@B25]\] or inappropriate implantation/ invasion associated with the development of preeclampsia \[[@B24]\] or gestational trophoblastic tumors \[[@B27]\]. To the best of our knowledge, our study is the first to report the effect of forskolin on MMPs in first trimester trophoblastic cells. In addition, this study also distinguishes between the contributions of each of the gelatinases to the invasive capacity enhanced by forskolin in trophoblastic cells. Epidermal Growth factor (EGF) plays a major role in placental implantation, growth and differentiation and is regarded a paracrinic factor modifying the implantation process. EGF acts on trophoblasts via a specific receptor (EGFR) from the tyrosine kinase receptor family \[[@B28]\]. EGF is secreted from the endometrium during the implantation window, in which the embryo also expresses EGFR \[[@B29]\], and expressed in placenta from 1^st^throughout third trimester \[[@B30]\] In this study EGF stimulated secretion of proMMP-2 in JAR cells, 6--8 w and 9--12 w trophoblasts and also enhanced the secretion of proMMP-9 in JAR, in 6--8 w trophoblasts and in 9--12 w trophoblasts. Our results therefore indicate, that MMP-2 and MMP-9 secretion by trophoblastic cells may be stimulated through the PTK pathways during the first trimester. We noted that EGF at a high concentration (80 ng/ml), in contrast, decreased proMMP-2 and -9 secretion in JAR cells. This result corresponds with previous published data regarding a dual, concentration dependent effect of EGF on cell functions \[[@B31],[@B32]\]. In order to examine the role of the gelatinases in the implantation process, we examined cell invasion after PTK stimulation in JAR cells, 6--8 and 9--12 w trophoblasts. Our results showed a significant increase in invasion ability in JAR cells and in early and late 1^st^trimester trophoblasts, after stimulation with EGF. In EGF-stimulated JAR cells and in 6--8 w and 9--12 w trophoblasts inhibitory MMP-2 antibody decreased cell invasion, whereas inhibitory MMP-9 antibody caused a significant decrease in invasion only in 9--12 w trophoblasts. We thereby showed, that in EGF stimulated cells as well, MMP-2 is the key-enzyme in the invasion process *in vitro*in JAR cell and in early 1^st^trimester trophoblasts, whereas in late 1^st^trimester trophoblasts both MMP-2 and MMP-9 have a role. EGF was found to induce changes in morphology and to increase invasive capacity of first trimester trophoblasts, whereas later gestational cytotrophoblasts (2^nd^trimester), whose invasive capacity is diminished, are much less affected \[[@B29]\]. EGF was also found to increase MMP-9 secretion by cytotrophoblasts \[[@B33]\]. In JAR control cells inhibitory, MMP-2 antibody also decreased invasion, whereas MMP-9 antibody had no affect, indicating that the basic invasive ability of these choriocarcinoma cells is mainly due to MMP-2 and not to MMP-9. Inhibition of MMP-2 in control cells also decreased invasion in 6--8 w trophoblast, but not in 9--12 w, indicating again the importance of MMP-2 in early (6--8 w) trophoblast invasiveness. Surprisingly inhibition of MMP-9 in 6--8 w and 9--12 w trophoblasts without treatment caused an increase in invasion. We speculate that this may be due to a release of other proteinases, including other MMPs, since MMP-9 is known to dimerize \[[@B34],[@B35]\] All in all we found, that in early 1^st^trimester trophoblasts (6--8 w), MMP-2 is the major gelatinase participant in cell invasion, whereas in later 1^st^trimester trophoblasts (\>9 w) both MMP-9 and MMP-2 most probably participate in cell invasion, however we cannot exclude the possibility of other MMP family members participating in this process. Isaka et al \[[@B9]\] have shown that invasive ability of early first trimester trophoblast was inhibited by MMP-2 antibody in a dose dependent manner, thereby suggesting that the invasive ability of trophoblasts may be regulated by the enzyme activity of gelatinases, especially MMP-2. This study supports ours in the involvement of MMP-2 in trophoblast cell invasion. MMP-2 was found to be located in invasive evCTB in 1^st^trimester placenta \[[@B9],[@B10],[@B36]\], whereas MMP-9 was located in the non-invasive vCTB \[[@B9]\]. In contrast, several studies have found MMP-9 to be the key-enzyme in trophoblast invasion *in vitro*\[[@B1],[@B5],[@B7]\]. We speculate, that the main reason for this controversy of results comes from the dynamic gelatinase expression during the 1^st^trimester, as earlier discussed. The choice of pathway for stimulation of cell invasion may also contribute to a difference in results. We used stimulation through the PKA and PTK pathways; whereas other groups used the PKC pathway \[[@B7],[@B8]\]. Various stimulators, inducing different signal pathways, are likely not to affect the same enzymes in an identical manner, and thereby can result in varying dominant enzymes. We found, that MMP-2 is also the key-enzyme in JAR cell invasion; therefore JAR cells resemble early 1^st^trimester trophoblasts in cell invasive ability and in MMP secretion profile and differ from late 1^st^trimester trophoblast in these parameters. It has been documented, that JAR-trophoblast cells have the ability to invade in vivo \[[@B37],[@B38]\]. We chose this cell-line because it provides a large number of uniform cells and preserve the ability to differentiate into syncytiotrophoblast-like cell in vitro \[[@B39],[@B40]\]. Other studies showed different compartment in vitro between choriocarcinoma cell-lines and human first trimester trophoblast in the regulation of invasion \[[@B36],[@B37]\]. The study of JAR cell invasion may therefore represent only partly the aspects and mechanisms of the in vivo situation of invasion, where many cell types are involved. Conclusions =========== We showed that forskolin and EGF stimulate proMMP-2 and -9 secretion from trophoblasts, and that there is a differential, dynamic importance of each gelatinase in trophoblast invasion during 1^st^trimester. We suggest that MMP-2 is the key-enzyme in JAR and early 1^st^trimester (6--8 w) trophoblast invasion, whereas both MMP-2 and -9 are important for late (9--12 w) trophoblast invasion. Acknowledgement =============== We thank Desoye G, clinic of Obstetrics and Gynaecology, Austria for generously providing the protocol of trophoblast isolation.

PubMed Central

2024-06-05T03:55:47.473162

2004-8-4

PMC516042

Background ========== For many years, estrogen actions have been presumed to be mediated almost exclusively through the regulation of target gene transcription by a chromosomal bound estrogen receptor. These genomic estrogen effects are the well described interactions between the estrogen receptor and adapter transcription factors that result in activation or inhibition of the basal transcription protein machinery. However, there is a growing body of evidence that several rapid estrogen effects are non-transcriptional in nature. These rapid estrogen effects include changes of calcium flux in several cell types \[[@B1]-[@B3]\], MAPK activation \[[@B4],[@B5]\], cAMP levels \[[@B6],[@B7]\], and nitric oxide release \[[@B8]\]. That many of these effects are mediated by a membrane-localized estrogen receptor has been postulated for some time \[[@B9],[@B10]\], but the majority of evidence supporting this hypothesis is indirect, relying on the induction of these non-genomic effects using estrogen covalently conjugated to BSA by a 6 atom hydrocarbon tether (E~2~-BSA) \[[@B11],[@B12]\]. However, the relative binding efficiency of these conjugates is low and concern has been raised regarding the use of these conjugates as direct surrogates for estrogen \[[@B13]\]. A recent report added to this controversy by showing that commercially available E~2~-BSA is contaminated by unconjugated free E~2~and a series of binding experiments demonstrated that E~2~-BSA was unable to bind to ER after the contaminant E~2~was removed. \[[@B14]\]. These findings contradict studies where fluorescein-labeled E~2~-BSA (E~2~-BSA-FITC) specifically bound to a putative ER on the cell membrane \[[@B15]-[@B17]\]. Elucidation of novel membrane-associated ER effects is crucial to our understanding of the non-genomic signaling pathways of ER and other hormone receptors. Hormone-conjugated BSA is an important tool in this pursuit. We believe the contradictory results are explained by differences in the rates of binding of the bulky E~2~-BSA and E~2~with the ER. We show that pre-incubation of E~2~-BSA with ERα results in a highly significant decrease in the binding of ^3^H-E~2~. The binding of ^3^H-E~2~with ERα is unaffected by the simultaneous addition of E~2~-BSA. We also demonstrate that fluorescein conjugated E~2~-BSA binds to the membrane of cells that endogenously produce ERα and to HeLa cell lines stably expressing mERα. Results ======= E~2~-BSA binding to purified estrogen receptor ---------------------------------------------- Although E~2~is covalently attached to BSA using a relatively long six atom hydrocarbon tether, the bulky BSA moiety of E~2~-BSA still may be interfering with the binding between the estrogen molecule and the estrogen receptor. This would result in an increase in the energy of activation required for E~2~-BSA binding. If so, increasing the reaction time would allow for the establishment of an equilibrium between bound and free forms of E~2~-BSA, maximizing the amount of E~2~-BSA bound to the receptor. To test this hypothesis, E~2~-BSA free of contaminant E~2~was prepared by ultrafiltration. Competition between the purified E~2~-BSA and labeled E~2~for binding to purified ERα was determined after E~2~-BSA was pre-incubated with ERα and also when added at the same time as labeled E~2~. As shown in figure [1](#F1){ref-type="fig"}, concurrent addition of labeled E~2~and E~2~-BSA had no effect on labeled E~2~binding. However, a four-hour pre-incubation of E~2~-BSA with ER significantly decreased E~2~binding. These results suggest that the large BSA molecule retards, but does not prevent binding of E~2~-BSA. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Pre-incubation of purified hERα with E~2~-BSA competes for estradiol binding.**Purified ERα was incubated with E~2~(solid line) or E~2~-BSA (dotted line) for four hours before (a) or concurrently with (b) the addition of labeled E~2~. Incubation was continued for another 2 h at room temperature, and at the end of this period, specific binding was determined by adsorption, removal, and counting of free labeled E~2~. :::  ::: E~2~-BSA binding to ER in intact cells -------------------------------------- Non-genomic actions of the estrogen receptor are now well established. Several investigators have demonstrated that fluorescein labeled E~2~-BSA (E~2~-BSA-FITC) binds to the cell membrane, suggesting that a form of the estrogen receptor is present within the cell membrane and capable of binding to extracellular E~2~. Specific binding of E~2~-BSA-FITC to this membrane-localized form of the ER would further establish that E~2~conjugated to the BSA molecule is capable of binding to the ER. To examine this possibility, E~2~-BSA-FITC binding studies were performed with MC7 cells that contain endogenous ERα and with ER-deficient HeLa cells stably transfected with the ERα (HeLa-ERα). Expression of ERα within the HeLa cells was established by demonstrating specific binding of labeled E~2~to HeLa-ERα, but not native HeLa cells (figure [2](#F2){ref-type="fig"}). Scatchard analysis of the binding of E~2~to HeLa-ERα cells showed that although weakly expressed, the Kd for the expressed ERα was 7.04 nM, similar to published values (figure [3](#F3){ref-type="fig"}). HeLa-ERα cells, but not native HeLa cells, exhibited fluorescent staining of the cell membrane after incubation with E~2~-BSA-FITC (figure [4](#F4){ref-type="fig"}). The heterogeneous staining pattern reflected the low level of ERα expression. This fluorescence was not seen when HeLa-ERα cells were incubated with BSA conjugated to fluorescein alone (data not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Whole cell binding of estradiol to MC7 cells and HeLa cells stably transfected with ERα.**2 × 10^6^cells were incubated with ^3^H-17β-estradiol (10^-8^M) in the absence (solid) and presence (white) of 100 fold excess of unlabeled estradiol for 15 minutes at room temperature, washed, and placed on ice for 30 minutes. Cells were then pelleted, lysed and counted. Results are expressed as the mean +/- the SEM of 3 experiments (\* p \< 0.01) :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Estradiol binding to HeLa cells stably transfected with ERα.**Subconfluent HeLa-ERα cells were trypsinized and aliquots (2 × 10^6^cells) incubated with several concentrations of ^3^H-17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. a) Representative binding results of 3 independent experiments with total binding (solid box), non-specific binding (open box), and specific binding (triangle). b) Scatchard analysis of binding results. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Membrane localization of ERα.**HeLa cells stably transfected with ERα(a) and native HeLa cells (b) were incubated with fluorescein labeled membrane impermeable BSA conjugated to estradiol (E~2~-BSA-FITC) and visualized under phase contrast bright field and with UV light with an excitation filter for FITC. :::  ::: To establish the specificity of E~2~-BSA binding, MC7 cells and HeLa-ERα cells were incubated with E~2~-BSA-FITC after pre-incubation with various concentrations of E~2~and the anti-estrogen ICI-182,780. As shown in figure [5](#F5){ref-type="fig"}, fluorescence was lost in both cell types in a dose dependent manner with increasing concentrations of E~2~. Fluorescence was almost completely eliminated by pre-incubation with the specific competitor ICI-182,780. BSA conjugated to FITC alone did not bind. These results suggest that estrogen covalently bound to BSA can bind to ER in a biologically significant manner. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **E~2~competition with E~2~-BSA-FITC binding.**MC7 cells (black bars) or HeLa-ERα (white bars) were incubated with vehicle, various concentrations of E~2~, or ICI 182,780 (10^-8^M) for 30 minutes and then incubated an additional 30 minutes with E~2~-BSA-FITC or BSA-FITC alone (grey bar). Cells were fixed, and visualized by confocal microscopy. Digitized images were inverted to black on white and pixel density for each cell was determined by averaging the density across the cell membrane at four orthogonal points. Each bar represents \>20 cells counted +/- SEM. (\* p \< 0.05). :::  ::: The possibility that E~2~-BSA-FITC could be degraded during incubation with intact cells was examined using HPLC. E~2~-BSA-FITC was incubated in empty wells or wells containing MC7 cells under the same conditions employed for the binding studies described above. Media was removed from the cells and HPLC performed with a reverse phase column. Peaks were visualized using a scanning fluorescence detector. Aqueous solutions of E~2~-BSA-FITC produced a single peak with a retention time of 5.5 minutes using a methanol-water gradient from 80% to 50% over 30 minutes at 1 ml/minute. E~2~and E~2~-BSA did not fluoresce at the excitation and emission wavelengths used (data not shown). Spectra obtained from media containing E~2~-BSA-FITC alone and media containing E~2~-BSA-FITC incubated with MC7 cells are shown in figure [6](#F6){ref-type="fig"}. The average area under the curve for E~2~-BSA-FITC was the same (p \< 0.05) for solutions incubated in the presence (44,556 +/- 432) and absence (43,436 +/- 289) of MC7 cells (p, 0.05). These results demonstrate that E~2~-BSA-FITC is stable under the culture conditions employed for the binding experiments. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Stability of E~2~-BSA-FITC.**E~2~-BSA-FITC (10^-8^M in estrogen) was placed in empty wells or wells containing MC7 cells and incubated for 30 minutes at 4°C. E~2~-BSA-FITC was detected by reverse phase HPLC using a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at 1.0 ml/min. The assay was run in triplicate. Representative spectra are shown for E~2~-BSA-FITC alone (A) and E~2~-BSA-FITC incubated with MC7 cells (B). :::  ::: Discussion ========== The cellular effects elicited by estrogen \[[@B11],[@B12],[@B18]-[@B20],[@B3]\] testosterone \[[@B3],[@B21],[@B22]\] and progesterone \[[@B23]-[@B25]\] covalently conjugated to membrane impermeable BSA have been attributed to non-genomic actions mediated by membrane associated hormone receptors. The use of these reagents for this purpose remains controversial for several reasons. A recent report demonstrated that E~2~-BSA does not bind to purified ER in competition assays with labeled E~2~\[[@B14]\]. The studies were performed when E~2~-BSA or cold E~2~were added concurrently with labeled E~2~. We obtained similar results under these conditions. However, pre-incubation of E~2~-BSA with purified ER results in significant competition with labeled E~2~. These conflicting results may be explained by differences in the rate of binding between E~2~and E~2~-BSA. E~2~-BSA is a large, bulky molecule similar in size to the ER and is probably spherical in general structure as is the parent BSA molecule. The BSA protein conformation immediately adjacent to the covalently bound estrogen undoubtedly provides substantial steric hindrance to the proper presentation of conjugated E~2~to the binding pocket of ER. The increased size of the E~2~-BSA molecule would also reduce the rate of its diffusion compared with the smaller E~2~. Correct orientation of E2 in the ER binding pocket is also impeded by the restraint on three-dimensional movement imposed by the six atom spacer used to connect BSA and E~2~. Lastly, the use of E~2~-BSA solutions that are formulated in terms of the molarity of total bound-E~2~probably overestimates the amount of E~2~available for binding. The rate of binding between E~2~and ER can be expressed using the second order rate equation: rate = k \[E~2~\] \[ER\], where \[E~2~\] is the concentration of estradiol, \[ER\] is the concentration of the ER, and k the rate constant. Commercially available E~2~-BSA is commonly composed of approximately 10 molecules of E2 attached to every BSA molecule. An E~2~-BSA solution equimolar in estradiol to a solution of estradiol alone would contain one-tenth the molarity of E~2~-BSA with respect to the concentration of estradiol alone. However the rate of E~2~-BSA binding is dependent upon the concentration of E~2~-BSA (rate = k \[E~2~-BSA\] \[ER\]). Even if every collision between E~2~-BSA and ER produced binding as successful as collisions between E2 alone and the ER, an E2-BSA solution equimolar in E2 would have approximately one-tenth the rate. Taken together, these factors reduce the binding efficiency of E~2~-BSA to ER compared with free E~2~. However, once binding has occurred, the stability of the E~2~molecule in the ER binding pocket may be only modestly impaired. This may explain how pre-incubation with E~2~-BSA results in successful binding, whereas immediate addition of E~2~-BSA does not have sufficient time to establish successfully bound forms. A similar rationale may explain our results and those of other investigators \[[@B15]\] that demonstrate specific cell surface binding of E~2~-BSA-FITC only to cells that express ERα. These studies typically employ at least a 30-minute incubation time with E~2~-BSA-FITC, which may be sufficient to result in significant binding. These factors strongly suggest that the rate of binding is an important consideration in experiments assessing potential interactions between E~2~-BSA and ER. Although an estrogen receptor has not been directly isolated and characterized from the cell membrane, evidence other than E~2~-BSA activation of non-genomic effects has recently been reported that strongly supports the existence of a membrane ER. Immunocytochemistry using antibodies specific to several domains of the ERα stained only on the membrane of GH3 cells \[[@B26]\]. Membrane specific staining was prevented by treatment with antisense ERα mRNA or peptides that interfere with antibody binding. E~2~conjugated to peroxidase also bound only to the membrane of pancreatic islet cells and this binding was competed by E~2~\[[@B27]\]. The membrane impermeable E~2~-BSA-FITC was shown to stain only the membrane of ER deficient CHO cells transiently transfected with ERα and ERβ \[[@B15]\]. Moreover, ERα and ERβ interact directly with the membrane associated Src complex to trigger prostate cancer cell proliferation through the RAF-1/Erk-2 signal transduction pathway \[[@B5]\]. Lastly, we demonstrate that E~2~-BSA-FITC membrane staining is absent with ER deficient HeLa cells and present only on the membrane of cells that endogenously produce ER or HeLa cells that stably express mERα. Taken together these data strongly suggest that non-genomic effects of E~2~are at least partially mediated by a membrane associated ER. However, whether the receptor is the classical nuclear ER translocated to the membrane or an ER unique to the membrane remains unanswered. Conclusions =========== The results presented here suggest that E~2~-BSA can bind to the estrogen receptor but the rate of binding is impeded due to steric and other considerations. Commercially available forms of the reagent are contaminated with dissociable E~2~and should be purified prior to studies designed to demonstrate effects mediated through a membrane ER. Although we demonstrate that classical nuclear ERs can be translocated to the membrane, the conclusive identity of the endogenous membrane receptor awaits purification and sequencing of the putative membrane ER protein. Materials and Methods ===================== Establishment of ER stable transfectants ---------------------------------------- Full-length cDNA encoding the mouse ERα was cloned into a vector containing the CMV promoter driving the neomycin resistance gene (pcERα). HeLa cells maintained in MEM containing 10% fetal bovine serum under 5% CO2 were transfected with pcERα and successful transfectants (HeLa-ERα) were selected by survival in media containing the neomycin analog, G418 (400 ug/ml). Preparation of E~2~-BSA free of E~2~ ------------------------------------ 400 ul of E~2~-BSA (10^-5^M in estrogen dissolved in 50 mM tris, pH 8.5, Sigma) was added to a centrifugal filter unit with a MW cut-off of 3,000 (Millipore) and centrifuged at 14,000 × g until 50 ul of retentate remained. The retentate was washed 3 times with 350 ul of buffer, recovered and volume adjusted to 400 ul. Binding of estradiol to purified estrogen receptor -------------------------------------------------- ^3^H-labeled E~2~(NEN, specific activity 48 Ci/mmol, 10^-8^M) was incubated with recombinant ERα (.035 pM, Alexis Corp) for four hours at room temperature in binding buffer (10 mM tris, 10% glycerol, 2 mM DTT, and 1 mg/ml BSA). The binding of labeled E~2~to ERα was competed by various concentrations of ultrafiltered E~2~-BSA or E~2~(10^-9^to 3.5 × 10^-6^M in E~2~) added four hours prior to or concurrently with the addition of labeled E~2~. ERα was precipitated by the addition of a hydroxyapatite slurry (50% v/v in TE) and centrifugation at 10,000 × g. The pellet was washed three times with wash buffer (40 mM tris, 100 mM KCl, 1 mM EDTA, and 1 mM EGTA) and ^3^H-E~2~binding determined by liquid scintillation counting. E~2~-BSA-FITC binding to cell membranes of ER producing cells ------------------------------------------------------------- HeLa-ERα cells or mammary tumor cells (MC7, ATTC) were plated on glass cover slips and incubated with 500 ul of 10^-8^M (in estrogen) E~2~-BSA conjugated to FITC (E~2~-BSA-FITC, Sigma, 10 moles E2 and 3.5 moles FITC per mole BSA) or BSA-FITC (Sigma, equimolar to E~2~-BSA-FITC with respect to BSA) for 30 minutes at 4°C. Binding of E~2~-BSA-FITC to MC7 cells was competed by a 30 minute pre-incubation with E~2~, ICI-182,780, or E~2~-BSA (Sigma, 10^-7^to 10^-9^M). Cells were fixed and FITC staining visualized by confocal microscopy. Images were digitized, inverted to black on white, and pixel density for each cell determined by averaging the density across the cell membrane at four orthogonal points (Scion Image, Scion Corp). The stability of E~2~-BSA-FITC during the incubation with MC7 cells was assessed by HPLC. E~2~-BSA-FITC (500 ul, 10^-8^M in estrogen) was added to empty wells and to wells containing MC7 cells prepared as above for 30 minutes at 4°C. 10 ul of supernatant was resolved using a C-18 reverse phase column (Xterra C-18 RP, 5 um, 4.6 mm × 250 mm, Waters). A multiple solvent deliver system (BIO CM 4000, Milton Roy) provided a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at a flow rate of one ml/minute. Peaks were detected by a scanning fluorescence detector (model 747, Waters) at an excitation wavelength of 495 nm and emission wavelength of 519 nm. Area under the curve was calculated using standard algorithms (Millenium Software). Assays were performed in triplicate. Estradiol binding studies ------------------------- Subconfluent HeLa-ERα or native HeLa cells were trypsinized and aliquots (2 × 10^6^cells) incubated with several concentrations of ^3^H 17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. Data was analyzed by Scatchard analysis. Competing interests =================== None declared. Authors\' contributions ======================= DB wrote the manuscript and performed binding assays. MK generated the stable cell lines. YT performed binding assays.

PubMed Central

2024-06-05T03:55:47.475879

2004-8-19

PMC516043

Background ========== Although it has long been known that cellular memory, or epigenetic states, can be created by emergent properties of regulatory or metabolic networks (see Delbrück in the discussion of \[[@B1]\], and \[[@B2],[@B3]\]), the full implications of this type of inheritance have only recently been understood. To date, pertinent studies focused mainly on phenomena related to chromatin structure and DNA methylation, RNAi and other post-transcriptional gene silencing processes, and prions. However, virtually any physiological process can adopt a bistable (or multistable) behavior, as defined by the ability to adopt two (or more) stable states rather than a range of intermediate states, provided that a positive feedback loop exists (or its related counterpart, the mutually inhibitory double negative feedback loop) within the system \[[@B1],[@B3],[@B4]\]. The bistability may sometimes be associated with hysteresis, i.e., the ability of the system to maintain, in a sustained manner, a particular state despite the fact that the stimulus initiating this state is no longer present or is below the level that initially activated the system \[[@B5],[@B6]\]. In some cases, the hysteresis is sufficiently dominant to permit faithful transmission of the different states during mitosis and even meiosis, thus appearing as an epigenetic phenomenon \[[@B4],[@B7]\]. In the light of these concepts, alternative inheritable regulatory states were designated (reviewed in \[[@B8]\]) based on previous descriptions of regulatory or metabolic networks, permitting the definition of new types of inheritance units, the \'toggle switch\' and the \'positive feedback switch\'. More recently, mathematical models defined the conditions in which a system endowed with positive autoregulation can present bistability \[[@B9],[@B10]\]. However, most studies on bistable regulatory states are presently performed with a few well-known systems \[[@B7],[@B8]\], including the lactose operon, the lambda lysogenic/lytic decision switch, and the *Xenopus*mos MAP kinase cascade. Other relevant models are necessary for the comprehension of these phenomena. Fungi are an excellent source of well-defined bistable and multistable processes associated with hysteresis (reviewed in \[[@B4],[@B11]\]; see also \[[@B12]\]). In yeasts, such phenomena are quite common and are called phenotypic switches \[[@B11],[@B13]\] or prions \[[@B14]\]. In filamentous fungi, bistable phenomena seem to be particularly prevalent, as one-third of the species show at least one example of these \[[@B4]\]. In most cases investigated in filamentous fungi, bistability results from the appearance and subsequent spread of cytoplasmic and infectious factors. Apart from the HET-s and the Crippled Growth determinants of *Podospora anserina*, their molecular nature and their roles remain unknown. HET-s is a prion \[[@B15]\] and the Crippled Growth determinant seems to result from a positive autoregulation of a signal transduction cascade \[[@B16]\]. The widespread recognition of these infectious factors in fungi is probably due to the ease with which they are detected. The syncytial structure of the mycelium facilitates the propagation of factors from cell to cell. Usually, the presence of the infectious element results in a modification of thallic characteristics, such as fertility or morphology, and is thus easily scored visually as sectors in which the mycelium displays different properties. In the filamentous ascomycete *Nectria haematoccoca*, two bistable phenomena have been described \[[@B17]\]. *N. haematoccoca*is the teleomorph of *Fusarium solani*, a fungus encountered throughout the world as both saprophyte and pathogen. *N. haematoccoca*produces naphthoquinones exhibiting biological activities. Production of naphthoquinones is regulated by genetic and environmental but also epigenetic factors. The thallus of this species, observed just after ascospore germination, is a dense, grayish aerial mycelium, called Normal. Two kinds of morphological modification (the Anneau and the Secteur) frequently and randomly appear in the growth margin of the thallus as small brown areas in which hyphal elongation is reduced and secretion of pigments is drastically increased (see Figure [1A](#F1){ref-type="fig"} for the development of the Secteur). Once initiated, the modifications spread and progressively invade the margin of the culture, with a speed that dictates their form (20 times the hyphal elongation speed for the Anneau and twice for the Secteur). The new growth forms can be perpetuated with large pieces of mycelium excised from the growing margin of affected mycelia. The resulting modified subcultures, called ZiS (from a Secteur) and ZiA (from an Anneau), grow slowly and are pigmented in brown. However, fragmentation of a modified culture permits the restoration of the Normal morphology in a variable proportion of subcultures \[[@B18]\], showing that ZiS and ZiA cultures are probably mosaics of infected and uninfected hyphae. Each modification can be specifically transmitted to Normal cultures by contamination experiments, as shown in Figure [1C](#F1){ref-type="fig"} for Secteurs. The tangential propagation of the new growth forms is thus due to the transmission through hyphal fusion (anastomoses) of specific infectious cytoplasmic determinants, called σ for the Secteur and α for the Anneau. Mutagenesis experiments of wild-type conidiospores demonstrated that both the Secteur and the Anneau are under the control of nuclear genes \[[@B19]\]. In these early mutageneses, several kinds of mutation were detected. Five mutations prevent the expression of both the Secteur and Anneau, either spontaneously or after inoculation. They differed from wild type in producing more aerial mycelium, lacking pigmentation, and suppressing the sexual stage through self-fertilization. They mapped to at least four loci. Since this type of mutation prevents the generation of both infectious factors, they are now designated *nas*mutants (for \'no Anneau or Secteur\'). Several mutations that specifically prevent the formation of Anneaux were also selected \[[@B19]\]. The two different types of mutations recovered, *a*and *a*\*, suggested that the Anneau is under the control of a unique but complex locus. All *a*and *a*\* mutations map to the same locus, *Ans*(for \'Anneau-specific\'). Mutants carrying the *a*mutations are unable to generate the α factor, since they never present Anneau morphology and cannot be infected by that factor. Mutants carrying the *a*\* mutations never show Anneaux, spontaneously or after contamination, but display a red color. Surprisingly, when used as donor in contamination with the wild type as recipient, these mutants are able to trigger Anneaux in 100% of attempts, showing that these *a*\* mutants carry the α factor constitutively. It is noteworthy that the phenotype of *a*\* mutants suggests that the mere presence of this factor is sufficient to promote a red color but is insufficient to alter growth. To date, only one mutation that specifically prevents Secteur formation has been described \[[@B18],[@B19]\]. This *s*\*~789~(formerly 789) mutant acts similarly to *a*\* mutation in that it entails a red pigmentation (Figure [1B](#F1){ref-type="fig"}) and specifically prevents the expression of the Secteur, although it possesses the σ determinant throughout the thallus. This property is shown in Figure [1C](#F1){ref-type="fig"}, by the ability of small inocula harvested in the red *s*\* thallus to induce Secteurs on growing recipient mycelia lacking the σ determinant. Because of the similar properties of Anneaux and Secteurs, we decided to screen for more mutations that specifically prevent Secteur formation, to determine if *s*mutations (i.e., a mutation that specifically prevents the generation of σ) could be obtained. To this end, we developed novel mutagenesis procedures permitting the recovery of mutations that specifically prevent Secteur development. Most were *nas*mutations. However, a few *s*\* alleles and two *s*alleles (*s*~1~and *s*~2~) were obtained. Genetic analysis suggested that the σ factor, like the α factor, is under the specific control of a single complex genetic locus. Molecular analysis of the *s*~2~allele, obtained by insertional mutagenesis, permitted the cloning and characterization of the *Ses*locus. As expected from the genetic data, the locus is complex and encompasses two linked genes, one of which has similarity with putative esterase/lipase/thioesterase enzymes. These data provide evidence that two linked genes could generate a bistable differentiation. Results ======= Selection and genetic characterization of mutants affected in the expression of the Secteur phenomenon ------------------------------------------------------------------------------------------------------ To increase the number of mutants affected in Secteur expression, we decided to use a strategy based on the recovery of fast-growing sectors after mutagenesis of the slow-growing ZiS cultures (see Methods; Figure [2](#F2){ref-type="fig"}). We recovered 354 independent mutants (Table [1](#T1){ref-type="table"}), with a large majority of *nas*mutants (97%). Eleven mutants were specifically affected in Secteur expression. Ten exhibited an *s*\* phenotype identical to that of the *s*\*~789~strain. One mutant, *s*~1~, had a wild-type phenotype, except that it was unable to express the Secteur, spontaneously or after contamination tests (Figure [1D](#F1){ref-type="fig"}). Genetic analysis of the mutants defines a complex locus involved in Secteur expression -------------------------------------------------------------------------------------- The *s*~1~mutant and five *s*\* mutants were initially crossed with the *I*~4~*mod*reference strain. All behaved as single mutants, with about 50% wild type and 50% mutant type in the progeny (data not shown). In some progeny, the *s*\* and *s*~1~mutations were recombined with the *I*~4~*mod*markers. These strains were kept for further crosses. Crossing *s*~1~with *s*\*~789~or crossing the new *s*\* mutants with *s*~1~and with *s*\*~789~yielded progeny that were mostly segregants, with the parental phenotypes in a ratio 1:1 and few segregants with the wild-type phenotype. Their frequency ranged from 0.1 to 0.6% of the progeny (Table [2](#T2){ref-type="table"}), indicating that mutations affecting the Secteur were closely linked. In order to determine dominance/recessivity relationships of the mutated and wild-type alleles, forced heterokaryons were produced by pairing some mutants and wild-type strains marked with auxotrophic mutations (see Methods; Table [3](#T3){ref-type="table"}). The *s*~1~/+, *s*\*~789~/+, and *s*\*~84~/+ prototrophic mycelia were grayish and able to differentiate Secteurs, as did the control heterokaryon, which carried only the auxotrophic markers. Thus, the wild-type allele was dominant over its mutated counterparts. Combinations of *s*\* mutants with *s*~1~gave rise to heterokaryotic wild-type mycelia, indicating that complementation occurred. In contrast, no complementation was observed in pairings involving two different *s*\* alleles. The simplest interpretation of these results is that *s*~1~and *s*\* mutations could affect two different linked genes or two separate domains of a multifunctional protein encoded by a large gene. However, due to the low representation of heterokaryotic cells (about 10%) determined in previous studies \[[@B20]\], one must carefully interpret these data, deduced from the characteristics of the prototrophic heterokaryotic mycelium. Insertional mutagenesis identified the *Ses*locus ------------------------------------------------- Since the *s*~1~mutant allele appears recessive to the wild type in balanced heterokaryons, we tried to clone the *Ses*locus by complementation using the sib selection strategy \[[@B21]\]. Although two different cosmid banks, XSG and XN (see Methods), were used, none of the tested transformants exhibited Secteurs. As an alternative strategy, we therefore used insertional mutagenesis, which can rapidly lead to the cloning of the relevant gene. Transformation with a plasmid that carries a hygromycin B resistance marker was performed to identify new mutants affected in *Ses*. However, because the contamination tests necessary for this screen were labor-intensive and time-consuming, we preferred the *61a*~1~strain as a recipient for transformation, since it expressed Secteurs very early, and at high frequency, and never expressed Anneaux \[[@B22]\]. As illustrated in Figure [3A](#F3){ref-type="fig"}, the majority of transformants grown for 15 days at 18°C differentiated Secteurs, so that few transformants had to be tested by contamination experiments for their Secteur expression. During the course of these experiments, we also screened transformants harboring a variation in colony morphology or pigmentation (Figure [3B](#F3){ref-type="fig"}). These phenotypes could be maintained from conidia isolation, showing that the transformation procedure was an effective approach in the creation of mutants. Ten transformants among the 5000 recovered failed to express the Secteur. Nine exhibited a *nas*phenotype. One was probably affected in the *Ses*locus, as it was unable to differentiate Secteurs, either spontaneously or after contamination tests. This transformant, called *s*~2~, had a wild-type morphology and behaved as did the *s*~1~mutant previously isolated. The *s2*transformant carries a DNA fragment integrated in the *Ses*locus ------------------------------------------------------------------------ In order to prove that *s*~2~carries integration in the *Ses*locus, we crossed it with the wild-type (wt) and *s*~1~strains. When crossed with wt, the 100 tested ascospores segregated 1:1 for ability:inability to differentiate Secteurs. As expected, hygromycin B resistance always cosegregated with the inability to differentiate Secteurs. When *s*~2~was crossed with the *s*~1~strain, only seven ascospores among 1657 were unable to express the Secteur (Table [2](#T2){ref-type="table"}), indicating that *s*~2~also mapped at the *Ses*locus. Overall, the data strongly suggested that *s*~2~resulted from a single insertion at the *Ses*locus. To establish the integration pattern in *s*~2~, Southern blotting analysis was performed after digestion by *Cla*I, which did not cut the plasmid, and by *Bam*HI, which cut once in the vector. The probe was the pAN7-1 vector used for insertional mutagenesis. As shown in Figure [4A](#F4){ref-type="fig"}, the number and intensity of hybridizing bands in *Cla*I digest reflected a complex integration event, with at least two integrated copies. Southern blot analysis of 10 *s*progeny from an *s*~2~× wild-type cross did not show segregation of the two integration sites (data not shown), suggesting that the two integrated copies were linked. The size of the larger *Cla*I-fragment, LI, was compatible with an entire copy of the vector, whereas the smaller, SI, could be interpreted as a truncated copy. Further analyses with other restriction enzymes and probes (data not shown) established the restriction map of this region (Figure [4B](#F4){ref-type="fig"}). The sequence of a region flanking SI revealed that both LI and SI occurred at the same genomic site and were separated by a DNA fragment of unknown origin (Figure [4B](#F4){ref-type="fig"}). Molecular cloning of the *Ses*locus ----------------------------------- The restriction enzyme *Bam*HI was used to recover the genomic sequence flanking LI (Flank1 in Figure [4B](#F4){ref-type="fig"}) along with part of the integrated pAN7-1 vector. A part of Flank1 was then used as a probe to screen the XN cosmid library. One hybridizing cosmid, XN31E6, was identified and used to transform the *s*\*~789~, *s*\*~18~, *s*\*~27~, and *s*\*~4~mutants. Most of the transformants recovered the ability to express the Secteur, showing that the *s*\* mutations are probably recessive. However, different phenotypes were observed (Figure [5](#F5){ref-type="fig"}). The \'mild S\' exhibited the Secteur altered in its propagation and maintenance, and the \'S → s\*\' showed an s\* phenotype beyond the Secteur. The mild S was interpreted as having a reduced amount of σ in the modified areas because of insufficient expression of the transgene, and the S → s\* type was probably due to transgene sorting. Indeed, wild-type and *s*\* nuclei could be recovered through the isolation of microconidia in such transformants, before expression of a Secteur, indicating that the S → s\* thalli had a heterokaryotic structure. After the development of a Secteur, only the *s*\* cells that were not affected in their growth in the presence of σ could grow, as previously observed for balanced heterokaryons \[[@B20]\]. Interestingly, we also observed several transformants displaying an s phenotype (Figure [5](#F5){ref-type="fig"}). These might have resulted from an abnormal integration inactivating the *Ses*locus. Molecular analysis of five hygromycin-resistant (Hyg^R^) transformants (four with a wild-type phenotype, and one with a mild-S phenotype) revealed two patterns of integration (Figure [6](#F6){ref-type="fig"}). The pattern exhibited by the four wild-type transformants was wild type, except for an additional band corresponding to the pMoCosX vector. These transformants were thus interpreted as having integrated one copy of XN31E6 at the resident *Ses*locus. The mild-S transformant pattern was more complex, showing additional bands and a difference in fragment stoichiometry, suggesting a complex integration. This is consistent with a reduced amount of σ hypothesized for mild-S transformants. It is noteworthy that despite several attempts, transformation experiments done with the *s*~1~mutant as a recipient did not yield any transformants able to express the Secteur. This suggests that, contrary to the observations in balanced heterokaryons, *s*~1~is dominant in partial diploids. Dominance of *s*~1~was indeed established through transformation (see below). This dominance in diploids explains the failure to clone *Ses*by complementation. In order to further define the DNA fragment complementing the recessive *s*\* mutation, cosmid XN31E6 was subcloned into pBluescript and the plasmids were assayed for complementation. This yielded psecX5, a plasmid carrying a 5.5-kb *Xho*I fragment sufficient to confer Secteur expression (Figure [7](#F7){ref-type="fig"}) with the same efficiency as did XN31E6. Gene organization of the *Ses*locus ----------------------------------- A 9-kb-long region surrounding the integration site in the *s*~2~strain was sequenced (GenBank Accession no. AY572411). One interesting feature of the sequenced region concerned the presence of several inverted repeats ranging from 32 to 290 bp and organized in a symmetrical fashion (Figure [7](#F7){ref-type="fig"}). Inspection of the sequences revealed four open reading frames (ORFs) that were either larger than 200 amino acids or showed significant similarity with proteins in the databases (Figure [7](#F7){ref-type="fig"}). To begin with, we found an ORF encoding a putative large protein of unknown function that is also detected in the complete genome sequences available for the other filamentous ascomycetes (those of *Neurospora crassa*, *Magnaporthe grisea*, *F. graminearum*, *Aspergillus nidulans*, *Aspergillus fumigatus*, and *P. anserina*). This protein, NhHET-E-like, has a weak similarity (23% identity and 39% similarity over 455 amino acids) to the HET-E protein of *P. anserina,*which has been shown to be involved in heterokaryon incompatibility \[[@B23]\]. On the other side of the insertion site, a partial ORF of 165 amino acids carrying a PROSITE (PS00061), short-chain alcohol dehydrogenases/reductases family signature, was detected and designated ADHS. Two additional ORFs, *SesA*and *SesB*, were also identified. Functional analysis revealed that only these two ORFs were part of *Ses,*each having a different role. *SesA*is a 210-codon ORF (Figure [7](#F7){ref-type="fig"}) with no homologue in the genomes of *N. crassa*, *M. grisea*, *A. nidulans*, or *A. fumigatus*. However, it is significantly similar (30% identity and 42% similarity) to the N terminus of a large putative protein of *P. anserina*of unknown function but with 46% identity and 60% similarity with the Het-E protein in its C terminus (Figure [8A](#F8){ref-type="fig"}). It is also similar to two ORFs, of 214 and 201 codons, present in the genome of *F. graminearum*, with 41% identity and 54% similarity over 197 amino acids and 32% identity and 49% similarity over 199 amino acids, respectively (Figure [8A](#F8){ref-type="fig"}). *SesA*is interrupted by pAN7-1 in the *s*~2~transformant, and sequence analysis of *SesA*in the *s*~1~mutant showed a mutation at the end of the coding sequence, which changes a glycine into a glutamic acid (Figures [6](#F6){ref-type="fig"}, [7](#F7){ref-type="fig"}, [8A](#F8){ref-type="fig"}). *SesA*was expressed because a 480-bp-long RT-PCR product was obtained by using the 3\' RACE (rapid amplification of cDNA ends) procedure. The sequence of this product revealed that a polyadenylation signal existed 141 bp downstream from the *SesA*stop codon. *SesB*, a 386-codon ORF (Figure [7](#F7){ref-type="fig"}), is located on the opposite strand from *SesA*. *SesB*shows significant similarity with several putative proteins from filamentous ascomycetes (at least three in *A. fumigatus*and *A. nidulans*, six in *F. graminearum,*two in *N. crassa*, and four in *M. grisea*and *P. anserina*) and animals (*Caenorhabditis elegans*, *Drosophila melanogaster*, or mammals). It does not show similarity with hemiascomycetous yeast ORFs (*Saccharomyces cerevisiae*and 12 other partially sequenced yeasts), basidiomycete ORFs (*Coprinus cinereus*, *Ustilago maydis*, and *Phanerochaete chrysosporium*), or *Arabidopsis thaliana*ORFs. All the proteins similar to *SesB*show a significant PROSITE (PS50187) esterase/lipase/thioesterase active site serine domain. The sequence of *SesB*in *s*~1~,*s*\*~789~,*s*\*~4~,*s*\*~27~, and *s*\*~18~revealed that in the four *s*\* mutant alleles, *SesB*contains a single point mutation (Figures [7](#F7){ref-type="fig"} and [8B](#F8){ref-type="fig"}), but no mutation in the *s*~1~mutant. In *s*\*~789~, *s*\*~18~, and *s*\*~4~, the mutations are missense mutations, and in *s*\*~27~, the mutation corresponds to a one-base addition resulting in a frameshift 120 bp upstream of the stop codon. None affected highly conserved residues of the esterase/lipase/thioesterase domain, suggesting that a functional protein was expressed in the mutants. The expression of *SesB*was also investigated with the 3\'-RACE procedure. A 370-bp-long RT-PCR product was obtained, confirming that *SesB*is expressed. Sequencing showed that a polyadenylation signal is used 81 bp downstream from the *SesB*stop codon. Downstream of *SesB*is another potential short ORF (ORF-C), encoding a putative peptide of 157 amino acids with no homologue in the databanks. To show that ORF-C is not an exon of *SesB*, we cloned into pBluescript the 2.4-kb-long *Sma*I fragment carrying only *SesB*, i.e., without ORF-C or *SesA*. This yielded plasmid psecSm24 (Figure [7](#F7){ref-type="fig"}). Its transformation in four different *s*\* mutants showed that the insert restored the expression of the Secteur. The molecular analysis of transformants showed that the integration was ectopic (data not shown), supporting the hypothesis that *SesB*alone is per se a functional gene involved in Secteur development. Since homologues of both *SesA*and *SesB*are present in the genomes of *F. graminearum*and *P. anserina*, we investigated whether the gene arrangement was conserved in these two species. In *P. anserina,*for which a single *SesA*homologue is found, the gene arrangement is not conserved, since *SesA*and *SesB*homologues are on two different contigs. In *F. graminearum*, the putative 201-amino-acid homologue of *SesA*that displays the lowest percentage of identity is not associated with any of the *SesB*homologues. On the contrary, the 214-amino-acid homologue of *SesA*that displays the highest identity is associated with a *SesB*homologue in the same order (Figure [8C](#F8){ref-type="fig"}). This homologue is highly conserved between *N. haematococca*and *F. graminearum*(79% identity and 85% similarity over 387 amino acids) whereas the other *F. graminearum*homologues are less conserved (40% identity and 63% similarity over 273 amino acids for the next-best homologue). In *F. graminearum*, the inverted repeats 1, 2, and 3 are absent but the inverted repeat 4 is present. Overall, these data suggest an evolutionary conservation of *Ses*between *N. haematococca*and *F. graminearum*, both of which are in the genus *Fusarium*, but with a lack of conservation of the gene organization in more distant species, including the loss of *SesA*. There are no data concerning the potential development of Secteur in the *F. graminearum*strain used by the Fungal Genome Initiative for sequencing. *s1*is dominant in diploids --------------------------- To investigate the dominance of *s*~1~, we co-transformed the wild-type strain with pSec3-s~1~and pBC-Hygro. pSec3-s~1~was obtained by cloning into \'pGEM-T easy\' (Promega, Charbonnières, France), a PCR amplification product constructed with oligonucleotides im317 and ip2393c (see Figure [7](#F7){ref-type="fig"}) using the s~1~mutant genomic DNA as template. Thus, pSec3-*s*~1~contained the entire *Ses*locus present in the s~1~mutant strain. Among 17 tested Hyg^R^transformants, seven could not differentiate Secteurs. *Stu*I-restricted DNA from five of these transformants was analyzed by Southern blotting. Two integration patterns were observed (Figure [9](#F9){ref-type="fig"}). For three transformants, the expected wild-type *Stu*I fragment was absent, suggesting that integration altered the *Ses*locus, thus accounting for the lack of Secteur formation. For the two other transformants, the wild-type *Stu*I fragment was present, as was an additional band larger than 15 kb. As this band hybridized with psec3-s~1~and the *hph*hygromycin resistance marker (data not shown), these transformants could be interpreted as bearing a co-integration of pBC-Hygro and pSecs~1~at an ectopic position. Sequencing of the *Ses*locus in these two transformants that do not display Secteurs confirmed the presence of both alleles, i.e., the wild-type allele at the resident locus and the mutated allele *s*~1~at an ectopic position. This clearly demonstrated the dominance of the *s*~1~mutation, in contrast to the *s*\* mutations, which are recessive. Discussion ========== Although the Secteur and Anneau phenomena were described about 30 years ago \[[@B17]\], the molecular mechanisms able to generate these fascinating bistable differentiations are still mysterious. Early studies suggested peculiar epigenetic mechanisms \[[@B24]\]. In this study, we have initiated the molecular characterization of *Ses*, the primary locus controlling the development of Secteurs. The impact of mutagenesis procedures ------------------------------------ The experimental design, based on the differences in the growth rate between modified and normal cells, was very efficient in finding mutations that block the propagation of Secteurs after the action of both UV and NG (*N*-methyl-*N*\'-nitro-*N*-nitrosoguanidine). A vast collection of mutants affected in the Secteur expression was easily obtained. One mutant with an *s*phenotype was isolated, along with 11 *s*\* mutants. Apparently, NG (*N*-methyl-*N*\'-nitro-*N*-nitrosoguanidine) mutagenesis was more efficient than UV mutagenesis in recovering these specific mutants (9/105 and 3/250, respectively). Genetic analysis showed that the structure of the *Ses*locus mirrors the structure of the *Ans*locus, indicating that α and σ could be generated through similar mechanisms, in agreement with the fact that both factors require the same set of *nas*genes for propagation. Relationships between *s1*and wt alleles: recessivity in heterokaryons versus dominance in partial diploids ----------------------------------------------------------------------------------------------------------- Dominance/recessivity tests using balanced heterokaryons showed that *s*~1~/wt heterokaryons were able to differentiate Secteurs as wild type. The recessivity of *s*~1~, deduced from the heterokaryon tests was the initial point of our cloning strategy, i.e., the use of cosmid libraries from wild type to restore the ability to express the Secteur in an *s*~1~recipient strain. The screening of two representative cosmid libraries failed, and we examined various hypotheses to explain this failure, especially the possibility of the dominance of *s*~1~. In order to easily clone the *Ses*locus, we used an insertional mutagenesis strategy, a powerful method for gene isolation in filamentous fungi \[[@B25]\]. With the pAN7-1 vector, 5000 Hyg^R^transformants were screened for defects in mycelium pigmentation, colony morphology, and the expression of the Secteur and around 4% of mutant phenotypes were detected. This frequency is similar to the 0.4 to 1.4% reported for loss of pathogenicity in different fungal species \[[@B25]\]. Among them, 9 *nas*mutants and 1 *s*mutant were obtained. Given the proportion observed for classical mutagenesis (12 *s*\* or *s*mutants among 355 *nas*), the recovery of the *s*~2~mutant was an unexpected outcome. Although we may have been lucky, the recovery of this transformant may reflect a preference for plasmid integration in the region of the *Ses*locus, which is compatible with the high recombination frequency observed at *Ses*. Cloning of *Ses*permitted the construction by transformation of a partial diploid containing both the wild-type and *s*~1~mutant alleles. The fact that this partial diploid has a *s*phenotype indicates that *s*~1~allele is indeed dominant over the wild-type allele in partial diploids. Similar contradictions have been observed in *A. nidulans*by comparing diploids and heterokaryons for complementation between mutations in the regulatory and the cognate structural genes. These data were interpreted as being due to a limitation of regulatory gene products to the nucleus or by a stringent dose effect when combined with a nonrandom distribution of nuclei in heterokaryons \[[@B26],[@B27]\]. In our study, a dose effect is also probable. In *N. haematococca*, as reported earlier \[[@B20]\], only 1--10% of hyphal fragments containing 3--4 cells are heterokaryotic, the others corresponding to both homokaryons in similar proportions. In addition, phase-contrast microscopic observation of the prototrophic mycelia revealed the presence of rows of uninucleate cells disrupting the heterokaryotic association. This structure does not prevent the stability of balanced heterokaryons with regard to metabolic requirements (hence the complementation of the auxotrophic mutations), but might severely affect the dosage of the *SesA*products expressed from the wild-type and *s*~1~nuclei in different portions of the thallus. Because *s*~1~is dominant and corresponds to a missense mutation, it is likely that the product expressed from *SesA*in *s*~1~is a dominant negative. If its concentration is insufficient, especially in hyphae homokaryotic for wild-type *SesA*nuclei, the propagation of σ and Secteur formation is favored. In contrast, in diploids an even ratio of the two products is always achieved, preventing propagation of σ and Secteur formation. Integrated model of functional elements at the *Ses*locus --------------------------------------------------------- The molecular characterization of *Ses*shows that it is composed of two linked and expressed genes, *SesA*and *SesB*, both of which are necessary for Secteur expression. Each has a different role. The *s*~1~and *s*~2~mutants map to *SesA*. Because *s*~2~is an integration of a large segment of DNA at the beginning of *SesA,*the phenotype of the *s*~2~mutant probably results from a complete loss of function of *SesA*. The *s*~1~mutant carries a dominant missense mutation and therefore synthesizes the *SesA*mRNA, and probably a dominant negative protein. The SesAp protein has no evident homologue in the databanks yielding a clue to its function. As *s*~2~does not produce the σ determinant, cannot be contaminated by mycelia carrying σ, and never differentiates the Secteur morphology, *SesA*is probably necessary for the production and transmission of σ. All *s*\* mutations map to *SesB*and determine a red-pigmented phenotype associated with the generation of the σ determinant throughout the thallus, but without any growth alteration. It is noteworthy that all the *s*\* mutations are probably not simply preventing the production of a functional mRNA, but are also not preventing the production of an active protein, as they map outside the evolutionarily conserved region of the protein. The function of *SesB*and its homologue remains unknown in fungi; however the inactivation by RNA interference of the most similar gene in *C. elegans*is lethal during embryogenesis, suggesting that the protein produced by *SesB*could be essential (accession NP\_510177; \[[@B28]\]). Based on the analysis of the mutant phenotype triggered by *s*\* mutation, the wild-type *SesB*product is involved in the repression of pigmentation and σ generation. To date, this situation has not been described for any of the previously reported systems responsible for bistable or multistable switches in fungi. Previous systems implicated prions \[[@B23],[@B29]\], chromatin silencing \[[@B12]\], hysteresis in a MAP kinase cascade \[[@B16]\], and possible membrane inheritance \[[@B30],[@B31]\]. At present, it is premature to propose a molecular model to explain the Secteur, because *SesA*and *SesB*display weak similarity only with proteins of unknown function. However, from our data we can infer that *SesA*most likely encodes a regulatory factor for *SesB*and that the regulation probably takes place at the protein level and probably not at the RNA level, since *s*~1~and all *s*\* mutation are point mutations that are not likely to affect RNA transcription and stability. Two formal models could explain how the regulation might work. Firstly, the product of *SesA*(SesAp) could exist in two states: one, A~N~, characterizing the Normal morphology and the other, A~S~, determining the Secteur morphology (Figure [10A](#F10){ref-type="fig"}). The transition A~N~to A~S~may be initiated by a rare event in cells of the growing margin of the thallus. Once formed, the A~S~state becomes predominant by directing other A~N~molecules to adopt the same state. This positive feedback loop allows A~S~propagation from hyphae to hyphae through anastomoses. A~S~alone would induce the hyperproduction of pigments (hence the absence of both σ and pigmentation in the *s*mutants, as they are mutated in *SesA*). In addition, the product of *SesB*(SesBp) can adopt the normal B~N~state, which can be transformed into a B~S~state in the presence of A~S~. B~S~would be responsible for the growth alterations observed in the Secteur and would follow the propagation of A~S~(hence the lack of growth impairment presented in the *s*\* mutants, as they are mutated in *SesB*). In the second model, SesAp may negatively regulate SesBp but also be negatively regulated by SesBp (Figure [10B](#F10){ref-type="fig"}). In the normal mycelium, SesBp, which probably has a catalytic activity, would be active in promoting healthy growth. The appearance in some cells from the growing margin of a sufficient amount of SesAp would displace the equilibrium, resulting in the inhibition of SesBp. In order for this to happen, the equilibrium constants must be in favor of SesAp. This would result in growth impairment (because SesBp would not be present) and in a red pigmentation (because SesAp would be present). In the *s*mutants, SesAp would never be present, thus making the presence of SesBp constitutive (hence the absence of Secteurs and the healthy growth). In the *s*\* mutant, the interactions between the two proteins would be abolished, permitting the activity of both SesAp and SesBp (hence the presence of a constitutive σ, the red pigment, and healthy growth). This model could explain the dynamic equilibrium found in the ZiS area, as well as the dose effect for SesAp, explaining the difference of heterokaryons and partial diploids. Conclusions =========== Secteur, one of the two bistable and hysteretic differentiations exhibited by the filamentous fungus *N. haematococca*, is controlled by a complex, two-gene locus. Each gene has a distinct role in defining bistability, and atypical regulatory relations between the two proteins coded by the two genes may account for the hysteresis of Secteur differentiation. The data exemplify the diversity of mechanisms that generate differentiation in fungi, and, more generally, in eukaryotes. Methods ======= Fungal strains and growth conditions ------------------------------------ The homothallic *N. haematococca*strain wt (Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands) was used as the standard wild-type strain in this study. The origin and relevant characteristics of all strains used in this study are listed in Table [4](#T4){ref-type="table"}. The a~1~s~1~I~4~mod and 61a~1~strains used as recipients in transformation experiments were constructed by crossing the s~1~I~4~mod strain with a~1~mutant and the 61 mutant with a~1~I~4~mod strain, respectively (unpublished data). The marker segregation was recorded after three weeks. This included (1) mycelium and perithecium pigmentation, (2) fertility, and (3) the ability to express the Secteur and the Anneau as tested by transferring the progeny onto potato dextrose agar (PDA) and by inoculating 3-day cultures with s\*~789~(for Secteurs) and a\*~58~(for Anneaux). General culture conditions and manipulations were as described by Daboussi-Bareyre \[[@B20]\]. Strains were purified through microconidia and maintained on PDA at 26°C. Long-term stocks were stored as plugs under mineral oil at 12°C. The medium selective for transgenes was PH8, i.e., PDA containing 8 μg/ ml of hygromycin B from Sigma-Aldrich (Saint Quentin Fallavier, France). Mating and ascospore recovery ----------------------------- The general methods for crossing the homothallic *N. haematococca*have been described elsewhere \[[@B19],[@B32]\]. Since the strains used in this study were homothallic, detection of hybrid perithecia in crosses were performed using as a partner in crosses the double mutant strain I~4~mod. This strain developed white, self-sterile perithecia whereas wild-type and fertile mutants developed self-fertile, red perithecia \[[@B32]\]. Thus, all the white, fertile perithecia, which appeared on the crossing plates, were hybrid. The progeny of crosses was analyzed in accordance with the following procedure: two to nine white, fertile perithecia from each cross were freed from mycelium and conidia. Each was opened to spread the ascospores on one Petri dish containing water plus 3% agar. To control viability, 35 ascospores from each perithecium were transferred to PDA and incubated for 4 days at 26°C, while the water-agar stock dishes were kept at 2°C to prevent ascospores from germinating. Then, the water-agar dishes containing many viable ascospores (usually those with more than 70% of germination were selected) were returned to 26°C to allow germination overnight. A sample of about 400 germinating ascospores from each dish was transferred to PDA. The phenotypes were recorded after a week at 26°C in the dark, except for the perithecium color, which was evaluated after an additional period of two weeks. Heterokaryon test ----------------- Heterokaryons were constructed as described in \[[@B20]\]. Initially, strains s~1~, s\*~84~, and s\*~18~were crossed with the two strains carrying either of the markers, arg~154~and lys~255~, that promote auxotrophy for arginine and lysine, respectively. Auxotrophic marked strains, which were recovered in the progeny, were paired on a cellophane membrane in the combinations appropriate to restore prototrophy. After 48 hours of growth on complete medium, the membrane was transferred to minimal medium. Widely inclusive inocula from prototrophic mycelia observed at the junction of the two partners were transferred to minimal medium and examined for their pigmentation and their ability to express the two modifications, either spontaneously or after inoculation. Standard mutagenesis -------------------- Plugs taken from the growing margin of a culture modified by the Secteur were inoculated on cellophane discs on PDA and grown for 4 days at 26°C. The modified slow-growing cultures (ZiS) were submitted to mutagenesis by UV or NG. For UV mutagenesis, cultures were exposed at 450 J/m^2^, with a UV light source (254 nm), and then kept in the dark for 72 hours. For NG mutagenesis, 0.1 ml of a 200γ/ml solution of NG was deposited under the cellophane disc. After 1 hour, the treated cultures were exposed to light for NG degradation. Fast-growing sectors were clearly visible a week after mutagenesis. For the estimation of the number of sectors recovered from each treated thallus, only sectors harboring different phenotypes were considered. A plug of these independent sectors was inoculated on PDA and the resulting cultures were examined for pigmentation and ability to express the Secteur and/or the Anneau at 26°C. The majority of the recovered mutants corresponded to *nas*mutants, defined by (1) the inability to differentiate both Anneaux and Secteurs, (2) an exuberant white mycelium, and (3) the inability to differentiate perithecia. Some of these mutants could express the modifications at other temperatures, as was previously observed for mutants 727 and 100 \[[@B33],[@B34]\]. *s*\* mutants were recovered as red, fast-growing sectors. They displayed the same properties as the *s*\*~789~mutant described previously \[[@B19]\], i.e., red thallus and inability to express the Secteur spontaneously or after inoculation. A unique *s*mutant was selected as a wild-type-growing sector. This mutant displayed a wild-type phenotype, except for the inability to express the Secteur spontaneously or after inoculation. Both *s*\* and *s*mutants could differentiate Anneaux. Insertional mutagenesis ----------------------- Plasmids pAN7-1 (GenBank accession number Z32698) or pBC-Hygro \[[@B35]\], which had no sequence similarity with the *N. haematococca*genome, were used to transform the double mutant strain *61a*~1~. Both vectors carried a hygromycin resistance gene. Transformation occurred at a frequency of about 10 transformants/μg plasmid. Southern analysis on 10 independent hygromycin-resistant transformants (Hyg^R^) indicated that the transforming DNA inserted generally at one or two genomic sites, in some cases in a tandem fashion (data not shown). This pattern of integration was suitable for recovery of tagged genes. To screen for mutant phenotypes, 5000 Hyg^R^independent transformants were transferred to PH8 (12 per Petri dish) and grown for 15 days at 18°C, a temperature that increased the frequency of spontaneous Secteur formation. Thalli with mutant phenotypes were frequently recovered (Figure [3](#F3){ref-type="fig"}). These most likely resulted from the plasmid integrations, since a sample of regenerating protoplasts treated in the same way but without the transforming DNA did not show any phenotypic variation. As control, a sample of transformants with an altered morphology was purified through single conidial isolation. Data showed that the mutant phenotype was stable. Potential candidates affected in the expression of the Secteur were recovered as thalli that did not form spontaneous Secteurs. Inoculation experiments were used to confirm the mutant phenotypes. Transformation procedure ------------------------ Protoplasts were prepared as described in \[[@B36]\], with the following modifications. Petri dishes containing 25 ml of a solid PDA medium covered with a cellophane disk were inoculated with 5×10^6^spores and incubated for 20 hours at 26°C. Mycelia were collected (0.2 g/10 ml) in a lysis buffer (1.2 M MgSO~4~, 10 mM sodium phosphate, pH 5.8, 10 mg/ml Glucanex (Novozymes, Bagsvaerd, Denmark), 10 mg/ml mutanase (Interspex Products, San Mateo, CA, USA), and incubated for 2--3 hours at 26°C with gentle agitation. Protoplasts were separated from cell debris by addition of 5 ml of ST buffer (0.8 M Sorbitol, 100 mM Tris-HCl pH = 7.5) and centrifugation at 2000 g for 10 min. The protoplasts collected at the interface of the two solutions were harvested by centrifugation at 750 ***g***for 5 min, washed twice in ice-cold buffers, once in ST and then in STC (0.8 M Sorbitol, 50 mM CaCl~2~, 100 mM Tris-HCl pH = 7.5; \[[@B36]\]). They were resuspended in 4/5 STC, 1/5 50% PTC buffer (50% PEG 3350 \[Sigma-Aldrich\], 100 mM CaCl~2~, 100 mM Tris-HCl, pH 7.5) at a final concentration of 5--10 × 10^7^protoplasts/ml and kept on ice. For transformation, 5 μg or 10 μg of DNA suspended in a maximum of 50 μl TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) were mixed gently with 100 μl protoplasts and placed on ice. After 30 min, 900 μl of PTCS buffer (50% PTC containing 0.8 M sorbitol) was added and incubation continued at room temperature for 30 min. Aliquots of the protoplasts were mixed with 2.5 ml molten (37°C) top PDAS (PDA with 0.8 M sucrose and 0.3% w/v agar), and overlaid onto 30 ml of selective medium (PDAS containing 8 μg/ ml of hygromycin B. Plates were incubated at 26°C and colonies appeared within 2--4 days. Numerous small colonies stopped growing after 2--3 days. These were interpreted as abortive transformants. Only the colonies that grew after this delay were considered resistant to hygromycin. When co-transformation experiments were performed, 5 μg of the transforming plasmid was mixed with 5 μg of pBC-Hygro. DNA manipulation ---------------- All the nucleic acid manipulations were performed using standard methods \[[@B37]\]. For Southern blot analysis, genomic DNA was extracted as described in \[[@B38]\]. The cosmid libraries were made in the pMoCosX vector as recommended by Orbach \[[@B39]\]. The library XSG was obtained with *Xho*I partially digested wt genomic DNA. Determination of gene representation in the XSG library was performed by estimating the number of clones containing conserved genes (*NhNia*encoding the nitrate reductase, *NhHsp70*and *NhHsp90*encoding molecular chaperones, *NhTUB1*encoding β-tubulin, *NhTEF1*encoding the translation initiation factor eEF1A) by PCR, Southern blot hybridization, or direct cloning using a complementation screen. Each tested gene was represented by 2 to 5 independent clones, indicating that the library contained at least four *N. haematococca*genome equivalents. Library XN was made with *Sal*I partially digested wt genomic DNA following the same protocol as for the XSG library. The sequence of the mutant alleles was performed on two independent PCR amplification products, obtained with oligonucleotides im317 (5\'-TGATCAACCTCCACGCACAT-3\') and ip2393c (5\'-AAGGAGATATCGCGCAGGCT-3\'). Identification of the 3\' end of the *SesA*and *SesB*genes was done by 3\'-RACE using the 5\'/3\' RACE kit (Roche Diagnostics, Meylan, France) on polyA^+^mRNA extracted from 2-day-old mycelium, using for *SesA*oligonucleotide ip238 (5\'-AAGAGGCCGTGAGACAAGAG-3\'), and for *SesB*ip974c (5\'-CCAAGACGACCGATAGAAGA-3\'). GenBank accession ----------------- The genBank accession no. for cosmid XN31E6 sequences is AY572411. Authors\' contributions ======================= SG performed all the experimental work and contributed to the interpretation of the data. PS and MJD performed part of the phylogenetic analysis, contributed to the interpretation of the data, and supervised the work. The writing of the manuscript was done in teamwork. All authors read and approved the final manuscript. Acknowledgements ================ We thank Emilie Robillard for expert technical assistance. This work was supported by ACC on ESST and Prions ref 2000-37. Stéphane Graziani was a PhD student, recipient of a \'Bourse de Docteur Ingénieur du Centre National de la Recherche Scientifique\' training grant. Philippe Silar is a professor at the University of Paris 7, Denis Diderot. The work was done in compliance with the current laws governing genetic experimentations in France. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **The Secteur and its transmission through contamination tests in *Nectria haematococca.*(A)**Wild-type strain exhibiting three spontaneous Secteurs (1, 2, 3). **(B)***s*\*~789~mutant. **(C)**Contamination tests, using plugs from a Secteur or from *s*\*~789~, on wild type (wt), result in one Secteur at each infection point. **(D)**Contamination tests on *s*~1~mutant did not result in the induction of any Secteur at the infection points. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Selection of *Nectria haematococca*mutants affected in the expression of the Secteur.(A)**Compares the morphology of a Normal growing culture (N) and of a ZiS culture (modified culture obtained by excision from a Secteur). **(B)**A 4-day ZiS culture submitted to UV mutagenesis. Note the emergence of four fast-growing sectors, which correspond to *nas*mutants. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Phenotypic characterization of transformants.(A)**Detection of a *nas*mutant phenotype among 12 transformants. Arrows indicate some Secteurs. **(B)**Summary of the different mutants recovered among the 5000 transformants: 152 were altered in pigmentation (99 reddish, 16 white/beige/yellow, 37 greenish), 45 were altered in colony morphology (12 irregular, 12 compact, 19 colonial, 2 fluffy), and 10 were affected in the expression of the Secteur (9 *nas*and 1 *s*). :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Analysis of pAN7-1 integration pattern in *s*~2~.(A)**Southern blot analysis of total genomic DNA of *s*~2~digested with *Cla*I, which does not cut the plasmid (lane 1), and *Bam*HI, which cuts once in pAN7-1 (lane 2), and probed with the pAN7-1 vector. Sizes are indicated in kilobases (kb). **(B)**Restriction map of the pAN7-1 integration point in *s*~2~. Wild-type genomic DNA is shown as a thick line, pAN7-1 sequences are represented by the open bar, and the dotted line stands for unknown sequences. The fragment LI is inserted at the bacterial replication origin (ORI). SI is truncated and carries a part of *hph*and of *tTrpC*. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Types of transformants recovered in transformation experiments using *s*\* mutants with XN31E6 and psecX5.**As the respective proportion of each type of transformant was similar with both vectors, the proportion observed is the combined percentage computed with 60 tested transformants. S, transformants differentiating Secteurs as wild type; mild S, transformants differentiating a Secteur that propagates more slowly than wild type and that frequently reverts to a normal morphology. S → s\*, transformants differentiating the Secteurs but showing an *s*\* phenotype beyond the modified area; s, transformants unable to differentiate Secteurs. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Southern blot analysis of XN31E6 transformants.**XN31E6 cosmid DNA along with total genomic DNAs of wild type (wt) *s*~2~, and five transformants obtained by transforming *s*\*~789~with XN31E6 were digested by *Xho*I and hybridized with an XN31E6 probe. Wild type, Tr1, Tr2, Tr3, and Tr4 differentiate wild-type Secteurs (S), Tr5 has a mild S phenotype, and *s*~2~is not able to differentiate Secteur(s). The 7-kb band corresponding to pMoCosX is marked by dark connecting lines. The arrow points to the 5.3-kb fragment, which shifts in *s*~2~. :::  ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Schematic representation of the *Ses*locus of *Nectria haematococca.*(A)**Open reading frames larger than 200 aa or showing sequence similarity to other genes are represented by gray arrows. Black arrows represent sequences in inverted repeated orientation: repeat 1 is 290 bp long; repeat 2 is 79 bp long; repeat 3 is 32 bp long; and repeat 4 is 93 bp long. Thick lines: subclones able to restore the Secteur modification in *s*\* mutants. **(B)**Detailed map of the region between oligonucleotides im317 and ip2393c. Vertical bold lines: position of the indicated mutations. Insertion: integration point of pAN7-1 in *s*~2~. Black lozenge: polyadenylation site. :::  ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### ***SesA*and *SesB*conservation during evolution.(A)**Alignment of SesAp with its homologues in *Fusarium graminearum*(F. gra 214 and F. gra 201) and *Podospora anserina*(P. ans). The amino acid change in *s*~1~is boxed. **(B)**Alignment of SesBp with representative homologues from *F. graminearum*(Fg1 and Fg2; the sequence was obtained from the Fungal Genome Initiative, <http://www-genome.wi.mit.edu>), *P. anserina*(Pa; <http://podospora.igmors.u-psud.fr>), *Homo sapiens*(Hs; GENBANK: BC001705), *Drosophila melanogaster*(Dm; GENBANK: AAF5369), and *Caenorhabditis elegans*(Ce; PIR: T21079). Arrowheads indicate the amino acid changed in *s*\*~4~, *s*\*~789~, and *s*\*~18~; arrow indicates the position of the frameshift in *s*\*~27~. **(C)**Conservation of Ses organization in *F. graminearum*. *SesA*corresponds to F. gra 214 of **(A)**and *SesB*corresponds to Fg1 of **(B)**. Percentages of identity with *Nectriahaematococca*SesAp and SesBp are 41% and 79%, respectively. In **(A)**and **(B)**, conserved amino acids are shaded. Color reflects the degree of conservation. :::  ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Southern blot analysis of *Stu*I-digested DNAs of wild-type *Nectria haematococca*and five transformants.**Total genomic DNAs of wild-type (wt) *Nectria haematococca*and five *s*transformants (Tr1-Tr5) that were obtained after co-transformation of wt with psec3-s~1~plasmid and pBC-Hygro vector, and probed with psec3-s~1~. All the transformants share a large band (\>15 kb), absent from wild type. Tr3 and Tr5 contain both the wild type and the *s*~1~allele. The faint bands noted \* correspond to repeated sequences homologous to those present at the *Ses*locus. :::  ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **Models integrating functional elements of Secteur expression in *Nectria haematococca.*(A)**First model: In the wild-type strain, the product of the *SesA*and *SesB*genes can exist in a normal state (A~N~and B~N~) and in a modified state (A~S~and B~S~). A~S~determines overproduction of pigments and B~S~is responsible for growth alteration. **(B)**Second model: The product of *SesA*, SesAp, is responsible for pigmentation. The product of *SesB*, SesBp, has a catalytic activity necessary for the normal growth. These two products negatively regulate each other, and the equilibrium constants favor SesAp. In juvenile thalli, only SesBp is produced, but SesAp can be randomly produced, switching to a new state with a majority of SesAp. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Mutagenesis efficiency and spectrum of mutants ::: Mutagen^a^ Total number of irradiated thalli Total number of fast-growing sectors Types of mutants^b^ ------------ ----------------------------------- -------------------------------------- --------------------- --------------- --------- UV~1~ 12 15 15 \- \- UV~2~ 26 53 53 (13) \- \- UV~3~ 21 40 39 (6) 1 (*s*\*~11~) \- UV~4~ 28 50 49 (16) 1 (*s*\*~27~) \- UV~5~ 44 75 74 1 (*s*\*~4~) \- UV~6~ 21 17 17 \- \- NG~1~ 28 25 22 2 1(s~1~) NG~2~ 22 38 37 1 (*s*\*~18~) \- NG~3~ 26 42 37 5 \- Total 228 354 343 11 1 1.5 per dish *97* *3* ^a^UV, 254 nm UV irradiation; NG, *N*-methyl-*N*\'-nitro-*N*-nitrosoguanidine. ^b^*nas*mutants differentiate neither Anneaux nor Secteurs at 26°C but some may do so at other temperatures. Their number are indicated in brackets; *s*\* mutants differentiate Anneaux but not Secteurs, though they have the σ determinant throughout the mycelium; *s*, cultures differentiate Anneaux but not Secteurs, and never contain the σ determinant. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Results of crosses involving mutants of the Secteur ::: Crosses Progency analyzed Wild-type progeny ----------------------- ------------------- ------------------- ------ *s*× *s*\*~27~ 3256 5 0.15 *s*~1~× *s*\*~18~ 3488 7 0.20 *s*~1~× *s*\*~789~ 3824 9 0.25 *s*~1~× *s*\*~4~ 2217 11 0.50 *s*~1~× *s*\*~84~ 1977 10 0.65 *s*~1~× *s*\*~11~ 1051 7 0.65 *s*\*~789~× *s*\*~84~ 2944 3 0.1 *s*\*~789~× *s*\*~4~ 1095 1 0.1 *s*\*~789~× *s*\*~11~ 1808 2 0.1 *s*\*~789~× *s*\*~27~ 2346 1 0.05 *s*\*~789~× *s*\*~18~ 2139 6 0.3 *s*~1~× *s*~2~ 1657 7 0.40 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Phenotypes of heterokaryotic mycelia from various pairings ::: Strains paired Prototrophic mycelium^a^ Conclusion ------------------------------------------------ -------------------------- -------------------- *arg*~154~(+) / *lys*~255~(+) Gray, AS *arg*~131~(*s*\*~789~)/*lys*~255~(*s*\*~789~) Red, As\* *arg*~154~(*s*\*~84~)/*lys*~255~(*s*\*~84~) Red, As\* *arg*~154~(*s*~1~) / *lys*~255~(~*s*1~) Gray, As *arg*~154~(*s*~1~) / *lys*~255~*(+)* Gray, AS s~1~recessive *arg*~154~(*s*\*~789~) / *lys*~255~*(+)* Gray, AS s\*~789~recessive *arg*~154~(*s*\*~84~) / *lys*~255~*(+)* Gray, AS s\*~84~recessive *arg*~154~(*s*~1~) / *lys*~255~(*s*\*~789~) Gray, AS Complementation *arg*~154~(*s*~1~) / *lys*~255~(*s*\*~84~) Gray, AS Complementation *arg*~154~(*s*~1~) / *lys*~255~(*s*\*~18~) Gray, AS Complementation *arg*~131~(*s*\*~789~) / *lys*~255~(*s*\*~84~) Red, As\* No complementation *arg*~154~(*s*\*~18~) / *lys*~255~(*s*\*~84~) Red, As\* No complementation ^a^AS differentiates both Anneaux and Secteurs and synthesize α and σ only in the respective modified areas. As\* differentiates Anneaux but not Secteurs, though they have the σ determinant throughout the mycelium; As differentiates Anneaux but not Secteurs and never contain the σ determinant. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### *Nectria haematococca*strains used ::: Characteristics^b^ ----------- ----------- ---------------------------------- -------------------------------- ------------------------------------- ------------------------------ Wild type CBS225/58 Gray FFF AS s\*~789~ NG Red F As\* Daboussi-Bareyre et al. 1979 s\*~27~ UV Red F As\* This study s\*~4~ UV Red F As\* This study s\*~11~ UV Red F As\* This study s\*~18~ UV Red F As\* This study s\*~84~ sp Red F As\* Parisot et al. 1981 s~1~ NG Gray FFF As This study s~2~ IM Gray FF As This study 61 UV Gray F AS with a high frequency of Secteur Daboussi 1985 a~1~ Sp Gray FFF aS Daboussi-Bareyre et al. 1979 a\*~58~ NG Red F a\*S Daboussi-Bareyre et al. 1979 arg~154~ NG Beige, Smooth arginine-auxotroph F AS Daboussi-Bareyre 1980 lys~255~ NG Beige, Smooth lysine-auxotroph F AS Daboussi-Bareyre 1980 I~4~mod cross Gray St, white and empty perithecia AS Babai-Ahary et al. 1982 ^a^CB: Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands. sp, spontaneous mutation; NG, UV and IM recovered after *N*-methyl-*N*\'-nitro-*N*-nitrosoguanidine, ultraviolet irradiadiation (254 nm) or insertional mutagenesis, respectively. cross: constructed by crossing. ^b^Main phenotypic characteristics. FFF, very fertile; F, fertile; St, sterile. AS differentiates both Anneaux and Secteurs and synthesizes α and σ only in the respective modified areas. As\* differentiates Anneaux but not Secteurs, though they have the σ determinant throughout the mycelium; As differentiates Anneaux but not Secteurs and never contain the σ determinant; a\*S differentiates Secteurs but not Anneaux, though they have the α determinant throughout the mycelium; aS differentiate Secteurs but not the Anneaux and never contain the α determinant. :::

PubMed Central

2024-06-05T03:55:47.477920

2004-8-16

PMC516044

Background ========== Pendred syndrome is a relatively common autosomal-recessive disorder characterized by deafness and goiter \[[@B1]\]. The syndrome is caused by mutations of the *PDS*gene SLC26A4, which codes for the protein pendrin \[[@B2]\]. Deafness is congenital and generally profound although sometimes late in onset and provoked by light head injury. Vestibular dysfunction is uncommon. Goiter is variable and generally develops around puberty \[[@B3]\]. The cause of goiter appears to be an impairment of iodide fixation in the follicular lumen due to a reduced rate of iodide transport across the apical membrane of thyroid gland epithelial cells \[[@B4]\]. A positive perchlorate discharge test and an enlarged vestibular aqueduct appear to be the most reliable clinical signs of Pendred syndrome \[[@B5]\]. Pendrin is an anion exchanger that can transport Cl^-^, I^-^, HCO~3~^-^and formate \[[@B6],[@B7]\]. Expression has been found in the inner ear and thyroid gland consistent with the clinical signs of deafness and goiter \[[@B2],[@B3],[@B8]\]. In addition, pendrin expression has been found in the kidney \[[@B9]\], mammary gland \[[@B10]\], uterus \[[@B11]\], testes \[[@B12]\] and placenta \[[@B13]\]. No expression was found in fetal or adult brain, consistent with a peripheral cause of deafness \[[@B2],[@B11]\]. Expression of pendrin mRNA in the inner ear has been found in several places including the cochlea, the vestibular labyrinth and the endolymphatic sac \[[@B8]\]. The precise location of pendrin protein expression, however, has not yet been determined. The variability of deafness in Pendred syndrome and the observation that deafness is sometimes late in onset suggest that pendrin dysfunction may not be the direct cause of deafness. It is conceivable that pendrin dysfunction favors changes in the expression levels of proteins that are critical for the maintenance of the hearing function. Detailed studies aimed at identifying the direct cause of deafness in Pendred syndrome have recently become possible due to the generation of a pendrin-specific polyclonal antibody \[[@B9]\] and the development of *Slc26a4*^-/-^mice, which bear a targeted disruption of the mouse *Slc26a4*gene \[[@B14]\]. The aim in the present study was to determine the location of pendrin and the cause of deafness in *Slc26a4*^-/-^mice. Methods ======= The endocochlear potential and the endolymphatic and perilymphatic K^+^concentrations were measured in young adult mice (1--4 month of age) that either have (*Slc26a4*^+/+^) or lack (*Slc26a4*^-/-^) a functional gene for pendrin \[[@B14]\]. The mouse strain 129Sv/Ev (Taconic, Germantown, NY) was chosen as the source of *Slc26a4*^+/+^controls, since *Slc26a4*^-/-^mice were propagated in the this strain and generated using a stem cell line derived from 129Sv/Ev. *Slc26a4*^+/+^and *Slc26a4*^-/-^were agouti. They did not differ in coat color. Differences in pigmentation were verified using coisogenic age-matched *Slc26a4*^+/+^and *Slc26a4*^-/-^that were bred in parallel. Expression of key proteins involved in the generation of the endocochlear potential and the transport of K^+^was studied using confocal immunocytochemistry and quantitative RT-PCR. K^+^secretion and the generation of the endocochlear potential were measured using electrophysiological techniques. All experiments were approved by the Institutional Animal Care and Use Committee of Kansas State University. Confocal immunocytochemistry ---------------------------- Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and transcardially perfused with a Cl^-^-free phosphate-buffered Na-gluconate solution containing 4% paraformaldehyde. Temporal bones were removed and cochleae fixed by perilymphatic perfusion, decalcified in EDTA, processed through a sucrose gradient and infiltrated with polyethylene glycol. Mid-modiolar cryosections (12 μm, CM3050S, Leica, Nussloch, Germany) were blocked in PBS with 0.2% Triton-X (PBS-TX) and 10% bovine serum albumin. Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1--3% BSA \[rabbit anti-pendrin, 1:500 (h766--780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 (Alomone, Jerusalem, Israel); rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)\]. Slides were washed in PBS-TX and incubated for 1 h at 25°C with appropriate secondary antibodies at a 1:1000 dilution in PBS-TX with 1--3% BSA \[donkey anti-rabbit Alexa 488, chicken anti-rat Alexa 594, and chicken anti-goat Alexa 594 (Molecular Probes, Eugene, OR)\]. Actin filaments were visualized by staining with Alexa 488 conjugated phalloidin (Molecular Probes). After incubation, slides were washed with PBS-TX, mounted with FluorSave (Calbiochem, La Jolla, CA), and viewed by confocal microscopy (LSM 5 Pascal or LSM 510 Meta, Carl Zeiss, Jena or Göttingen, Germany). Laser scanning brightfield images were collected to document structural preservation, for morphometric analysis and for analysis of pigmentation. Quantitative RT-PCR ------------------- Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Kidneys and brain were removed and pulverized in liquid N~2~. Temporal bones were removed and two preparations of inner ear tissues were obtained by microdissection: (1) stria vascularis without spiral ligament, and (2) spiral ganglia including the surrounding bone and the organ of Corti. The dissection medium was changed twice during the microdissection and isolated tissues were washed three times to minimize cross-contamination. The *Slc26a4*^-/-^genotype was verified by the observation of large otoconia in the utricular macula \[[@B14]\]. Total RNA was isolated and residual DNA contamination was removed by enzymatic digestion (RNeasy micro, Qiagen, Valencia, CA). Quality and quantity of 18S rRNA obtained from kidneys and brain were determined (Nano Assay, 2100 Bioanalyzer, Agilent, Palo Alto, CA). Further, the quality of RNA preparations obtained from stria vascularis and spiral ganglia was assessed (Pico Assay, 2100 Bioanalyzer). Real time RT-PCR was performed on RNA obtained from individual animals (OneStep RT-PCR, Qiagen; Smart Cycler, Cepheid, Sunnyvale, CA) in the presence of 0.2× SYBR green I (Molecular Probes). Transcripts of 18S rRNA and mRNA for the K^+^channels KCNJ10, KCNQ1 and KCNQ4 were amplified using gene-specific primers (Table [1](#T1){ref-type="table"}). RT was performed for 30 min at 50°C and 15 min at 95°C. PCR consisted of 50 cycles of 1 min at 60°C, 1 min at 72°C, 7s heating to hot-measurement temperature, 13s hot-measurement at 3--5°C below product melting temperature (Table [1](#T1){ref-type="table"}) and 1 min at 94°C. Hot-measurements were performed to eliminate the detection of primer-dimers that had melting temperatures between 72 and 75°C \[[@B15]\]. PCR was followed by a melt (60 to 95°C). The generation of a single product of appropriate size was routinely checked by the presence of a single melt peak and by agarose gel-electrophoresis. Product identity was confirmed by sequencing. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Primers ::: Template Primers Product Length Melting Temperature Hot Measurement Fidelity n ---------- ------------------------------------------ ---------------- --------------------- ----------------- ------------- ---- 18S gag gtt cga aga cga tca ga (*sense*) 316 bp 83.2 ± 02°C 78°C 1.89 ± 0.02 17 tcg ctc cac caa cta aga ac (antisense) KCNJ10 tgg tgt ggt gtg gta tct gg (*sense*) 411 bp 83.2 ± 02°C 78°C 1.86 ± 0.02 15 tga agc agt ttg cct gtc ac (*antisense*) KCNQ1 ttt gtt cat ccc cat ctc ag (*sense*) 239 bp 82.5 ± 02°C 79°C 1.85 ± 0.02 17 gtt gct ggg tag gaa gag (*antisense*) KCNQ4 ccc gga aac cct tct gtg tc (*sense*) 245 bp 83.2 ± 02°C 80°C 1.87 ± 0.01 17 aaa gat gag cac cag gaa cc (*antisense*) ::: Template molecules (T) were quantified according to T = P / (F \^C~t~) where P is the number of product molecules, F is the fidelity of the reaction and C~t~is the cycle at which the number of product molecules reaches a chosen threshold \[[@B15]\]. Fidelity (F) was obtained from the slope (S) of the log-linear phase of the growth curve via a best-fit fifth-order polynomial: F = 7.39 + 3.80 × S + 1.05 × S^2^+ 0.15 × S^3^+ 11.38 × 10^-3^\* S^4^+ 3.39 × 10^-4^× S^5^. The number of product molecules at threshold (P~Ct~) was determined by amplification of known amounts of 18S rRNA according to P~Ct~= T × F \^C~t~. Quantifications of 18S rRNA were used to compare tissue amounts. Genomic contamination of inner ear samples was assessed to be \<0.02% by omission of the RT step. In vitro electrophysiology -------------------------- Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Stria vascularis without spiral ligament was obtained by microdissection. Currents generated by the stria marginal epithelium were recorded \[[@B16]\]. A Pt-Ir wire microelectrode with a Pt-black tip was positioned 20--30 μm from the apical surface of the epithelium and vibrated at 200--400 Hz by piezo-electric bimorph elements (Applicable Electronics, Forest Dale, MA; ASET version 1.05, Science Wares, East Falmouth, MA). A Pt-black electrode (26-gauge) served as reference in the bath chamber. The signals from the phase-sensitive detectors were digitized (16 bit) at a rate of 0.5 Hz. The output was expressed as current density at the electrode. In situ electrophysiology ------------------------- Animals were anesthetized with inactin (thiobutabarbital sodium salt, 140 mg/kg ip). The endocochlear potential and the endolymphatic \[K^+^\] were measured with double-barreled microelectrodes \[[@B17]\]. Measurements were made in the basal turn by a round-window approach through the basilar membrane and in the apical turn after thinning the bone over the stria vascularis and picking a small hole (\~30 μm). K^+^-selective electrodes were calibrated in solutions of constant cation (K^+^and Na^+^) concentration of 150 mM. The minor selectivity of the K^+^electrodes for Na^+^produced a nonlinearity in the calibration curve, which was closely fitted by the Nicolski equation using nonlinear curve-fitting software (OriginLab, Northampton, MA): V = V~i~+ *S*× log (\[K^+^\] + *A*× \[Na^+^\]), where *V*~i~is an offset term, *S*is slope, *A*is selectivity, and \[Na^+^\] is Na^+^concentration. Calibrations were made immediately after withdrawal of the electrodes from the cochlea. Plasma K^+^concentrations were obtained using a blood analyzer (Stat Profile M, Nova Biomedical, Waltham, MA). Data are presented as mean ± sem; n denotes the number of experiments. Differences were considered significant when p \< 0.05. Results and Discussion ====================== *In situ*hybridization in the cochlea has suggested that pendrin mRNA is expressed in cells that reside immediately beneath the spiral prominence on the lateral wall of the external sulcus \[[@B8]\]. To determine the location of pendrin protein expression, we performed confocal immunocytochemistry on cryosections prepared from temporal bones of normal (*Slc26a4*^+/+^) mice using an established antibody \[[@B3]\]. Staining was absent when the primary antibody was pre-absorbed with the antigenic peptide (*data not shown*). Strong expression of pendrin was observed not only in outer sulcus epithelial cells, as predicted from *in situ*hybridization data, but also in root cells, in apical membranes of spiral prominence surface epithelial cells and in apical membranes of spindle-shaped cells that are part of stria vascularis (Fig. [1a,1b,1c](#F1){ref-type="fig"}). The presence of pendrin in spindle-shaped cells suggests that these cells secrete HCO~3~^-^into endolymph. Pendrin-mediated HCO~3~^-^transport has previously been shown in the kidney \[[@B9]\]. HCO~3~^-^in the cochlea may be generated from CO~2~by carbonic anhydrase located in strial intermediate cells \[[@B18],[@B19]\]. CO~2~may be supplied by the metabolically highly active stria marginal cells. It is conceivable that pendrin dysfunction interrupts HCO~3~^-^secretion and leads to an accumulation of HCO~3~^-^in stria vascularis. Preliminary data (Wangemann et al., *unpublished*) support a role for pendrin in HCO~3~^-^secretion into endolymph. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Protein localization of pendrin, KCNQ1, ZO-1 and F actin in cochlea and vestibular labyrinth of *Slc26a4*^+/+^and *Slc26a4*^-/-^mice. **a:**Overview of cochlea; bar = 100 μm. **b-f:**Detail of cochlear lateral wall; bar = 10 μm. **g:**Detail of utricle; bar = 10 μm. **h-i:**Detail of ampulla; bar = 10 μm. RM, Reissner\'s membrane; SC, spindle-shaped cells, SMC, strial marginal cells; SV, stria vascularis; SL, spiral ligament; LIM, spiral limbus. BC, basal cells; SP, spiral prominence epithelial cells; RC, root cells; OS, outer sulcus epithelial cells; VHC, vestibular hair cells; VTC, vestibular transitional cells; VDC, vestibular dark cells; arrows, basal cells at the top and bottom of stria vascularis form tight junctions with surface epithelial cells. :::  ::: The extent of pendrin expression in spiral prominence cells and stria vascularis was determined by labeling KCNQ1, a K^+^channel that is expressed in strial marginal cells, and ZO-1, a tight junction protein that labels basal cells and thereby delineates the boundaries of stria vascularis. Dual labeling experiments demonstrated that pendrin is expressed in spindle-shaped cells, which are surface epithelial cells in stria vascularis adjacent to strial marginal cells near the borders of both the spiral prominence and Reissner\'s membrane (Fig. [1c](#F1){ref-type="fig"}). *In situ*hybridization in the vestibular labyrinth suggested that pendrin mRNA is expressed in non-sensory cells \[[@B8]\]. Using confocal immunocytochemistry on cryosections, we observed strong expression of the pendrin protein in the apical membrane of vestibular transitional cells in the utricle and ampullae (Fig. [1g,1h,1i](#F1){ref-type="fig"}). Dual labeling with KCNQ1 demonstrated that pendrin expression was clearly limited to vestibular transitional cells and did not extend to other non-sensory cells such as vestibular dark cells, which were clearly identified by the expression of KCNQ1 and KCNE1 in their apical membranes. The onset of pendrin expression during development of the mouse inner ear has been determined to be embryonic day (ED) 13 \[[@B14]\]. Morphologically detectable differences in the inner ears of *Slc26a4*^+/+^and *Slc26a4*^-/-^mice become evident as early as ED 15, when *Slc26a4*^-/-^mice start to develop an enlarged endolymphatic space that persists into adulthood \[[@B14]\]. Interestingly, sensory hair cells in the cochlea appear normal until postnatal day (PD) 7 but show clear evidence of degeneration by PD 15 \[[@B14]\]. These observations suggest that the cochlear environment supports the survival of sensory hair cells in spite of the enlargement of the endolymphatic duct. A normal endolymphatic K^+^concentration, which is critical for hair cell survival \[[@B20]\], is established at PD 3 \[[@B21]\] and may persist in *Slc26a4*^-/-^at least until PD 7. The time period between PD 7 and 15 is the time when the endocochlear potential develops at the onset of hearing \[[@B22]\]. We hypothesized that a lack of a normal endocochlear potential or an alteration of the endolymphatic K^+^concentration could account for deafness in *Slc26a4*^-/-^mice. Measurements revealed that the endocochlear potential was absent but that the endolymphatic K^+^concentration was normal in adult *Slc26a4*^-/-^(Fig. [2a](#F2){ref-type="fig"}). No significant differences between *Slc26a4*^+/+^and *Slc26a4*^-/-^mice were found in perilymphatic (Fig. [2a](#F2){ref-type="fig"}) or plasma K^+^concentrations (*Slc26a4*^+/+^, 4.9 ± 0.3 mM, n = 6; *Slc26a4*^-/-^, 5.1 ± 0.3 mM, n = 6). These observations suggest that a primary event leading to deafness in *Slc26a4*^-/-^mice, and potentially in patients suffering from Pendred syndrome, is the loss of the endocochlear potential. Degeneration of hair cells is probably a response to the loss of the endocochlear potential. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Potential, K^+^concentrations and pigmentation of stria vascularis in *Slc26a4*^+/+^and *Slc26a4*^-/-^mice. **a:**Endocochlear potential and K^+^concentrations in endolymph and perilymph at the apex (A) and base (B) of the cochlea. Numbers adjacent to symbols denote number of measurements. **b-d:**Pigmentation of stria vascularis in *Slc26a4*^+/+^and *Slc26a4*^-/-^mice. **b:**View of stria vascularis through the bony capsule of the cochlea. OW, oval window, RW, round window; arrows, stria vascularis. **c-d:**Whole-mounts of stria vascularis isolated from age-matched mice. **c:**Laser-scanning images, bar = 10 μm, **d:**Quantification of pigmentation based on optical density. :::  ::: The endocochlear potential is generated by stria vascularis in the lateral wall of the cochlea \[[@B17],[@B23]\]. The potential is generated across the basal cell barrier of stria vascularis by the K^+^channel KCNJ10 located in intermediate cells \[[@B24]\], which are connected to basal cells by a high density of gap junctions \[[@B25]\]. Marginal cells of stria vascularis, which form the barrier toward endolymph, transport K^+^from the intrastrial space into endolymph and keep the K^+^concentration low adjacent to the KCNJ10 K^+^channel \[[@B26]\]. To determine the cause of the loss of the endocochlear potential in *Slc26a4*^-/-^mice, we first determined whether intermediate cells are present in stria vascularis, since a loss of intermediate cells is known to lead to a loss of the endocochlear potential \[[@B27],[@B28]\]. Intermediate cells of stria vascularis were visualized by their pigmentation. Pigmentation was present in stria vascularis of *Slc26a4*^-/-^mice (Fig. [2b](#F2){ref-type="fig"}), which suggests that intermediate cells are present. Interestingly, pigmentation of stria vascularis was much stronger in *Slc26a4*^-/-^than in *Slc26a4*^+/+^mice. To determine in greater detail the cause of the loss of the endocochlear potential in *Slc26a4*^-/-^mice, we isolated total RNA from stria vascularis and spiral ganglia of young (1--4 month) *Slc26a4*^+/+^and both young and old (\~12 month) *Slc26a4*^-/-^mice, assessed amounts of isolated tissues by quantification of 18S rRNA, and quantified the expression of KCNJ10 mRNA. Quantities of stria vascularis isolated from these different mice were similar, since no significant differences were found in the numbers of 18S rRNA molecules (log(rRNA) = 9.46 ± 0.08, n = 17, *pooled data*). In contrast, quantities of spiral ganglia isolated from young and old *Slc26a4*^-/-^mice (log(rRNA) = 9.04 ± 0.18, n = 4 and 9.29 ± 0.15, n = 6) were significantly smaller than in *Slc26a4*^+/+^mice (log(rRNA) = 9.48 ± 0.19, n = 7), consistent with morphometric data (*see below*). Expression of KCNJ10 mRNA was normal in stria vascularis and spiral ganglia of young *Slc26a4*^-/-^mice but significantly reduced in old *Slc26a4*^-/-^mice (Fig. [3](#F3){ref-type="fig"}). Quantifications of KCNQ1 and KCNQ4 mRNA were used to assess possible cross-contamination between the stria vascularis and spiral ganglia preparations based on the assumptions that (1) KCNQ1 is expressed in cells of the stria vascularis but not the spiral ganglia preparation and (2) KCNQ4 is expressed in cells of the spiral ganglia but not the stria vascularis preparation. The number of KCNQ1 mRNA molecules in stria vascularis was 24-fold greater than in spiral ganglia, and the number of KCNQ4 mRNA molecules in spiral ganglia was 5-fold greater than in stria vascularis. These observations validate our measurements of KCNJ10 and KCNQ1 by demonstrating that the microdissected preparations of stria vascularis and spiral ganglia were 98% and 78% pure, respectively. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Quantification of KCNJ10 and KCNQ1 mRNA expression in stria vascularis and spiral ganglia of *Slc26a4*^+/+^and *Slc26a4*^-/-^mice. **a:**Electropherogram of total RNA isolated from stria vascularis microdissected from one mouse. The amount of total RNA was obtained from the total integral (*shaded*) and the amount of 18S rRNA was obtained from the integral of the 18S peak. Sharp peaks representing 18S and 28S rRNA demonstrate the high quality of RNA. **Insert:**Genotype of *Slc26a4*^-/-^mice was verified by the observation of one or few very large rhomboedric otoconia in the utricular macula (*arrow*). A, crista ampullaris; U, utricular macula. Scale bar: 100 μm. **b:**Example of real-time RT-PCR data used for quantification of 18S, KCNJ10, KCNQ1 and KCNQ4. Known quantities of 18S rRNA were used to calibrate the threshold. SV, stria vascularis; SG, spiral ganglia. **c:**Quantification of KCNJ10 and KCNQ1 mRNA in young *Slc26a4*^+/+^and young and old *Slc26a4*^-/-^mice. :::  ::: The presence of KCNJ10 mRNA in stria vascularis of *Slc26a4*^-/-^mice supports the finding that intermediate cells are present. Expression of the KCNJ10 protein was assessed in young *Slc26a4*^-/-^mice by confocal immunocytochemistry. Interestingly, expression of the KCNJ10 protein was absent in stria vascularis but normal in spiral ganglia (Fig. [4](#F4){ref-type="fig"}). The absence of the KCNJ10 K^+^channel in stria vascularis is sufficient to explain the loss of the endocochlear potential \[[@B17]\]. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Protein localization of KCNJ10 in the cochlea of *Slc26a4*^+/+^and *Slc26a4*^-/-^mice. **a:**Overview of cochlea; bar = 100 μm. Compare to Fig. 1a to note the enlarged scala media and the distended Reissner\'s membrane. **b-c:**Detail of lateral wall and spiral ganglia (*insert*); main bar: 10 μm, insert: 5 μm. Expression of KCNJ10 in *Slc26a4*^-/-^mice was absent in stria vascularis but unchanged in spiral ganglion cells. RM, Reissner\'s membrane, SV, stria vascularis; SP, spiral prominence; SL, spiral ligament; LIM, spiral limbus; SG, spiral ganglion. :::  ::: Histological evaluation of cryosections revealed an enlargement of scala media with a large bulging of Reissner\'s membrane and an apparent degeneration of the organ of Corti, as described earlier \[[@B14]\]. Tissue height of stria vascularis was normal (Fig. [5](#F5){ref-type="fig"}), consistent with the similar numbers of 18S rRNA molecules in isolated preparations (*see above*). The absence of a change in tissue height is consistent with the presence of intermediate cells \[[@B28]\]. Further, we observed an apparent loss of tissue masses in areas that are normally occupied primarily by type I and II fibrocytes. Spiral prominence in *Slc26a4*^-/-^mice was less prominent, spiral ligament thinner and spiral limbus flatter (Fig. [5](#F5){ref-type="fig"}). The observation that tissue masses were lost in the spiral limbus region is consistent with the finding of a reduced number of 18S rRNA molecules in the spiral ganglia preparation (*see above*). In addition, we observed an apparent degeneration of stria vascularis, including an increase in pigmentation and an irregular pattern of the tight junctions of marginal cells (Fig. [2c](#F2){ref-type="fig"}, [6](#F6){ref-type="fig"},[7](#F7){ref-type="fig"}). Tight junctions were visualized by F-actin expression. Marginal cells appeared to form a continuous layer. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Morphometric analysis of cochlear tissue masses in *Slc26a4*^+/+^and *Slc26a4*^-/-^mice. **a:**locations of measurements. Thickness of stria vascularis (SV) was obtained as average of three distance measurements perpendicular to the surface of marginal cells. Thickness of spiral prominence (SP) was measured perpendicular to a tangential line (*dashed*) that connects the surface of the outer sulcus (OS) with the basal layer of stria vascularis. Thickness of spiral ligament (SL) was measured perpendicular to the tangential line as distance between the surface of spiral prominence and the interface between spiral ligament and bone (B). Thickness of spiral limbus (LIM) was obtained perpendicular to the surface of the bone as a tangential line that touches the inner sulcus (IS) and reaches from the surface of the spiral limbus to the interface between spiral limbus connective tissue and bone. **b:**Summary. Data from 7--8 animals contributed to each column. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Analysis of marginal and basal cell barriers by in *Slc26a4*^-/-^mice. Tight junctions were visualized by F actin. Whole-mounts of stria vascularis were viewed either from the basal cell side (**a-f**) or from the marginal cell side (**g-l**). Bright field images verify that the same area was viewed from either side (**b**and **h**). Colored bright field images were mixed with images of F actin staining to indicate the position of pigment granules (**d**, **j**, **f**and **l**). Focus was varied to either visualize the marginal cell barrier (SMC, **c-d**and **i-j**) or the basal cell barrier (BC, **e-f**and **k-l**). Both the marginal cell (**e-f**) and the basal cell barrier (**i-f**) appeared to be intact. It was critical for this finding that pigmentation did not block the path of the laser. Blockage of the laser by pigmentation produces the untrue impression of a discontinuous marginal cell barrier (**c-d**) or basal cell barrier (**k-l**). Comparison of images is aided by marking a significant area with a *star*. Bars = 10 μm. :::  ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Analysis of marginal and basal cell barriers in *Slc26a4*^+/+^and *Slc26a4*^-/-^mice. Tight junctions were visualized by F actin in whole-mounts of stria vascularis. Bright field images were taken to evaluate pigmentation (**a**, **d**and **g**). Note the intact marginal cell (**b**) and basal cell (**c**) barriers in *Slc26a4*^+/+^mice. Minimal pigmentation of *Slc26a4*^+/+^mice did not compromise F actin localization. Whole-mounts of stria vascularis from *Slc26a4*^-/-^mice were viewed either from the basal cell side (**e-f**) or from the marginal cell side (**h-i**). Bright field images verify that the same area was viewed from either side (**d**and **g**). Focus was varied to either visualize the marginal cell barrier (SMC, **b**,**e**and **h**) or the basal cell barrier (BC, **c**,**f**and **i**). Both the basal cell (**f**) and the marginal cell (**h**) barriers appeared to be intact. Blockage of the laser by pigmentation produces the untrue impression of \'holes\' in the marginal cell barrier (**e**) or basal cell barrier (**i**). Comparison of images is aided by marking three significant areas with colored *stars*. Bars = 10 μm. :::  ::: Pendrin-expressing surface epithelial cells in the spiral prominence region are located in an area where basal cells, which are interconnected by tight junctions, form additional tight junctions with surface epithelial cells \[[@B29]\]. A discontinuity of this complex junction in *Slc26a4*^-/-^mice would explain the absence of the endocochlear potential. To evaluate the presence of this connection, we determined by confocal immunocytochemistry the expression of ZO-1 and of F-actin, which associate with tight junction complexes. ZO-1 and F-actin expression revealed a continuous layer of basal cells, including a junction of basal cells with surface epithelial cells in *Slc26a4*^-/-^mice, as observed in normal mice (Fig. [1c,1d,1e,1f](#F1){ref-type="fig"},[6](#F6){ref-type="fig"},[7](#F7){ref-type="fig"}). These observations make it unlikely that the primary cause of the loss of the endocochlear potential is a compromise in the basal cell barrier. The observation that endolymphatic and perilymphatic K^+^concentrations were normal in *Slc26a4*^-/-^mice suggests that stria vascularis was able to secrete K^+^in spite of the apparent signs of degeneration. The rate of K^+^secretion necessary to maintain endolymphatic K^+^concentration, however, may be less than necessary in *Slc26a4*^+/+^mice given the reduced numbers of sensory hair cells, which provide a major pathway for K^+^exit from endolymph \[[@B30],[@B31]\]. In order to substantiate the view that stria vascularis in *Slc26a4*^-/-^secretes K^+^, we measured the magnitude of the bumetanide-sensitive current exiting stria vascularis across the apical membrane of strial marginal cells and determined the expression of the proteins KCNQ1, KCNE1 and SLC12A2, which are essential for K^+^secretion \[[@B20],[@B32],[@B33]\], and of GJB2 (Cx26), which is thought to contribute to K^+^cycling \[[@B23]\]. K^+^secretion is known to be sensitive to 10^-5^M bumetanide, a loop-diuretic that inhibits the Na^+^/2Cl^-^/K^+^cotransporter SLC12A2 \[[@B26]\]. Bumetanide-sensitive currents were found in both *Slc26a4*^+/+^and *Slc26a4*^-/-^mice although the magnitude of the current was significantly smaller in *Slc26a4*^-/-^mice (22 ± 6 μA/cm^2^, n = 4 versus 14 ± 2 μA/cm^2^, n = 4). KCNQ1 and KCNE1, subunits of the secretory K^+^channel, were co-localized in the apical membrane of strial marginal cells; the Na^+^/2Cl^-^/K^+^cotransporter SLC12A2, which is located in the basolateral membrane of strial marginal cells, was found in stria vascularis; and the gap junction protein GJB2 was found in spiral ligament of *Slc26a4*^+/+^and *Slc26a4*^-/-^mice (Fig. [8](#F8){ref-type="fig"}). Co-localization of KCNQ1 and KCNE1 proteins was also observed in vestibular dark cells (Fig. [1i](#F1){ref-type="fig"}). Expression of KCNQ1 protein in stria vascularis of *Slc26a4*^-/-^mice is consistent with the finding of KCNQ1 mRNA expression (Fig. [3](#F3){ref-type="fig"}). These observations make it unlikely that the primary cause of the loss of the endocochlear potential is a compromise of K^+^secretion by strial marginal cells or a compromise of gap junction mediated K^+^cycling. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Protein localization of KCNQ1, KCNE1, SLC12A2 and GJB2 in the cochlear lateral wall of *Slc26a4*^+/+^and *Slc26a4*^-/-^mice. **a-f:**bars: 10 μm. SMC, strial marginal cells; SV, stria vascularis; BC, basal cells; SL, spiral ligament. :::  ::: Conclusions =========== We described locations of pendrin expression in the cochlea and vestibular labyrinth and detected normal endolymphatic K^+^concentrations in spite of an enlargement of this fluid compartment. We found that *Slc26a4*^-/-^mice lack the endocochlear potential because they do not express KCNJ10 protein. Intermediate cells protect stria vascularis by converting CO~2~to HCO~3~^-^and by detoxifying free radicals (Fig. [9](#F9){ref-type="fig"}). CO~2~and free radicals are generated by the large numbers of mitochondria in the metabolically highly active strial marginal cells. Intermediate cells employ carbonic anhydrase to convert CO~2~to HCO~3~^-^\[[@B18],[@B19]\] and catalase to detoxify free radicals. To protect themselves from free radical damage, intermediate cells generate glutathione and melanin pigment \[[@B34]-[@B37]\]. It is conceivable that loss of pendrin, which may secrete HCO~3~^-^into endolymph, results in an accumulation of HCO~3~^-^and an alkalinization of the intrastrial spaces. This extracellular alkalinization may enhance free radical stress, since it may inhibit the uptake of cysteine and thereby limit production of the protective glutathione \[[@B38]\]. Support for the hypothesis of enhanced free radical stress comes from the observed hyperpigmentation in mice lacking pendrin. Strial hyperpigmentation has also been observed in other conditions that are associated with free radical stress, such as acoustic trauma \[[@B37]\]. Alterations in the cytosolic pH in conjunction with free radical stress may lead to the loss of KCNJ10 protein expression in strial intermediate cells. Function and expression of other K^+^channels has been shown to be controlled by the cytosolic pH and free radicals, which encode the metabolic state of the cell \[[@B39]\]. Suppression of the KCNJ10 K^+^channel in strial intermediate cells, which is essential for the generation of the endocochlear potential, is probably the direct cause of deafness in *Slc26a4*^-/-^mice and patients suffering from Pendred syndrome. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Model for the loss of KCNJ10 in the absence of pendrin expression in stria vascularis. Cys, cysteine, Glu, glutamate, Gly, glycine, CA, carbonic anhydrase, GST, glutathione-S-transferase, GSH, glutathione. :::  ::: Competing interests =================== None declared. Authors\' contributions ======================= PW designed and coordinated the immunocytochemical, morphometrical and molecular biological experiments. EMI and BA carried out confocal immunocytochemistry on cryosections. SJ and SVJ carried out confocal microscopy on whole-mounts of stria vascularis. SVJ and RJM carried out quantitative RT-PCR. DCM designed and coordinated electrophysiological experiments. TW carried out measurements of the endocochlear potential and the endolymphatic K^+^concentration. JHL carried out current measurements on strial marginal cells. SMW, LAE, IER and EDG provided the mice and the pendrin-specific antibody. PW and DCM conceived the study. PW wrote the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1741-7015/2/30/prepub> Acknowledgements ================ This work was supported by NIH-R01-DC01098 (PW), NIH-R01-DC00212 (DCM), NIH-R01-DK52935 (SMW) and Core facilities funded by NIH-P20-RR017686 (Confocal Microfluorometry and Microscopy Core, Molecular Biology Core) are gratefully acknowledged.

PubMed Central

2024-06-05T03:55:47.484204

2004-8-20

PMC516045

Background ========== Genetic-neuropathological correlation indicates that the apolipoprotein E type ε4 isoform specifies increased cerebral \[[@B1],[@B2]\] and cerebrovascular \[[@B3]\] accumulation of amyloid β-protein (Aβ). In addition, the apolipoprotein E ε2 isoform can apparently prevent the expression of clinical Alzheimer-type dementia which is otherwise typically associated with amyloidogenic mutations in the amyloid-β protein precursor \[[@B4]\]. Since the apolipoprotein E ε4 allele contributes to the genetic susceptibility underlying a large proportion (\~40--60%) of typical, sporadic Alzheimer disease, it is important to search for relevant interactions(s) between apolipoprotein E ε4 and Aβ in order to clarify the biological role for apolipoprotein E ε4 in Alzheimer disease. Currently proposed mechanisms include differential activities of apolipoprotein E isoforms in modulating Aβ fibrillogenesis \[[@B5]-[@B7]\] and/or Aβ clearance \[[@B8],[@B9]\]. Many studies of apolipoprotein E modulation of Aβ fibrillogenesis have utilized denatured apolipoprotein E, purified from the serum of human apolipoprotein E homozgotes following extraction in organic solvents \[[@B10]\]. While providing a convenient source of pure apolipoprotein E protein, this preparation does not represent native apolipoprotein E as it exists *in vivo*. Using a thioflavine T (ThT)-based assay \[[@B11]\] we have investigated the effects of native human apolipoprotein E isoforms on the kinetics of Aβ fibrillogenesis. Methods ======= Synthetic Aβ~1-40~or Aβ~1-42~(Keck Foundation Protein Facility, Yale University, New Haven CT) was freshly prepared for each assay at a concentration of 16 mg/ml in distilled, deionized water and diluted with phosphate-buffered saline (PBS) to generate a 5 mg/ml working solution. The \"aggregation step\" consisted of a reaction mixture containing 8 μl Aβ peptide (1 mg/ml final conc) and 32 μl of either apolipoprotein E ε3- or ε4-containing conditioned medium or control conditioned medium from SV40 empty vector-transfected cells. For the investigation of native apolipoprotein E preparations, apolipoprotein E isoforms were generated in the conditioned medium of stably-transfected SV40-apolipoprotein E ε3-, or SV40-apolipoprotein E ε4-, expressing Chinese hamster ovary (CHO) cells (CHO cells lack detectable endogenous apolipoprotein E; data not shown). All conditioned media were prepared using Dulbecco\'s minimal essential medium supplemented with 0.2% (wt/vol) bovine serum albumin only (no fetal bovine serum). Apolipoprotein E isoform levels were determined by quantitative immunoblotting of conditioned medium and apolipoprotein E-containing serum standards, the latter having been kindly provided by Dr. Petar Alaupovic of the Oklahoma Medical Research Foundation (Oklahoma City OK). Conditioned medium apolipoprotein E concentrations were then standardized using control medium conditioned by SV40 empty vector-transfected cells as diluent, yielding a final concentration of apolipoprotein E of 14 μg/ml, within the range of that reported in human cerebrospinal fluid. Since the final concentration of Aβ peptide was 1 mg/ml, the Aβ/apolipoprotein E stoichiometry (molar ratio) was \~500:1, suggesting models for the Aβ/apolipoprotein E interaction based either on a \"catalytic\" \"pathological chaperoning\" model of apolipoprotein E action on Aβ, or with a \"seeding\" model of Aβ folding. Detailed biochemical characterization of this native apolipoprotein E preparation has been reported \[[@B9]\]. The \"aggregation step\" fibrillogenesis reaction \[[@B11]\] was incubated at 37°C until the time of the ThT fluorescence measurement, which was performed from 1 to 7 days later. For the \"measurement step\" \[[@B11]\], 960 μ1 of 10 μM ThT (Nakarai Chemicals, Kyoto, Japan) in 50 mM phosphate buffer (pH 6.0) was added to the \"aggregation step\" reaction mixture. Within 30 minutes after addition of ThT, fluorescence was measured with a Millipore Cytofluor (Bedford MA) in each of five successive 200 μl aliquots of the reaction mixture, using an excitation filter of 450 nm and an emission filter of 482 nm, and a temperature of 25°C. In order to standardize the ThT assay in our Laboratory, we performed studies of Aβ fibrillogenesis following 1--7 day incubations of Aβ~1-40~, either in physiological phosphate buffer alone or in the presence of metal ions (Zn^2+^, Fe^2+^, or A1^3+^; 1 mM final conc \[[@B12]\]. ThT fluorescence and ultrastructural features were measured daily (not shown). Profibrillogenic activities of the metal ions tested were in agreement with a published report \[[@B12]\] (e.g., Al^3+^stimulated ThT fluorescence of Aβ~1-40~by 3.6 ± 1.1- to 5.7 ± 1.4-fold; p \< 0.01), indicating that our Aβ preparations were capable of metal ion-induced fibrillogenesis. Metals were not present during assessment of profibrillogenic effects of apolipoprotein E isoforms. Results and discussion ====================== No obvious profibrillogenic activity was detected in Aβ~1-40~-based assays of any native apolipoprotein E isoform (Table [1](#T1){ref-type="table"}). However, when ThT assays were repeated using Aβ~1-42~, modest, but statistically significant, profibrillogenic activity was detected in both apolipoprotein E ε3- and apolipoprotein E ε4-containing media and was similar in magnitude for the two isoforms (Table [1](#T1){ref-type="table"}). The observation of a profibrillogenic effect of apolipoprotein E specifically for Aβ~1-42~has been noted \[[@B5]\] and is of particular interest in light of biophysical and molecular neuropathological evidence suggesting that \"long\" Aβ peptides ending at positions N-42 or N-43 are apparently crucial for the initiation (\"seeding\") of Aβ deposition \[[@B13]\]. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effects of native apolipoprotein E isoforms on fibrillogenesis of Aβ~1-40~and Aβ~1-42~. Fold-effects represent means ± SEM of the quotients of ThT fluorescence values obtained for each Aβ peptide in the presence of apolipoprotein E-isoform-containing conditioned medium divided by ThT values obtained in the presence of conditioned medium lacking apolipoprotein E, derived from empty vector-transfected cells (n = 5--6). ::: ----------------------------------- ------------------------- ----------------- ------------- Aβ~1-40~ [1 day co-incubation]{.underline} CHO apolipoprotein E ε3 1.0 ± 0.l-fold N.S. CHO apolipoprotein E ε4 1.0 ± 0.l-fold N.S. [7 day co-incubation]{.underline} CHO apolipoprotein E ε3 1.0 ± 0.l-fold N.S. CHO apolipoprotein E ε4 1.2 ± 0.l-fold N.S. Aβ~1-42~ [4 day co-incubation]{.underline} CHO apolipoprotein E ε3 1.7 ± 0.27-fold p \< 0.01 CHO apolipoprotein E ε4 1.6 ± 0.18-fold p \< 0.005 [7 day co-incubation]{.underline} CHO apolipoprotein E ε3 1.7 ± 0.22-fold p \< 0.005 CHO apolipoprotein E ε4 1.8 ± 0.19-fold p \< 0.0005 ----------------------------------- ------------------------- ----------------- ------------- ::: These data demonstrate that native apolipoprotein E possesses \"pathological chaperone\"-type activity for Aβ: in other words, the data indicate that a chaperone-like misfolding reaction can occur between native apolipoprotein E and Aβ, at least at the concentrations and proportions evaluated herein. However, the equipotent activities of the apolipoprotein E ε3 and ε4 isoforms suggests the possibility that either extended co-incubation of apolipoprotein E and Aβ, or, perhaps, the inclusion in the reaction of other fibrillogenesis-modulation co-factors (such as metal ions, or inflammatory mediators such as reactive oxygen species, α~1~-antichymotrypsin, heparin sulfate-protcoglycan, non-Aβ component, apolipoprotein J, complement, etc.) may be required for modeling *in vitro*the apolipoprotein E-isoform-specific-regulation of extracellular Aβ accumulation that occurs *in vivo*. Alternatively, other events, such as differential apolipoprotein E-isoform-mediated clearance of Aβ or of apolipoprotein E/Aβ complexes \[[@B8],[@B9],[@B14]\] may contribute to apolipoprotein E-isoform-dependent Aβ accumulation. Differential anti-inflammatory activity might also play a role. Further investigation will be required in order to elucidate the precise mechanism(s) which specify how apolipoprotein E ε4 promotes Aβ accumulation in human brain and cerebral vessels *in vivo*. List of abbreviations ===================== ThT, thioflavine T; Aβ, amyloid-β peptide; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary cells. Competing interests =================== None declared. Authors\' contributions ======================= DS performed all assays, including the ThT assay, which was originated by HL. HL also oversaw the transfer of the assay from his lab to ours. RM prepared standard conditioned media from transfected cells provided by JDS. SG oversaw the project, supported the project as noted below, and wrote the manuscript. Acknowledgements ================ This research was supported by USPHS PPG grant AG10491 to S.G. We thank Jan Naslund and Christer Nordstedt (Karolinska Institute, Stockholm, Sweden) for critical comments and helpful discussion.

PubMed Central

2024-06-05T03:55:47.486705

2004-8-16

PMC516168

Background ========== Vacuolar H^+^-ATPases are large multi-heteromeric protein complexes located at endomembranes of all eukaryotic cells. Plant V-ATPase has been identified at the vacuolar and various other endomembranes, and also at the plasma membrane \[[@B1]-[@B3]\]. The total molecular mass of V-ATPase is estimated to surpass 700 kDa. V-ATPase pumps protons into membrane-surrounded intracellular compartments at the expense of hydrolysis energy of ATP \[[@B4]\]. V-ATPases share a common structure composed of a ball-like head, a membrane-intrinsic part and connecting stalks similar to ATP-producing F-ATP synthases. Biochemical examinations of the subunit composition revealed that V-ATPases are built from up to 14 subunits. The protein subunits are denominated VHA-A through H for soluble components protruding into the cytoplasm (V~1~-part), and VHA-a, c, c\', c\", d and e for membrane-associated subunits (V~0~-part) \[[@B5]\]. In plants, the molecular characterisation of VHA-subunits is only beginning to include also the genes that were identified recently \[[@B6],[@B7]\]. Following the cloning of cDNA sequences coding for VHA-A, -B, -c, and -E until 1995, VHA-D, -F, -C and -G were cloned from *A. thaliana*, oat and barley \[[@B5],[@B7]\]. However, the first complete set of subunit sequences only became available with the sequencing of the genome of *A. thaliana*\[[@B9]\]. The second set was cloned from the halotolerant, facultative CAM plant *Mesembryanthemum crystallinum*\[[@B10]\], and a third set is now available from rice. A comparative analysis of the genes/ESTs revealed that *Arabidopsis*and *Mesembryanthemum*express a similar set of subunits \[[@B7]\]. A detailed analysis of the sequences from both species showed that all examined subunits share the same properties in binding of ATP and conducting protons through the proteolipid ring \[[@B10]\]. An important question concerns the composition of the plant V~0~-sector since its subunit composition is not resolved yet. VHA-a is the subunit with the highest molecular mass within the V-ATPase and reveals a bipartite structure. As first shown in yeast, VHA-a consists of a 50 kDa hydrophilic N-terminal domain and a hydrophobic, membrane-spanning C-terminus \[[@B11]-[@B13]\]. Interestingly, Li and Sze \[[@B14]\] could not observe the VHA-a subunit in functional V-ATPase of oat. Therefore, they suggested a role of VHA-a in assembly of plant V-ATPase. The conclusion is in contrast to results in yeast where site directed mutagenesis had allowed to identify amino acids involved in the mechanism of proton translocation across the membrane. Mutant yeast cells devoid of VHA-a or complemented with modified VHA-a variants exhibited the conditional phenotype of V-ATPase deficiency \[[@B15]\]. Another novel subunit in plants that was only addressed recently is VHA-H. VMA13p being the yeast orthologue of VHA-H was cloned and represents the only crystallized subunit of V-ATPase at present \[[@B16]\]. VHA-H is considered to activate and regulate V-ATPase by functionally coupling ATP hydrolysis to proton flow through the V~o~-domain \[[@B17]\]. The aim of the study was to improve the understanding of V-ATPase distribution in plant cells with emphasis on the localization of VHA-a and VHA-H in the V-ATPase structure. Three specific questions were answered using different methodology: (i) Is VHA-a part of the V-ATPase structure? (ii) Where are different subunits localized within plant cells? (iii) Is FRET a suitable method to investigate subunit interaction within the complex, for example between VHA-a and VHA-c, and VHA-H and VHA-B. Results ======= Primary structure of VHA-a -------------------------- From the database dbEST (NCBI, USA) a cDNA fragment from *M. crystallinum*was identified (AI822404) with similarity to known genes coding for VHA-a in yeast. Using RACE-PCR a full length cDNA was obtained with a size of 2783 bp. Its open reading frame encoded a hypothetical protein of 93.1 kDa. Database search with the program FASTA revealed the highest similarities to the entries At2g21410 (77%), At4g39080 (76 %) and At2g28520 (60 %) from the *A. thaliana*genomic database and also significant similarity on the amino acid level to the genes Stv1 (34 %) and Vph1 (38 %) from *S. cerevisiae*. These two latter genes are coding for isoforms of the yeast 100 kDa subunit whereas all other subunits of the yeast vacuolar ATPase are encoded by one gene each. The sequence alignment between the newly cloned amino acid sequence from *M. crystallinum*, the three isogenes from *A. thaliana*and the two isogenes from yeast (Fig. [1](#F1){ref-type="fig"}) indicates a structure with two distinct segments distinguished by their degree of similarity: the N-terminus with a low degree of sequence conservation (amino acid 1 to 400) and the C-terminus (amino acid 401 to 816) with a higher degree of conservation between the sequences. This corresponds to the above mentioned domain structure of *S. cerevisiae*VHA-a with a cytoplasmic and a membrane-integrated domain. The heterogeneity in the N-terminus is characterised through a high number of deletions and insertions most remarkably between the amino acid positions Gly84 to Ile85, and Gly141 to Gln142. In contrast to all other VHA-sequences, in yeast-Stv1 this region contains additional sequence insertions of approximately 20 amino acids. Sequence variation between VHA-a from distinct species and paralogues within same species continues until Glu193. Other domains of the N-terminus vary to a lesser extent. The heterogeneity decreases in direction to the membrane-spanning C-terminus. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Amino acid sequence alignment of plant and yeast VHA-a.**Comparison of amino acid sequences deduced from the three coding regions detected in the *A. thaliana*genome, the sequence of *M. crystallinum*and the two yeast gene products Vph1 and Stv1 using ClustalW-algorithm. Identical amino acids are marked in dark grey, those similar with *M. crystallinum*in light grey. The programs JPRED (EMBL, Hinxton) and THMM (EBS, Denmark) were used to predict transmembrane domains (labelled with +). Amino acids identified as essential for H^+^-pumping in yeast are marked with \*. :::  ::: The N-terminal amino acids of VHA-a that are conserved in all species are characterised by an above average portion of amino acids with an acid or aromatic character. A comparison of the first 400 amino acids of VHA-a from 10 different species showed the presence of 34 amino acids conserved throughout all species, 10 of which have acidic and 8 aromatic residues. In all sequences compared the representation of aromatic amino acids is higher than the average. In yeast it was shown that sequence motifs with aromatic amino acid residues are involved in the targeting of proteins \[[@B18]\]. An assignment of VHA-a sequences from *A. thaliana*and *M. crystallinum*to the distinct yeast isoforms Vph1 and Stv1, in order to define orthologous genes, was not possible on basis of the amino acid sequence alignment. The comparison of the C-termini revealed a higher degree of sequence conservation interrupted through various short insertions or deletions. The sequence was analysed for secondary structures (Predict Protein, EMBL, Heidelberg) and membrane topology (THHM, Denmark). The prediction correlated regions of high sequence conservation with the localisation of putative transmembrane domains (Fig. [1](#F1){ref-type="fig"}, marked with +). Furthermore, these hypothetical transmembrane domains are in accordance with the membrane-topology suggested by Leng et al. \[[@B19]\] for the yeast Vph1 protein. Mutations in single amino acids of Vph1 have previously allowed the identification of 5 charged amino acids in the membrane spanning helices of the C-terminus whose mutation strongly affected (Lys734, His743, Glu789 and Arg799) or fully inhibited (Arg735) transmembrane H^+^-transport, although they did not affect the assembly of the vacuolar ATPase \[[@B19]-[@B23]\]. These functional amino acids are also conserved in Mc-VHA-a. Detection of VHA-a in membrane preparations in vitro ---------------------------------------------------- For further examination of McVHA-a, antisera were raised against two polypeptide fragments, i.e. a N-terminal 42 kDa polypeptide and a 13 kDa domain located between predicted transmembrane helices 3 and 4. Both were expressed heterologously in *E. coli*using the primer combination a-nterm-f and a-nterm-r, and a-memb-f and a-memb-r, respectively. The derived PCR-products were cloned into the expression vector pCRT7-NT (Invitrogen, Holland), introduced and expressed in *E. coli*. The 42 kDa and 13 kDa polypeptides were purified to homogeneity by chromatography on Ni-NTA and by preparative SDS-PAGE. The 42 kDa polypeptide was recognized by a purified antibody against yeast Vph1 \[[@B13]\] (not shown). The antiserum produced in guinea pig reacted with the heterologously expressed protein a~N-term~(not shown). The presence of VHA-a was investigated in plant endomembranes and soluble fractions rapidly prepared from 5 week old *M. crystallinum*plants. In the soluble and the membrane-fraction, the serum against McVHA-a~N-term~identified two polypeptides with apparent molecular masses between 65 and 70 kDa (Fig. [2](#F2){ref-type="fig"}). After increasing the NaCl concentration to 500 mM, the apparently full size 95 kDa band was detected in the membrane fraction corresponding to the expected size of untruncated McVHA-a (Fig. [2](#F2){ref-type="fig"}). To test whether the addition of NaCl to the membrane preparation affected the purification efficiency of other V-ATPase subunits from the V1-sector, membranes and soluble fractions purified with buffer containing either 100 or 500 mM NaCl were also reacted with antibodies against VHA-E \[[@B24]\] and anti VHA-D~i~recognizing VHA-B and D~i~\[[@B25]\]. The latter authors demonstrated that this antibody marked both VHA-A and -B. The increased NaCl concentration had no effect on the labelling strength of VHA-E and VHA-A/B (not shown). Interestingly, also McVHA-A, -B and -E were detected both in the membrane and soluble fractions. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Immunodetection of VHA-a in membrane and soluble fractions of *M. crystallinum*leaves.**Membranes were isolated in the presence of 100 (1, 2), 500 mM NaCl (3, 4), and without (5) or with (6) protease inhibitor complete^®^, respectively. Membranes and soluble fraction were separated rapidly for (1)--(4) whereas the standard membrane isolation procedure with ultracentrifugation was undertaken for (5) and (6). The 95 kDa band is seen as the dominant protein in the membrane fraction isolated at high salt and to some part in the isolate obtained in the presence of protease inhibitor. :::  ::: To examine the sensitivity of VHA-a against proteases, membranes were isolated in second approach, in the absence and presence of a protease inhibitor cocktail (complete^®^, Roche, Mannheim, Germany). Without protection from proteolysis, a band at about 50 kDa and a doublet band above 60 kDa were predominant. In the sample with protease inhibitor, the 95 kDa band appeared although as one of five bands of similar intensity if decreasing transfer efficiency of high molecular mass polypeptides from the gel to the membrane is assumed. Apparently, VHA-a is prone to degradation. However, the results also indicate that VHA-a is part of V-ATPase. To further prove that tentative conclusion, immunoprecipitation was performed using anti VHA-a~N-term~and anti VHA-a~memb~followed by Western blot analysis with anti VHA-E and anti-VHA-A (Fig. [3](#F3){ref-type="fig"}). Each control, i.e. immunoprecipitation without serum and with preimmune serum, gave the expected results of no response (Lanes 1 and 2 in Fig. [3A,3B,3C](#F3){ref-type="fig"}). With serum, the 55 kDa band of VHA-A and the dimer of VHA-E was seen. Apparently, immunoprecipitation with antibody directed against the N-terminus as well as the membrane part of VHA-a pulled down a complex also containing subunits of the V~1~section, and thus probably the holocomplex. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Co-immunoprecipitation of VHA-E and VHA-A with VHA-a.**Tonoplast enriched membranes of *M. crystallinum*were solubilized in buffer supplemented with 2% (v/v) Triton X-100. Antibody directed against VHA-a, either the N-terminal or membrane part, was added and immunoprecipitation was performed. The pellet samples and part of the supernatant (10--15% of total) were loaded on a SDS-gel, and Western blot was performed with anti VHA-E or A. The band intensities related to loaded sample size indicate that only a fraction of total V-ATPase was immuno-precipitated with the anti-VHA-a antibodies. As controls, immunoprecipitation was performed without added serum and with preimmune serum. With anti-E, monomeric and dimeric band of 30 and 60 kDa was detected by Western blotting in the precipitate obtained with anti VHA-a~N-term~, with anti VHA-A, a 65 kDa band was labelled in separations of both, the precipitates obtained with anti VHA-a~N-term~and anti-VHA-a~Memb~, respectively. :::  ::: Immunochemical analysis of VHA-distribution ------------------------------------------- A more detailed analysis of the distribution of the VHA-subunits in the plant cell was performed by immuno-labelling of maize root tip cells. To highlight the distribution of the different VHA-subunits in the cell, squashed maize root cells were incubated with anti VHA-A, anti VHA-E or anti VHA-a~N-term~. Fig [4](#F4){ref-type="fig"} shows the staining patterns of the antibodies in young cells devoid of large vacuoles (Fig. [4A,4D,4G](#F4){ref-type="fig"}), cells with beginning vacuolization (Fig. [4B,4E,4H](#F4){ref-type="fig"}) and older cells with many vacuoles of 2--5 μm diameter (Fig. [4C,4F,4I](#F4){ref-type="fig"}). In cells devoid of large vacuoles, anti VHA-E as well as anti-VHA-A marked punctuated compartments whereas in cells with developed vacuoles a nearly complete staining of the tonoplast and an unsteady staining of other small cellular compartments was observed. In a converse manner, anti-VHA-a~N-term~did not stain the tonoplast of cells of various vacuolisation state. The staining with anti-VHA-a~N-term~revealed a distinct reticulate pattern and a staining of the nuclear membrane reminding of ER-labelling. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Localization of VHA-subunits in plant cells, using anti-VHA-A, anti-VHA-E and anti-VHA-a~Nterm~-antibody**Confocal images represent single images of isolated maize root cells. Immuno-staining was performed on maize root cells. Secondary fluorescently labelled antibodies used were anti-rabbit-FITC (A-F) or anti-guineapig (Cy5). Images were colour coded with Adobe Photoshop. Scalebars are 10 μm. Labelling of tonoplast membranes with anti VHA-A (A to C) and anti VHA-E (D-F) in root cells of increasing age, i.e. non-vacuolized to vacuolized (left to right). Anti-VHA-a~N-term~labelling of root cells of increasing age (G-I). Note that the staining pattern of VHA-a~N-term~is distinct from the tonoplast labelling with VHA-A and VHA-E in all cases. :::  ::: A double immuno-labelling-technique \[[@B26]\] was then employed to identify the antibody-marked intracellular compartments in detail. The selected antibodies were directed against the aquaporin γ-Tip located in the tonoplast of the lytic vacuole (anti γ-Tip; \[[@B27]\]) and against marker components of the endoplasmic reticulum (ER) (anti-calreticulin). The results of this immuno-staining are shown in Fig. [5](#F5){ref-type="fig"}. In cells with developed vacuoles, anti-γ-Tip-labelling of the tonoplast (Fig. [5A](#F5){ref-type="fig"}) co-localised completely with anti-VHA-A (Fig. [5B,5C](#F5){ref-type="fig"}). Anti VHA-E labelled similar structures as anti-VHA-A and co-localised also with anti-γ-Tip on the tonoplast (not shown). A co-staining of root cells with anti-VHA-A and the ER-marker anti-calreticulin was then performed. Anti-calreticulin marks a specific ER-network including the nuclear membrane (Fig. [5D,5G,5J](#F5){ref-type="fig"}). The double labelling with antibodies against VHA-A (Fig. [5E](#F5){ref-type="fig"}) or VHA-E (Fig. [5H](#F5){ref-type="fig"}) showed no significant co-localisation of the typical tonoplast staining with the ER-marker. When performing a co-labelling of maize root cells with anti-calreticulin (Fig. [5J](#F5){ref-type="fig"}) and anti-VHA-a~N-term~(Fig. [5K](#F5){ref-type="fig"}) revealed a complete co-labelling of the two markers (Fig. [5L](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Co-localization of VHA-subunits with the tonoplast marker γ-Tip and calreticulin, a marker for the ER.**Confocal images represent single images of isolated maize root cells which were immuno-probed with antibodies directed against marker polypeptides of the vacuolar membrane (γ-Tip) and endoplasmatic reticulum (calreticulin) and simultaneously treated with anti-VHA-antibodies. Secondary fluorescently labelled antibodies used were anti-rabbit-FITC (A), anti rabbit-Cy3 (B, E, H), anti-mouse-FITC (D, G, J) or anti-guineapig-Cy5 (K). Images were colour coded with Adobe Photoshop. Scalebars are 10 μm. In each row, the immuno-decoration with the marker, with the VHA-subunit specific antibody and the superposition of both is shown. (A-C) Root cell labelled with γ-Tip (A) and VHA-A (B). Note the complete co-localisation of the both markers on the tonoplast of small vacuoles (C). (D-F) Double-staining with calreticulin (D) and VHA-A. (G-I) Double-labelling of a root cell with calreticulin (G) and VHA-E (H) reveals a similar result as with VHA-A. Tonoplast labelling and ER-staining are distinct. (J-L) Co-labelling with calreticulin (J) and VHA-a~N-term~reveals a complete co-localisation of the two signals on the ER. :::  ::: For high resolution, immunogold labelling with anti VHA-a~N-term~and anti VHA-A was performed on ultra-thin cross sections of maize root cells (Fig. [6](#F6){ref-type="fig"}). With anti VHA-a pronounced label with gold particles was detected in ER membranes (Fig. [6D](#F6){ref-type="fig"}), and occasionally a weak labelling of the Golgi apparatus (Fig. [6C](#F6){ref-type="fig"}). A labelling of tonoplast membranes was not found, indicating that VHA-a~N-term~is predominantly located on the ER. For comparison, immuno-gold analysis of ultrathin cross sections with anti-VHA-A. revealed labelling of the tonoplast (not shown) and a labelling of the Golgi apparatus significantly stronger than with anti-VHA-a (Fig. [6A](#F6){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Immunogold-based localisation of VHA-a.**Ultra-thin cross sections of maize root cells in 1 mm distance to the tip were decorated anti VHA- (A), premmune serum, (B), anti VHA-a~N-term~-antibody (C,D), respectively. Sections were washed, treated with secondary antibody linked to 15 nm gold particles and visualized in an electron microscope. :::  ::: Fluorescence resonance energy transfer between VHA-subunits in vivo ------------------------------------------------------------------- FRET allows to investigate protein-protein interaction *in vitro*and *in vivo*. Both partners have to carry fluorescent labels with overlapping emission (donor fluorophore) and excitation spectra (acceptor) and need to be situated in close proximity. Half maximum energy transfer takes place at distance of the Förster radius R~0~. Cyan and yellow fluorescent proteins constitute such a FRET pair and were fused to the C-termini of various subunits of V-ATPase. Under the assumption of freely rotating fluorophores, R~0~is close to 5 nm. The size of the V-ATPase complex is about 15 nm (diameter) × 25 nm (length from lumen side to tip of head). *Arabidopsis*protoplasts were co-transformed with vector constructs of VHA-a fused to YFP and VHA-c fused to CFP under the control of the 35S promotor. Upon excitation of doubly labelled protoplasts at 458 nm both, CFP and YFP showed strong fluorescence (Fig. [7A](#F7){ref-type="fig"}). Fluorescence emission spectra were recorded using a double dichroic mirror which exhibits high reflectivity at \~514 nm. Therefore, the two emission maxima were separated by a minimum (Fig. [7C](#F7){ref-type="fig"}). This fact renders the quantitative analysis of the FRET efficiency due to the decrease in donor and increase in acceptor fluorescence more difficult. Alternatively, the method of acceptor bleaching can be used to verify energy transfer \[[@B28]\] and can be seen in Fig. [7B](#F7){ref-type="fig"}, the fluorescence intensity of YFP decreased rapidly within 10 scan cycles upon excitation at 514 nm due to photobleaching to a residual intensity attributable to auto-fluorescence. Simultaneously, the donor fluorescence increased substantially, thus providing direct evidence for energy transfer from CFP to YFP. From the increase in CFP fluorescence upon acceptor bleaching an overall FRET efficiency of \~0.45 is estimated. Assuming freely rotating fluorophores (K^2^= 2/3) this corresponds to a distance between VHA-a, and VHA-c of \~5.4 to 7.2 nm (mean 6.3 ± 0.8 nm). Here it has to be pointed out that each ring of the rotor V~0~contains 5 VHA-c-subunits and one VHA-c\"-subunit. Hence, dependent on the position of the VHA-c/CFP-subunits in the ring different distances between CFP-labelled VHA-c subunits and the YFP-labelled VHA-a subunit will result. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **FRET between VHA-subunits co-expressed in protoplasts of *A. thaliana*and onion epidermis cells**. (A) Mesophyll protoplasts of *A. thaliana*were simultaneously transformed with p35S::VHA-a/YFP and p35S::VHA-c/CFP using the polyethylene glycol method. After 20 h, fluorescence emission from protoplasts was measured following excitation at 458 nm and 514 nm, and image analysis in the range of 470 -- 500 nm for CFP and 560 -- 585 nm for YFP fluorescence, respectively. (B) Acceptor bleaching in dependence on scan numbers. For this experiment, protoplasts expressing VHA-a-YFP were excited at 514 nm and emission was recorded between 550 and 600 nm. (C) Emission spectra of VHA-c-CFP and VHA-a-YFP before (solid line) and after (broken line) acceptor bleaching. (D) FRET between VHA-A-YFP and VHA-B-CFP after co-expression in onion epidermis cells. Excitation was achieved at 458 nm, and 2D emission images were recorded in the range of 470 to 510 nm (CFP), and 550 to 600 nm (YFP). :::  ::: Similar experiments were performed with onion epidermis cells (Fig. [7D](#F7){ref-type="fig"}) co-transformed with VHA-A/YFP and VHA-B/CFP, and VHA-B/CFP and VHA-H/YFP, respectively. This system was employed for two reasons, (i) to confirm and extent the results from protoplasts and (ii) to work in turgescent cells, not previously subjected to a protoplast isolation stress. Emission spectra were recorded before and after photobleaching of acceptor. Decreases in acceptor and increases in donor fluorescence intensities, respectively, were smaller for these pairs of VHA-fusions than for VHA-a-YFP and VHA-c-CFP (shown in Fig. [7C](#F7){ref-type="fig"}). Nevertheless there was significant FRET in the case of VHA-A/YFP and VHA-B/CFP and some indication of FRET in the case of VHA-B/CFP and VHA-H/YFP, whereas the co-transformed pair of VHA-A/CFP and VHA-H/YFP gave no FRET. It should be noted that in addition to the highly expressed fusion proteins, untagged endogenous subunits still are present in the cell. Under such condition, formation of partial subcomplexes with possibly varied FRET properties may not be ruled out. Discussion ========== The set of subunits that assemble plant V-ATPase has recently been completed by similarity searches in plant genomic and EST sequences using information on yeast VMA and other orthologues \[[@B5],[@B6]\]. The presence of a subunit of about 100 kDa in the functionally active plant vacuolar ATPase has been under discussion for a long time \[[@B14]\]. Here, for the first time, a cDNA coding for a plant VHA-a was cloned and characterised. McVHA-a as well as the homologous *Arabidopsis*gene products contain all charged amino acids that have been shown to be essential for proton-translocation at conserved positions in the membrane spanning region of the C-terminus (Fig. [8](#F8){ref-type="fig"}). Additional evidence for an essential function of VHA-a in V-ATPase was first obtained for V-ATPase of *Bos bovis*. The specific V-ATPase-inhibitor bafilomycin \[[@B29]\] was shown to bind to the 100 kDa VHA-a subunit and not, as previously suggested to the proteolipid VHA-c. The results were confirmed for all tested species \[[@B30]\]. Bafilomycin also is a potent inhibitor of plant V-ATPase and is routinely used to distinguish V-ATPase-dependent ATP hydrolysis from background activity \[[@B31]\]. In 1999, Li and Sze \[[@B14]\] detected two unknown polypeptides with apparent molecular masses of 63 and 54 kDa in purified catalytically active V-ATPase but no polypeptide with a molecular mass of about 100 kDa. The authors hypothesized that plant V-ATPase is active in the absence of the 100 kDa subunit. Another explanation for this observation could be the sensitivity of VHA-a to degradation through proteases \[[@B32]\], and the detected unknown polypeptides could result from limited proteolysis of VHA-a, producing subunit-fragments still capable of transporting protons. The immunoblots with soluble fractions and membrane-preparations of *M. crystallinum*(Fig. [2](#F2){ref-type="fig"}) incubated with anti VHA-a~N-term~support this hypothesis. The 65 kDa fragment in the cytoplasmic fraction is likely to derive from a proteolytic processing, releasing the soluble portion of McVHA-a. Accordingly, high NaCl concentration in the purification medium could inhibit involved proteases. The hypthesis is supported by the experiment with protease inhibitor cocktail, where the 95 kDa subunit was detected as band with intermediate intensity, although only among other bands that were immuno-responsive to anti-VHA-a antibody. Thus even the protease inhibitors could not fully suppress proteolysis. Immunoprecipitation with the antibodies anti VHA-a~N-term~and anti VHA-a~memb~further proved that both the N-terminal hyrophilic portion and the membrane sector of VHA-a are associated with a complete V-ATPase complex. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Membrane topology of plant VHA-a and location of the C-termini of individual subunits based on the FRET data.**Based on amino acid sequence analysis and similarity with Vph1 (Leng et al. 1999), the topology of Mc-VHA-a is depicted in (A). The relative location of amino acid residues essential for proton pumping or structure are indicated with boxes. The numbers indicate the amino acid positions. In (B), the results from the FRET experiments are summarized: Asterisks tentatively mark the positions of the C-termini where the GFP variants have been fused to. :::  ::: In plants, immuno-cytochemical examinations of the subcellular localisation of single subunits or holocomplexes (mostly using antibodies directed against VHA-A) have previously indicated a distribution of V-ATPase among nearly all endomembranes of the secretory pathway including the plasmalemma \[[@B1]-[@B3]\], \[[@B33]-[@B35]\]. These findings were supported by measurement of bafilomycin-sensitive ATPase activity in selectively purified endomembranes of plants \[[@B36]-[@B38]\]. In our examination the antisera against VHA-A and -E showed a staining of the tonoplast and to a lesser extent also of the ER and Golgi-Apparatus (data not shown). VHA-A and E are part of the cytoplasmically exposed V~1~-structure of V-ATPase. The presence of both subunits on all these membranes shows the presence of fully assembled V-ATPase. The presence of the active V-ATPase in plants is not only necessary at the tonoplast, since Matsuoka et al. \[[@B38]\] could show that the activity of the vacuolar ATPase on the ER and the GA is necessary for correct targeting of soluble storage proteins to the vacuole. The presence of active V-ATPase on the prevacuolar compartment was concluded from the acid pH-optimum of enzymes involved in vacuolar transport, for example the activity of the vacuolar sorting receptor BP-80 whose action is strictly pH dependent \[[@B21]\]. For this reason a similar staining pattern of all used anti-VHA antisera would have been anticipated, but our results showed distinct staining patterns of anti-VHA-A and -E on the one hand and anti-VHA-a~N-term~on the other hand. Immuno-labelling indicates that VHA-a~N-term~antiserum exclusively labels the ER with some rare association on Golgi stacks. These results are surprising since the sequence features of McVHA-a suggest an essential involvement of VHA-a in proton transport. A possible explanation might be the presence of three different isoforms in *A. thaliana*and *O. sativa*which all share the localisation of the charged amino acids responsible for proton-translocation (Fig. [1](#F1){ref-type="fig"}). Based on our results, we suggest that the isoform of VHA-a recognised by anti VHA a-~Nterm~(an antibody which was generated against the isoform-specific N-terminus) is exclusively associated with V-ATPase localised on the ER. The hypothesis of a compartment-specific localisation of VHA-a subunits is supported through several findings in plants and yeast. In yeast all known subunits and chaperones of the V-ATPase are encoded by one gene. Only the 100 kDa VHA-a is encoded by two different isoforms (Vph1 and Stv1) \[[@B13]\]. In general the subunit-isogenes of the V-ATPase have a very high degree of similarity within each species \[[@B16],[@B39]\]. In a converse manner, the sequences of the VHA-a isogenes are very heterogenic in *S. cerevisiae*and *A. thaliana*(Fig. [1](#F1){ref-type="fig"}) especially in the cytoplasmic region of the N-terminus \[[@B40]\]. A differential localisation was shown for the two yeast isoforms (Vph1 and Stv1) \[[@B12]\]. A detailed examination through Kawasaki-Nishi et al. \[[@B40]\] showed a localisation of Vph1 on the tonoplast, while Stv1 was detected on the late Golgi-apparatus and the prevacuolar compartment of yeast. By expressing chimeric proteins composed of the N-terminus of Stv1 and the C-terminus of Vph1, and vice versa, the authors were able to show that the N-terminus defines subcellular sorting. Following selective enrichment of the differentially localised complexes, both types of V-ATPases were shown to differ in their stability of the V~0~/V~1~-complex and in their coupling efficiency \[[@B41]\]. The N-terminus was responsible for the differential coupling activity, whereas the C-terminus mediated the differential dissociation. In plants, Matsuoka et al. \[[@B38]\] were able to distinguish between two different V-ATPase activities through their differential response to the V-ATPase inhibitor bafilomycin. These V-ATPase activities were localised in distinct membrane fractions of the secretory pathway including the vacuolar compartments. An antibody directed against the V-ATPase holoenzyme revealed significant differences in immuno-staining of endomembrane and vacuolar fractions. These and our results on sequence properties of the different plant VHA-a isoforms and the intracellular localisation of VHA-a support the hypothesis that at least two different V-ATPase activities exist in plants, differing in intracellular localisation and sensitivity to bafilomycin. The target of bafilomycin is VHA-a \[[@B29]\]. Thus, both types of V-ATPases might be distinguished through the presence of different isoforms of VHA-a. Our results from FRET analysis also allows to make some structural assignments: From crystal structure of F-ATP synthase, the C-termini of subunit β, homologous to VHA-A, and subunit α, homologous to VHA-B, are located in close vicinity and oriented to the membrane \[[@B42]\]. Occurrence of FRET between VHA-A/YFP and VHA-B/CFP supports the same structural arrangement in V-ATPase. Following crystalization of isolated yeast VHA-H \[[@B16]\], the structure was fitted in 3D reconstructions of plant V-ATPase based on electron microscopic analysis \[[@B43]\] and suggests localization of the C-terminus of VHA-H to the head structure in proximity to VHA-B. *In vivo*-FRET in protoplasts expressing VHA-B/CFP and VHA-H-YFP confirms the orientation of the C-termini of VHA-B and H in close vicinity. It should be noted that co-expression of other pairs of chimeric donors and acceptors such as VHA-E/CFP and VHA-c/YFP did not elicit FRET after excitation of CFP (not shown). The assumed location of the C-terminus of VHA-c in the lumen of the endomembrane compartments and the C-terminus of VHA-E most likely in the vicinity of the head is in agreement with the negative result, i.e. the absence of FRET between VHA-E/CFP and VHA-c/YFP. The studies exemplify the suitability of FRET to analyse structural features of V-ATPase *in vivo*. The efficient but variable FRET in cells expressing VHA-c/CFP and VHA-a/YFP allows two conclusions: First, the C-termini of VHA-c and VHA-a are likely to be located on the same, i.e. luminal, side of the endomembrane compartments supporting the topological model with nine transmembrane-domains of VHA-a in plants as previously suggested for yeast \[[@B22],[@B23]\] (Fig. [8](#F8){ref-type="fig"}). Second, a significant portion of total VHA-a is located in the neighbourhood of VHA-c. The calculated distance of 5.4 to 7.2 nm between donor and acceptor fluorophore corresponds to the diameter of the proteolipid-ring of the rotor, consisting of 5 subunits of VHA-c and 1 VHA-c\", respectively. The results confirm that VHA-a is part of the functional complex and not only involved in V-ATPase assembly. More than half (cf. Fig. [2](#F2){ref-type="fig"}), and possibly all V-ATPase complexes contain the holopolypeptide of 95 kDa. Conclusions =========== The analysis of the primary structure of plant VHA-a revealed the presence of amino acid residues that are essential for proton pumping in yeast. Employing immuno-co-precipitation and FRET it could be demonstrated that subunits VHA-a and VHA-H are part of the V-ATPase complex of plants. Furtheron it is shown that one distinct VHA-a subunit isoform is localized on the ER. The study also shows the usefulness of FRET to study multisubunit protein structures in vivo and in vitro. Methods ======= Plant growth ------------ *Mesembryanthemum crystallinum*and *Arabidopsis thaliana*were grown in hydroponics and soil culture, respectively, as described in \[[@B10],[@B44]\]. Growth conditions were 120 μmol quanta m^-2^s^-1^, 60% relative humidity, 20°C, and a daily photoperiod of 12 h duration. Rosette leaves from 3- to 5-week-old *Arabidopsis*plants were taken for protoplast transfection. *Zea mays*and *Hordeum vulgare*were germinated on filter paper in the dark at 25°C for 48 h. Cells were isolated from the first 2 mm of the growing root tip. Onion epidermis was stripped from onion bulbs obtained from a local market. Membrane isolation ------------------ Leaves (50 g) of *M. crystallinum*were homogenized in a buffer containing 250 mM sucrose, 50 mM Tris-Cl, pH 8.0, 4 mM ethylenediamine tetraacetic acid (EDTA), 4 mM dithiothreitol and a few crystals of phenylmethylsulfonylfluoride \[[@B45]\]. As indicated in a set of experiments, either NaCl was added at 100 or 500 mM concentration or complete protease inhibitor^®^cocktail (Roche, Mannheim, Germany) was added throughout the procedure. Following differential sedimentation and gradient centrifugation, tonoplast enriched membranes were recovered from a 30%/35% sucrose interphase, sedimented, frozen in liquid nitrogen and stored at -80°C. Gel electrophoresis and Western blot detection ---------------------------------------------- Membrane proteins were separated on 12.5% sodium dodecylsulfate polyacrylamide gels, transferred to nitrocellulose and probed with anti-VHA-E \[[@B46]\], anti-VHA-A (kind gift of Dr. R. Ratajczak and Prof. U. Lüttge, TU Darmstadt, Germany) raised in rabbit or anti-VHA-a raised in guinea pig. Following incubation with primary and secondary antibody conjugated with peroxidase, detection was achieved with the lumilight^®^system according to the supplier (Roche, Mannheim, Germany). Immunoprecipitation ------------------- For immunoprecipitation, membranes were solubilised in 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA and 2% (v/v) Triton X-100, 5 μl anti VHA-a antiserum was added, and the samples were shaken at room temperature for 45 min. Then 150 μl protein A-sepharose equilibrated in the same buffer was added. After 15 min, the suspension was placed on a cushion of 1 ml of 40% sucrose and spun at 10,000 × g for 1 min. The sediment was washed thrice with 50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100 and 0.1 % (w/v) SDS, and finally once in 125 mM Tris-Cl, pH 6.8. The sediment was boiled in loading buffer and analysed by Western blot using rabbit antisera raised against VHA-E or A. Anti-VHA- a antibody preparation and other antibodies used in this study ------------------------------------------------------------------------ Two antibodies against specific domains of VHA-a were raised in rabbits, and denominated anti-VHA-a~N-term~and anti-VHA-a~Memb~. For both the corresponding cDNA fragments of Mc-VHA-a were amplified by PCR using primer combinations a-nterm-f (ATG CGA TCG GAG CCG ATG CAA) and a-nterm-r (TTC ACC CAA CTC ATC GGT GG) encoding the 42 N-terminally located fragment, and a-memb-f (CTT CCA AAG CCC TTT ATT ATG) and a-memb-r (TCA CTC ATG TCC ACC ATG TCA ATC) encoding the polypeptide loop of about 13 kDa located between transmembrane domain 3 and 4 according to the topological model of Vph1p of *S. cerevisiae*\[[@B47]\]. The gene fragments were cloned into the vector pCR-T7-NT-Topo (Invitrogen, The Netherlands) and transformed into *E. coli*JM109. The 6x-his-tagged proteins were expressed, purified by chromatography on Ni-nitrilotriacetate columns, separated by preparative SDS-PAGE, excised as protein bands, eluted and used for immunization (Pineda, Berlin). In addition, antisera against subunits VHA-A (kind gift of Dr. R. Ratajczak and Prof. U. Lüttge, TU Darmstadt, Germany), VHA-E, calreticulin (\[[@B48]\], kindly provided by Andrew Smith, Oxford, UK), Jim 84 (\[[@B49]\] kindly provided by Chris Hawes, Oxford, UK), γ-TIP (\[[@B27]\]; kindly provided by John C Rogers, Washington State University, USA) were used in the co-localization studies. Construction of fusions between VHA subunits and variants of green fluorescence protein (GFP) --------------------------------------------------------------------------------------------- Mc-VHA-a and -c were cloned into the vectors pECFP/pEYFP (Clontech, Palo Alto, USA) in a site-directed manner after amplification from cDNA \[[@B10]\] using the primers a-ges-BamHI-f (AAA AGG ATC CAT GCG ATC GGA GCC GAT GCA A) and a-ges-NcoI-r (AAA AAC ATG GCC TCT TCT TCT TCA CCA ATC GT), McVHA-c with c-ges-BamHI-f (AAA AGG ATC CAT GTC AAC CGT CTT CAA TGG) and c-ges-NcoI-r (AAA ACC ATG GCT GCC CTT GAC TGT CCA GCT CG). Mc-VHA-A and Mc-VHA-H were cloned as described in \[[@B10]\]. The constructs were introduced into the vector p35SGFP \[[@B50]\], so that the chimeric genes were placed under control of the 35S promoter and the original GFP gene was lost. The same strategy was used to produce Mc-VHA-A, -B and -H gene fusions with variants of GFP. Protoplast isolation and transformation methods ----------------------------------------------- Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium (0.4 M mannitol, 15 mM MgCI~2~, 4 mM morpholinoethane sulfonic acid, KOH , phl 5.7) and checked for sufficient intactness in the microscope. In short, 1 mm leaf slices of 3- to 5-week-old *Arabidopsis*plants were vacuum-infiltrated and cell walls were digested in media containing 1.5 % (w/v) cellulase R10 and 0.4 % (w/v) macerozyme R10. Protoplasts were gently sedimented by centrifugation, resuspended in W5 medium, sedimented again, resuspended in MMG medium (0.4 M mannitol, 15 mM MgCl~2~, 4 mM morpholinoethane sulfonic acid, KOH, pH 5.7) and checked for sufficient intactness in the microscope. 110 μl PEG-medium (4 % (w/v) polyethylene glycol 4000, 0.2 M mannitol, 0.1 M CaCl~2~) and 20 μl plasmid DNA (3 μg/μl) were added to 100 μl protoplast suspension. The samples were incubated at room temperature for 15 min and then consecutively diluted with 0.5, 1, 2 and 4 ml W5-medium with 15 min incubation steps in between (154 mM NaCl, 125 mM CaCl~2~, 5 mM KCl, 2 mM morpholinoethane sulfonic acid, KOH, pH 5.7). Following 24 h incubation at 25°C, sedimented protoplasts were used for analysis. Cells of onion epidermis were placed on filter paper soaked with one-strength MS basal medium in petri dishes and were transiently transformed with a biolistic approach. Gold particles (1.6 μm, 60 mg/ml) were suspended in 50 % glycerol. 8.33 μl of the suspension were mixed with 8.33 μl plasmid DNA (1 μg/ μl), 8.33 μl 2.5 M CaCl~2~, 3.33 μl 0.1 M spermidine. Sedimented gold particles were consecutively washed with 70 % and 100 % ethanol and resuspended in 8 μl 100 % ethanol, loaded on a macro carrier for transformation with the Particle Delivery System using a rupture disc of 1100 psi (PDS-1000/He, Biorad, Hercules, USA). The distance between macrocarrier and tissue was 12 cm. The epidermis tissue was incubated for about 20 h at room temperature in the dark prior to analysis. Immuno-fluorescence labelling and image acquisition by confocal laser scanning microscopy (CLSM) ------------------------------------------------------------------------------------------------ Immuno-labelling was performed according to \[[@B26]\]. In brief, cells were fixed in 3.7 % para-formaldehyde (10 mM MgSO~4~, 10 mM EGTA, 1 × phosphate buffered saline, pH 6.8), washed, permeabilised in 0.5% Triton X-100 and washed again. Following blocking of non-specific binding sites with 1 % bovine serum albumin, primary antibody was added for over night at 4°C. Washed samples were incubated with secondary antibody labelled with Cy3, Cy5 or FITC for 1 h. Double labelling was performed by combined application of primary antibodies from rabbit and guinea pig. Slides were mounted with Citifluor Mounting Medium. Fluorescence analysis was performed with a confocal laser scanning microscope Leica TCS-SP2 (Leica, Heidelberg, Germany) equipped with three lasers and excitation wavelengths of 458, 476, 488, 514, 568 and 633 nm. The double dichroic mirror DD488/543 was used for fluorescein isothiocyanate (FITC), and for Cy5 the triple dichroic mirror TD488/543/633 was used. Background was controlled and photomultiplier voltage (800 V) selected for maximum sensitivity in the linear range. Immunogold-labelling and electron microscopy -------------------------------------------- Cells were fixed in 2.5% glutaraldehyde in EM buffer (50 mM KH~2~PO~4~, 50 mM NaH~2~PO~4~, pH 7.0) for 45 min, washed with EM buffer and dehydrated with a series of increasing concentration of acetone. Samples were embedded in epoxyresin (Transmit EM, TAAB laboratories equipment, Berkshire, Great Britain), cut into ultra-thin cross-sections of 60--70 nm and immobilized on 200 mesh gold nets. Immuno-decoration was performed with antibody diluted in Tris-buffered saline (TBS, 10 mM bovine serum albumin and 0.05 % (w/v) NaN~3~) for an hour. Samples were washed five times and incubated with secondary antibody conjugated to 15 nm gold particles. The samples were stained with 0.1 % (w/v) uranyl acetate for 5 s and afterwards with 2 % lead citrate. The samples were analysed with an electron microscope (H500, Hitachi, Japan) at 75 kV. Confocal microscopy of GFP-fusion proteins and FRET-measurement --------------------------------------------------------------- Transformed protoplasts and onion epidermis cells were examined for the localisation of the CFP/YFP-fused proteins using the same CLSM set-up as mentioned above. Autofluorescence of 10 protoplasts, as well as reference spectra of YFP and CFP-derived fluorescence were recorded in the spectral range of 480 to 700 nm, averaged and used for corrections. Excitation was recorded at 458 nm (CFP and FRET) and 514 nm (YFP), respectively. Scan speed was 800 Hz. Acceptor dye was bleached with 100 % laser intensity. Emission spectra were recorded and averaged from 20 transformed protoplasts. For a first estimate of transfer efficiency, a Foerster radius for green fluorescence protein variants of R~o~= 5 nm \[[@B52]\] was used to calculate the donor/acceptor distance via the equations E = (I~CFP/bleached~- I~CFP/unbleached~)/I~CFP/unbleached~and R = ((R~o~^6^/E)-R~o~^6^)^1/6^, where E is the transfer efficiency, and I~CFP~the fluorescence emission intensity in the CFP peak. Abbreviations ============= CFP: cyan fluorescence protein; CLSM: confocal laser scanning microscope; FRET: (Förster) fluorescence resonance energy transfer; PAGE: polyacrylamide gel electrophoresis; Stv1: VHA-a subunit isogene in yeast; VHA: vacuolar H^+^-ATPase; Vph1: VHA-a subunit isogene in yeast; YFP: yellow fluorescence protein Authors\' contributions ======================= CK: VHA-a sequence analysis, immuno-cytochemistry, transient expression systems, preparation of anti VHA-a~N-term~; TS: co-transfection of protoplasts and epidermis cells, CLSM analysis; SB: immuno-co-localisation and discussion; SS: co-immuno-precipitation; MH: preparation of anti VHA-a~Memb~; BS-J: immuno-co-localisation and discussion; JR and MS: FRET analysis; DG: transformation and construct design, KJD: project design and supervision. Acknowledgements ================ We are grateful to Dr. Uwe Kahmann for immuno-gold labelling. Technical assistance by Ulrike Windmeier and Martina Holt in preparing membranes and isolating V-ATPase and by Marie-Thérèse Crosniere (plate-forme deimagerie IFR87 \"la plante et son environnement\", CNRS, Gif sur Yvette, France) for the help with the immunochemistry is gratefully acknowledged. The whole work was performed within the framework of the SFB 613, TP A5.

PubMed Central

2024-06-05T03:55:47.487865

2004-8-13

PMC516236

Background ========== In living systems, extracellular information is processed through signal transduction machinery to appropriately regulate cellular function. This information processing machinery is made up of a complex web of enzyme cascades, allosteric interactions and feedback loops. Depending on their regulatory design these signaling networks elicit diverse responses, but display many common operating principles. A recurring theme in signaling systems is switch-like responses to graded or transient input signal. Various mechanisms are known to generate such all-or-none responses \[[@B1]\]. Bistability is one such system level property, in which, the system switches between two discrete stable steady states without being able to rest in an intermediate state. Bistable systems exhibit hysteresis wherein, the value of input stimulus required for system transition from one state to another is quite different from the value required for reverse transition. Both computational and experimental analyses have shown that bistability plays a significant role in cellular differentiation and cell cycle progressions \[[@B2]-[@B5]\], production of biochemical memory \[[@B6]\], microbial metabolic systems \[[@B7]\], lateral signal propagation \[[@B8]\] and protein translocations \[[@B9]\]. Existence of bistability in cellular regulation has been attributed to nonlinearity embedded in positive feedback loop or double negative feedback loop \[[@B10]\]. Here, we present steady state simulation results of metabolic insulin signaling pathway comprising of positive feedback loops and show that this system can convert graded inputs into switch-like bistable output response. Insulin is the most potent anabolic peptide hormone known that elicits myriad biological responses by specifically binding to insulin receptor and simultaneously stimulating multiple signaling pathways to regulate growth, differentiation and metabolism. Insulin maintains glucose homeostasis by stimulating the uptake, utilization and storage of glucose in muscle and adipose tissue, and inhibits hepatic glucose production \[[@B11]\]. Defects in any of the pathway components lead to disturbance in growth, differentiation, and in the homeostasis of glucose and lipid levels. This leads to disease conditions such as type 2 diabetes, hypertension, obesity and a cluster of abnormalities characterized by insulin resistance or deficiency. In such a condition, normal circulating concentration of insulin is insufficient to elicit appropriate response \[[@B12],[@B13]\]. Studies over the last century have identified the major insulin signaling components involved in the regulation of glucose uptake into cells and its various defects in diseased states. A wide family of glucose-transporter proteins localized in the plasma membrane, facilitate uptake of glucose from the blood into tissues. Among different isoforms, only glucose transporter isoform-4 (GLUT4) is specifically expressed to promote glucose uptake in insulin sensitive tissues, viz. muscle and adipose, and in response to insulin, GLUT4 gets translocated to the plasma membrane from intracellular vesicles \[[@B14]\]. The biological action of insulin is initiated by binding to the tyrosine kinase receptor and its subsequent activation. The activated tyrosine kinase receptor undergoes autophosphorylation and catalyzes the phosphorylation of several intracellular substrates including the insulin-receptor substrate (IRS) proteins (Fig. [1](#F1){ref-type="fig"}). The activated IRS isoform-1 protein further activates downstream components to elicit translocation of GLUT4 \[[@B11]\]. There are several downstream kinases like PI-3 kinase, Akt (or protein kinase B) and protein kinase C-ζ (PKC-ζ) demonstrated to be potentially capable of phosphorylating upstream proteins like IRS-1 and tyrosine phosphatase 1B (PTP1B) thus serving as negative and positive feedback loops respectively \[[@B15]\]. Other than feedback loops, crosstalk between multitudes of signal transduction pathways have also been reported, thus making the insulin-signaling pathway a highly intricate network \[[@B11]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Simplified representation of molecular mechanism involved in insulin signaling pathway that regulates glucose transporter (GLUT4) translocation to cell membrane.**Some of the details like, other isoforms of insulin receptor substrate and multiphosphorylation of insulin receptor substrate are not shown here. Nomenclature: GLUT4: Glucose-transporter isoform 4; IRS-1: Insulin receptor substrate-1; PI3K: Phosphatidylinositol-3-kinase; PI (3, 4, 5) P3: Phosphatidylinositol (PI)-3, 4, 5-tiphosphate; PDK1: phosphosinsositide-dependent kinase 1; Akt: Protein kinase Akt or protein kinase B (PKB); PKC: Protein kinase C-ς; PTP1B: Protein tyrosine phosphatase 1B; PTEN: 3\' lipid phosphatase; SHIP2: 5\' lipid phosphatase; Detailed description of signaling events are given in the methods section. Letter \'P\' indicates phosphorylated species. :::  ::: Although studies on various cell lines, transgenic and knock-out mice, have helped to uncover and characterize the different components involved in insulin signaling pathway, there are many voids in our understanding of the precise molecular mechanisms of signal transduction and cellular effects of insulin \[[@B16],[@B17]\]. The major hurdles are complexity of insulin signaling pathway and technical problems like experimental methodology employed for system level quantification. For example, depending upon different techniques employed, quantification of GLUT4 translocation in response to insulin binding yielded different results in the same cell type \[[@B18]\]. Recent technical developments however have helped in studying the localization and translocation of signaling proteins and overall quantification of signaling processes in single cells has been possible \[[@B19]\]. In such a scenario, it is pertinent to ask questions regarding the design principles involved in intracellular regulation. For example, what does a particular regulatory structure accomplish and how does it help in exhibiting different physiological responses. Based on available experimental data, computational and mathematical analysis can answer some of these questions and possibly propose new experiments and hypotheses. Earlier mathematical modeling studies of insulin signaling pathways have focused on subsystems of the pathway, like insulin receptor binding kinetics \[[@B20],[@B21]\], receptor recycling \[[@B22]\] and GLUT4 translocation \[[@B23]-[@B25]\]. Recently a comprehensive dynamic model of metabolic insulin signaling pathway was presented, which involved most of the known signaling components \[[@B26]\]. Although the model correlated well with the published experiment data, authors did not discuss the system level regulatory design of insulin signaling system. In the present work, we have developed a steady state model of insulin signaling to generate dose response curves for fractional translocation of GLUT4 to varying input insulin stimuli. One of the main objectives was to investigate the effect of inherent signaling structure made up of phosphorylation cycles, allosteric interactions and feedback loops on the system level response of insulin on GLUT4 translocation. Furthermore, we were interested in examining whether the regulatory design consisting of positive feedback loops in insulin signaling pathway exhibits bistable response. We solved the steady state equations for the entire metabolic insulin pathway including the positive feedback loops numerically, and found that GLUT4 gets translocated to the plasma membrane in an all-or-none manner in response to a varying concentration of input insulin stimuli. We show that GLUT4 translocation switches between the on-state and off-state and exhibits hysteresis in its response to increasing and decreasing input insulin concentration. This input-output relationship was then studied at various concentration of signaling components and system parameters in order to monitor the range over which this response persisted. We discuss these results by comparing with the known specific defects in regulation of the system (insulin dependent diseases) that lead to improper glucose uptake into the cell. Methods ======= Figure [1](#F1){ref-type="fig"} shows a simplified representation of molecular mechanisms involved in insulin signaling pathway. The metabolic insulin-signaling pathway used for the steady state simulation in the present work is shown in Fig. [2](#F2){ref-type="fig"}. This schematic representation is a compilation of various interactions in insulin pathway which have been very well reviewed \[[@B11]-[@B27]\]. We have used the framework of Goldbeter and Koshland \[[@B28]\] to model the insulin system at steady state and accordingly an equivalent rate constant and Michaelis-Menten constant nomenclature scheme is applied. The detailed list of the steady state equations for covalent modification cycles, equilibrium relationships for allosteric interactions, mass balance equations for total species and parameters used in the simulations are provided in Appendix. All component enzyme concentrations are represented with respect to whole cell volume. Most of the kinetic/equilibrium constants are taken from the literature. In this analysis, the reactants like ATP and PPi concentrations are assumed to be constant. In the following paragraphs we present the system considered and assumptions made during the analysis. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Schematic representation of metabolic Insulin signaling pathway used for the steady state analysis.**Nomenclature: I, Insulin; X, unbound surface insulin receptor; XI, unphosphorylated once-bound surface receptor; XIP, phosphorylated once-bound surface receptor; XI~2~P, phosphorylated twice-bound surface receptor; X~i~represents intracellular receptor pool; XIP~i~and XI~2~P~i~are internalized form of XIP and XI~2~P; phosphatase PTP catalyzes the dephosphorylation of AP, XIP, XIP~i~and XI~2~P~i~. A, unphosphorylated IRS-1; AP, phosphorylated IRS-1; B, inactive PI3-kinase; APB, phosphorylated IRS-1 and PI3-kinase complex; CP3, lipid PI\[3,4,5\]P~3~; CP2, lipid PI\[4,5\]P~2~; CP2\', lipid PI\[3,4\]P~2~; phosphatase SHIP2 catalyzes dephosphorylation of CP3 to form CP2\', phosphatase PTEN catalyzes dephosphorylation of CP3 to form CP2; F, inactive Akt and PKC-ς; FP, phosphorylated Akt and PKC-ς; E~8~dephosphorylates FP; E~6~phosphorylates CP2\' to form CP3; FP activates GLUT4 from intracellular location to plasma membrane. G~C~and G~M~represent GLUT4 in cytoplasm and on plasma membrane respectively. Kd~1~to Kd~3~are dissociation constants; Kd~4~and Kd~5~are distribution coefficients; Km~r~, Km, Km~1~to Km~8~are Michaelis-Menten constants; k, k~p~, k~d~, k~s~, k~0~, k~1~to k~13~are reaction rates as shown in the figure. :::  ::: Insulin initiates its biological action by interacting with the insulin receptor, which belongs to a superfamily of tyrosine kinase receptors. On binding to the first insulin molecule, the receptor gets auto-phosphorylated and is dephosphorylated by phosphatase PTP1B \[[@B12]\]. The phosphorylated insulin receptor can either bind with another insulin molecule or undergoes dissociation. Binding of the second insulin molecule does not affect the phosphorylation state of the receptor. Here we have assumed that the concentration of unbound phosphorylated receptor is negligible. Thus, phosphorylated receptors can exist as species bound to either singly or doubly bound molecules of insulin. Insulin bound phosphorylated receptor rapidly gets internalized into the endosomal apparatus of the cell before it gets dephosphorylated by PTP1B and incorporated into intracellular receptor pool \[[@B29]\]. However recent studies indicate that, PTP1B might interact with insulin receptor directly and deactivate it without internalization \[[@B30]\]. We have assumed that, the membrane bound phosphorylated insulin-receptor and its internalized form, both get dephosphorylated by PTP1B. The rate equation for intracellular receptor at steady state is represented as  where k~p~is rate constant and Km~r~is Michaelis-Menten constant for dephosphorylation of internalized insulin receptors XIP~i~and XI~2~P~i~. The term k~d~is first order degradation rate constant and k~s~is zero order synthesis rate constant of intracellular receptor X~i~. The receptor exocytosis and endocytosis are assumed to be at quasi-equilibrium because of their faster time scales than the synthesis and degradation of receptors \[[@B26]\]. The phosphorylated active receptors further catalyze phosphorylation of several intracellular substrates including the IRS proteins, GAB-1, Shc and c-Cab1 \[[@B16]\]. Among these, IRS-1 protein is known to participate in the regulation of GLUT4 translocation. In the present study we have assumed that, at steady state the twice-bound phosphorylated receptor catalyses the phosphorylation of IRS-1 protein while neglecting the activation of GAB-1, Shc, c-Cab1. The phosphorylated active IRS-1 further binds and activates PI3 kinase and this association is assumed to occur with a stoichiometry of 1:1. Activated PI3 kinase further phosphorylates phosphatidylinositol-(4,5)-bisphosphate (PI-4,5-P2) to form phosphatidylinositol -3,4,5-triphosphate, (PIP3). The dephosphorylation of PIP3 to form PI-4,5-P2 is catalyzed by phosphatase PTEN, whereas, PIP3 is dephosphorylated to form PI-3,4-P2 by phosphatase SHIP2. Active PIP3 then is known to interact allosterically with phosphosinsositide-dependent kinase 1 (PDK1) and which in turn appears to phosphorylate kinase Akt (or protein kinase B) and protein kinase C-ζ (PKC-ζ) \[[@B11]\]. However, as the interaction due to PDK1 is unclear, active PIP3 is assumed to play a role in phosphorylation of Akt and PKC-ζ. Since the parameters affecting the modification-demodification of Akt and PKC-ζ are considered to be similar, their modification is represented as a single enzyme cascade (Fig. [2](#F2){ref-type="fig"}). The downstream elements of Akt and PKC-ζ, which effect GLUT4 translocation, are also unknown \[[@B11]-[@B13]\]. Therefore, we have assumed that phosphorylated Akt and PKC-ζ directly activate the GLUT4 translocation to the plasma membrane. In the basal state, GLUT4 slowly recycles between the plasma membrane and intracellular vesicular compartment. The phosphorylated Akt and PKC-ζ favor GLUT4 translocation (exocytosis) to the plasma membrane and thus increase glucose uptake as a response to insulin binding to the receptor \[[@B14]\]. Here, total GLUT4 (G~t~) is assumed to be sum of GLUT4 concentration in the cytosol (G~C~) and on the membrane (G~M~). The rate equation for GLUT4 species in cytoplasm at steady state is represented by,  where, k~9~is the basal zero order synthesis rate of GLUT4, k~10~is basal first order degradation rate, k~11~is the insulin-activated GLUT4 exocytosis, k~12~and k~13~are basal first order rate of exocytosis and endocytosis, respectively. As assumed by Sedaghat, *et al*. \[[@B26]\], the basal equilibrium distribution of cell surface GLUT4 and GLUT4 in the intracellular pool are taken as 4% and 96%. The insulin signaling pathway has been shown to consist of multiple feedback loops \[[@B15]\]. Active Akt is known to phosphorylate and thereby negatively regulate the upstream phosphatase PTP1B. This phosphorylation impairs the ability of PTP1B to dephosphorylate insulin receptor and IRS-1 by 25% \[[@B31]\]. This represents overall positive feedback loop as Akt inhibits signal attenuation enzyme PTP1B. The resulting circuit also represents a double negative feedback loop, in which phosphorylated protein negatively regulate the phosphatase that dephosphorylates it. To incorporate these feedback loops we assumed that active Akt affects the total active PTP1B enzyme and thus inhibits the dephosphorylation of the receptor and IRS-1. The feedback effect of Akt on PTP1B was incorporated by following relationship  where, \[PTP\]~max~is maximum PTP1B concentration, PTP~t~is the total active PTP1B concentration after incorporating the effects of feedback, AktP represents the phosphorylated Akt concentration influencing the PTPase activity, and k~f~represents the half saturation constant quantifying feedback. The value of k~f~was estimated based on the assumption that 25% of PTP1B is inactivated by total AktP \[[@B31]\]. Thus, k~f~is appropriately calculated so that the first term \[k~f~/\[k~f~+ AktP\]\] is equal to 0.75. In absence of feedback effects, PTP~t~equals PTP~max~. The set of equations given in \'appendix\' and in \'methods\' section were solved numerically using fsolve program of Matlab (The MathWorks Inc. USA). The accuracy of the simulation was verified by numerically checking the mass balance of all species. The steady state modeling of entire insulin signaling was evaluated including the feedback loops and estimating the fractions of GLUT4 translocated to the plasma membrane for a particular concentration of insulin. Thus, the overall action of insulin on GLUT4 translocation is quantified as,  where, *f*is fractional GLUT4 on plasma membrane, G~M~is GLUT4 concentration on plasma membrane and G~t~is total GLUT4 concentration with respect to whole cell volume. Results ======= Bistability in GLUT4 translocation to plasma membrane ----------------------------------------------------- Fig. [3A](#F3){ref-type="fig"} shows the predicted dose response curve of steady state fraction of GLUT4 bound to the plasma membrane at different concentrations of insulin. The predicted dose response curve indicates that, there are three steady states exist between 0.01 nM and 0.05 nM of insulin for GLUT4 translocation (curve b, Fig [3A](#F3){ref-type="fig"}). Out of these three steady states, GLUT4 gets distributed between two discrete stable steady states, either at plasma membrane or in the cytosol without settling in an intermediate unstable state, thus showing a typical hysteresis response. Due to hysteresis, the dose response curve appears to split and we obtain two distinct half-maximal concentrations (K~0.5~, insulin concentration required for 50% of GLUT4 to reside on the plasma membrane). This represents two threshold concentrations of insulin required for GLUT4 translocation switching on (GLUT4 translocation to plasma membrane at 0.05 nM) and switching off (GLUT4 translocation from to plasma membrane at 0.01 nM). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Hysteresis and bistability in insulin-stimulated GLUT4 translocation.*A***. Dose response curve of insulin stimulated fractional GLUT4 on plasma membrane. Curve \'a\' is sigmoidal dose response curve \[\~Hill coefficient of 3.1\] obtained in absence of feedback loop. Curve \'b\' represents hysteresis in insulin-stimulated fractional GLUT4 on plasma membrane in presence of feedback loop which impairs the ability of PTPase by 25%. Arrows indicate the switching-on \[up arrow\] and switching-off \[down arrow\] GLUT4 translocation. ***B***. A simulated type 2 diabetic condition represented by dose response curve of insulin-stimulated fractional GLUT4 on plasma membrane at higher phosphatase PTP1B concentration. Curve \'a\' is typical bistable response obtained in presence of positive feedback loops \[PTP1B conc. 0.039 nM\]. Curve \'b\' represents dose response curve when PTPase concentration was increased by 3 fold \[PTP1B conc. 0.098 nM\]. A 3-fold increase in the PTPase concentration increased the half-maximal concentration by 100 fold and the response looses bistability. :::  ::: The observed hysteresis is characteristic of a bistable response obtained due to negative feedback regulation of upstream signal attenuation enzyme PTP1B by downstream kinase Akt. Experimental data available in the literature indicates a subsensitive response of insulin, requiring \~130 fold change in insulin concentration for the maximal GLUT4 translocation to plasma membrane \[[@B32]\]. Our results show an ultrasensitive response in insulin-stimulated GLUT4 translocation due to bistability (\~4-fold change in insulin concentration); however, the half saturation values match with that of experimental data. The response was ultrasensitive (Hill coefficient \~3.1) and not bistable in absence of feedback loops (curve a, Fig [3A](#F3){ref-type="fig"}). Effect of system component concentration on GLUT4 translocation --------------------------------------------------------------- To examine the influence of pathological conditions arising due to variations in protein expression levels on final output response of insulin, we varied the concentration of individual signaling components IRS-1, PI3K, lipids, PKC-ζ, Akt and phosphatases, PTP1B, PTEN and SHIP2 over a wide range. For each case, the dose response curve of fractional GLUT4 on the plasma membrane at various insulin concentrations was obtained and the response was quantified in-terms of half saturation constant. To illustrate this, we consider a case of increase in PTP1B concentration. Fig. [3B](#F3){ref-type="fig"} shows the dose response curves for insulin stimulated GLUT4 translocation at plasma membrane at two different concentrations of PTP1B. At high PTP1B concentration, the bistable dose response curve becomes monostable (but, still ultrasensitive) and shifts to the right. This indicates a nullifying effect of negative feedback regulation on PTP1B by Akt and higher requirement of insulin for maximal translocation of GLUT4. Thus, in Fig [3B](#F3){ref-type="fig"} curve \'a\' and curve \'b\' can be characterized by two and one half saturation values respectively. Fig. [4A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"} show the distinct half saturation constant values obtained for switching-on and switching-off of GLUT4 translocation at various concentrations of IRS-1 and Akt respectively. Such an increase or decrease in the half-maximal concentration of insulin characterizes the decrease and increase in insulin sensitivity found in diseased conditions. The threshold concentration of insulin required for switching-on GLUT4 translocation decreases with increase in IRS-1 concentration. This implies that, increase in IRS-1 concentration amplifies the input signal and beyond a certain concentration of IRS-1 \[\~3 nM\], the system looses bistability. Similar results were obtained for variations in lipid, PI3K and insulin receptor concentration (results not shown). GLUT4 translocation at various concentrations of Akt shows that the system becomes monostable when Akt concentration is decreased. However, the degree of bistability (i.e., difference between half maximal concentrations for switch-on and off) increases with increase in Akt concentration and furthermore, the threshold value to activate GLUT4 translocation decreases. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Half-maximal concentration of insulin required for 50% GLUT4 translocation at elevated levels of signaling components.**Curve \'a\' shows half maximal concentration of insulin required to switch-on GLUT4 translocation. Curve \'b\' shows half maximal concentration of insulin required to switch-off GLUT4 translocation. Arrow indicates physiological concentration of particular signaling components. ***A***. Half saturation constant at various concentration of IRS-1. Simulated results indicate increased insulin sensitivity when IRS-1 overexpressed. ***B***. Half saturation constant at various concentration of Akt. Simulated results indicate increased insulin sensitivity when Akt overexpressed and loss of bistability when Akt concentration decreased below 0.01 nM. ***C***. Half saturation constant at various concentration of PTP1B. Simulated results indicate decreased insulin sensitivity when PTP1B overexpressed. ***D***. Half saturation constant at various concentration of PTEN. Simulated results indicate decreased insulin sensitivity when PTEN overexpressed. :::  ::: To study the effect of signal attenuation enzymes such as phosphatases on the output response, the concentrations of PTP1B, PTEN and SHIP2 were altered over a wide range, keeping other parameters constant. Fig. [4C](#F4){ref-type="fig"} and [4D](#F4){ref-type="fig"} show the influence of variation in concentrations of PTP1B and PTEN on half saturation constant of insulin. Increase in PTP1B and PTEN concentration results in a drastic increase in the threshold concentration of insulin required to switch-on or switch-off GLUT4 translocation. This illustrates that more insulin than physiological concentration is required at higher phosphatase (PTP1B or PTEN) concentrations to translocate GLUT4 from cytoplasm to plasma membrane. For example, around 16-fold change in the insulin concentration is observed for a 1.5-fold increase in PTP1B concentration from 0.039 nM to 0.06 nM. The system looses bistability beyond a narrow range of PTP1B concentration between 0.02 nM to 0.05 nM. Thus, the response of GLUT4 translocation to insulin is particularly sensitive to PTP1B concentration. Influence of feedback on GLUT4 translocation -------------------------------------------- The feedback effect of active Akt on PTP1B was studied by increasing the Akt concentration (Fig. [5A](#F5){ref-type="fig"}) and by changing the percentage feedback at a fixed Akt concentration (Fig. [5B](#F5){ref-type="fig"}). As shown in Fig. [5A](#F5){ref-type="fig"}, increase in Akt concentration amplifies the signal by maintaining bistable response. Similarly, by increasing the percentage feedback at a fixed Akt concentration, (Fig. [5B](#F5){ref-type="fig"}) the degree of bistability dramatically increased, while not influencing the threshold concentration required for switching-on the response. The bistable response was not observed when percentage feedback was smaller or in absence of feedback loops. In absence of receptor internalization, 65% inhibition of PTP1B by Akt was required to display a bistable response, whereas, inclusion of receptor internalization demonstrated bistability even at 25% inhibition of PTP1B. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Influence of feedback effects on bistable insulin-stimulated GLUT4 translocation.*A***. Bistable response with increase in the concentration of Akt representing increased non-linearity due to zero order ultrasensitivity. Dose response curves obtained at different Akt concentrations: Curve \'a\', 0.01 nM; Curve \'b\', 0.03 nM; Curve \'c\', 0.05 nM. ***B***. Influence of percentage of feedback effects on dose response curve of insulin-stimulated GLUT4 translocation. The percentage feedback represents the percentage by which the dephosphorylation ability of PTP1B is impaired. Dose response curves obtained: Curve \'a\' in absence of feedback; Curve \'b\' 25% feedback effect; Curve \'c\' 67% feedback effect; Curve \'d\' 90% feedback effect. :::  ::: The steady state analysis of metabolic insulin-signaling pathway demonstrated signal amplification as signal propagates down the cascades. The amount of insulin required for 50% activation of insulin receptor, IRS-1, PIP3, Akt, PKC-ζ and GLUT4 was estimated to decrease in presence or absence of feedback loops (results not shown). Effect of system parameter values on GLUT4 translocation -------------------------------------------------------- In addition to genetic variation at the protein expression levels in diseased conditions, mutational changes can also alter the system parameters and thereby modify the final output response. To examine the influence of system parameter values on insulin-stimulated GLUT4 translocation, we have analyzed the performance of insulin signaling pathway to variations in key parameter values such as, dissociation constant and Michaelis-Menten constant. Increase in dissociation constant quantifying the interaction between insulin-receptor and phosphorylated IRS-1-PI3K shows an increase in the half saturation constant indicating higher requirement of insulin over the physiological concentration (Fig. [6A](#F6){ref-type="fig"} and [6B](#F6){ref-type="fig"}). The system becomes monostable at very low values of dissociation constants. Similarly, decrease in the Michaelis-Menten constant of the dephosphorylation cycles, also increases the half saturation constant, thus decreasing the insulin sensitivity (Fig. [6C](#F6){ref-type="fig"}). Simulation results indicate that, the alterations in binding constant of allosteric interactions and Michaelis-Menten constants in modification-demodification cycles in the insulin-signaling pathway can result in insulin resistance or diabetes. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Effect of key system parameter values on GLUT4 translocation.**Curve \'a\' shows half maximal concentration of insulin required to switch-on GLUT4 translocation. Curve \'b\' shows half maximal concentration of insulin required to switch-off GLUT4 translocation. Arrow indicates parameter used in the simulation. ***A***. Half maximal concentration of insulin required for GLUT4 translocation at different values of dissociation constant \[Kd~2~\] for binding of second molecule of insulin to phosphorylated insulin bound receptor. Simulated results indicate decreased insulin sensitivity when Kd~2~increased. ***B***. Half maximal concentration of insulin required for GLUT4 translocation at different values of dissociation constant \[Kd~3~\] for binding of phosphorylated IRS-1 to PI3K species. Simulated results indicate decreased insulin sensitivity when Kd~3~increased. ***C***. Half maximal concentration of insulin required for GLUT4 translocation at different values of Michaelis-Menten constant \[Km~2~\] for dephosphorylation of phosphorylated IRS1 by PTP1B. Simulated results indicate decreased insulin sensitivity when Michaelis-Menten constant \[Km~2~\] was decreased due to increased affinity with dephosphorylating enzyme. :::  ::: Discussion ========== In this work we have demonstrated that, the dose response curves of fractional GLUT4 concentration on plasma membrane at various concentration of insulin exhibit hysteresis-a property of bistable systems. The analysis of bistable response in presence of feedback loops was done at varying concentration of signaling components and system parameters in physiological range. The overall response of insulin demonstrated signal amplification as the signal propagates down the cascade, thus requiring less insulin for GLUT4 translocation. The insulin sensitivity increased by increasing the concentration of proteins that amplify the insulin action and decreasing the concentration of proteins that attenuate insulin-signaling pathway. This indicates that the bistability and the half saturation constant are dependent on the component concentrations and system parameters. It is known that defects in insulin signaling pathway leads to pathological conditions like diabetes, wherein normal or elevated levels of insulin produces impaired biological response. This characteristic decrease or increase in insulin sensitivity is mainly attributed to post-receptor defects including mutational changes in protein expression levels or other parameters like dissociation constants and Michaelis-Menten constants \[[@B13],[@B33]\]. Numerous experimental studies like targeted deletions/mutations of signaling components have yielded insights about the disease states. In the present work, to study the influence of pathological conditions on final output response of insulin, the concentration of individual signaling components was varied over a wide range, by keeping other parameters constant. The predicted results are consistent with various reported experimental observations and thus validate our steady state model. (*i*) Decreased concentration of phosphorylated insulin receptor and IRS-1 are observed in muscle from morbidly obese subjects \[[@B34]\] and those with diabetes \[[@B35]\]. This decreased phosphorylation can be either due to decrease in protein concentration itself or impaired phosphorylation event. (*ii*) Increase in the level and activity of several tyrosine phosphatases (PTP1B) was found to be associated with insulin resistance and reduced insulin sensitivity \[[@B12],[@B13],[@B33],[@B36]\]. (*iii*) Overexpression of PI3K and its downstream targets Akt and PKC in cell culture models increased GLUT4 translocation \[[@B12]\]. (i*v*) Decrease in the association of PI3K with phosphorylated IRS-1 and subsequent activation appears to be a characteristic abnormality in type 2 diabetes and obesity \[[@B13],[@B33]-[@B35]\]. (*v*) Single gene knockout experiments have shown that, mice with PTP1B knockout \[[@B37]\], mice with SHIP2 knockout \[[@B38]\] and targeted deletion of PTEN in murine lever \[[@B39]\], all results in hypersensitivity towards insulin. In the present work, though we have not done *in-silico*perturbation analysis by deleting a particular protein, we have changed the concentration of specific protein over wide range to bring about the similar effect of deficiency. Thus, our simulation results show that the insulin sensitivity dramatically increased when we decreased the concentration of phosphatases like PTP1B, PTEN and SHIP2. Increase in the concentration of Akt, makes the signal amplification increased along with slight increase in the degree of bistability. This effect is brought about by the enhanced nonlinearity in the feedback loop due to zero order ultrasensitivity \[[@B28]\] imposed by increasing the concentration of Akt or percentage feedback. At high Akt concentration (or when overexpressed), the system can respond in constitutively active mode or might also function as a memory module. That is, once insulin switches on the system, the high Akt concentration or percentage feedback by itself can keep the switch on even after insulin is removed. This may be the reason for the experimental observation of insulin independent GLUT4 translocation to plasma membrane when Akt is overexpressed \[[@B12],[@B40]\]. This insulin independent translocation of GLUT4 is thought to be due to activation of PI3K independent pathway or by amplification of residual signal. Our analysis indicates that the inherent feedback structure present in the insulin-signaling pathway by itself can induce this phenomenon. Does GLUT4 translocation show a bistable response to insulin in-vivo? --------------------------------------------------------------------- Bistability has been shown to be the outcome of regulatory structure with feedback loops and non-linearity in the system \[[@B41]\]. The non-linearity in the system is brought about by an ultrasensitive response typically obtained through enzyme cascades. Such ultrasensitive responses exhibit steep dose response curves with Hill coefficient greater than one \[[@B1]\]. The cascade systems use energy for their operation and are optimally operated under zero order sensitivity (i.e., cascades operating under saturating interconvertable enzymes) requiring minimum energy \[[@B42],[@B43]\]. Presence of feedback loops may further optimize the requirement of energy. Enzyme cascades and multiple positive feedback loops are observed in insulin-signaling pathway. Experimental results have shown that the dose response curve of insulin-stimulated glucose uptake is subsensitive with a Hill coefficient of about 0.8 \[calculated from ref. \[[@B32]\]\]. Thus the question arises as to what may be the significance of the cascade signaling system with positive feedback loops in insulin signaling pathway. The reason for this discrepancy may be because, the experimental data represents a profile of glucose uptake in ensemble of cells \[[@B32]\], and furthermore, glucose uptake may not be proportional to the amount of GLUT4 on cell surface \[[@B18]\]. Recently, bistability has been experimentally demonstrated in many cellular regulation systems \[[@B10]\]. Experiments on cellular differentiation and cell-cycle progression have shown that, to demonstrate bistability it is essential to measure the amount of input required to switch-on the system from a low activity state to a high activity state, and amount of input required to hold-on the system in high activity state \[[@B3]-[@B5]\]. Reynolds *et al*. \[[@B8]\], have shown experimentally that, the reaction network of PTPase inhibition by activated epidermal growth factor receptor (EGFR, a receptor tyrosine kinase, RTK) through reactive oxygen species, can generate highly amplified and switch like bistable response to a threshold concentration of ligand stimulus. In this system, PTPase is a negative regulator of active RTK and thus, PTPase inhibition by reactive oxygen species serves as a positive feedback loop. Our simulation results indicate that similar bistable response can be obtained in insulin-stimulated GLUT4 translocation because of the positive feedback loops (inhibitory action of Akt on PTP1B). Though experimental verification of this property is awaited, there are indications that insulin signaling pathway possesses many requisite components to exhibit bistable response. The simulation results presented here showed that, the ultrasensitivity in absence of feedback effects and the regulatory structure of feedback loops are main reasons for a bistable response. Other than the positive feedback loops considered in the present work, Insulin signaling system is also known to contain many feedback loops which have not been entirely characterized \[[@B15]\]. One possible reason for having so many interlocking positive feedback and negative feedback loops may be to cause oscillations in GLUT4 translocation or to make the bistability of GLUT4 translocation -- more robust. Recently, it has been shown that insulin stimulation in a variety of cell types elicit a rapid production of hydrogen peroxide and which reversibly inhibit PTP1B enhancing propagation of the early insulin signal \[[@B44]\]. This regulatory mechanism was also found to be essential for PI3K mediated Akt activation, GLUT4 translocation to plasma membrane and insulin-stimulated glucose uptake \[[@B45]\]. However, unlike EGFR system \[[@B8]\] existence of bistable behavior is yet to be shown in insulin signaling system. In insulin signaling pathway other than GLUT4, proteins like Akt and PKC get translocated to plasma membrane and such inter-compartmental translocations can also exhibit switch like bistable response \[[@B9]\]. It is becoming clear that quantification studies have to be performed in single cell rather than cell populations \[[@B19]\]. This is true especially while addressing the system level questions like ultrasensitivity, bistability and oscillations \[[@B4]-[@B7],[@B46]\]. Recently, this was also found to be of immense value in case of insulin signaling pathway to uncover the PIP3 activation mode \[[@B47]\]. Simultaneous measurement of PIP3 production and GLUT4 insertion in individual 3T3L1 adipocytes suggested that a threshold for PIP3 signals exists. Below this threshold, GLUT4 transporters are minimally inserted into the plasma membrane and their insertion increases once this threshold is overcome. In summary, it is essential to show through experiments that, the stimulus-response curve for insulin-stimulated GLUT4 translocation exhibits hysteresis, -- a distinctive splitting in stimulus response curve. There should be a particular concentration of insulin, which is not sufficient to translocate GLUT4 to plasma membrane, but is sufficient to maintain GLUT4 on plasma membrane. What may be the significance of such a bistable behavior in GLUT4 translocation? Though there is no obvious answer to this question yet, it is known that, bistability can maintain a biological response even when the input stimulus is brief and the high activity level is maintained only as long as the system requires. Insulin binding to its cell surface receptor is rapidly followed by internalization of insulin-receptor complex. This internalization of receptor has been implicated in receptor down regulation, attenuation of insulin sensitivity and insulin clearance from the circulation \[[@B12],[@B13]\]. Thus a brief input stimulation should be sufficient to balance the translocation of GLUT4 to plasma membrane and its reversal depending on glucose concentration. Additionally, due to small absolute stimulus concentrations, the probability of noise occurrence is high. A bistable response having distinct threshold concentration to switch-on and switch-off offers advantage in handling noise. In cellular regulation, different structural motifs such as enzyme cascades, feedforward control and multiple feedback loops yield complex regulatory networks. These are shown to be responsible for exhibiting system level properties including bistability and robustness \[[@B10],[@B48],[@B49]\]. Other than multiple feedback loops, structural regulatory motifs like multisite covalent modification cycles have been shown to induce bistability \[[@B50]\]. The interconnections between these regulatory motifs at the system level may elicit a multistable response to particular stimuli, which have to be theoretically uncovered and demonstrated through experiments. Abbreviations used ================== GLUT4: Glucose-transporter isoform 4; IRS: Insulin-receptor substrate; PI3K: Phosphatidylinositol-3-kinase; PIP3: Phosphatidylinositol (PI)-3,4,5-tiphosphate (PI-3,4,5-P3); Akt: Protein kinase Akt or protein kinase B (PKB); PKC: Protein kinase C; PTP1B: Protein tyrosine phosphatase 1B; PTEN: 3\' lipid phosphatase; SHIP2: 5\' lipid phosphatase; Competing Interests =================== None declared. Author\'s Contributions ======================= LG, VKM and KVV conceived and designed the experiments. LG performed the experiments. LG, VKM and KVV analyzed the data. VKM and KVV conceptualize the manuscript. All authors have read and approved the final manuscript Appendix ======== **Equilibrium relationships, rate equations, mass balance equations and model parameters used for simulation of metabolic insulin signaling system at steady state (refer Fig.**[2](#F2){ref-type="fig"}**for nomenclature and interaction details).**Equations were solved simultaneously, for evaluating fractional GLUT4 translocation at a particular insulin concentration, using fsolve function of Matlab (The MathWorks Inc. USA). Most of the values of model parameters for steady state analysis are taken from Sedaghat *et al*. \[[@B26]\]. Nomenclature, parameter values are: Component concentrations ------------------------ I~t~, total insulin concentration varied over a wide range; X~t~, total receptor = 0.003 nM; A~t~, total IRS-1= 0.01 nM, B~t~, total PI3-Kinase = 0.5 nM, PTEN~t~, total PTEN= 0.007 nM; CP2~t~, total lipid = 0.01 nM; SHIP2~t~, total SHIP2 = 0.001 nM; F~t~, total Akt+PKC-ξ = 0.02 nM, PTP~max~, total PTP1B= 0.039 nM; G~t~, total GLUT4 = 9 nM; E~6t~, total E~6~= 0.001 nM; E~8t~, total E~8~= 0.001 nM; Rate constants -------------- k~0~= 2500 min^-1^; k = 0.2 min^-1^; k~1~= 4.16 min^-1^; k~2~= 1.4 min^-1^; k~3~= 50 min^-1^(assumed); k~4~= 42.1 min^-1^; k~5~= 2.8 min^-1^; k~6~= 3 min^-1^; k~7~= 20 min^-1^(assumed); k~8~= 6.9 min^-1^; k~9~= 0.11 min^-1^; k~10~= 0.0012 min^-1^; k~11~= 3.47 min^-1^(assumed); k~12~= 6.96\*10^-3^min^-1^; k~13~= 0.17 min^-1^; k~p~= 0.461 min^-1^; k~d~= 1.67 × 10^-18^min^-1^; k~s~= 1.67\*10^-25^nM min ^-1^; Dissociation constants ---------------------- Kd~1~= 3.33 nM; Kd~2~= 333.3 nM; Kd~3~= 0.014 nM; Distribution coefficients ------------------------- Kd~4~= 9 nM; Kd~5~= 0.1 nM; Michaelis-Menten constants -------------------------- Km~r~, Km~1~to Km~8~= 10^-4^nM The total molar balances for different species are given below. *I*~*t*~= *I*+ *XI*+ *XIP*+ 2*XI*~2~*P*+ *PTP*.*XIP*+ *A*.*XI*~2~*P*+ *XIP*~*i*~+ 2*XI*~2~*P*~*i*~+ *PTP*.*XIP*~*i*~+ *PTP*.*XI*~2~*P*~*i*~    \[A1\] *X*~*t*~= *X*+ *X*~i~+ *XI*+ *XIP*+ *XI*~2~*P*+ *XIP*~i~+ *XI*~2~*P*~i~+ *PTP*.*XIP*+ *A*.*XI*~2~*P*+ *PTP*.*XIP*~i~+ *PTP*.*XI*~2~*P*~i~    \[A2\] *A*~*t*~= *A*+ *AP*+ *APB*+ *A*.*XI*~2~*P*+ *PTP*.*AP*+ *APB*.*CP2*    \[A3\] *CP*2~*t*~= *CP*2 + *CP*3 + *CP*2\' + *APB*.*CP*2 + *PTEN*.*CP*3 + *SHIP*2.*CP*3 + *SHIP*2.*CP*3 + *E*~6~.*CP*2\' + *F*.*CP*3     \[A4\] *F*~*t*~= *F*+ *FP*+*CP*3*F*+ *E*~8~.*FP*    \[A5\] *SHIP*2~*t*~= *SHIP*2 + *SHIP*2.*CP*3     \[A6\] *PTEN*~*t*~= *PTEN*+ *PTEN*.*CP*3     \[A7\] *PTP*~*t*~= *PTP*+ *PTP*.*XIP*+ *PTP*.*AP*+ *PTP*.*XIP*~*i*~+ *PTP*.*XI*~2~*P*~*i*~    \[A8\] *B*~*t*~= *B*+ *APB*+ *CP*2.*APB*    \[A9\] *E*~6*t*~= *E*~6~+ *E*~6~.*CP*2\'     \[A10\] *E*~8*t*~= *E*~8~+ *E*~8~.*FP*    \[A11\] *G*~*t*~= *G*~*M*~+ *G*~*C*~    \[A12\] Equilibrium relationships      Rate expression with pseudo-steady state representation of complexes for modification-demodification cycles Receptor autophosphorylation and dephosphorylation cycle  IRS-1 phosphorylation and dephosphorylation cycle  Phosphorylation and Dephosphorylation of PI-4,5-P2, PI-3,4-P2 and PIP3   Phosphorylation and Dephosphorylation of Akt + PKC 

PubMed Central

2024-06-05T03:55:47.491994

2004-8-3

PMC516237

Background ========== The mechanism by which \[Ca^2+^\]~i~is increased in excitable cells differs from that obtaining in non-excitable cells. Excitable cells exhibit an action potential, a substantial general depolarization of the plasma membrane, in response to depolarizing stimuli; influx of Ca^2+^occurs via plasma membrane Ca^2+^channels and/or release from sarco (endo) plasmic reticulum via ryanodine-receptor Ca^2+^channels which regulate the excitation -- contraction coupling \[[@B1],[@B2]\]. The factors that determine the extent of Ca^2+^entry are **(i)**magnitude of the membrane potential and (**ii)**magnitude of the transmembrane Ca^2+^gradient. These two factors also determine whether Ca^2+^or Mg^2+^enters and the time (probably milliseconds) that elapses between channel opening and termination of Ca^2+^or Mg^2+^transport \[[@B3]\]. In non-excitable cells, the increase in \[Ca^2+^\]~i~results from influx of Ca^2+^across the plasma membrane and Ca^2+^release from the endoplasmic reticulum. Ca^2+^release from the SER depends on the binding of inositol 1,4,5-triphosphate (InsP~3~) to its receptor Ca^2+^channels, and also on Ca^2+^binding to ryanodine receptor -- Ca^2+^channels. Ca^2+^is removed from the cell by the following means. **i:**the sarco (endo) plasmic reticular Ca^2+^pump ATPase (SERCA), which transports Ca^2+^from the cytoplasm into the SER lumen (\~70% of the activator Ca^2+^); **ii:**The plasma membrane Ca^2+^pump ATPase (PMCA), which exports Ca^2+^across the plasma membrane (\~1% of the activator Ca^2+^); **iii:**Mitochondrial Ca^2+^Uniporters (mCa^2+^uniporters), which transport Ca^2+^into mitochondria (\~1% of the activator Ca^2+^);**iv:**the Na^+^/Ca^2+^exchanger (28% of the activator Ca^2+^). This last transport system is reversible but under normal physiological conditions, in the Ca^2+^extrusion mode, it exhibits a stoichiometry of 3 Na^+^influx/1 Ca^2+^efflux \[[@B4]\]. Ca^2+^enters animal cells via **(i)**voltage-operated Ca^2+^channels **(VOCC),**(**ii)**ligand gated non-specific cation channels **(LGCCS),**and (**iii)**stretch/receptor activated non-specific Ca^2+^channels **(RACC)**\[[@B4],[@B5]\]. A \"receptor operated Ca^2+^channel\" **(ROCC)**is defined as a plasma membrane Ca^2+^channel other than VOCC or RACC. VOCC opening depends on membrane depolarization, whereas RACC opening depends on both direct and indirect activation of membrane bound receptors. In contrast, ROCC opening depends solely on agonist-receptor interaction. It has also been suggested that mobile intracellular messengers such as elevated \[Ca2+\]i play a role in ROCC opening \[[@B5],[@B6]\]. Different types of ROCC are activated (opened) by diverse cell signaling mechanisms such as ligand specificity, increase in \[Ca^2+^\]~I~, increase in \[cAMP\]~i~\[[@B7]\] and activation/inactivation of specific trimeric G proteins \[[@B8]\]. Opening of Ca^2+^channels must be a highly regulated event involving physical movement of channel components inclusive of the alteration in channel protein conformation; Also, an extracellular source of free energy (ΔG) could be of critical importance. This might be supplied by E-NTPase/E-NTPDase mediated hydrolysis of NTP/NDP. Co-ordination of this process might play a role in the opening of Ca^2+^channels, independently of membrane depolarization or other factors. The biochemical, structural, and functional properties of E-type nucleotidases have been covered in several excellent reviews: **i**. Extracellular metabolism \[[@B9]\]; **ii**. purine signalling \[[@B10],[@B11]\]; **iii**. adhesion \[[@B12]\]; **iv**. transporter functions \[[@B13]\]; **v**. pathophysiology \[[@B14],[@B15]\]. Rationale for the proposed hypothesis: E-NTPase/E-NTPDase mediated Ca^2+^/Mg^2+^transport ----------------------------------------------------------------------------------------- It has been suggested that Ca^2+^entry during the slow inward current in normal myocardium involves membrane-bound channels potentially controlled and/or regulated by metabolic energy transfer from unknown sources, though Ca^2+^enters the cell down its concentration gradient \[[@B16]\]. Electrical stimulation and membrane phosphorylation by cAMP-dependent protein kinase have been shown to increase E-NTPase/E-NTPDase activity. Metal ions such as Mn^2+^, Co^2+^, Ni^2+^and La^2+^that attenuate Ca^2+^influx also inhibit the E-NTPase. In the late stages of heart failure the E-NTPase is down regulated. Activation of E-NTPase by various concentrations of Ca^2+^has been shown to correlate linearly with cardiac contractile force development \[[@B17]\]. **\"Calcium paradox\"**is defined as irreversible functional and structural protein loss in the isolated heart that is first perfused with Ca^2+^-free buffer and then reperfused with Ca^2+^-containing buffer \[[@B18]\]. E-NTPase activity is highest during the initial phases of reperfusion, which might favour the initial Ca^2+^influx that causes Ca^2+^overload. During the later stages of reperfusion with Ca^2+^-containing buffer there is a loss of E-NTPase activity. During mild stages of Ca^2+^paradox, E-NTPase retains its function and continues to favour Ca^2+^influx, resulting in the development of intracellular Ca^2+^overloads. However, during severe stages of calcium paradox, impaired E-NTPase activity may contribute to irreversible failure of contractile force recovery \[[@B19]\]. To date there is no report describing the detailed mechanism of E-NTPase/E-NTPDase-mediated channel gating and its role in Ca^2+^/Mg^2+^transport. In this article an attempt is made to delineate the molecular mechanism of Ca^2+^/Mg^2+^transport, identifying the source of energy and the activation and termination of the process. The central issues are: **a**. How the metabolic energy from nucleotide hydrolysis is effectively utilized in channel opening; **b**. What stage of the opening/closing cycle requires energy; **c**. By what (probable) mechanism the proposed scheme is completed; **d**. How, if at all, homeostasis is affected The current hypothetical proposal is set out in three sections with appropriate illustrations. Phase I: Activation ------------------- identifies the evidence that leads to the current proposal and describes how the metabolic energy from nucleotide triphosphate hydrolysis is utilised to assemble a functional homo-oligomer of the E-NTPase/E-NTPDase, forming a channel that is subsequently opened. Phase II: Suggested: Ca^2+^/Mg^2+^Transport ------------------------------------------- Describes, with supporting evidence, how the energy released from \[NTP\] o/ \[NDP\] o hydrolysis might be utilized for opening the channel formed by the homo-oligomeric ENTPase/E-NTPDase. Phase III: Termination of the transport processes ------------------------------------------------- outlines the intracellular and extracellular factors that would influence the termination of the Ca^2+^/Mg^2+^transport processes, and the experimental evidence obtained in favor of the whole proposal. Phase I: Activation of E-NTPase/E-NTPDase and channel formation --------------------------------------------------------------- Membrane depolarization could locally alter protein conformation. This in turn could potentially induce post-translational modification in the (intracellular) monomer subunits of the E-NTPase/E-NTPDase, followed by translocation to the membrane (depending on the tissue type(s) and functional requirement(s)) (Fig. [1](#F1){ref-type="fig"}). Fig. [2](#F2){ref-type="fig"} shows the proposed functional state of the E-NTPase/E-NTPDase after oligomerization and assembly in the membrane to form a gated Ca^2+^/Mg^2+^channel. Fig. [3](#F3){ref-type="fig"}, indicates that the oligomerized E-NTPase/E-NTPDase is likely to possess sensors to control the opening and closing of the Ca^2+^/Mg^2+^channel gate. Fig. [4](#F4){ref-type="fig"}, represents an interior view of the E-NTPase/E-NTPDase in the functional state after oligomerization and assembly in the membrane. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Phase I: Activation.**Based on direct experimental evidence, suppose that in response to electrical stimuli, an increased phosphatidylinositol turnover leads to elevated intracellular phospholipid. This in turn could induce post-translational modification of the monomer subunits of E-NTPase/E-NTPDase in the intracellular milieu. Subsequently, the monomers are translocated to the membrane, depending on the tissue type(s) and functional requirement(s). :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Phase I: Activation.**Proposed model for E-NTPase/E-NTPDase in a functional state after oligomerization and assembly in the membrane, functioning as a gated channel. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Phase I: Activation.**The oligomerized E-NTPase/E-NTPDase would probably possess hypothetical sensors acting to open/close the gates. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Phase I: Activation.**Interior view of E-NTPase/E-NTPDase in a functional state in the membrane. :::  ::: Probable energy sources and other significant factors are as follows. The source of extracellular nucleotides could be spontaneous release from dead cells or exocytosis from live/damaged cells \[[@B20]\]. In ocular ciliary epithelial cells, ATP is released in hypotonic conditions, and this release is inhibited by **NPPB**(5-nitro-2-(3-phenyl propylamine benzoic acid), a potent inhibitor of CFTR (cystic fibrosis transmembrane receptor) and p-glycoprotein mediated ATP release \[[@B21]\]. On the other hand, the endogenous CD39 of oocytes transforms under hypertonic conditions to a conformation mediating ATP transport to the extracellular environment, either by exocytosis or by acting as an ion channel \[[@B22],[@B23]\]. However, under what conditions (hyper-or hypotonic) might CD39 assume an extracellular nucleotide hydrolyzing activity; and under those conditions, can this property be coupled to ion influx? This question remains unanswered. At normal physiological temperature in presence of divalent succinyl CoA, Con A mediates the oligomerization of E-NTPase monomers/dimers to form a holoenzyme with enhanced activity. Eosin iodoacetamide (EIAA), a fluorescein iodoacetamide that forms thioester bonds with cysteine at neutral pH, enhances chicken gizzard ecto-ATPase activity \[[@B24]\]. There are ten conserved cysteine residues in E-NTPase (with additional cysteine residues in the N-terminal region that are known to mediate disulfide bond formation, essential in oligomerization). CD39, an ecto-Ca^2+^/Mg^2+^apyrase that hydrolyses ATP and ADP \[[@B25]\], forms tetramers and might act as a bivalent cation channel. However, the precise mechanism and functional properties are not known at present. CD39 expression is associated with ATP release; it was speculated that ATP release (along with drugs) into the extracellular milieu is followed by the hydrolysis of the extracellular nucleotides by CD39 \[[@B26]\]. Furthermore, native CD39 (ecto-ATP/Dase/ apyrase) forms tetramers upon oligomerization. Loss of either of the two transmembrane domains of rat CD39 ecto-ATP/Dase impairs enzyme activity. It has been suggested that the functional (holoenzyme) E-NTPase/E-NTPDase is a homotrimer in mammals. Differences in enzyme activity among different species have been attributed to variations in the interaction among the monomers resulting in homotrimeric holoenzyme formation (66 kDa-ATPase) \[[@B27]\]. It seems clear that changes in the conformation of the E-NTPase/E-NTPDase could mediate changes in the channel transport function. Phase II: Ca^2+^/Mg^2+^Transport -------------------------------- Fig. [5a](#F5){ref-type="fig"}, illustrates the possible utilization of the energy released from \[NTP\] o /\[NDP\] o hydrolysis (-7.3 kcal mol^-1^or by formation of AMP, -10.9 kcal/mol^-1^) for opening the channel formed by the homo-oligomeric E-NTPase/E-NTPDase. This channel is postulated to open and close in response to energy availability (Fig. [5b](#F5){ref-type="fig"}). Fig. [6A](#F6){ref-type="fig"}, is an artist\'s impression of the three-dimensional configuration of the E-NTPase/E-NTPDase in vivo. Ca^2+^might enter the cell and excess Mg^2+^might leave by the influx and efflux mechanisms depicted in Fig [6b](#F6){ref-type="fig"}. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Phase II: Ca^2+^/Mg^2+^Transport.**(A) Free energy released from ATP hydrolysis by E-NTPase on the outer membrane surface would yield -7.3 kcal mol^-1^**or**by formation of AMP by E-NTPDase would yield -10.9 kcal mol^-1^. (B) The energy is utilized for opening the channel formed by the E-NTPase/E-NTPDase, by altering the conformation of the sensors. This altered conformation has an inherent channel-opening effect; loss of the energy source causes the sensors to revert to the resting state, which corresponds to channel closing. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Phase II: Ca^2+^/Mg^2+^Transport.**(A) Three-dimensional impression of the E-NTPase/E-NTPDase in vivo. (B) It is possible that Ca^2+^can enter the cell and excess Mg^2+^can leave via the influx/efflux mechanisms depicted in the figure. :::  ::: The opening of the slow inward Ca^2+^current channel in cardiac sarcolemma during the plateau phase of the action potential requires ATP \[[@B28]\]. Furthermore, protein kinase-A (PKA) dependent phosphorylation appears to mediate the increase in Ca^2+^influx in hormonal modulation of that process \[[@B29]\]. A similar model has been proposed for sodium channels in nerve membranes, in which a cycle of phosphorylation and dephosphorylation is proposed for opening and closing \[[@B30]\]. Other corroborating evidence implicating E-NTPase in Ca2+/Mg2+ transport via the gated channel is briefly summarised. Rat cardiac sarcolemmal E-NTPase has considerable sequence homology with the human platelet thrombospondin receptor CD36 \[[@B31]\]. An antibody directed against the purified E-NTPase blocked the increase in intracellular calcium concentration, implying that the E-NTPase plays an unknown but significant role in the delayed Ca^2+^influx or Mg^2+^efflux during the plateau phase of the action potential (Unpublished observation). Activation of E-NTPase by millimolar concentrations of Ca^2+^and electrical stimulation is linearly related to the contractile force developed in the myocardium \[[@B32]\]. Gramicidin S inhibits the E-NTPase activity and it attenuates the slow channel efflux in perfused frog left ventricles. Based on these observations, we propose that E-NTPase might be involved in providing energy for Ca^2+^/Mg^2+^influx-efflux in the cardiac sarcolemma, opening the channel formed by the E-NTPase/E-NTPDase protein by altering the conformation of the sensors. The altered channel sensor conformation opens the channel; loss of the energy source allows the sensors to revert to the resting state, which corresponds to channel closing. There are at least two Mg^2+^transport systems: (a) rapid transport down the concentration gradient and (b) efflux in low Ca^2+^Ringer during ventricular perfusion in vitro. In rat liver mitochondria, 50 nM cAMP or 250 μM ADP induced rapid loss of 6 mmol of Mg^2+^/mg protein coupled with the stimulation of ATP efflux. This effect was specific and was blocked by adenosine nucleotide translocase inhibitors. Evidently cAMP acts as a mobilizer of Mg^2+^in isolated rat liver mitochondria. Adenine nucleotide translocase is the cAMP target \[[@B33]\]. Myocardial Mg^2+^content is maintained at physiological level by the sarcolemmal transport system, which pumps Mg^2+^across the plasma membrane when the extracellular \[Mg^2+^\]~o~concentration is \<1 mM and restores \[Mg^2+^\]~i~when the heart is perfused with Ringer buffer containing 5 × 10^-7^M Mg^2+^. Failure of either of these two transport mechanisms may result in a rise in \[Mg^2+^\]~i~, impairing the contractile machinery of the myocardium \[[@B34]\]. Gramicidin S inhibits total Mg^2+^efflux in the myocardium, while epinephrine restores Mg^2+^efflux and contractile force development in the frog ventricle perfused with 10 mM Mg^2+^. It should be pointed out that both E-NTPase activity and myocardial contraction and relaxation are inhibited by gramicidin S \[[@B35]\]. In the light of the evidence surveyed here, there would appear to be a significant functional role for activated E-NTPase in Ca^2+^influx and Mg^2+^efflux (or vice versa) in the myocardium. Phase III: Termination of the transport process ----------------------------------------------- Fig. [7](#F7){ref-type="fig"} summarizes the possible means by which the transport process is terminated. There are several potential contributing factors that can be grouped into two categories, extracelluar and intracellular. Additional experimental evidence is indicated. Based on the heterologous expression of ecto-apyrase in COS cells in the presence of tunicamycin, glycosylation might be required for homo-oligomerization and nuclotidase activity. Conversely, deglycosylation might impair the E-type nucleotidase activity by weakening the monomer-monomer interaction and altering the tertiary and quaternary structures, result in the loss of holoenzyme. Essentially, glycosylation and deglycosylation of the ecto apyrase (HB6) monomer and the consequences for homodimer formation have been regarded as an on-off switch for ecto nucleotidase activity \[[@B36]\]. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Phase III: Termination of the transport processes.**(A) Several factors might contribute to the termination of Ca^2+^/Mg^2+^transport via channel gating by E-NTPase/E-NTPDase: extracelluar and Intracellular. Additional experimental evidence is mentioned. Decreased flow of Ca2+/Mg2+ due to closing of the channel gate. :::  ::: Fig. [8a](#F8){ref-type="fig"} is a three-dimensional impression of the ecto-ATPase in vivo at the termination of ion transport. Fig. [8b](#F8){ref-type="fig"} illustrates how biochemical modifications such as deglycosylation of the E-NTPase/E-NTPDase oligomers might cause dissociation of the homo-oligomers to individual monomers This is a potential mechanism for the disassembly of the functional channel and closure of Ca^2+^influx and Mg^2+^efflux. Also, an increase in membrane fluidity induced by cholesterol oxidation might cause defective association or disassociation due to weak interaction among the E-NTPase monomers, whereas increased membrane cholesterol might sustain higher E-NTPase activity. Oligomerization of E-NTPase and associated increase of activity could also be responsible for the rapid termination of the purinergic response mediated by extracellular ATP \[[@B37]\]. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Phase III: Termination of the transport processes.**(A) Three-dimensional impression of the E-NTPase/E-NTPDase in vivo when termination of the ion transport function commences. (B) Biochemical modifications of the E-NTPase/E-NTPDase oligomers such as deglycosylation would probably cause instability, leading to dissociation of the homo-oligomers. Disassembly of the functional molecule would ensue, closing the Ca^2+^influx and Mg^2+^efflux processes, as portrayed in the figure. :::  ::: The extracellular nucleotide mediated activation of channel gating could be terminated by ecto (extracellular)-adenylate kinase, which catalyzes trans-phosphorylase activity (ADP+ADP→ ATP+AMP). This enzyme has a higher affinity for extracellular nucleotides than the dephosphorylating enzyme (E-NTPase/E-NTPDase) or ecto-nucleotide pyrophosphatase/phospho-diesterase (ATP→ AMP +ppi) \[[@B38]\]. As the transport process winds down, ecto-adenylate kinase mediated ATP generation might maintain the extracellular nucleotide level. However, the precise biochemical kinetic process by which this process is completed remains to be elucidated \[[@B39]\]. Pathophysiological Significance of E-type nucleotidase mediated Ca^2+^/Mg^2+^transport -------------------------------------------------------------------------------------- Impairment of E-Type nucleotidases during Ca^2+^paradox in isolated rat heart model warrants investigation of the molecular mechanism(s) involved. Knowledge obtained from these studies will elucidate the observed protective effects of anti-rat cardiac Ca^2+^/Mg^2+^-ecto-ATPase antibodies in ischemia reperfusion induced damage, which is a corollary of organ transplantation. Furthermore, the antiproliferative effect(s) of these antibodies in left anterior descending coronary artery smooth muscle cell(s) emphasize the need to explore more fully the hypothesis proposed in this article. Authors\' contributions ======================= HMS participated and provided the hypothetical scheme of the gating mechanism with appropriate literature. SK conceived and carried out experimental part of the investigation and formulating the hypotheses. Abbreviations ============= **E-NTPase**= Ecto or Extracellular Nucleotide triphosphatase; **E-NTPDase**= Ecto or Extracellular Nucleotide triphosphate diphosphohydrolase; **\[Ca^2+^\]~i~**= Intracellular Ca^2+^; **\[NTP\]~0~**= Extracellular Nucleotide triphosphate; **\[NDP\]**~0~= Extracellular Nucleotide diphosphate; **ROCC**= Receptor Operated Ca^2+^channel; **SER**= Sarco (Endo) plasmic reticulum; **\[cAMP\]~i~**= Cytoplasmic or intracellular cAMP; **PMCA**= Plasma membrane Ca^2+^pump ATPase.**VOCC**= voltage-operated Ca^2+^channel; **LGCCS**= Ligand gated non-specific cation channels. **RACC =**Stretch/Receptor activated non-specific Ca^2+^channels. **SUR**= Sulfonylurea Receptor Proteins. **CD36**= Thrombospondin receptor on platelets. **CD39**= Ecto Ca^2+^/Mg^2+^apyrase. Acknowledgements ================ Data were obtained during graduate (Ph.D.) work (1993--1998) supported by a graduate fellowship (to S.K) from St. Boniface General Hospital Research Foundation, Winnipeg, Canada. T.R. Smith, Medicinal Chemistry, University of Warwick, Leamington Spa, and Warwickshire, United Kingdom prepared most of the models. This manuscript was prepared during the tenure of a post-doctoral fellowship (September 2000 -- January 2001) supported by grants DK-52216 and DK-44237 from the National Institutes of Health, Bethesda, MD, and USA. T.L. Kirley, A.F. Knowles, L. Plesner, A.R. Beaudoin, A.Z. Herzberg, M. Handa, K.A. Jacobson, A. Froese, P. Zahradka, L.J. Murphy, N.N. Tandon, N. Abumrad, A. Ibrahimi, R. Lipsky, D. Perlmutter, S.H. Lin, H. Zimmermann and N.N. Tandon are acknowledged for donating reagents, advises, and expert opinion on investigation during the period 1991--1998. Dr. Hans M. Schreiber has passed away during the preparation of this manuscript. The authors do not have any competing financial or intellectual properties interests with CIBA-GEIGY Canada or Novartis Pharmaceuticals Inc, Switzerland. Modified reagents are part of the impending U.S. or International patent application(s).

PubMed Central

2024-06-05T03:55:47.495350

2004-8-12

PMC516238

1. Background ============= We all talk, more or less knowingly, about Mendelian genetics. But four questions need to be asked and answered. 1\. Do we understand Mendel\'s work? To judge from nearly all modern accounts of genetics, we do not. Mendel\'s paper of 1866 has been persistently misrepresented ever since it was rescued from obscurity in 1900. 2\. Do we teach our students a rational description of the inheritance of traits? The answer is again no. Why? Because our current depiction of the inheritance of traits or characteristics is based on false statements, inconsistent arguments and an implausible assertion. 3\. Does the current description of Mendelian genetics account for his observations of dominant and recessive traits? No, for the reasons given in answering question 2. 4\. Do we account rationally for Mendel\'s observation of a 3(dominant):1(recessive) trait ratio in some but not all of his experiments? The answer is again no. The reasons will become clear in this article and its successor. A survey of the relevant literature for the period from 1900 to 2003 shows that the various misrepresentations of Mendel\'s first paper \[[@B1]\] are of long standing. This is not the place to review all the accumulated historical evidence. The present article concentrates on demonstrating that the currently favoured depiction of elementary Mendelian genetics is untenable; it fails to achieve its intended purpose. A change in the concepts and notation for the interpretation (and teaching) of elementary genetics is suggested. There are two long-established tests of the validity of any hypothesis or proposed explanation for the results observed by experiment. The first test asks: Are all the arguments employed consistent, one with all the others? The second test asks: Are all the proposed mechanisms plausible? Could they be confirmed by experiment, i.e. by a \"real\"experiment or by a logical \"thought experiment\". Both tests must be passed if the proposed explanations for the observations are to be accepted. If judgement is being passed on work carried out in the distant past, allowance must be made for the availability or lack of availability of tests of plausibility at that time. On the other hand, we should not hesitate to criticise a current explanation that fails tests of plausibility that are now available but were not available in the past. These two tests of validity (consistency and plausibility) will be applied to Mendel\'s explanation for his observations and to the currently favoured explanation for his observations. We must first re-establish what experiments Mendel performed and what he wrote in his published accounts of these experiments in order to correct the various false textbook descriptions of Mendel\'s work. For this purpose it is necessary to study authentic reprints of his two papers \[[@B1],[@B2]\]. The first paper is the one we are concerned with here; it was reprinted \[[@B3]\] and in a version \[[@B4]\] correcting several type-setting errors that occurred when Mendel\'s manuscript was set in typescript. The translation into English by Sherwood \[[@B5]\] avoided several errors in earlier attempts to translate Mendel\'s *Versuche*paper \[[@B1]\]. There may be other sound translations, but Sherwood\'s version is strongly recommended. It is accurate and also captures Mendel\'s literary style. 2. Mendel\'s experiments and his conclusions ============================================ 2.1. Why did Mendel carry out his experiments? ---------------------------------------------- Many earlier biologists had noted the appearance of hybrid plants but their findings did not show how hybrids arose, whether there was any regularity in their occurrence, or how their properties were related to those of their parents. Mendel showed that there was a general rule for the appearance of hybrid plants and that an exact relationship existed between the traits displayed by hybrids and those displayed by their parents. Hence the title of his first paper: *Versuche über Pflanzen-hybriden*(Investigations on plant hybrids). 2.2. Mendel\'s preliminary work and his conditions for successful experimentation --------------------------------------------------------------------------------- Mendel recognised five preconditions for success in his experiments on the origin of hybrids: \(i) He needed suitable plants for his experiments. He chose *Pisum sativum*(the edible pea plant) for most of his work because many established varieties were readily available; and because the flowers enclose the reproductive organs, so minimising accidental cross-fertilisation by insect-or air-borne pollen. \(ii) *Pisum sativum*, like all leguminosae, is androgynous. The flowers contain both male (pollen or sperm) and female (germinal or ova) cells and are therefore normally self-fertilising. This provided experimental advantages, as we shall see. \(iii) It was necessary to have stocks of true breeding plants for his cross-fertilisation experiments. He therefore spent much time establishing that 22 varieties of edible pea plants were in fact true breeding. He discarded those plants that were not true breeding before starting his experiments on hybridisation. \(iv) He had to ensure that any cross-fertilisations were strictly under his control. To achieve this control, he removed all the immature pollen-bearing stamens from a true-breeding pea plant that displayed a particular trait, e.g. green seeds, then transferred pollen to these emasculated flowers from another true breeding pea plant that displayed an alternative form of the same trait (e.g. yellow seeds). \(v) Success depended on meticulous enumeration of the occurrence of hybrids, and of alternative traits, in the populations of plants that arose from his cross-and self-fertilisation experiments; and on repetition of each cross-fertilisation and self-fertilisation experiment in order to obtain reliable, average, results. Table [1](#T1){ref-type="table"} reveals the magnitude of Mendel\'s undertaking and records his observations on the occurrence of hybrids, and of plants displaying either dominant or recessive traits (see further descriptions in the following section). Reciprocal crosses gave the same results; Mendel thus established that male and female sex cells contributed equally to the final outcomes. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Mendel\'s novel observations summarised. Mendel demonstrated that crossing parental plants bearing alternative forms (*A*) and (*a*) of any one of seven traits generated a F1 population of plants (not shown) all of which were hybrids (*Aa*). Each of these F1 hybrid plants displayed only one of the two alternative parental traits, defined as the dominating trait (*A*). When these F1 hybrid plants were allowed to self-fertilise, the ratio of dominant to recessive traits in the F2 population was always close to 3:1. ::: ------------------------------------------------------------------------------------------------ Pairs of parental plants Their F2 progeny --------------------------- ---------------------------- ----------------- --------------------- Dominant traits (***A***) Recessive traits (***a***) Number of F2\ Dominant:recessive\ plants examined trait ratio in the\ F2 population Green pods Yellow pods 580 2.82:1 Axial flowers Terminal flowers 858 3.14:1 Red flowers White flowers 929 3.15:1 Long stems Short stems 1064 2.84:1 Inflated pods Constricted pods 1181 2.95:1 Round seeds Wrinkled seeds 7324 2.96:1 Yellow seeds Green seeds 8023 3.01:1 ------------------------------------------------------------------------------------------------ ::: 2.3. Bateson\'s notation for successive stages in breeding experiments ---------------------------------------------------------------------- The following account uses the notation proposed by Bateson \[[@B6]\] for successive generations arising from sexual reproduction:- P = the original male and female parental generations; F1 = the first filial progeny population arising from crosses between plants of the P generation; F2 = the second filial generation that arises from sexual reproduction by members of the F1 generation -- and so on. The advantage of Bateson\'s notation is that it does not depend on any preconceived ideas about the mechanisms of inheritance of traits during sexual reproduction. It can therefore be used to describe the stages in Mendel\'s experiments without misrepresenting any of his observations, arguments or conclusions. 2.4. Mendel\'s initial observations summarised ---------------------------------------------- Table [1](#T1){ref-type="table"} shows the results of seven different cross-fertilisations between parental (P) plants displaying alternative forms of the same trait, e.g. red rather than white forms of the trait \"flower colour\"; all individual plants in the F1 population displayed only one of the two parental trait forms. Also shown are the results observed by Mendel when he allowed these F1 plants to self-fertilise; the ratio of (*A*) form to (*a*) form plants was, in every case, close to 3:1. Mendel also carried out experiments in which he cross-fertilised plants displaying concurrently two or three trait differences, and then recorded the occurrence of each trait in the F1 and F2 generations. These results are not shown here but they were consistent with the findings exemplified in Table [1](#T1){ref-type="table"}. These initial findings led Mendel to a remarkable generalisation and a definition. \(i) All plants in the F1 population displayed only one of any two differing trait forms (*A*) and (*a*) displayed by the parental (P) plants. \(ii) He defined the trait form that was displayed in the F1 plants as *das dominirende Merkmal*(*A*) -- the dominating trait *(A)*. He defined the alternative trait form, which did not appear in any of the F1 plants, as *das recessivem Merkmal (a)*-- the recessive trait (*a*). 2.5. Further experiments ------------------------ Mendel now faced the problem of explaining how the 3(dominant):1(recessive) trait ratio arose in the F2 population of plants (Table [1](#T1){ref-type="table"}). In further experiments on each of the seven crosses shown in Table [1](#T1){ref-type="table"}, he was able to show that those F2 plants he had identified by the symbol (*a*) were \'constant form\' (true-breeding) plants; i.e., when they were allowed to self-fertilise, all their F3 progeny displayed the same parental trait (*a*). On the other hand, when F2 plants initially identified by the symbol (*A*) were allowed to self-fertilise *some*proved to be \'constant form\' plants because, when they were allowed to self-fertilise, they produced F3 progeny that again displayed this same parental trait (*A*). But other plants initially identified by the symbol (*A*) in the F2 population were not \'constant form\' plants. Some of their F3 progeny did display the original parental trait (*A*). Other plants in the same F3 population displayed the alternative parental trait (*a*). Yet other plants in this F3 population were again not \'constant form\' plants. They were like the F1 plants (their \"grandparents\") and like the F2 parents from which they were immediately derived. When they were allowed to self-fertilise, some of their progeny displayed the (*A*) form, some the (*a*) form of trait and some were again like the F1 plants. The experimental procedures Mendel used to make these distinctions are readily understood by reading a reprint of the original paper or a reliable translation. Given this ability to distinguish, by experiment, between those plants initially designated (*A*) and those now designated (*Aa*), Mendel was able to state the average distribution of trait forms among the plants of the F2 population as (one dominant: two hybrid: one recessive) or, in his notation, *(A +*2*Aa + a)*; i.e. the 3:1 trait ratio factored into the proportions 1:2:1. Mendel was now able to add a further generalisation: When F1 plants were allowed to self-fertilise, 1/4 of the F2 population displayed the \'constant form\' parental trait *(A)*that was displayed by the F1 plants, 1/4 displayed the \'constant form\' parental trait *(a)*that did not appear in any of the F1 plants (Table [1](#T1){ref-type="table"}), and 1/2 were hybrids (*Aa*) that displayed only the dominant trait (*A*) but were not \'constant form\' plants. 2.6. Mendel\'s notation ----------------------- Mendel used upper case and lower case italicised letters throughout his paper to denote, by definition, dominant and recessive *traits*. Examples have already been given of the use of letters (*A*) and (*a*) when only one trait difference between parental plants was tested (Table [1](#T1){ref-type="table"}). Mendel made similar use of the letters (*B*) and (*b*), (*C*) and (*c*) when he described experiments in which two or three trait differences were displayed concurrently. For reasons given in Section 2.5 these single letters also designated what Mendel called \'constant forms\' of traits. Plants displaying these traits were \'true-breeders\'; they were the parental plants he used in cross-fertilisations (Table [1](#T1){ref-type="table"}). There is one further crucial feature of Mendel\'s single letter notation for \'constant form\' traits. These letters (*A, a, B, b, C, c*) did not represent the structure or composition of the traits. All the traits shown in Table [1](#T1){ref-type="table"} obviously had complex compositions. But, irrespective of such complexity, each dominant trait was denoted by (*A*) and each recessive trait by (*a*) in Table [1](#T1){ref-type="table"}. The traits were what Mendel could see with his own eyes. He distinguished a dominant trait from a recessive trait by qualitative observations. He was not concerned with and did not analyse the structural composition of the traits. The letters (*A, a, B, b, C, c*) represented classes of traits -- a dominant class represented by an upper case letter, and a recessive class of trait represented by the corresponding lower case letter (Table [1](#T1){ref-type="table"}). It is necessary to recognise these facts if a rational explanation for Mendel\'s observation is to be obtained; and if gross misrepresentations of Mendel\'s paper are to be detected. Why then did Mendel use a combination of letters (e.g. *Aa*) to represent hybrid plants? This will become clear in section 2.7. 2.7. Postulates and arguments; Mendel\'s explanations of his observations ------------------------------------------------------------------------- Mendel accounted for the two generalisations (section 2.4) by the following postulates and arguments; they were based on his further experiments (section 2.5):- \(1) All the F1 plants were hybrids (*Aa*) *in welcher beide Merkmale vereinigt sind*-- in which both (parental) traits (*A*and *a*) were united; trait (*a*) was not displayed by these hybrids, so that these hybrids displayed what he had defined as the dominating trait (*A*) only. \(2) The traits (*A*) and (*a*) in the F1 hybrids (*Aa*) segregated into traits (*A*) and (*a*) during formation of the male pollen (sperm) cells and also during formation of the female germinal cells (ova). Thus, each pollen cell and each germinal cell carried only one trait -- either (*A*) or (*a*) but not both. \(3) Fertilisation of one germinal cell by one pollen cell was a random event. \(4) When a pollen cell bearing trait (*A*) fertilised a germinal cell bearing the same trait (*A*), all their progeny displayed the trait (*A*). Likewise, when a pollen cell bearing a trait (*a*) fertilised a germinal cell bearing the same trait (*a*), all their progeny displayed the trait (*a*). But when a pollen cell bearing trait (*A*) fertilised a germinal cell bearing the alternative trait (*a*), the resulting plant was the hybrid (*Aa*); if the pollen cell displaying a trait (*a*) fertilised a germinal cell displaying the alternative trait (*A*), the outcome was again a hybrid (*Aa*). In either event, the hybrid (*Aa*) displayed only the dominant trait (*A*). \(5) Mendel illustrated these postulates and explanations in a diagram (Figure [1](#F1){ref-type="fig"}) showing the consequences of self-fertilisation of F1 hybrids (*Aa*), given that traits (*A*) and (*a*) in the hybrid (*Aa*) first segregated into individual pollen cells (sperm) and individual germinal cells (ova) before recombining, in random fashion, during formation of the F2 population. The arrows in Figure [1](#F1){ref-type="fig"} represent the fertilising event. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Mendel\'s diagrammatic explanation for the formation of the F2 population of plants produced by self-fertilisation of his F1 hybrids. Mendel proposed that F1 hybrids (*Aa*) contained a dominant trait (*A*) that was displayed and a recessive trait (*a*) that was not displayed. Self-fertilisation of F1 hybrids (*Aa*) then involved segregation of the component traits (*A*) and (*a*) into individual male pollen and female germinal cells, as shown in his diagram. Mendel proposed that if a male pollen cell carrying a trait (*A*) fertilised a female germinal cell carrying the same trait (*A*), the progeny would display trait (*A*). He used the analogous argument for the generation of progeny bearing trait (*a*). Only if male and female sex cells carried differing forms of a given trait (*A*or *a*but not both) would the progeny be hybrids (*Aa*). Thus random recombination of the segregated traits during self-fertilisation of hybrids would yield (on average) the F2 population of plants represented by the trait series (*A*+ 2*Aa*+ *a*) shown below Mendel\'s original diagram. :::  ::: Note two crucial points:- \(i) Mendel observed and recorded the occurrence of traits (*die Merkmale*) or the characters *(die Charaktere)*in his plants and their seeds, not the mechanisms underpinning these occurrences. These mechanisms could not have been investigated in 1866. \(ii) All Mendel\'s explanations were based solely on observations of the changes in the occurrence of alternative traits in successive populations that arose from cross-or self-fertilisations and back-crosses. 2.8. Comment ------------ Mendel was a well-trained scientist \[[@B7]\], an astute thinker, a careful and systematic experimentalist, an expert hybridiser and an exemplary writer but he was not the first geneticist. That title should go, possibly, to Bateson \[[@B6],[@B8],[@B9]\] for advocating Mendel\'s experimental methods, for showing that Mendel\'s findings could be repeated in animals, and for emphasising that *combination*, *segregation*and *recombination*of *traits*during gametogenesis was the most important feature of Mendel\'s work. Moreover, Bateson realised \[\[[@B6]\]; in a footnote on page 133\] that the occurrence of alkaptonuria, one of the \"Inborn Errors of Metabolism\" first reported by Garrod \[[@B10]-[@B12]\], was an example of Mendelian recessivity of a trait or character. Bateson, incidentally, coined the word \"genetics\". Another leading contender for the title \"the first geneticist\" was the Danish biologist, Johannsen \[[@B13],[@B14]\], an equally enterprising experimentalist and astute thinker. Johannsen \[[@B14]\] was the first to define the term *\"das gen*; (plural) *die gene\"*as the determinant of a trait; he was also the first to make a clear distinction between the genotype *(der Anlagetypus)*and the phenotype *(der Erscheinungstypus*) on the basis of his experiments with self-fertilising bean plants. In Johannsen\'s experiments the weights of individual beans were the characteristics or traits. He had, in effect, repeated Mendel\'s experiments but by measuring a trait (individual bean weights in successive populations of plants) he was able to introduce three new concepts (gene, genotype and phenotype) that were the most significant, after Mendel\'s concepts of combination, segregation and recombination of traits during gametogenesis, in understanding the origin of genetic phenomena (the origin of changing traits). Failures to recognise the significance of Johannsen\'s work \[[@B13],[@B14]\] prevented the development of rational concepts in genetics for at least the first two decades of the 20^th^century. This failure is, surprisingly, still evident in current depictions of elementary Mendelian genetics (Section 3). 2.9. The tests of validity applied to Mendel\'s explanation for his observations -------------------------------------------------------------------------------- It is clear that Mendel\'s experimental procedures (sections 2.2, 2.5) were sound; his notation was simple, unambiguous and consistently applied (section 2.6). His arguments (section 2.7) for a combination of traits in forming the F1 hybrids (*Aa*) are consistent with his arguments for the segregation of the component traits of the hybrid into separate gametes, and their random recombination in generating the F2 population (*A*+ 2*Aa*+ *a*). Mendel\'s arguments pass the test of consistency. It is equally clear (but hitherto not noticed) that Mendel\'s explanations failed the test of plausibility. Mendel postulated that a F1 hybrid (*Aa*) was formed by combining the two differing traits (*A*) and (*a*) of their parents. He did not explain how a F1 hybrid (*Aa*) displayed only trait (*A*) and how it did not display trait (*a*), even when some F2 plants, like one of the two original parental (P) plants, did display trait (*a*). What explanation could we now give for this selective display of only one of two traits that are said to be combined in a hybrid? It may be (and has been) argued by some that trait (*A*) was displayed by the hybrids (*Aa*) because (*A*) was a dominant trait and (*a*) was a recessive trait. Such statements do not even qualify as a circular argument. They are illogical. Such statements fail to distinguish between an arbitrary definition and a plausible explanation. Mendel\'s definition of a dominant trait should be seen as an arbitrary device that accounts for his observation (by experiment) that his hybrids (*Aa*) in the F2 populations (*A*+ 2*Aa*+ *a*) displayed trait (*A*) but not trait (*a*). A word of caution is necessary. Mendel\'s formulation (*Aa*) for a hybrid was crucial in establishing his consistent arguments; it was also the basis for Bateson\'s recognition that the essential features of Mendel\'s work were the concepts of combination, segregation and recombination of alternative traits (i.e., components of the phenotype). If we now wish to replace Mendel\'s implausible formulation (*Aa*) for a hybrid by a plausible formulation, we face the prospect of abandoning the rest of Mendel\'s arguments. That is not to say that we abandon admiration for Mendel\'s work. For its time, it was unsurpassed and should be recognised as one of the important steps in the development of experimental procedures in what became known as genetics. We should take care not to misrepresent Mendel\'s experiments and his arguments. It will become clear that misrepresentations of Mendel\'s paper have served only to sustain untenable concepts in current biology. In the post-Mendel era we assert that it is not components of the phenotype that segregate and recombine. It is the alleles (i.e., components of the genotype) that combine, segregate and recombine. May we then anticipate that modern explanations of Mendel\'s observations will pass the tests of consistency and plausibility? 3. Current accounts of elementary Mendelian genetics ==================================================== 3.1. Explanations of Mendel\'s observations ------------------------------------------- The currently favoured explanation for Mendelian heredity in general, and in particular for the occurrence of Mendel\'s 3(dominant):1(recessive) trait ratio, is shown in Figure [2](#F2){ref-type="fig"}. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The currently favoured depiction of Mendelian inheritance following self-fertilisation of F1 hybrids represented by the allele pair (*Aa*). Section 3.1 of the text records the arguments commonly used in attempts to account for the alleged F2 trait series (*AA*+ 2*Aa*+ *aa*) and for Mendel\'s 3(dominant):1(recessive) trait ratio. Sections 3.2 and 3.3 discuss the faults in these arguments. :::  ::: The assertions and descriptions generally attached to Figure [2](#F2){ref-type="fig"} are as follows. \(i) Mendel explained his experimental results by assuming that particles or factors (now called alleles) determined or specified the observed traits. \(ii) (*A*) is a dominant allele;*(a)*is a recessive allele. \(iii) The alleles in the male and female heterozygous somatic cells *(Aa)*segregate into separate gametes. Each gamete then contains only one dominant allele *(A)*or only one recessive allele *(a).* \(iv) Fertilisation is a completely random event. Given a large number of fertilisation events, the possible recombinations of alleles are those displayed in the four squares. \(v) Therefore the average distribution of the alleles at one diploid locus in the resulting progeny population of individual plants will be (*AA*+ 2*Aa*+ *aa*). It is then argued that: \(vi) The dominant allele pair (*AA*) will give rise to a dominant trait (*AA*). \(vii) The recessive allele pair (*aa*) will give rise to a recessive trait (*aa*). \(viii) In the heterozygote (*Aa*), the recessive allele is ineffective, or is suppressed by the dominant allele (*A*), so that only the dominant allele (*A*) is expressed in the heterozygote. Expression from one (*A*) is as effective as that from two dominant alleles (*AA*). Thus the heterozygote (*Aa*) expresses a dominant trait. \(ix) Therefore the allele series (*AA*+ 2*Aa*+ *aa*) is expressed (in a population of the progeny plants, animals or cells) as the trait series (*AA +*2*Aa + aa*). \(x) This trait series gives rise to Mendel\'s 3(dominant):1(recessive) trait ratio (by the arguments in vi, vii, viii). 3.2. Faults in these currently favoured descriptions of Mendelian genetics -------------------------------------------------------------------------- There are seven faults in the descriptions and arguments attached to Figure [2](#F2){ref-type="fig"}. \(i) Mendel is misrepresented; he did not assume that particles or factors specified the observed traits. It is historically inaccurate and scientifically misleading to suppose that he made any such assumption. \(ii) The letters (*A*) and (*a*) are Mendel\'s notation for dominant and recessive *traits*(Figure [1](#F1){ref-type="fig"}, Table [1](#T1){ref-type="table"}). If we are to continue to discuss Mendelian genetics, these notations (and the nomenclature *dominant*and *recessive*) should refer to traits alone. \(iii) Figure [2](#F2){ref-type="fig"} fails to distinguish between the components of the genotype and the components of the phenotype (Johannsen, Section 2.8) because it asserts that alleles are dominant or recessive; and uses the same notation (*A*and *a*) and the same nomenclature (*dominant*and *recessive*) for both. \(iv) Because we must not confuse alleles with traits, we could reasonably write an allele series as (*UU*+ 2*Uu*+ *uu*); this states that a given locus, in three genetically related diploid cells, comprises a pair of two normal alleles (*UU*), or one normal and one mutant allele (*Uu*), or a pair of two mutant alleles (*uu*). Mutations change the allele constitution or composition at a locus. The modern (non-Mendelian) notation (*AA +*2*Aa + aa*) in Section 3.1 (items vi, vii) then states explicitly that a dominant trait (*AA*) comprises two aliquots (*A*+ *A*) of some material substance or of two doses of dominance (*A + A*); likewise that a recessive trait (*aa*) is composed of two entitities (*a*+ *a*) or two doses of recessivity. This is simply not true. It was not true in Mendel\'s time and it is not true today. Furthermore, it is not what Mendel\'s notation meant. It was pointed out (Sections 2.5, 2.6) that Mendel\'s notation (*A*) and (*a*) distinguished *classes*of traits, specifically \'constant form\' classes of traits (Table [1](#T1){ref-type="table"}). To substitute (*AA*) for (*A*) and (*aa*) for (*a*) in a trait series is illogical and indicates a regrettable failure to read Mendel\'s paper with the care that should be given to one of the classic papers in biology. \(v) If the arguments attached to the homozygotes in Figure [2](#F2){ref-type="fig"} are sound, they should also apply to the heterozygote. It is argued in Figure [2](#F2){ref-type="fig"} that two dominant alleles (*AA*) generate a dominant trait (*AA*); and that two recessive alleles (*aa*) generate a recessive trait (*aa*). In other words, it is asserted that there is a direct, positive, linearly proportional (or additive) relationship between the allele constitution at a gene locus and the constitution of the trait expressed from that locus. If we are to be consistent, the same arguments should apply to the heterozygote (*Aa*). On the contrary, the arguments in section 3.1 (item viii) state that one dominant allele (*A*) in a heterozygote (*Aa*) is as effective as two dominant alleles (*AA*) in the homozygote. The arguments in item (viii) are therefore inconsistent with arguments in items (vi) and (vii). Item (viii) also transfers Mendel\'s implausible assertion that a hybrid (*Aa*) displays only trait (*A*) to the equally implausible assertion that one allele (*A*) in a heterozygote (*Aa*) is as good as two such alleles in a homozygote (*AA*). The argument in item (viii) that allele (*a*) is ineffective is an extreme case; it is therefore not generally applicable. The alternative argument, that allele (*a*) in a heterozygote is suppressed by the dominant allele (*A*), lacks any experimental support or rational theoretical justification. Items vi, vii and viii attached to Figure [2](#F2){ref-type="fig"} are arbitrary, irrational and implausible devices applied to the heterozygote alone; they seem to have been introduced solely in order to arrive at the desired result. \(vi) Figure [2](#F2){ref-type="fig"} and the attached arguments thus fail to give rational explanations for the occurrence of dominant and recessive traits and for Mendel\'s 3(dominant):1(recessive) trait ratio. \(vii) Figure [2](#F2){ref-type="fig"} does not and cannot account for the observation that dominance and recessivity are not observed for all traits. The assertion in Figure [2](#F2){ref-type="fig"} that the alleles are themselves \"dominant\" or \"recessive\" (and thus determine that traits are dominant or recessive) conflicts with inability of Figure [2](#F2){ref-type="fig"} to explain why dominant and recessive traits are not always observed; nor does Figure [2](#F2){ref-type="fig"} account for the observation that, when dominance and recessivity do occur, they do not always exhibit a 3:1 trait ratio. 3.3. Comments on these faults ----------------------------- It is necessary to restate fault (iii) in section 3.2 in more widely applicable terms. It is illegitimate to use the same notation and nomenclature for a *parameter*and a *variable*in the same system. Parameters are those components of any system that are directly accessible to the experimentalist; they can be changed and *maintained*by the experimentalist at the new value, at least for the duration of an experiment. Variables are those components of the same system that are not directly accessible to the experimentalist; they can be changed and maintained at a new value only by making a finite change in at least one parameter of the system or of its immediate environment. The magnitudes of individual variables, in any system, respond to changes in the magnitude of one or more parameters of the system or of the immediate environment. In the case under discussion, the alleles are parameters (and part of the genotype); the traits are variables (and part of the phenotype). If the parameters and variables of any system of interacting components are represented by the same notation and the same nomenclature, confusion will inevitably result -- as illustrated by Figure [2](#F2){ref-type="fig"}, by the assertions (i) and (ii) and by the false arguments (vi) to (x). Traits may be dominant or recessive \[[@B1]\]; alleles cannot also be dominant or recessive. Figure [2](#F2){ref-type="fig"}, and the arguments attached to it, fail all tests of consistency and plausibility (Section 2.9); they also fail the test of historical accuracy. 3.4. Another example of the improper transfer of dominance/recessivity from traits to alleles --------------------------------------------------------------------------------------------- The primary error in Figure [2](#F2){ref-type="fig"} is the illegitimate transfer of Mendel\'s terms \"dominant\" and \"recessive\" from traits (variables) to alleles (parameters), followed immediately by the reverse (and perverse) argument that the traits specified by the alleles must be dominant or recessive *because the alleles are dominant or recessive*. This habit is unscientific. It also occurs in discussion of mutations of non-catalytic proteins. When haemoglobin A (HbA) is mutated to the sickle cell haemoglobin (HbS), the three possible trait forms are correctly depicted as follows: (A/A) -- the homologous, *normal/normal*protein, condition; (A/S) -- the heterologous, *normal/mutant*protein (sickle cell), condition; (S/S) -- the homologous, *mutant/mutant*protein, condition. Contrast these depictions with those sometimes found: (A/A) -- the *dominant*condition; (A/S) -- the *sickle cell*condition; (S/S) -- the *recessive*condition. These latter statements depend solely on the illegitimate transfer of Mendel\'s terms *dominant*and *recessive*from traits (variables) to alleles (parameters) and the contention that, if alleles are themselves dominant or recessive, their expressed traits must always be dominant or recessive. If changes in the composition of non-catalytic proteins do explain the occurrence of Mendel\'s dominant and recessive traits, we require a demonstration that does not depend on these illogical notions. The sickle cell trait (A/S) in humans is significantly different from the normal trait (A/A). Those carrying the sickle cell (A/S) condition enjoy an advantage in areas *where malaria is endemic*. They do not die *from malaria*as frequently as those in the population with the (A/A) condition. The sickle cell condition (A/S) is debilitating but, provided it is not too debilitating, the frequency in the local population of those carrying the (A/S) protein pair is greater than it would be in malaria-free areas. This higher frequency of the sickle-cell (A/S) condition in areas where malaria is endemic is often said to be an example of \"over-dominance\". The term \"over-dominance\" is inappropriate. It presumably arose from the illegitimate transfer of the terms *dominant*and *recessive*noted above. The appropriate term is \"heterozygous superiority\". The \"superiority\" indicates the better chance of surviving in regions where malaria is endemic. 4. Conclusions: beginning a rational explanation for Mendel\'s observations =========================================================================== The illegitimate use in Figure [2](#F2){ref-type="fig"} of the same notation (*A*and *a*), and the same nomenclature (dominant and recessive), to describe an allele series and a trait series can be traced to Sutton \[[@B15]\]. Sutton asserted that the proportions of the chromosome pairs in the F2 population \"would be expressed by the formula *AA*:2*Aa*:*aa*which is the same as that given for any character in the Mendelian case.\" Mendel\'s expression (*A*+ 2*Aa*+ *a*) gave the proportions of characters in his F2 population as *A*:2*Aa*:*a*. Sutton gave no justification for rewriting these proportions in the form *AA*:2*Aa*:*aa*. By writing the expression for chromosome pairs as *AA*:2*Aa*:*aa*and the expression for the proportions of F2 characters as *AA*:2*Aa*:*aa*, Sutton established a direct, one-for-one, relationship between pairs of chromosomes and the traits arising from them. This false relationship also persists in the currently favoured depiction of Mendelian genetics (Figure [2](#F2){ref-type="fig"}). Sutton\'s notation for pairs of chromosomes (*AA*:2*Aa*:*aa*) was later transferred to pairs of alleles (what Sutton described as subunits of the chromosomes). It would be easy to blame Sutton for our present confusions. We should remember that Sutton, and those in the early years of the 20^th^Century who copied his error, were struggling to understand the hereditary origin of traits. We may more reasonably ask: Why, one hundred years later, are these obvious errors still one of the features of Figure [2](#F2){ref-type="fig"}? Have these errors not been noticed before or, if they have been noticed, why they have not been corrected? Why also has the inconsistency and the implausibility of the arguments attached to Figure [2](#F2){ref-type="fig"} not been noticed or corrected? Why (in both of the examples given in sections 3.3 and 3.4) are alleles (components of the genotype) not distinguished, as they surely should be in genetics, from traits (components of the phenotype) by using different notations and nomenclatures for each? Traits (variables) may be dominant or recessive, as defined by Mendel. Alleles (parameters) are, always have been, and can only be normal or abnormal (mutant). Harris (pages 143--157 in reference \[[@B16]\]), for example, referred consistently to normal and abnormal alleles (not to dominant and recessive alleles), whereas, as noted above, alkaptonuria was a Mendelian recessive trait or character (page 133 in reference \[[@B6]\]; page 19 in reference \[[@B16]\]). A review of 13 textbooks of genetics showed that in 12 instances, dominance and recessivity were defined specifically as properties of genes or alleles. These texts, published between 1982 and 2002, were intended for student use; their definitions of dominance and recessivity ignore Mendel\'s definition of dominance and recessivity as properties of the traits (sections 2.4, 2.5, 2.6, 2.7); they take no account of the need to distinguish between the parameters and variables of a system of interacting components (section 3.2). In one of these 12 texts, it was further claimed that: \"Mendel proposed the existence of what he called particulate unit factors for each trait\". In another, that: \"Mendel realised that some genes (dominant genes) expressed themselves when present in only one copy\". In a third that: \"Mendel imagined that during the formation of pollen and egg cells, the two copies of each gene in parents segregate\". Of these three quoted texts: The first misrepresented Mendel; he did not \"propose the existence of particulate unit factors for each trait\". The second misrepresented Mendel by transferring his term *dominirende*(\"dominating\") from traits to genes; the second and the third quoted texts ignored the fact that the term \"*das gen*\" (plural \"*die gene*\") was first used and its role as the determinant of traits postulated by Johannsen, 43 years after Mendel\'s paper was published (Section 2.8); Mendel did not mention the word gene (Section 3.2). Of the 13 texts examined, only one gave a definition of dominance and recessivity that would have been recognised by Mendel. Even so, this author contradicted his correct definition of dominance and recessivity as properties of components of the *phenotype*by giving an explanation of elementary Mendelian genetics that employed Figure [2](#F2){ref-type="fig"} and its associated arguments. All 13 of the texts examined ignored or contradicted the verifiable historical evidence (sections 2.2--2.7) and failed to make the obligatory distinction between the functions of alleles and the properties of traits. The correct nomenclature for alleles used by Harris (pages 143--157 in reference \[[@B16]\]) is, unfortunately, rarely if ever employed by other authors. Pasternak \[[@B17]\], for example, accepted that \"in strict genetic terms, dominance and recessivity are descriptions of the phenotype and not of the genes.\" but then continued: \"However, few textbooks bother to make the distinction, because it was both convenient and highly ingrained for geneticists and others to refer to dominant and recessive alleles.\" Ingrained it may be, convenient (and scientifically legitimate) it is not. If we continue to propose Figure [2](#F2){ref-type="fig"} and the attached arguments as an explanation of Mendel\'s work, we deceive ourselves and encourage irrational thinking in our students at a time in their education when they are most vulnerable. It is extraordinary that an \"explanation\", like Figure [2](#F2){ref-type="fig"}, should still be found in textbooks intended for student instruction; it exposes our own confusion but explains nothing of scientific value in genetics. Any student who criticised Figure [2](#F2){ref-type="fig"} and the attached arguments in an answer to an examination question would have shown commendable scientific insight but, according to current teaching, would be deemed to have failed that question. Barker \[[@B18]\], writing on another topic, suggested that it might take 50 rather than 25 years for textbooks \"to get it right\". On the evidence presented here, Barker was too optimistic. The four errors introduced by Sutton \[[@B15]\] remain uncorrected (Figure [2](#F2){ref-type="fig"}) 100 years later. To be fair to authors of textbooks of genetics, every author inevitably relies on what has been written by preceding authors. However that may be, we are faced with an uncomfortable question. Are we content to continue to deceive ourselves, to give our students a false picture of what Mendel achieved, and to provide them with untenable \'explanation\' of his remarkable observations (Figure [2](#F2){ref-type="fig"})? Presumably not, especially when we can very easily begin, in this article, to construct a rational explanation for Mendel\'s observations and for other observations of current interest in genetics. A fresh approach to the origins of dominant and recessive traits is needed. As a first step, we need to represent normal and mutant *alleles*by symbols that differ from those (*A, a, B, b, C, c*) used by Mendel to represent *traits*. We must replace symbols (*A*and *a*) for *alleles*in Figure [2](#F2){ref-type="fig"} by quite different symbols; e.g. (*U*) to represent a normal allele, *not*a \"dominant allele\"; and (*u*) to represent a mutant or abnormal allele, *not*a \"recessive allele\"*.*The F2 allele series in Figure [2](#F2){ref-type="fig"} would then be, on average, *UU*+ 2*Uu*+ *uu.* Similarly, the *trait*series in Figure [2](#F2){ref-type="fig"} must be replaced by Mendel\'s notation *(A*+ 2*Aa*+ *a)*because, as explained earlier, Mendel was concerned (as we are, first and foremost) only with understanding the origin of two *classes*of trait -- the dominant class (*A*) and recessive class (*a*). We will later be concerned with the quantitative composition of traits. We have, however, already identified an implausibility in Mendel\'s notation (*Aa*) for a hybrid that, allegedly, displayed the trait (*A*). An implausibility, like an inconsistency, must be eliminated if we are to arrive at an internally consistent and plausible account of Mendel\'s observations. The implausible notation (*Aa*) can be eliminated by replacing it by the single symbol (*H*) for a hybrid. We have now adopted a stance that, in sharp contrast to Figure [2](#F2){ref-type="fig"}, distinguishes clearly between determinants and that which is determined. We have allocated a nomenclature and notation for alleles that is distinct from that allocated to traits. We have differentiated clearly between the *parameters*of the system (in this particular case, the components of the genotype) and the *variables*of the system (in this particular case, the components of the phenotype). Mendel found, by experiment, that the proportions of different plant forms in his F2 populations were 1(dominant trait):2(hybrids):1(recessive trait) or, in his notation, (*A*+ 2*Aa*+ *a*). Replacing Mendel\'s notation (*Aa*) for a hybrid by the single symbol (*H*) does not alter Mendel\'s experimental observation of the proportions of trait forms in the F2 populations (section 2.5). It does mean that we can avoid Mendel\'s implausible postulate that, although recessive trait plants did display trait (*a*), his hybrids (*Aa*) did not. We have, of course, to discover an experimentally verifiable mechanism that would explain why hybrids (*H*) display a trait that is sometimes indistinguishable and sometimes distinguishable from trait (*A*). Our remaining task is to explain rationally how this series of *normal*and *mutant*alleles *(UU*+ 2*Uu*+ *uu)*in the F2 population is expressed as the trait classes (*A*+ 2*H*+ *a*) in that population, where all that we have done is to replace Mendel\'s implausible (*Aa*) by a plausible (*H*). Note also that we have now also eliminated the illegitimate use of paired symbols for Mendel\'s dominant (*A*) and recessive (*a*) traits. Most of the clues that facilitate this task are present in this article. One clue is missing, but it can be inferred by asking how one allegedly dominant allele (*U*) in a heterozygote (*Uu*) could be as effective as two such alleles (*UU*) in a homozygote. A further article will provide the answers, but in the interval readers may like to rise to the challenge of explaining: (1) how *dominant and recessive traits*arise from *normal and mutant alleles*, and (2) why Mendel\'s 3:1 trait ratio, though not uncommon, does not always occur.

PubMed Central

2024-06-05T03:55:47.497447

2004-8-16

PMC516255

Background ========== Integral membrane proteins are the primary choice as targets when developing new drugs and although clearly of medical relevance, forming 20% -- 30% of the gene products of most genomes, these proteins have been structurally determined in only about thirty cases \[[@B1],[@B2]\]. Where high levels of sequence homology exist, an unknown protein\'s structure and hence, the location of its membrane interactive segments, can sometimes be deduced by direct comparison to known protein structures. However, where sequence information alone is available, the identification of transmembrane *α*-helical structure requires a bioinformatics approach to understanding the structure / function relationships of these *α*-helices. A number of *α*-helical properties have been used as models to study transmembrane *α*-helices and their structure / function relationships but the most commonly used are those based on the amphiphilicity of protein *α*-helices with the hydrophobic moment used as a measure of amphiphilicity \[[@B3]\]. To quantify the amphiphilicity of protein secondary structures, Eisenberg and co-workers \[[@B4]\] introduced the hydrophobic moment, *μ*(*θ*), which provides a measure of the structured partitioning of hydrophilic and hydrophobic residues in a regular repeat structure of period *θ*. For a structure comprising *L*consecutive residues, the general form of *μ*(*θ*) is given by:  where *H*~*j*~is the hydrophobicity of the *j*^th^residue within the sequence, and *θ*is the angular frequency of the amino acid residues forming the structure. Eisenberg *et al*., \[[@B4]\] assumed that for an *α*-helix, *θ*is fixed at 100°, and that a segment of eleven consecutive residues, equivalent to three turns of an *α*-helix, could be used to represent amphiphilic *α*-helices. These assumptions led to the more generally used measure of *α*-helix amphiphilicity, the mean hydrophobic moment \<*μH*\>, where \<*μH*\> = *μ(100°)/11* As a major extension to the use of the hydrophobic moment, Eisenberg *et al*., \[[@B5]\] introduced hydrophobic moment plot methodology, which provides a graphical technique for the general classification of protein *α*-helices. Using this methodology, a putative protein *α*-helix is characterised according to its maximum \<*μH*\> and corresponding mean hydrophobicity, \<*H*\>, where this is defined by:  The parameters \<*μH*\> and \<*H*\> are then plotted on the hydrophobic moment plot diagram (figure [1](#F1){ref-type="fig"}) and the location of the resulting data point used to classify the putative *α*-helix. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Conventional hydrophobic moment plot analysis of the transmembrane protein data set.**Figure 1a shows the hydrophobic moment plot diagram \[5\] with protein classification boundaries. Figure 1b shows the results of hydrophobic moment plot analysis of the 403 transmembrane sequences of our data set using the conventional values of *L*= 11 and *θ*= 100° \[4\]. :::  ::: The mean hydrophobic moment is widely used and generally regarded as a good predictor of *α*-helix amphiphilicity but the results of statistical analyses have shown the efficacy of hydrophobic moment plot methodology as a predictor of *α*-helical class to be less certain \[[@B6]\]. A number of authors have observed that the methodology can erroneously classify *α*-helices in cases where the hydrophobic moment for a particular amino acid sequence is greatly affected by the spatial arrangement of a few extreme amino acids, thus masking the overall nature of an *α*-helix \[[@B3]\]. However, a more fundamental source of erroneous classification could come from the questionable assumption made by hydrophobic moment methodology with respect to angular periodicity. It is known that in naturally occurring *α*-helices, *θ*can vary over the range (95° ≤ *θ*≤ 105°) and between consecutive residues \[[@B7]\]. Clearly, assuming a fixed value of *θ*= 100° for all *α*-helices is an approximation and could lead to classification difficulties for the methodology. Furthermore, classification difficulties could arise from the arbitrary choice of window length made by the methodology as window length is known to have a profound effect on the relationship between \<*μH*\> and \<*H*\>\[[@B7]\]. It would seem that the optimisation of *θ*and window length are crucial to the classification of amphiphilic *α*-helices yet the values chosen for these parameters by hydrophobic moment plot analysis are not optimal for the classification of any single subclass. A number of studies have considered the significance of \<*μH*\> in relation to structure / function relationships of the *α*-helical classes described by hydrophobic moment plot methodology with common examples including: surface active *α*-helices, transmembrane *α*-helices and oblique orientated *α*-helices \[[@B8]-[@B10]\]. However, if different *α*-helical classes have differing optima for *θ*and window length, not only does this question the validity of results obtained in these studies but also questions the validity of *α*-helix classification according to hydrophobic moment plot methodology. In this paper we examine the criteria upon which the methodology is based and, in view of their medical relevance, we use transmembrane *α*-helices as a test data set. These *α*-helices possess central regions, which are predominantly formed by hydrophobic residues and interact with the membrane lipid core, and end regions, which are primarily formed by hydrophilic residues and reside in the membrane surface regions \[[@B8]\]. For the *α*-helices of our data set, we analyse the relationships for the mean hydrophobic moment and window size, angular frequency and the robustness to varying angular frequency. Correlations between the mean hydrophobic moment and mean hydrophobicity of transmembrane *α*-helices are established, verified and analysed to appraise biological function using resampling Bootstrap and Monte Carlo techniques \[[@B11],[@B12]\]. Results ======= A data set of 84 transmembrane proteins were identified within Swiss-Prot and used to generate a set of 403 transmembrane sequences (see [Additional file 1](#S1){ref-type="supplementary-material"}). All sequences within the data were of 21 residues in length and showed less than 5% homology (data not shown). For the sequences of this data set, the maximum mean hydrophobic moment, \<*μH*\>, and its corresponding mean hydrophobicity, \<*H*\>, were determined and used to generate the hydrophobic moment plot shown in figure [1](#F1){ref-type="fig"}, based on the generally used 11 residue window (*L*= 11) introduced by Eisenberg *et al*., \[[@B4]\]. It can be seen that data points representing the sequences of our data set cluster around the transmembrane region identified by Eisenberg *et al*., \[[@B5]\] but as previously noted \[[@B6]\] there are a significant number that fall outside the boundaries of this region. In particular, many of this number possess \<*H*\> values less than 0.5 and would not be classified as transmembrane *α*-helices according to the hydrophobic moment plot taxonomy of Eisenberg *et al*., \[[@B5]\]. Even allowing for the diffuse nature of these boundaries on the hydrophobic moment plot diagram \[[@B5]\], these results clearly question the efficacy of hydrophobic moment methodology for the prediction of transmembrane *α*-helices. The above analysis was repeated except that window sizes varying in the range (7 ≤ *L*≤ 20) were employed. The values for \<*μH*\> and corresponding \<*H*\> were plotted as above and the results for window sizes of 7, 9, 16 and 20 are shown in figure [2](#F2){ref-type="fig"}. It can be seen that a weak negative correlation exists between \<*μH*\> and \<*H*\> for smaller window sizes but that the level of correlation appears to reduce as window size increases. The sample correlation coefficients for the various window sizes are given in table [1](#T1){ref-type="table"}. To conduct standard statistical tests to determine whether the population correlation coefficients do differ from zero, it is necessary to establish if these data are bivariate Normal. The P-values obtained from Anderson-Darling and Kolmogorov-Smirnov tests for Normality for the various window sizes with *θ*= 100° are shown in table [2](#T2){ref-type="table"}. These results present clear evidence that the populations for the variates for each window size are not bivariate Normal. These findings prompted the use of the bootstrap procedures to estimate the confidence intervals for the population correlation coefficient values for the window sizes in the range (7 ≤ *L*≤ 20). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Hydrophobic moment plot analysis of the transmembrane protein data set with varying window size.**Figure 2 shows the 403 transmembrane sequences of our data set, which were analysed according to hydrophobic moment plot methodology but with varying window size (L). In comparison to *L*= 1 (figure 1b), here in figure 2a, *L*= 7; in figure 2b, *L*= 9; in figure 2c, *L*= 16; and in figure 2d, *L*= 20. In each case, *θ*= 100°. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Sample correlation coefficients between \<*μH*\> and \<*H*\> for window sizes (7 ≤ *L*≤ 20). ::: **Window size ([L]{.underline})** **Sample correlation coefficient (r)** **Window size ([L]{.underline})** **Sample correlation coefficient (r)** ----------------------------------- ---------------------------------------- ----------------------------------- ---------------------------------------- **7** -0.57648 **14** -0.34654 **8** -0.45020 **15** -0.31280 **9** -0.30316 **16** -0.17998 **10** -0.40110 **17** -0.15074 **11** -0.47663 **18** -0.21843 **12** -0.33693 **19** -0.20038 **13** -0.30354 **20** -0.15653 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Confidence Intervals for regression coefficient from bivariate Normality goodness-of-fit for window size *L*. \* 93% Confidence Interval ::: **Window size ([L]{.underline})** **95% Confidence Interval** **99% Confidence Interval** ----------------------------------- ----------------------------- ----------------------------- **7** (1.077, 1.112) (1.072, 1.176) **8** (1.051, 1.084) (1.046, 1.089) **9** (1.067, 1.091) (1.061, 1.095) **10** (1.091, 1.171) (1.078, 1.184) **11** (1.078, 1.134) (1.068, 1.149) **12** (1.046, 1.075) (1.042, 1.080) **13** (1.054, 1.110) (1.047, 1.167) **14** (1.055, 1.124) (1.044, 1.135) **15** (1.050, 1.087) (1.044, 1.093) **16** (1.036, 1.045) (1.030, 1.051) **17** (0.976, 1.001) (0.977, 0.999)\* **18** (0.959, 0.980) (0.956, 0.983) **19** (0.957, 0.967) (0.955, 0.968) **20** (0.950, 0.960) (0.948, 0.962) ::: The results of this investigation for *θ*= 100° are presented in figure [3](#F3){ref-type="fig"}. It would appear that the smaller window sizes do show correlations between \<*μH*\> and \<*H*\> and if this reflects a biological property of transmembrane sequences, it could be of use in the analysis and prediction of these motifs. It is known that angular frequency for a transmembrane *α*-helix varies between 95° and 107° \[[@B16]\], rather than being fixed at 100° as proposed by the methodology of Eisenberg *et al*., \[[@B4]\]. For each window size in the range (7 ≤ *L*≤ 21) residues, to accommodate the findings of Cornette *et al*., \[[@B16]\], the fixed value of *θ*was therefore varied from 95° to 108° in increments of 1°. Once the optimal window had been obtained, to observe the discriminating effect of *θ*on \<*μH*\>, the \<*μH*\> values, denoted by Σ\<*μH*\>, were summed for the 403 sequences for each *θ*. Figure [4](#F4){ref-type="fig"} shows the optimal *θ*, based on the maximum values of Σ\<*μH*\> for each window length. It can be seen that as the window size increases the total \<*μH*\> reduces approximately linearly until the intermediate size of eleven residues in length. For subsequent larger window sizes, we observe a further near linear reduction trend but at a reduced rate. The optimal angular frequency corresponding to each window size (7 ≤ *L*≤ 21) is also given in figure [5](#F5){ref-type="fig"}. The overall relationship between Σ\<*μH*\>, the window size, *L*, and the angular frequency, *θ*, is finally depicted in figure [6](#F6){ref-type="fig"} as a response surface diagram. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Confidence intervals for the Correlation Coefficient.**Figure 3a shows the 99% BCa confidence intervals for the correlation coefficients estimated from 4000 bootstrap replicates. Figure 3b shows the 99% ABC confidence intervals for the correlation coefficients. Figure 3c shows the 99% Delta Method confidence intervals for the correlation coefficients. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Σ\<*μH*\> for the transmembrane protein data set for variable window sizes with optimised angular frequency.**Figure 4 shows the variation of Σ\<*μH*\> for the 403 transmembrane sequences of our data set with window size (7 ≤ *L*≤ 20) for optimised *θ*(95° ≤ *θ*≤ 108°). :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **The variation of optimal angular frequency with window size for the transmembrane protein data set.**Figure 5 shows the variation of optimal angular frequency, [*θ*]{.underline}, (95° ≤ *θ*≤ 108°) with window size (7 ≤ *L*≤ 20) for the 403 transmembrane sequences ofour data set :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Response surface diagram for the transmembrane protein data set.**Figure 6. Response surface diagram for the Σ\<*μH*\> for window sizes (7 ≤ *L*≤ 20) and angular frequency (95° ≤ *θ*≤ 108°). :::  ::: To assess the robustness of \<*μH*\> to this fixed angular frequency assumption, and thus, the accuracy of the hydrophobic moment plot analysis for candidate transmembrane sequences, Monte Carlo simulation studies were conducted. Initially, the angular frequency, *θ*, was assumed to have a mean value, E(*θ*), fixed at 100° and the angle for each successive residue varied about E(*θ*). The random variation, X, followed a Normal distribution and six separate simulations were undertaken with X\~N(100, *σ*^2^), where the standard deviation, *σ*, was set at 0.1°, 0.3°, 0.5°, 0.7°, 0.9° and 1.1° respectively for each. The process was repeated with the mean value being set at the identified optimal angular frequency for the window size, again, for each of the window sizes in the range (7 ≤ *L*≤ 20). Hydrophobic moment plots for variable angular frequency were obtained for E(*θ*) = 100° for each window size in the range (7 ≤ *L*≤ 21) residues and for the separate standard deviation values, *σ*= 0.1°, 0.3°, 0.5°, 0.7°, 0.9°, 1.1°. These were compared visually with the original plots obtained under the fixed angular frequency assumption (*θ*= 100°). In all cases, the bulk properties of the plots were similar irrespective of the level of dispersion introduced by the different values of the standard deviation. The hydrophobic moment plot for *L*= 15; *θ*= 100° is provided in figure [7](#F7){ref-type="fig"}. This is to be contrasted with the plots for *L*= 15; E(*θ*) = 100°, *σ*= 0.1°, *σ*= 0.7° and *σ*= 1.1°, also present in figure [7](#F7){ref-type="fig"}. Similar results were obtained for all other values, confirming, at least visually, that \<*μH*\> is robust to slight random perturbations about a fixed value. These properties were also observed for the simulation study with the fixed angular frequency assumption being violated about the optimum frequency for each of the window sizes in the range (7 ≤ *L*≤ 20) and for each corresponding level of dispersion. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Hydrophobic moment plot analysis of the transmembrane protein data set with varying standard deviation of *θ*about *θ*= 100°.**Figure 7 shows hydrophobic moment plot analysis of the 403 transmembrane sequences of our data set using *L*= 15 and: In figure 7a, *θ*= 100°; in figure 7b, *θ*is from a Normal Distribution with E(*θ*) = 100° and standard deviation of 0.1° ; In figure 7c, *θ*is from a Normal Distribution with E(*θ*) = 100° and standard deviation of 0.7° and in figure 7d, *θ*is from a Normal Distribution with E(*θ*) = 100° and standard deviation of 1.1°. :::  ::: A more rigorous assessment of the variation was provided by analysis of the sample correlations. These were calculated in each case and compared to the empirically derived 99% confidence intervals established for window sizes in the range (7 ≤ *L*≤ 20) under the fixed angular frequency assumption of *θ*= 100°. The calculated sample correlation coefficients were also compared to the point estimates for the original data. In all cases, the values were within the appropriate confidence intervals and were always close to the original sample correlation coefficient values, again providing evidence that \<*μH*\> is robust to random variation in angular frequency. The results of this investigation are given in table [3](#T3){ref-type="table"}. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Sample correlation coefficients for optimum \<*μH*\> for *θ*= 100°, *θ*\~N(100, *σ*^2^) and window sizes, *L*= 7, 11, 15, 16, 20. ::: **Window size (*L*)** ***θ*= 100; *σ*= 0** ***σ*= 0.1** ***σ*= 0.3** ***σ*= 0.5** ***σ*= 0.7** ***σ*= 0.9** ***σ*= 1.1** ----------------------- ---------------------- -------------- -------------- -------------- -------------- -------------- -------------- **7** -0.576465 -0.576557 -0.576118 -0.574907 -0.577803 -0.575951 -0.577435 **11** -0.476666 -0.476109 -0.475923 -0.476820 -0.476131 -0.475736 -0.475371 **15** -0.312882 -0.312924 -0.312973 -0.313221 -0.313488 -0.312796 -0.311120 **16** -0.180014 -0.180160 -0.180679 -0.180656 -0.179292 -0.178218 -0.180065 **20** -0.156516 -0.156837 -0.156606 -0.156546 -0.158868 -0.158272 -0.155921 ::: To test whether these correlations are artefactual, hydrophobic moment plots were obtained for the \<*μH*\> and \<*H*\> derived from the 403 artificial randomisation sequences generated by random re-ordering or randomisation \[[@B20]\] of each of the original optimum window sequences. The plot for a window size of *L*= 11 is given in figure [8](#F8){ref-type="fig"}. These analyses were undertaken for all those window sizes with previously identified statistically significant correlation coefficients between \<*μH*\> and \<*H*\> and were designed to test the importance of the spatial arrangement of the amino acids within the optimum sequences. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Hydrophobic moment plot analysis of the transmembrane data set using randomised sequence arrangements.**Figure 8 Hydrophobic moment plot analysis of our data set was performed using sequences generated by a) random rearrangement of sequences for the optimal windows, b) random sequences formed with amino acid relative frequencies the same as those of the optimal windows. In all cases, *L*= 11 and *θ*= 100°. :::  ::: Similar plots were obtained from Monte Carlo simulated data derived from the 403 sequences that had been generated by random sampling using the relative abundancies of residues found in the set of optimal windows. These analyses were therefore designed to look at the importance of relative amino acid composition for the correlations between \<*μH*\> and \<*H*\> and the results can be seen for a window size of *L*= 11 in figure [8](#F8){ref-type="fig"}. Again, analyses were performed for all window sizes with associated statistically significant correlations (data not shown). It is worth noting that since the effect of varying window size had a significant effect on the correlation between \<*μH*\> and \<*H*\>, varying *L*was observed to vary the optimal sequence identified within the transmembrane domain. Clearly this was not unexpected. Conclusions =========== It can be seen from figure [5](#F5){ref-type="fig"} that the most discriminating angular frequency for a fixed window size varies within the range, (95° ≤ *θ*≤ 104°) for window sizes (7 ≤ *L*≤ 20). There is an obvious damped oscillation present, which can be seen to correspond to the assumed intrinsic periodicity of *α*-helical secondary structure i.e. 3.6 residues per turn. Figure [5](#F5){ref-type="fig"} clearly demonstrates that the fixed 100° angular frequency, assumed when modelling *α*-helices in general, is no more than a representative average with a value nearer 102° providing a maximum for an optimum *L*= 11 residue window in a transmembrane *α*-helical sequence. From figure [4](#F4){ref-type="fig"}, it is also evident that the degree of discrimination possible using \<*μH*\> declines in a near linear fashion with increasing window size with the optimum *L*= 11 residue window appearing to provide approximately average discrimination for transmembrane *α*-helices. The bootstrap derived 99% confidence intervals for the correlation coefficients between \<*μH*\> and \<*H*\> for window sizes in the range (7 ≤ *L*≤ 20) showed that there are highly significant linear associations for the smaller window sizes in the range (7 ≤ *L*≤ 16). As the magnitude of each of the corresponding sample coefficients is small (table [1](#T1){ref-type="table"}), this should be interpreted as evidence of a strong (negative) association but with high variability being present. These correlations become weaker, on average, with increasing window size until they are not statistically significant at the 1% level and we have no compelling evidence that the variates are not independent. The choice of window size therefore, becomes paramount if \<*H*\> and \<*μH*\> are to be used to classify transmembrane *α*-helices. More importantly, the variation in correlation between these parameters and the effect of varying window size on the location of the sequence identified as optimal for *α*-helix classification brings into question the relevance of using the mean hydrophobic moment for comparison between varying window sizes. However, \<*μH*\> has been shown to be robust to departures from the fixed angular frequency assumption for a large range of window sizes appropriate for transmembrane proteins and for a range for levels of dispersion. There were no substantial differences between the plots for relative abundance sample data and those for the randomisation sequences (figure [8](#F8){ref-type="fig"}) except for a few chance negative \<*H*\> observations from the former. This suggests that there are no serial correlations between residue types, where presence in the identified section of the penetrating transmembrane stretch is determined predominantly by relative abundance. This is to be contrasted with the distribution of observations for the original transmembrane sequences for a window size of 11 residues (figure [1](#F1){ref-type="fig"}). Most noticeable is the difference in \<*μH*\> over the range of \<*H*\> values. There appears to be a lower bound for \<*μH*\> for the original sequence, which is clearly not present for the randomisation data. Furthermore, whilst the negative correlation would appear to be an artefact, as it is exhibited in all cases, the dispersion around any optimal fitted line through the data such as a least squares fit also is clearly different. It appears similar and quite spread out for the two randomised sequence data but considerably less so for the transmembrane sequences. This provides evidence that within the optimum window, whilst residue composition is not influential, order is. It would appear that this ordering is leading to both organisation and biological function for at least segments of the interacting portions of transmembrane proteins. This is consistent with the belief that the hydrophobic moment is a good predictor of amphiphilicity \[[@B8]\] although it can be unduly influenced by relatively few amino acid residues within a sequence \[[@B21]\]. In summary, our analyses confirm previous studies, which have shown limitations to the ability of hydrophobic moment plot methodology to assign function to membrane interactive *α*-helices \[[@B6]\]. More importantly, our investigation leads to a questioning of the logic of comparing mean hydrophobic moments, in general, for transmembrane proteins. This is due to the effect of window size on both, the correlation of mean hydrophobic moment with mean hydrophobicity and the identified sensitivity of the optimum window. Comparisons of the hydrophobic moment are seemingly only meaningful for separate transmembrane proteins with identical window sizes. Despite these limitations, \<*μH*\> has been shown to be robust to departures from the fixed angular frequency assumption for transmembrane proteins. Given the severe lack of structural information for transmembrane proteins, the identification of transmembrane *α*-helices using hydrophobic moment based analyses, and other bioinformatic approaches, seems likely to continue for the foreseeable future. Nonetheless, the results of such analyses should only be taken as a guide, and where possible, obtaining corroborative experimental data is essential. On the positive side, our results have demonstrated the importance of amino acid residue sequence order in establishing organisation and biological function for the transmembrane *α*-helices of proteins. With the ongoing development of predictive techniques, these results should be useful in furthering this development and helping to improve drug target identification. Methods ======= The selection of transmembrane, *α*-helix forming segments ---------------------------------------------------------- The primary structures of 96 transmembrane proteins were selected from the Swiss-Prot data bank (<http://us.expasy.org/sprot/>; accessed 25.05.04) and confirmed as transmembrane by extensive analysis of the literature. The sequences were analysed for homology using the sequence alignment program BLAST (Basic local alignment search tool) \[[@B13]\] and twelve homologous sequences were rejected. From the remaining 84 primary structures, a data set comprising 403 putative transmembrane *α*-helical sequences, each of 21 residues, was established using the algorithm, Top Pred2 (\[[@B14]\]; <http://www.sbc.su.se/~erikw/toppred2>; accessed 25.05.04). Hydrophobic moment plot analysis of transmembrane, *α*-helix forming segments ----------------------------------------------------------------------------- In the present study, all hydrophobic moment plot analyses were performed using the consensus hydrophobic scale of Eisenberg \[[@B4],[@B5]\]. To identify putative transmembrane *α*-helix forming segments using hydrophobic moment plot methodology, hydropathy plot analysis \[[@B15]\] is initially undertaken to identify the primary amphiphilicity of candidate sequences. These sequences are selected using a 21 residue window as this is sufficiently long for an *α*-helix to span the bilayer. Once a putative transmembrane domain has been identified, an eleven residue window is considered to progress along the amino acid sequence and for each window, the hydrophobic moment at 100° is calculated. Based on the assumption that a protein sequence will adopt its most amphiphilic arrangement, the window with the maximum mean hydrophobic moment, \<*μH*\>, is taken as the most likely to form an amphiphilic *α*-helix \[[@B5]\]. The location of the optimum window was observed accordingly for window sizes of seven through to twenty consecutive residues. Optimal angular frequency and window length for \<*μH*\> -------------------------------------------------------- For window sizes ranging from 7 to 20 amino acid residues \<*μ*~*H*~\> were computed for the range of angular frequency values (95° ≤ *θ*≤ 108°). In each case, the value of *θ*, which maximises \<*μ*~*H*~\>, i.e. the value of *θ*which produces \<*μH*\>, was determined and is referred to as the optimal angular frequency for that window size. These procedures were based on previously published work, which identified variations in *θ*for *α*-helices \[[@B16]\]. Hydrophobic Correlation ----------------------- For window sizes ranging from 7 to 20 amino acid residues, scatterplots of \<*μH*\> versus \<*H*\> (hydrophobic moment plots) with *θ*= 100° were obtained. The corresponding sample correlation coefficients were calculated to identify the effect of window size on the relationship between these variates and hence on their ability to act as discriminators in the prediction of transmembrane *α*-helices. In addition, for each window size in the range (7 ≤ *L*≤ 20) residues and for *θ*in the range (95° ≤ *θ*≤ 108°), the response surface diagram for \<*μH*\> was constructed. Confidence intervals for the Correlation Coefficient ---------------------------------------------------- Statistical confidence intervals were established for the Pearson (Product-Moment) Correlation Coefficient between \<*μH*\> and \<*H*\> for both cases where window size was varied for a fixed value of the angular frequency, and the angular frequency was varied for a fixed window size. The resulting mean hydrophobicity measures were checked for bivariate Normality and non-parametric bootstrap procedures \[[@B11]\] were used to estimate confidence intervals for the Correlation Coefficients \[[@B17]\]. To provide evidence of the statistical significance of any linear association, the bootstrap bias-corrected and accelerated technique (BCa) \[[@B18]\] and an analytical extension of this, the ABC \[[@B19]\]. In addition, the bootstrap Delta method was employed, which although another bootstrap based method, was developed specifically for estimating the variance of a function of sample means. As the sample Correlation Coefficient can be readily expressed as such a statistic, it is also well suited to the estimation of confidence intervals for these Correlation Coefficients \[[@B12]\]. As both main approaches differ substantially, a more informed assessment of statistical significance could therefore be made. Variable angular frequencies ---------------------------- To assess the robustness of \<*μH*\> to the fix angular frequency assumption, e.g., *θ*= 100°, *θ*was varied randomly about 100° and \<*μH*\> was calculated for each of the optimal windows for window sizes (7 ≤ *L*≤ 20) for the 403 transmembrane proteins. These calculations were also obtained for similar random variations about the observed optimum angular frequencies, again, for the various window sizes (7 ≤ *L*≤ 20). In all cases, it is assumed that the variation follows a Normal distribution with the mean value set at the desired value for *θ*and with the standard deviation, *σ*, set at: 0.1°, 0.3°, 0.5°, 0.7°, 0.9° and 1.1° respectively for six separate Monte Carlo simulation studies. The sample correlation coefficients for each simulation were calculated and compared to the empirically derived 99% confidence intervals for the corresponding population values and, in particular, with the point estimates for the original sequences. Causality and biological function --------------------------------- Given that these data are from an observational study, it is necessary to assess whether any linear associations between \<*μH*\> and \<*H*\> for the *α*-helix forming sequences of our data set are likely to be causal or merely an artefact of amino acid composition. To investigate these possibilities, two additional simulation studies were undertaken. The first looked at spatial arrangements of residues within the primary sequences and the second focused on the effect of amino acid composition on correlations between \<*μH*\> and \<*H*\>. To assess if positional or sequential correlational properties existed for the amino acids within the sequences, the sequence of residues for each of the optimum windows was re-ordered randomly. Artificial sequences were thus generated by random rearrangement or randomisation \[[@B20]\] of the primary sequences within the 403 optimal windows. Hence, each window associated with \<*μH*\> was used to generate a random arrangement. To further investigate whether correlations between \<*μH*\> and \<*H*\> were dependent on sequence composition and not on spatial or sequential correlation, an additional parametric bootstrap simulation study was conducted. Here 403 artificial sequences were created. Each was randomly generated where, for each position, selection was based on the relative abundance of all the residues for the complete 403 optimum windows. In both cases the corresponding \<*μH*\> and \<*H*\> from these newly created sequences were calculated, the associated hydrophobic moment plots obtained and sample correlations calculated. These were inspected to assess whether any linear associations for the original transmembrane data were thus likely to be causal or merely artefactual and whether, from inspection of variation, there was evidence of increased organisation, which could be interpreted as an indication of biological function. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Transmembrane sequence data set ::: ::: {.caption} ###### Click here for file :::

PubMed Central

2024-06-05T03:55:47.501899

2004-8-16

PMC516440

Background ========== Intestinal epithelial cells serve as a barrier between the luminal microflora and the body and as such are perfectly positioned to monitor the approach/invasion of pathogens. These intestinal epithelial cells (IECs) serve as innate immune sentinels and monitor their environment and constantly give out innate host defense instruction to local immune effector cells \[[@B1],[@B2]\]. Pathogens such as *Salmonella*and other enteroinvasive pathogenic bacteria such as enteroinvasive *E. Coli*, *Shigella*and *Yersinia*upon infection of IECs leads to the up-regulation of the expression of host genes, the products of which activate mucosal inflammatory and immune responses and alter epithelial cell functions \[[@B3]-[@B6]\]. Previously we and others demonstrated that IKK via NF-κB and the SAPK signaling pathways via Jun N-terminal kinase (JNK) and p38 kinase were key regulators of the up-regulation of the proinflammatory gene program \[[@B3],[@B7]-[@B9]\], with NF-κB appearing to be the most critical \[[@B3]\]. Typically *Salmonella*infects thirty-forty percent of IECs in culture models of infection \[[@B10]\], however, we and others have found that *Salmonella*infection activates NF-κB DNA binding activity to levels equivalent to that of TNFα which activates NF-κB in all of the cells \[[@B3]\]. Previous studies examining NF-κB activation by *Salmonella*in HT29 colonic intestinal epithelial cells, which serve as model colonic epithelial cells in culture, indicated that delivery of *Salmonella*proteins into the host cell via its type III secretion system (TTSS), such as SopE and SopE2, the bacterially encoded exchange factors for the Rho-family members Rac1 and CdC42, result in exchange factor activation, cytoskeletal rearrangements and activation of the MAPK, SAPK and NF-κB signaling pathways \[[@B7],[@B8],[@B11]-[@B15]\]. Recent observations that utilized *Salmonella*strains that were defective in invasion and delivery of invasion proteins by the TTSS but not attachment indicated that additional factors other than those delivered by the TTSS could lead to NF-κB activation \[[@B16]\]. Presently it is not clear what protein(s) dictate the activation of key signaling pathways that lead to the temporal expression of the proinflammatory gene program, although the SopE proteins have been given extreme attention recently \[[@B7],[@B8],[@B15]\]. In searching for additional *Salmonella*proteins that could activate the proinflammatory gene expression program, bacterial flagellin was recently found to be such a protein \[[@B16]-[@B19]\] and had been shown previously to activate IL-8 expression in monocytes \[[@B19]-[@B21]\]. Flagellin was found to activate NF-κB in polarized epithelial cells only when flagellin was present on their basolateral surface \[[@B22]\] consistent with the idea that a cell surface receptor was present there and could recognize it. The toll-like receptors (TLRs) have been found to recognize pathogen associated molecular patterns (PAMPs) reviewed in \[[@B23]-[@B26]\]. TLR2 interacts with TLR1 and TLR6 to recognize bacterial lipopeptides and zymosan respectively \[[@B27],[@B28]\]. TLR4 recognizes LPS only when associated with its co-receptor MD2 and CD14 \[[@B29]-[@B32]\]. Recently, flagellin was demonstrated to be recognized by TLR5 and activate an innate host response \[[@B22],[@B33]\]. However, little was known or demonstrated about the endogenous levels of TLR5 in cells used in those studies and why those cells failed to respond to flagellin. Here we have identified flagellin as the primary initiator and temporal regulator of not only the major signaling pathways activated during *Salmonella*infection but also of key target genes of the proinflammatory gene program too. We have also found flagellin expression to be required for *Salmonella*bacterial invasion. Independently we found TLR5 recognizes flagellin but its signaling activity toward this PAMP is consistent with either the aid of another flagellin-recognizing co-receptor (as TLR4 utilizes for LPS) or the use of another adapter protein, perhaps similar to MyD88, that is absent or present at low levels in flagellin non-or low-responding cells. Results ======= Salmonella infection leads to a minority of cells invaded but activates NF-κB in nearly all cells ------------------------------------------------------------------------------------------------- Previously, we have noted that pathogenic *Salmonella sp*. infection leads to potent IKK and NF-κB activation and activation of the proinflammatory gene program \[[@B3]\]. Previous studies suggest that about thirty-forty percent of the intestinal epithelial cells are infected during a typical *Salmonella*infection in cultured intestinal epithelial cells \[[@B10]\]. We wished to address the question of how bacterial infection of about thirty percent of the host cells could give rise to NF-κB DNA binding activity equivalent to activation of NF-κB in nearly all of the host cells as TNFα treatment of the cells does. To examine this phenomenon in detail HT29 cells either mock-infected or infected at a MOI of fifty for one-hour with wild-type *S. typhimurium*that had been transformed with the plasmid pFM10.1, that encodes green fluorescent protein (GFP) under the control of the *Salmonella*ssaH promoter and only functions once the bacteria has invaded the host cell \[[@B34]\]. As can be seen in Fig. [1A](#F1){ref-type="fig"}, GFP expression occurs in about thirty to forty percent of the cells. We next examined the localization of the NF-κB subunit p65 (RelA) in non-treated (mock-infected), *Salmonella*infected or TNFα (10 ng/ml) stimulated cells and found that p65 (RelA) was localized to the cytoplasm in non-treated cells whereas, in *Salmonella*infected cells or in TNFα treated cells p65 (RelA) had localized to the nucleus (Fig. [1B](#F1){ref-type="fig"}). These results demonstrate that *Salmonella*infection activates NF-κB in virtually all of the cells even though only a minority of them become infected and is consistent with and aids in explanation of our previous results examining *Salmonella*infection and NF-κB activation \[[@B3]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Salmonella*infection leads to NF-κB nuclear localization even in non-infected cells. HT29 cells were grown on glass coverslips and either mock-infected, left untreated, infected with *Salmonella typhimurium*, or treated with TNFα (10 ng/ml). Cells fixed after 30 min (TNF) and 1 h (*Salmonella*) as described in Experimental Procedures and *Salmonella*that had invaded HT29 cells were detected by direct fluorescence microscopy of GFP expression, p65(RelA) localization was monitored by indirect immunoflourescence of rabbit anti-p65 antibody detected with FITC-conjugated donkey anti-rabbit antibody. DAPI was used to stain nuclei. A, HT29 cells were mock-infected or infected at an MOI of 50 with *Salmonella typhimurium*strain SJW1103G which expresses GFP from the ssaH promoter that is only active inside infected host cells \[10,34\]. Cells were photographed using bright field microscopy (BF), and immunoflourescence to detect GFP or DAPI staining as indicated. Images were merged (overlay) to reveal cells that were infected. B, HT29 cells were left untreated, infected with *Salmonella typhimurium*strain 1103 or treated with TNFα. NF-κB p65(RelA) localization under various conditions as indicated was monitored by indirect immunofluorescence. Cells were visualized by bright field microscopy (BF), cell nuclei were stained with DAPI and p65(RelA) was visualized with FITC. DAPI staining was falsely colored red to make visualization of the merge (overlay) easier to distinguish. :::  ::: Soluble bacterial product identified as flagellin can activate NF-κB in intestinal epithelial cells --------------------------------------------------------------------------------------------------- Since *Salmonella sp*. infection of intestinal epithelial cells in culture led to only roughly thirty percent infection but activation of NF-κB in nearly all of the cells, we anticipated that NF-κB activation was in response to host cell recognition of bacteria structural components or products produced by the bacteria and not by the invasion process. Invasion itself has been demonstrated not to be required for activation of the proinflamatory gene program as had previously been thought \[[@B16]\]. To investigate this possibility sterile-filtered *S. dublin*culture broth left either untreated or boiled for twenty minutes was used to challenge HT29 intestinal epithelial cells and NF-κB DNA binding activity was monitored by electromobility shift assays (EMSAs) of whole cell extracts (WCE) prepared forty-five minutes after exposure \[[@B3],[@B35]\]. Potent activation of NF-κB in response to the broth under both conditions was observed indicating the activating factor was heat-stable (AD, TT and JD, personal observations) and is not LPS since HT29 cells are not responsive to LPS \[[@B3],[@B35]\]. The native sterile-filtered concentrated broth was subsequently treated with DNase, RNase, proteinase K or crudely size fractionated on 100 kDa centricon filters. The variously treated broths were then used to challenge HT29 intestinal epithelial cells and WCEs were prepared after forty-five minutes and NF-κB DNA binding activity was analyzed by EMSA (Fig [2A](#F2){ref-type="fig"}). Direct infection of HT29 cells by *S. typhimurium*1103 or exposure to the culture broths (supt), as indicated, induced NF-κB DNA binding activity, while the activity-inducing factor was found to be sensitive to protease digestion and was retained by a 100 kDa filter (Fig. [2A](#F2){ref-type="fig"}). To further determine the identity of the NF-κB inducing activity, sterile-filtered concentrated broth culture was fractionated by Superose 12 gel permeation chromatography (Fig. [2B](#F2){ref-type="fig"}) and by anion exchange chromatography (Fig. [2C](#F2){ref-type="fig"}). Aliquots of chromatography fractions were assayed for their ability to activate NF-κB in HT29 cells and analyzed by EMSA. As can be seen from the Coomassie blue stained gel (Fig. [2B](#F2){ref-type="fig"}, top panel) increased NF-κB DNA binding activity (Fig. [2B](#F2){ref-type="fig"}, lower panel lanes 4--6) corresponded to the increased abundance of an approximately 55 kDa protein. Anion exchange chromatography on POROS HQ matrix and elution of bound proteins with an increasing salt gradient as indicated (Fig. [2C](#F2){ref-type="fig"}) demonstrated that NF-κB DNA binding-inducing activity corresponded to chromatographic fractions containing an increased abundance of the 55 kDa protein (Fig. [2C](#F2){ref-type="fig"} top panel, and data not shown). Eluted fractions observed in Fig. [2C](#F2){ref-type="fig"} were concentrated and fractionated on preparative 12% SDS-PAGE gels and bands corresponding to B1-B6 were cut from the gels and the proteins eluted, precipitated and renatured as described in Experimental Procedures and used to stimulate HT29 cells. Whole cell extracts from these cells were assayed for NF-κB DNA binding-inducing activity by EMSA and only band 2 (B2) corresponding to the 55 kDa protein (Fig. [2C](#F2){ref-type="fig"} lower panel) was able to elicit NF-κB DNA binding activity while buffer from the beginning or end of the salt gradient failed to activate NF-κB DNA binding activity. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Protein factor in *Salmonella*culture broth leads to NF-κB activation. A, *Salmonella dublin*culture broth concentrated 100-fold was treated as indicated or infectious bacteria, as indicated was used to challenge HT29 cells. NF-κB DNA binding activity was assayed by EMSA from whole cell extracts prepared 45 min after treatment. Authenticity of the NF-κB DNA:protein complex was determined using p65(RelA)-specific and p50-specific antibody supershifts. B, Concentrated *Salmonella dublin*culture broth (IN) was chromatographed by gel permeation on a Superose 12 column. Eluted protein fractions were analyzed by fractionation on 10% SDS-PAGE and visualized by Coomassie blue (CB) staining. Molecular weight markers for chromatography and on the gels are indicated. Aliquots of each fraction as indicated was used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. C, Concentrated *Salmonella dublin*culture broth (IN) was chromatographed by anion exchange chromatography on POROS HQ matrix. Proteins were eluted with an increasing NaCl gradient as indicated and analyzed on 10% SDS-PAGE and visualized by Coomassie blue (CB) staining. Input and aliquots of each fraction as indicated was used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. Eluted material corresponding to protein bands B1-B6, a blank portion of the gel was isolated from a duplicate 10% SDS-PAGE gel as described in Experimental Procedures along with buffer samples from the beginning and end NaCl buffer gradient and used to stimulate HT29 cells and resultant WCEs were analyzed by EMSA for NF-κB DNA binding activity. :::  ::: Proteins corresponding to protein bands B1-B6 and blank areas of the gel were further processed for peptide sequencing as described in Experimental Procedures. Trypsin digestion of the protein corresponding to B2 and analysis by electrospray ion trap LC/MS identified the amino acid sequence of twenty-one peptides. Flagellin (seventy-five percent coverage by the twenty-one peptides) was unambiguously identified as the protein consistent with inducing NF-κB DNA binding activity (Fig [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Identifcation by mass spectrometry of flagellin as the NF-κB activating factor in *Salmonella*culture broth. Microcapillary HPLC tandem mass spectrometry of Band 2 digested by trypsin. Peaks corresponding to *Salmonella*peptides are numbered and identified with the corresponding numbered peptide sequence to the right. :::  ::: Flagellin is required to activate NF-κB in intestinal epithelial cells ---------------------------------------------------------------------- To determine if flagellin was indeed the factor that was responsible for triggering activation of NF-κB after exposure of intestinal epithelial cells to direct bacterial infection or to filtered culture broths of pathogenic *Salmonella sp*. we prepared infectious bacteria and boiled and filtered culture broths from the non-flagellated E. Coli DH5α, pathogenic *S. dublin*strain 2229, an isogenic *S. dublin*2229 SopE^-^mutant, isogenic *S. dublin*2229 SopB^-^mutant, isogenic *S. dublin*2229 double SopE^-^/SopB^-^mutant (strain SE1SB2), *S. typhimurium*strain 1103, and isogenic *S. typhimurium*fliC^::^Tn*10*insertion mutant (strain 86) and a *S. typhimurium*1103 isogenic double mutant fliC^-^/fljB^-^and were used to challenge HT29 cells. Bacteria and culture broths were used to challenge HT29 intestinal epithelial cells and WCE extracts were prepared after forty-five minutes and analyzed for NF-κB DNA binding activity by EMSA. *Salmonella*strains could activate NF-κB, while *Salmonella*strains failing to produce flagellin (fliC and fliC^-^/fljB^-^mutants as indicated) also failed to activate NF-κB (Fig. [4A](#F4){ref-type="fig"} &[4B](#F4){ref-type="fig"}). *E. Coli*DH5α is non-flagellated and does not produce flagellin failed to activate NF-κB. We also noticed through numerous experiments that *S. dublin*direct infections always activated NF-κB to a greater extent than *S. typhimurium*as observed in Fig. [4A](#F4){ref-type="fig"} while culture broths from both species activated NF-κB almost equally well (Fig. [4B](#F4){ref-type="fig"}). We believe this difference is due perhaps to *S. dublin*releasing more flagellin into the cell culture media than *S. typhimurium*during infection since purification of flagellin from both *S. dublin*and *S. typhimurium*and addition of equivalent amounts of chromatographically purified flagellin gave similar NF-κB activation profiles (TT & JD, unpublished observations). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Flagellin mutants fail to activate NF-κB. EMSAs assaying for NF-κB DNA binding activity in WCEs prepared 45 min from non-infected cells (UN) and after direct infection of HT29 cells with wild-type *E. coli*DH5α, wild-type *Salmonella dublin*or SopE^-^mutant, SopB^-^mutant, the SopE^-^/SopB^-^double mutant, wild-type *Salmonella typhimurium*strain 1103, the fliC^-^mutant (fliC::Tn*10*), the fliC^-^/fljB^-^double mutant as indicated at an MOI of 50. B, EMSAs assaying for NF-κB DNA binding activity in WCEs prepared 45 min after challenge of HT29 cells from non-infected cells (UN) or with sterile-filtered concentrated culture broths from wild-type and mutant bacteria as indicated. :::  ::: Of note is the total failure of the double flagellin gene mutants to activate NF-κB as compared to the very minor activation observed in the single Phase I flagellin fliC::Tn*10*insertion mutant (next to last lanes in Fig. [4A](#F4){ref-type="fig"} &[4B](#F4){ref-type="fig"}) which likely is due to the extremely limited expression of the phase II flagellin (from fljB), although the strains of *Salmonella*used here genetically are unable or rarely shift phases of flagellin production. These results are consistent with previous reports identifying flagellin as a potent inducer of the proinflammatory response and IL-8 production \[[@B16]-[@B19]\]. Since flagellin appears required for activation of the NF-κB pathway upon direct infection of intestinal epithelial cells it appeared possible that flagellin may also be the major determinant of other major mitogenic and stress activated signaling pathways activated upon pathogenic *Salmonella*infection of intestinal epithelial cells. Previously others and we have demonstrated that direct *Salmonella*infection of intestinal epithelial cells results in JNK activation \[[@B8]\] and also the activation of NF-κB via IKK \[[@B3]\]. The identification of flagellin as a potent NF-κB activator is significant since SopE had previously been shown to be a pathogenic *Salmonella*bacteriophage encoded protein that is injected into the host cell and acts as an exchange factor for the small Rho GTPases Rac1 and CdC42 initiating cytoskeleton rearrangements and eventual activation of the MAPK, SAPK and NF-κB pathways \[[@B7],[@B15]\], while SopB is a *Salmonella*protein that functions as an inositol phosphate phosphatase and participates in cytoskeletal rearrangements and stimulates host cell chloride secretion \[[@B36]\]. Flagellin triggers activation of the mitogen activated protein kinase, stress activated protein kinase and IKK signaling pathways --------------------------------------------------------------------------------------------------------------------------------- Intestinal epithelial cells act as sentinels for invasion of luminal surfaces and orchestrate the attraction of effector immune cells to the area by production of chemokine genes like IL-8 and macrophage chemoattractant protein 1 (MCP1) proinflammatory cytokine genes such as TNFα, IL-1 and IL-6 \[[@B1],[@B4]-[@B6]\]. Expression of these genes primarily depends upon the action of transcription factors that are activated in response to the transmission of signals via the MAPK, SAPK and IKK signaling pathways. Since NF-κB is considered a central regulator/activator of the proinflammatory gene program we decided to examine the effect that non-flagellin producing mutant strains of *Salmonella*had on activation of the MAPK, SAPK and IKK signaling pathways compared to infection of intestinal epithelial cells with wild-type *Salmonella*or by exposure of the intestinal epithelial cells to purified flagellin. Infection of HT29 cells with wild-type *S. typhimurium*resulted in activation of MAPKs ERK1&2, the SAPKs p38, JNK and IKK (Fig. [5](#F5){ref-type="fig"}) as determined by use of activation-indicating phospho-specific antibodies in immunoblot (IB) analysis or antibody-specific immuno-kinase assays (KA) for JNK and IKK using their respective substrates GST-cJun 1--79 and GST-IκBα1--54 \[[@B37]-[@B39]\]. Interestingly, MAPK stimulation is transient in nature as activation declines beginning at forty-five minutes while p38, JNK and IKK activity increases with time through one hour. As seen in Fig. [4](#F4){ref-type="fig"}, the fliC^-^/fljB^-^double mutant *Salmonella*also failed to induce IKK and NF-κB activity (Fig. [5](#F5){ref-type="fig"} as indicated). Surprisingly, the fliC^-^/fljB^-^double mutant *Salmonella*failed to induce the SAPKs p38 and JNK and only briefly (fifteen minutes) activated MAPK. This result is puzzling since other *Salmonella*proteins such as SopE and SopE2 can activate the small GTPases Rac and CdC42, and these Rho family GTPases have been linked to JNK and p38 activation \[[@B7],[@B8],[@B14],[@B15]\] yet appear not to function in the flagellin minus strain. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Flagellin is required for activating multiple signaling pathways during *Salmonella*infection and leads to nuclear localization of NF-κB. HT29 cells were left untreated, stimulated with TNFα (10 ng/ml) or a cocktail of anisomycin \[An\] (20 μg/ml)/PMA (12.5 ng/ml) for 15 min, or infected with either wild-type (WT) *Salmonella typhimurium*strain 1103 or the *Salmonella typhimurium*double fliC^-^/fljB^-^mutant strain 134 as indicated. WCE were prepared at the indicated times or at 10 min for TNF-treated cells or 15 min for anisomycin/PMA treated cells and used in EMSAs to analyze NF-κB DNA binding activity, or in immuno-kinase assays (KA) using anti-IKK or anti-JNK antibodies to measure IKK and JNK kinase activity on their respective substrates GST-IκBα 1--54 and GST-cJun 1--79 (as indicated). Immunoblot (IB) analysis of equivalent amounts (40 μg) of protein from each extract was fractionated on SDS-PAGE gels and transferred to PVDF membranes and probed with the indicated antibodies to detect bulk IKK, JNK, ERK and p38 as indicated. Immunoblot analysis using phospho-specific antibodies for ERK and p38 to detect activated ERK and p38 are indicated. B, Immunofluorescence demonstrating that flagellin mutant Salmonella fail to infect HT29 cells and that purified flagellin stimulation of HT29 cells leads to NF-κB nuclear p65 (RelA) localization as determined by indirect immunofluorescence. Imaging of the treatment indicated HT29 cells grown on coverslips was essentially the same as in Fig. 1A & 1B. False coloring of the DAPI stain was used to enhance the visualization of both DAPI stained nuclei and p65 nuclear localization. :::  ::: The fliC^-^/fljB^-^double mutant *Salmonella*failed to invade HT29 cells compared to the wild-type *Salmonella*strain as determined by gentamycin protection/invasion assay (see Experimental Procedures). The flagellin fliC^-^/fljB^-^double mutant displayed a four orders of magnitude difference in its ability to invade HT29 cells (TT & JD, unpublished observations). To demonstrate this point further, we infected HT29 cells with either wild-type *Salmonella*or the fliC^-^/fljB^-^double mutant *Salmonella*(strain 134), both strains were transformed with the plasmid pFM10.1 that encodes GFP under the control of the Salmonella ssaH promoter and only functions once the bacteria has invaded the host cell \[[@B10],[@B34]\]. The wild-type *Salmonella*clearly was able to infect HT29 cells (GFP, Fig. [5B](#F5){ref-type="fig"}) while the flagellin mutant bacteria failed to invade HT29 cells as evidenced by the lack of GFP expression (Fig. [5B](#F5){ref-type="fig"}). To determine if flagellin is sufficient or that other bacterially produced proteins are required for invasion, we added either purified flagellin or sterile-filtered culture broths or a combination of both to HT29 cells that were challenged with the *Salmonella*fliC^-^/fljB^-^double mutant and assayed for invasion. Intestinal epithelial cells failed to be invaded using all tested combinations of purified flagellin and/or culture broths with the fliC^-^/fljB^-^double mutant strain (TT & JD, unpublished observations). To our knowledge there is no known direct connection between expression of flagellin genes and the effectiveness of the type III secretion system to deliver bacterially produced proteins such as SopE, SopE2 and SipA or other Sip or Sop proteins \[[@B7],[@B14],[@B15],[@B40],[@B41]\] that play important roles in initiating bacterial internalization. Furthermore, to evaluate the effectiveness of flagellin to stimulate p65 (RelA) nuclear localization in intestinal epithelial cells we challenged HT29 cells with purified flagellin and examined p65 (RelA) localization using indirect immunofluorescence and found p65 (RelA) nuclear localization in nearly every cell (Fig. [5B](#F5){ref-type="fig"} as indicated). Purified flagellin (0.5 μg/ml) was added to the culture media of HT29 cells and WCE were prepared at various times as indicated after exposure and assayed for NF-κB DNA binding activity in EMSAs (Fig. [6A](#F6){ref-type="fig"}). Flagellin potently activated NF-κB in a time dependent manner similar to that observed for TNF (10 ng/ml) treatment of HT29 cells (Fig. [6A](#F6){ref-type="fig"}). Analysis of the MAPK, SAPK and IKK signaling pathways (Fig. [6B](#F6){ref-type="fig"}) at various times after flagellin treatment of HT29 cells using activation-specific phospho-antibodies to monitor MAPK and p38 kinase activation or antibody-specific immunoprecipitation kinase assays for JNK and IKK activities demonstrated that JNK and IKK activity increased through time to one-hour while p38 and MAPK (ERK1&2) activity peaked at thirty minutes and began to decline to noticeably lower levels by one-hour (Fig [6B](#F6){ref-type="fig"} as indicated). The activation profile of the MAPK, SAPK and IKK signaling molecules ERK1&2, p38, JNK and IKK in intestinal epithelial cells in response to purified flagellin exposure remarkably resembled that of intestinal epithelial cells infected with wild-type *Salmonella*(Fig. [5A](#F5){ref-type="fig"}). From these observations we conclude that the temporal activation of the signaling pathways examined here (MAPK, SAPK and IKK), which reflect early events in *Salmonella*infection, are determined almost exclusively by recognition and response of intestinal epithelial cells to flagellin. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Purified flagellin activates signaling pathways and proinflammatory gene expression in intestinal epithelial cells mimicking that of wildtype a wild-type *Salmonella*infection. HT29 cells were left untreated or treated with TNFα (10 ng/ml) or a cocktail of anisomycin \[An\] (20 μg/ml)/PMA (12.5 ng/ml) for 10 min, or with flagellin (1 μg/ml) for the indicated times. WCE were prepared and analyzed by EMSA for NF-κB DNA binding activity, immuno-kinase assays (KA) or immunoblot analysis using phospho-specific antibodies for ERK or p38 to detect activation and with kinase-specific antibodies as described in Fig. 5A to detect bulk kinase abundance as indicated. A, EMSA to detect NF-κB DNA binding activity. Authenticity of the NF-κB bandshift was tested with supershift of the complex with p65(RelA)-specific antibody (α p65), normal rabbit serum (NRS) served as an irrelevant antibody control. B, immunoblot and kinase assays to detect IKK, JNK, ERK and p38 kinase activities and protein abundance as in Fig. 5A. C, semi-quantitative RT-PCR of proinflammatory gene expression of non-treated, wild-type and flagellin double mutant Salmonella typhimurium infected, TNFα (10 ng/ml) or flagellin (1 μg/ml) stimulated cells. HT29 cells were harvested at the indicated times after the indicated treatments and isolated RNA was used to make first strand cDNA that subsequently used in RT-PCR reactions (as described in Experimental Procedures) using gene-specific primers for IL1α, IL1β, IL-8, TNFα, MCP1 and β-actin. β-actin was used as a standard for normalizing expression patterns. Resulting PCR products were fractionated on 2% agarose gels and visualized by eithidium bromide staining. :::  ::: We wished to further examine the effect of purified flagellin and flagellin present on *Salmonella*on the temporal pattern of proinflammatory cytokine gene expression in intestinal epithelial cells in order to differentiate the effects of flagellin alone vs. flagellated *Salmonella*or non-flagellated *Salmonella*infection. HT29 cells were left untreated, stimulated with TNFα (10 ng/ml), or stimulated with flagellin (0.5 ug/ml), or infected with wild-type *Salmonella*typhimurium or the *Salmonella*fliC/fljB double mutant (at MOI of 50). After the indicated times after treatment or infection, HT29 cells were harvested in ice-cold PBS and the cell pellets lysed in Trizol and RNA was purified and used to prepare first-strand cDNA (see Experimental Procedures). Aliquots of the cDNA were used in semi-quantitative RT-PCR reactions using IL1α, IL-1β, IL-8, TNFα, MCP1 and β-actin gene specific primers (sequences available upon request) and the products were fractionated on ethidium bromide containing 1.2% agarose gels. Expression of the known NF-κB target genes IL-1β, IL-8, TNFα and MCP1 was increased in response to TNFα or purified flagellin exposure (Fig. [6C](#F6){ref-type="fig"}). Wild-type *Salmonella*infection also led to activation of these same genes although the expression of TNFα and MCP1 was transient in comparison and occurred immediately after infection. The *Salmonella*fliC^-^/fljB^-^double mutant failed to induce IL-1β, IL-8 and TNFα expression, however MCP1 expression was induced, although at lower levels than that induced by wild-type *Salmonella*, and also, the expression of MCP1 was not transient in nature and continued throughout the time course (9 h) (Fig. [6C](#F6){ref-type="fig"}). The expression level of β-actin served as an internal standard for comparison. Interestingly, IL-1α, which is not an NF-κB target gene was stimulated in response to HT29 cell challenge by all of the treatments. Obviously, the *Salmonella*fliC^-^/fljB^-^double mutant can activate other signaling pathways leading to IL-1α expression. We presently do not know what these signaling pathways are. Flagellin activates NF-κB DNA binding in a MyD88-dependent manner ----------------------------------------------------------------- Flagellin was capable of activating the requisite signaling pathways consistent with proinflammatory gene activation similar to that of a cytokine like TNFα that activates all cells on which a functional cell surface receptor for it is present (see p65 \[RelA\] nuclear localization in Fig. [1](#F1){ref-type="fig"} and Fig. [5C](#F5){ref-type="fig"}) we decided to examine the potential of the Toll-like receptors, to activate the NF-κB pathway in response to flagellin exposure. To quickly test this hypothesis we examined the effect that a dominant-negative MyD88 (aa 152--296) \[[@B42]\] expressing adenovirus had on flagellin-mediated NF-κB activation in HT29 cells. MyD88 is an adapter protein utilized by the IL-1 receptor and all of the known TLRs, which share homology to IL-1 through their cytoplasmic signaling domain and is required for immediate activation of the NF-κB pathway \[[@B43],[@B44]\]. We found that expression of DN-MyD88 in HT29 cells blocked the activation of NF-κB DNA binding activity assayed by EMSA analysis in response to IL-1 or flagellin exposure, consistent with the action of a TLR-mediated activation of NF-κB (TT & JD, unpublished observations). To examine this possibility further we initially used wild-type, MyD88^-/-^and TLR2^-/-^/TLR4^-/-^MEFs (a gift of S. Akira, Univ. of Osaka, JA) to verify the role of MyD88 and to examine the potential role of two of the TLRs to respond to flagellin or to direct wild-type *Salmonella*infection and lead to NF-κB activation (Fig. [7](#F7){ref-type="fig"}). Wild-type *Salmonella*infection activates NF-κB potently in both the wild-type and TLR deficient MEFs (lanes 2 & 15) but this activation is somewhat defective in the MyD88 deficient MEFs (lane 10). Challenge of all three types of cells with concentrated sterile-filtered wild-type *S. dublin*or the double SopE^-^/SopB^-^isogenic mutant *S. dublin*strain SE1SB2 culture broths activated NF-κB in wild-type MEFs and TLR2/4 double deficient cells but failed to activate NF-κB in MyD88 deficient cells (compare lanes 11 and 12 with lanes 3, 4, 6, 7, 16 and 17). NF-κB was potently activated in wild-type MEFs by exposure to purified flagellin (0.5 μg/ml) and therefore eliminated the possibility that LPS played a role in NF-κB activation in these experiments. The exclusion of LPS as a major contributor to NF-κB activation is also provided by the potent activation of the TLR2/4 double deficient MEFs (lanes 16 & 17). TLRs 2 and 4 respond to bacterial lipopeptides, peptidoglycans, certain LPSs and gram negative LPS respectively \[[@B45]-[@B47]\]. IL-1 stimulation verified the functional requirement of MyD88 in transmission of IL-1 and flagellin-mediated signals. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Flagellin-mediated activation of NF-κB is MyD88 dependent. Infectious wild-type *Salmonella Dublin*(MOI of 100), IL-1 (20 ng/ml), purified flagellin (1 μg/ml) (as indicated), sterile-filtered and concentrated 100 kDa filter retentate supernatant (spt) from wild-type *Salmonella dublin*and SopE^-^/SopB^-^double mutant *Salmonella dublin*strain SE1SB2 (S2, as indicated) was used to challenge wild-type, MyD88^-/-^knockout or TLR2^-/-^/TLR4^-/-^double knockout MEFs as indicated. WCEs were prepared 45 min after treatments and examined by EMSA to analyze NF-κB DNA binding activity. IL-1 (20 ng/ml) was used as a positive control to monitor MyD88 function. :::  ::: To further define a possible role for the TLRs in flagellin recognition we assayed for the ability of overexpressed TLRs to activate NF-κB in cells that normally respond poorly to flagellin exposure. Choosing cells that responded slightly to purified flagellin ensured that the signaling components and adapters that flagellin uses were present and functional and that the limiting factor was likely only to be the receptor that responds to flagellin. We found that HeLa cells and HEK293T cells activated NF-κB DNA binding activity in response to IL-1 stimulation but poorly to flagellin exposure (TT & JD, unpublished observations) (but see Fig. [9B](#F9){ref-type="fig"}) and we chose HEK293T cells to use further because of their greater transfection efficiency. Amino-terminus FLAG epitope-tagged TLRs 1--9 (kind gifts of R. Medzhitov, Yale Univ. and R. Ulevitch, TSRI) \[[@B48],[@B49]\] were overexpressed in HEK 293T cells in transient transfections along with the 2×-NF-κB-dependent promoter driven luciferase reporter gene \[[@B50]\] and the expression of luciferase in response to no treatment, flagellin (0.5 μg/ml) or TNFα (10 ng/ml) was determined. TLR5 was the only TLR whose expression resulted in a noticeable response to flagellin challenge of the cells (Table [1](#T1){ref-type="table"}). ::: {#F9 .fig} Figure 9 ::: {.caption} ###### TLR5 is expressed in numerous cell types and has variable responses to flagellin. A, whole cell extracts were prepared from non-stimulated T84, HT29, A549, HeLa, 293T and T98G cells and fractionated on a 8% SDS-PAGE gel, proteins were transferred to PVDF membrane and probed with anti-TLR5 antibody for immunoblot analysis (IB). Protein loading was examined by probing with anti-actin antibody. B, HT29, A549, HeLa, 293T and T98G cells were left untreated (\--), treated with flagellin (F) or TNFα (T) and WCEs were prepared after 45 min and used in EMSA to monitor NF-κB DNA binding activity. Authenticity of the NF-κB bandshift was tested with supershift of the complex with p65(RelA)-specific antibody (αp65), normal rabbit serum (NRS) served as an irrelevant antibody control. C, HT29, A549, HeLa, 293T and T98G cells WCEs (50 μg) were fractionated on a 8% SDS-PAGE gel, proteins transferred to Immobilon P and immunoprobed with anti-muc1 (1:450, Santa Cruz). Size markers are listed and muc1 position is indicated with an arrow. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### *TLR*5 reponds to flagellin and activates NF-κB ::: **[No Stim]{.underline}** **[TNF]{.underline}** **[Flic]{.underline}** -------- --------------------------- ----------------------- ------------------------ Vector 1 13.5 4.9 TLR1 1.7 ND 5.1 TLR2 1.6 ND 5.3 TLR3 1.5 ND 5.0 TLR4 1.8 ND 5.4 TLR5 1.6 ND 9.2\* TLR7 1.5 ND 5.2 TLR8 1.4 ND 5.0 TLR9 1.5 ND 5.1 293T cells were transfected with empty vector (pCDNA3.1) or the individual listed wild-type TLR alleles in triplicate in 6-well dishes. Cells were left untreated (No Stim), TNFα (10 ng/ml) or flagellin (1 μg/ml). NF-κB reporter activity was adjusted by normalizing expression to control Renilla luciferase activity and fold induction was calculated as reporter gene activity in treated cells/reporter gene activity in non-stimulated cells. ND is not determined. ::: To further determine the likelihood of TLR5 being the TLR through which flagellin activated NF-κB, we constructed dominant-negative signaling mutations by deletion of the carboxyl portion of each TLR to a conserved tryptophan in the TIR domain (see Materials and Methods). A similar mutation in the IL-1 receptor abrogates its ability to lead to NF-κB activation \[[@B51],[@B52]\]. Each DN-TLR along with a reverse cloned TLR5 (AS-TLR5) were cloned into the mammalian expression vector pCDNA3.1 (Invitrogen, Carlsbad, CA). All mutant proteins were expressed well (TT & JD unpublished observations). Each DN-TLR mammalian expression vector and empty expression vector along with 2× NF-κB Luc was transfected as previously described \[[@B3]\] into HT29 cells which respond very well to flagellin. The transfected cells were left untreated, stimulated with TNFα (10 ng/ml) or with purified flagellin (0.5 μg/ml). Reporter gene expression was observed not to be affected by DN-TLR expression in response to TNFα stimulation of transfected cells (Fig. [8A](#F8){ref-type="fig"}) however, only expression of either the DN-TLR5 or an antisense TLR5 construct resulted in a nearly fifty percent and twenty-five percent inhibition of flagellin-mediated reporter gene activation respectively (Fig. [8B](#F8){ref-type="fig"}), while DN-TLR2 also was found to mildly inhibit flagellin-mediated reporter expression. These results imply that TLR5 takes part in cell surface recognition of flagellin and initiates the signaling pathway leading to NF-κB activation. The effect of DN-TLR2 on NF-κB-dependent reporter gene activation may be non-specific since its expression also inhibited TNFα-mediated reporter activation as compared to the other DN-TLRs. DN-TLR2 may also compete for an unknown adapter protein that both TLR2 and TLR5 might share. In any event, TLR2 and TLR4 were shown by the results presented in Fig. [7](#F7){ref-type="fig"} not to be required for flagellin-mediated activation of NF-κB. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### TLR5 inhibits flagellin-mediated NF-κB reporter gene activity. HT29 cells were transfected in triplicate in 6-well dishes using the indicated DN-TLR mammalian expression vectors or antisense TLR5 (AS TLR5) (2 μg/well), 2× NF-κB Luc reporter gene (100 ng/well), pRL-TK Renilla luciferase for normalization (50 ng/well) adjusted to 4 μg total DNA/well with empty vector pCDNA3.1 DNA. A, Fold-induction of 2× NF-κB Luc reporter gene in non-stimulated cells (light shading) and in TNFα (10 ng/ml) treated cells (dark shading). Lysates were prepared 12 h after stimulation. Results of a representative experiment are shown. B, HT29 cells transfected as in A were treated with flagellin (1 μg/ml) and cell lysates were prepared and analyzed as in Fig. 8A. Results of a representative experiment are shown. :::  ::: Flagellin-mediated activation of NF-κB in intestinal epithelial cells leads to increased and decreased expression of a subset of TLRs ------------------------------------------------------------------------------------------------------------------------------------- Stimulation of intestinal epithelial cells with *S. typhimurium*or with purified flagellin led to activation of the proinflammatory gene program (Fig. [6C](#F6){ref-type="fig"}). We wished to examine whether or not expression of TLR genes would also be altered in flagellin stimulated cells. HT29 cells were treated or not with purified flagellin (0.5 μg/ml) or with TNFα (10 μg/ml) and total RNA was isolated from non-treated and treated cells three hours after stimulation and used to make first-strand cDNA. Real-time RT-PCR using gene-specific primers for each of the TLRs (Superarray, Frederick, MD) and first-strand cDNA prepared from non-stimulated or flagellin stimulated cells was used to generate SYBR-green (Perkin-Elmer) labeled DNA products that were detected in an iCycler™ (Bio-Rad). Interestingly, flagellin only mildly activated the expression of TLR2, while expression levels of TLRs 5, 6, 9 and 10 were decreased by 2-fold (Table [2](#T2){ref-type="table"}). Contrastingly, TNF stimulation led to increased expression of TLRs 3 and 4 (1.6- and 3.5-fold respectively), while TLRs 2, 9 and 10 were decreased by approximately 2-, 5- and 3-fold respectively. GAPDH expression served as comparative standard. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Change in TLR mRNA levels following TNFα or FliC stimulation. ::: **Fold change** **Fold change** ------- ----------------- ----------------- TLR1 ND ND TLR2 0.6 1.3 TLR3 1.6 0.6 TLR4 3.5 1.1 TLR5 0.9 0.5 TLR6 0.9 0.6 TLR7 M M TLR8 ND ND TLR9 0.2 0.5 TLR10 0.3 0.5 GAPDH 1.0 1.0 ND None detected by RT^2^PCR M None detected above level of minus RT control. Reverse Transcription and Real Time PCR (RT^2^PCR)-RNA was prepared from cells left untreated, stimulated with TNFα (10 ng/ml) or flagellin (1 μg/ml) for 3 h. RT^2^PCR was performed with an iCycler (Bio-Rad) to quantify SYBR-green labeled products generated from PCR products of 1^st^strand cDNA prepared from TLR1 through TLR10 mRNA, 18S rRNA, and GAPDH mRNA. RT^2^PCR (25 ul reaction volume) was performed with the appropriate primers (SuperArray) in triplicate with HotStart Taq DNA polymerase (SuperArray) at 95°C for 5 min to activate Taq and amplified for 40 cycles (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec). RT^2^PCR was performed on the minus RT controls with TLR5 primers to detect DNA contamination. Fold change in mRNA expression was expressed as 2^ΔΔCt^. ΔCt is the difference in threshold cycles for the TLR mRNAs and 18S rRNA. ΔΔCt is the difference between ΔCt non-simulated control and ΔCt stimulated sample. ::: TLR5 is expressed in cells that don\'t respond well to flagellin ---------------------------------------------------------------- This study and others \[[@B22],[@B33]\] have identified TLR5 as the likely TLR through which flagellin activates NF-κB. Previous reports made no determination on the presence or abundance of TLR5 in the cells that they used to ascertain its function \[[@B22],[@B33]\]. We wished to determine if TLR5 protein abundance was absent or greatly decreased in cells that failed to respond or responded poorly to challenge by flagellin. TLR5 abundance in a number of cell lines was examined by immunoblot analysis using a TLR5-specific antibody and compared with the ability of purified flagellin to induce NF-κB DNA binding activity of WCEs prepared from them. Intestinal epithelial cell lines T84 and HT29 were used as was the lung adenocarcinoma cell line A549, the human cervical adenocarcinoma cell line HeLa, the human embryonic kidney cell line expressing large T antigen HEK293T, and the glioblastoma cell line T98G. TLR5 protein was detected in all cell lines examined by immunoblot with TLR5-specific antibody (Fig. [9A](#F9){ref-type="fig"}). T84 cells exhibited the highest abundance while expression levels of the other cell lines were similar and appeared not to differ by more than two-fold (Fig. [9A](#F9){ref-type="fig"}). NF-κB DNA binding activity in non-stimulated, TNFα and flagellin stimulated cells was analyzed by EMSA assays of WCEs prepared from each cell type (Fig [9B](#F9){ref-type="fig"}). HT29 and A549 cells responded strongly to flagellin and to TNFα stimulation while HeLa, 293T and T98G cells responded poorly (HeLa, 293T) or not at all (T98G) to flagellin stimulation. The authenticity of the NF-κB DNA binding complex was determined using p65-specific antibody to supershift the NF-κB DNA:protein complex. It is of interest that some cells that express TLR5 either do not respond at all or do so very poorly. This may be due to either lack of receptor presence at the plasma membrane and intracellular localization, inactivating or detrimental mutations in the TLR5 gene in these cell lines or lack of or low abundance of a required co-receptor or adapter protein (as is the case in some cells for TLR4 and its co-receptor/adapter MD2 \[[@B30],[@B53],[@B54]\]). IL-1 can activate NF-κB DNA binding activity in all of the examined cell lines so it appears that the signaling apparatus downstream of MyD88 to NF-κB is intact. Recently Muc1 a secreted and membrane bound mucin protein was shown to serve as a receptor that bound *Pseudomonas aeruginosa*and its flagellin, leading to activation of the MAPK pathway \[[@B55],[@B56]\] although NF-κB activity was not examined. We examined the muc1 abundance levels in HT29 (strong flagellin responder), A549 (strong flagellin responder), HeLa, 293T (both weak flagellin responders) and T98G (no flagellin response) to determine if its expression correlated with the activation profile of NF-κB and MAPK in these cells in response to flagellin \[[@B55]\]. Should this be the case then muc1 might serve as a viable co-receptor for TLR5 in propagating activation signals leading to NF-κB activation. We observed that only HT29 cellular proteins gave a strong signal by immunoblot analysis using an muc1-specific antibody while muc1 was barely detectable in the other cell lines (Fig. [9C](#F9){ref-type="fig"}). These results suggest that muc1 does not serve the role of a TLR5 co-receptor that leads to NF-κB activation and likely plays little to no role activating MAPK pathways in A549 cells where we have observed similar temporal MAPK activation in response to flagellin exposure as we do in HT29 cells (TT and JD, unpublished results). Further examination of muc1\'s role in HT29 cells in regards to NF-κB and MAPK signaling using siRNA is warranted. Discussion ========== Intestinal epithelial cells at mucosal surfaces serve as innate immune sentinels controlling the innate host defense instruction to the immune effector cells inside the body in response to the external environment \[[@B1],[@B2]\]. Previous studies examining pathogenic *Salmonella*invasion of intestinal epithelial cells demonstrated activation of the proinflammatory gene program and invasion of only a minor portion of the cells \[[@B10]\]. We previously demonstrated that NF-κB is as potently induced in pathogenic *Salmonella .sp*infected cells similar to those treated with the proinflammatory cytokines that are potent NF-κB activators such as TNFα and IL-1β and that this activity was IKK-mediated \[[@B3]\]. Here we examined how bacterial invasion of only a third of the cells could give rise to NF-κB activity profiles consistent with activation of NF-κB in every cell such as the profile TNFα stimulation provides. We found that bacterial infection activates nuclear translocation of p65 (RelA) in nearly all of the intestinal epithelial cells consistent with the hypothesis that a cell surface receptor was recognizing either a soluble product that bacteria were producing, or a bacterial product on the bacteria, or both. We examined bacterial culture broths and found a bacterial product that was protein in composition and when used to challenge intestinal epithelial cells it potently activated NF-κB DNA binding activity (Fig. [2A](#F2){ref-type="fig"}). We further purified this protein by gel permeation and anion exchange chromatography and found the protein to be flagellin by electrospray ion trap mass spectroscopy (Fig. [2B](#F2){ref-type="fig"} &[2C](#F2){ref-type="fig"} and Fig. [3](#F3){ref-type="fig"}). While our studies were in progress, flagellin was identified as being a potent proinflammatory mediator leading to IL-8 production and secretion \[[@B16]-[@B18]\]. We demonstrate in this study that flagellin appears to be exclusively responsible for activating NF-κB in intestinal epithelial cells since flagellin mutant strains do not activate NF-κB (Fig. [4](#F4){ref-type="fig"}) nor lead to their internalization (Fig. [5B](#F5){ref-type="fig"}). Furthermore, flagellin challenge of intestinal epithelial cells leads to p65 (RelA) nuclear localization in nearly all of the treated cells (Fig. [5B](#F5){ref-type="fig"}). Transcription factors like activator protein 1 (AP-1) and NF-κB, which are key regulators/activators of the proinflammatory gene program \[[@B57],[@B58]\] are activated by engagement of the MAPK, SAPK and IKK signaling pathways. We demonstrate that the MAPK, SAPK and IKK signaling pathways activation fails to occur in host cells by infection/exposure to *Salmonella*strains devoid of flagellin or products in the culture broths derived from those mutant *Salmonella*strains (Figs. [5A](#F5){ref-type="fig"} and [6B](#F6){ref-type="fig"}). We also demonstrate here that combined mutants of both fliC and fljB exhibit a severe lack of invasion (10^-4^less than wild-type) and failure to activate stress response signaling, which has not been revealed previously. It is likely that the lack of flagellin production interferes with the functioning of the type III secretion system (TTSS) although flagellin is not known to effect expression of TTSS-required gene products. This hypothesis seems credible since supply of flagellin or bacterial culture components from wild-type *Salmonella*cultures in *trans*to the double fliC^-^/fljB^-^mutant bacteria fails to compliment their lack of infectivity in gentamycin invsion assays (TT & JD, unpublished observations and see Fig. [5B](#F5){ref-type="fig"}). In fact, we found the abundance of a subset of Sip and Sop proteins (SipA and SopD) released into the bacterial culture media to be drastically reduced in the flagellin mutant strains used here (TT & JD, unpublished observations). These two proteins have not previously been identified as activators of NF-κB nor are they considered as such here. The TTSS translocates the *Salmonella*invasion proteins (Sips) and the SopE proteins into the host cell initiating cytoskeletal rearrangements that ultimately lead to bacterial internalization, \[[@B11],[@B12],[@B41]\]. In any event, it is clear that purified flagellin activates a similar cadre of proinflammatory genes as does infection of intestinal epithelial cells with wild-type flagellated *Salmonella*. The temporal expression pattern of these genes was found to be remarkably similar (Fig. [6C](#F6){ref-type="fig"}) indicating that flagellin-mediated temporal activation of the MAPK, SAPK and IKK signaling pathways can suffice for signaling pathways activated by Sips or SopE and SopE2 and largely recapitulates the temporal activation of key proinflammatory genes as does infection of intestinal epithelial cells with wild-type flagellated *Salmonella*. The rapid, and potent activation of the MAPK, SAPK and IKK signaling pathways by flagellin was consistent with and indicative of the activation of a cell surface receptor. In this study and in other studies TLR5 has been demonstrated to play an integral role in the recognition of flagellin leading to activation of NF-κB and expression of the IL-8 gene (Fig. [6C](#F6){ref-type="fig"}) \[[@B22],[@B33]\]. Identification of TLR5 utilized transfection of TLRs 1 -- 9 into cell lines which responded poorly to flagellin (this study) or not at all \[[@B22],[@B33]\] and challenging the transfectants with flagellin to identify which TLR responded to this PAMP. Previous studies that identified TLR5 as the receptor for flagellin did not examine the abundance of TLR5 in these cells or account for the lack of TLR5-mediated signaling in response to flagellin \[[@B22],[@B33]\]. We demonstrate here that cells which respond poorly (HeLa and HEK293T) or not at all (T98G) contain TLR5 in at least equivalent abundance as HT29 cells which are highly responsive to flagellin. We propose at least three possibilities to account for this discrepancy, first, this may be due to either lack of TLR5 receptor presence at the plasma membrane and intracellular localization; second, inactivating or detrimental mutations in the TLR5 gene in these cell lines; and lastly, lack of or low abundance of a required co-receptor or adapter protein required for either efficient ligand recognition and/or signaling. These possibilities are currently being investigated. We favor the last possibility since surface biotinylation experiments indicate that TLR5 is present on the cell surface in both flagellin responding cells and in non-responders mentioned above (data not shown). Invocation of the second hypothesis would require inactivating mutations be present in three different cell lines, a highly improbable outcome. How do the findings presented here correlate with events during a \"normal\" *Salmonella*infection? We have indicated in this study that defective type III secretion system functioning leads to loss of host cell infectivity and underscores the importance of this system in the normal course of infection. In the *in vivo*setting, polarized epithelial cells express TLR5 on the basolateral surface \[[@B48]\] and flagellin can only reach the receptor either after either breaching the tight junction barrier by physical damage or by loosening of the junctions in response to Sips and Sops delivered into the intestinal epithelial cells by the TTSS or by delivery of flagellin across the intestinal epithelial cell by the bacteria itself \[[@B17],[@B59]-[@B61]\]. This scenario would imply the main function of the type III secretion system would be to trigger stress response signaling facilitating invasion and lead to loosening the tight junctions and result in flagellin/ flagellated bacteria to passing through the junctions and infected cells allowing access the basolateral surface and then systemic dispersion. TLR5 on the basolateral surface of the intestinal epithelial cells, in response to flagellin, could then lead to activation of NF-κB and the proinflammatory gene program and host protection. This model is consistent with activation of the proinflammatory gene program observed in response to flagellated *Salmonella sp*. infection in many reports too numerous to cite here and would allow the innate host defense system a fail-safe way to recognize pathogen exposure. In instances where infection of intestinal epithelial cells by naturally occurring non-flagellated *Salmonella*occurs, a strong proinflammatory response would not initially be presented but the *Salmonella*would instead lead to systemic infection as is the case in chickens with *S. galinarum*and *S. pollorum*and result in typhoid-like disease \[[@B62]\]. Infection of chicken epithelial cells does not lead to proinflammatory gene expression by these non-flagellated pathogens but does when infected with *S. typhimurium*or *S. dublin*\[[@B62]\]. Argument for the existence of an additional TLR5 co-receptor/adapter being in limited abundance or absent might be in evidence from the transfection results presented in Table [1](#T1){ref-type="table"} which demonstrated that overexpression of cell surface localized FLAG-tagged TLR5 only resulted in slightly over a two-fold increase in NF-κB reporter gene expression in response to flagellin. If only TLR5 was required for activation of the signaling pathway should not a much more robust response been observed? We have also used DN-TLR5 transfections and NF-κB-dependent reporter gene assays or overexpresssion of DN-TLR5 using recombinant adenoviruses and analysis of resulting NF-κB DNA binding activity in response to flagellin to examine its effectiveness to completely inhibit TLR5-mediated flagellin activation of NF-κB. We have found it difficult to gain more than a fifty-percent reduction in either reporter gene activation or NF-κB DNA binding activity in HT29 cells (TT, AD & JD, unpublished observations). These results suggest that the resting TLR5 signaling complex may be quite stable as has recently been suggested \[[@B63]\]. Should the endogenous TLR5 signaling complex be extremely stable it would therefore be expected that titration of a required pre-stimulus bound adapter or co-receptor away would be inefficient and this is what we have observed. Expression of a DN-MAL (TIRAP), a MyD88-related TLR adapter \[[@B64],[@B65]\] had little to no effect on flagellin-mediated NF-κB activity in transient transfection NF-κB reporter gene assays (TT & JD, unpublished observations). Recently, TLR5 has been shown to bind flagellin \[[@B66]-[@B68]\] and that this is likely a direct interaction due to failure of the human TLR5 to respond to a purified flagellin derived from a mouse-specific *Salmonella*strain \[[@B68]\]. These observations still do not preclude the existence of a co-receptor or adapter that is critical for signal transmission. Detailed biochemical characterization of the TLR5 signaling complex will resolve this issue. Muc1, a recently described flagellin interacting membrane protein by virtue of its ability to trigger activation of the MAPK pathway in response to flagellin exposure \[[@B55]\] was considered a viable candidate for such a co-receptor but our observations suggest that it can not serve as the putative TLR5 co-receptor as it is expressed at similar levels in flagellin non-responding cell lines examined here as it is in A549 cells which respond strongly to flagellin and both cell line types express similar levels of TLR5 (Fig. [9](#F9){ref-type="fig"}). Conclusion ========== In conclusion, our data clearly demonstrates that flagellin can act as the major determinant in activating key stress response signaling pathways and proinflammatory gene program expression in a temporal and qualitative fashion as observed during infection of intestinal epithelial cells by wild-type *Salmonella sp*. that express flagellin, a point that was not well established until this study. In addition, expression of the *fli C*gene appears to play an important role in the proper functioning of the TTSS since mutants that fail to express *fli C*are defective in expressing a subset of Sip proteins and fail to invade host cells. Flagellin added in *trans*cannot restore the ability of the *fli C*mutant bacteria to invade intestinal epithelial cells. Flagellin is \"sensed\" by TLR5 and in response propagates signaling pathways culminating in potent proinflammatory gene expression. Interestingly we found that TLR5 is expressed in weakly responding and also in some flagellin non-responding cells, 293T, HeLa and T98G cells respectively at levels similar to cells such as HT29 and A549 cells that respond strongly to flagellin and can be found on the cell surface, raising a strong possibility that productive TLR5 signaling may require an additional factor/adaptor other than those already known to be key in the IL-1 signaling pathway, which shares extensive similarities to the TLRs signaling pathways. Methods ======= Materials --------- Human tumor necrosis factor alpha (TNFα) and human IL-1 were purchased from R&D Systems (Minneapolis, MN). Tris \[hydroxymethyl\]aminomethane (Tris) was purchased from Fisher Scientific (Fairlawn, NJ). Fetal calf serum was purchased from US Biotechnologies Inc. (Parkerford, PA). Para-nitro-phenylphosphate (PNPP) was purchased from Aldrich Chemical (Milwaukee, WI). The Polyacrylamide gel electrophoresis (PAGE) supplies: acrylamide, bis-acrylamide, sodium dodecyl sulfate (SDS), TEMED, and ammonium persulfate were purchased from Bio-Rad Laboratories (Hercules, CA). Dulbecco\'s modified essential medium (DMEM), DMEM:F12, phosphate buffered saline (PBS), glutamine, penicillin G, streptomycin, amphotericin B, and Grace\'s Insect medium were purchased from Invitrogen (Carlsbad, CA). Luria Broth (LB) was purchased from Becton Dickson and Co (Sparks, MD). The protease inhibitors: aprotinin, bestatin, leupeptin, pepstatin A, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Cal Biochem (La Jolla, CA). Protease inhibitor cocktail contained 10 μg/ml aprotinin, 2.5 μg/ml leupeptin, 8.3 μg/ml bestatin, and 1.7 μg/ml pepstatin A. Phorbol 12-myristate 13 acetate (PMA), N-\[2-hydroxyethyl\]piperazine-N\'-\[2-ethanesulfonic acid\] (Hepes), anisomycin, and 2-\[N-morpholino\]ethanesulfonic acid (MES) were purchased from Sigma Chemical (St. Louis, MO). All other reagents were purchased from Sigma Chemical or Fisher Scientific unless stated otherwise. Cell culture ------------ HT29 human intestinal (colorectal adenocarcinoma) epithelial cells (ATCC HTB-38), HeLa cervical epithelial adenocarcoma cells (ATCC CCL-2), 293T kidney cells (CRL-11268), A549 lung carcinoma cells (ATCC-185), and T98G glioblastoma cells (ATCC CRL-1690) were cultured in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO~2~atmosphere. T84 colorectal carcinoma cells (ATCC CCL-248) were cultured in DMEM:F12 with 2 mM glutamine, 5% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO~2~atmosphere. H5 insect cells (Invitrogen) were cultured in Grace\'s medium with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, 100 μg/ml Streptomycin, and 0.25 μg/ml amphotericin B at 28°C. MyD88^-/-^& TLR2^-/-^/TLR4^-/-^double knockout cells were obtained from Shizuo Akira and Osamu Takeuchi (Univ. of Osaka, Japan) and grown in DMEM with 2 mM glutamine, 10% Fetal Calf Serum, 100 Units/ml Penicillin G, and 100 μg/ml Streptomycin at 37°C in a humidified 5% CO~2~atmosphere. Bacterial strains ----------------- *Salmonella typhimurium*strain SJW1103 (FliC, phase 1 flagellin, stabilized) \[[@B69]\] is a wild-type *Salmonella typhimurium*and can only express the Phase I fliC flagellin, SJW86 (SJW1103 FliC::TN10), and SJW134 (SJW1103 FliC and FljB deletions) were obtained from Robert Macnab (Yale Univ., Conn) and have been described \[[@B70]\]. *Salmonella*serovar dublin strain 2229, strain SE1 (2229 SopE mutant), strain SB2 (2229 SopB mutant), and SE1SB2 (2229 SopE and SopB mutant) were obtained from Edward Galyov (Compton Laboratory, Berkshire, UK) and have been described \[[@B14],[@B15]\]. *Salmonella*strains for stimulation were grown in LB at 37°C without agitation for 16 hours, centrifuged at 6,000 × g for 1 minute, gently washed with PBS, and gently suspended in DMEM to maintain cells with attached flagella. Plasmid pFM10.1 (ampicillin resistance), encodes a green fluorescent protein (GFP) expressed after the *Salmonella*host is internalized by mammalian cells, obtained from Stanley Falkow (Stanford Univ., Stanford, CA) \[[@B10],[@B34]\] and was transformed into strains SJW1103 and SJW134 by electroporation. Strains containing pFM10.1 were designated SJW1103G and SJW134G. Preparation and analysis of Salmonella cell free culture supernatant -------------------------------------------------------------------- Native flagellin was harvested from *S. dublin*2229 or *S. typhimurium*SJW1103. Starter cultures were grown in Luria broth (LB) for 18 hours at 37°C with aeration, diluted 1:5000 in fresh LB, and grown for 12 hours under the same conditions. All subsequent procedures were performed at 4°C. Cells were removed from the medium by centrifugation at 10,000 × g for 5 min and discarded. The supernatant containing flagellin was filtered through a 0.8 micron filter (Millipore, Bedford, MA) to remove residual cells. Supernatant was concentrated 100 fold using an Amicon 100 kiloDalton (kDa) cutoff membrane (Millipore). Initial studies used concentrated culture supernatant from *S. dublin*strain 2229 that was treated with DNase, RNase, Protease K, boiled for 20 min or 100 mM DTT at 37°C for 2 hours and used for stimulation of cultured cells. Concentrated *S. typhimurium*1103 bacterial culture supernatant was washed 4 times by 1:10 dilution with 50 mM MES, pH 6.0, 50 mM NaCl and re-concentrated. Material not retained by the 100 kDa membrane was discarded. Washed culture supernatant was fractionated by gel permeation or anion exchange chromatography for analysis. For long-term storage, washed culture supernatant was supplemented with protease cocktail and stored at -20°C. Fractionation by gel permeation chromatography was performed with a Superose 12HR column (Pharmacia) on a Bio-Logic system (Bio-Rad). One-half mililiter of 100× washed supernatant (equivalent of 50 ml original culture supernatant) was separated on the column at 0.4 ml/minute in 50 mM Hepes, pH 7.4, 200 mM NaCl. Fractions (0.5 ml) were collected, and 50 μl was fractionated by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Thirty microliters of each fraction was used for stimulation of HT29 cells (60 mm dishes) for 45 min and NF-κB DNA binding activity in the resulting whole cell extracts extracts were assayed by EMSA. The column was standardized with catalase (232 kDa), aldolase (158 kDa), abumin (67 kDa), ovalbumin (43 kDa), and Chymotripsinogen A (25 kDa), all obtained from Amersham-Pharmacia. Fractionation by anion exchange chromatography was performed with Poros HQ matrix (2 ml column, PerSeptive Biosystems, Farmingham, MA) on a Bio-Logic system. Five mililiters of 100× washed supernatant (equivalent of 500 ml original culture supernatant) was separated at 1 ml/minute in 50 mM Hepes, pH 7.4, and a NaCl gradient from 50--500 mM. Fractions were collected and 5 μl of each fraction was examined by 10% SDS-PAGE. Proteins were fractionated on duplicate 10% SDS-PAGE precast gels (BioRad). One gel was stained with Bio-Safe Coomassie (Bio-Rad) and the protein bands were isolated for Mass Spectroscopy analysis (CCF Mass spectroscopy core facility) from the other identical non-stained gel, by electro-elution with a whole gel eluter (Bio-Rad) and SDS was removed with SDS-Out (Pierce, Rockland, IL) per the manufacturers directions. Proteins isolated from bands B1 to B6 were acetone precipitated by addition of 20 μg Aprotinin and 5 μg of BSA to each eluted fraction, ice-cold acetone (-20°C) was added to 80%, mixed well and precipitated overnight at -20°C. Proteins were pelleted by centrifugation at 14,000 × g in the cold for 30 min, acetone/liquid was removed and the pellets washed 2× with 1 ml acetone (-20°C). After removal of the acetone, protein pellets were air dried and then resuspended and denatured in 5 μl of 6 M guanidinium hydrochloride (Gu-HCl) at room temperature for 30 min. Resuspended proteins were two-fold serially diluted in DMEM to a final Gu-HCl concentration of 55 mM to renature the proteins. Two hundred fifty microliters of individual renatured proteins/DMEM were added per ml to HT29 cells (60 mm dishes) and whole cell extracts were prepared 45 min after stimulation and were assayed for NF-κB DNA binding activity by EMSA. Purification of flagellin (purified flagellin) ---------------------------------------------- The washed and concentrated culture supernatant from *S. typhimurium*1103 containing flagellin was boiled for 20 minutes and precipitants removed by centrifugation at 15,000 × g. The supernatant containing flagellin was diluted 1:2 with 50 mM MES, pH 6.0, 50 mM NaCl and mixed with 2 ml Poros SP cation exchange matrix (PerSeptive Biosystems) per 1 liter of original culture. The Poros SP matrix was prepared as a 50% slurry and equilibrated with 50 mM MES, pH 6.0. The flagellin preparation and matrix were mixed on a roller at 12 to 14 RPM for 2 hours. The matrix along with bound contaminants was removed by filtration through a 0.85 micron filter and discarded, flagellin failed to bind to the cation exchange matrix at pH 6.0 and eluted in the flowthrough and was collected. The pH of the flowthrough was adjusted by five-fold dilution of the sample with 50 mM Hepes, pH 7.8, 50 mM NaCl, and loaded onto a Poros HQ anion exchange column (2 ml column, PerSeptive Biosystems) equilibrated with 50 mM Hepes, pH 7.4, 50 mM NaCl. The column was washed with 2 volumes 50 mM Hepes, pH, 7.4, 50 mM NaCl, and eluted with a 10 column volume linear gradient of 50--500 mM NaCl in 50 mM Hepes, pH 7.4. Flagellin eluted from the column between 200--275 mM NaCl. Fractions containing flagellin were pooled and concentrated. The preparation was determined to be pure by electrophoresis of 5 μg protein by SDS-PAGE and stained with Bio-Safe Coomassie (Bio-Rad). Samples were stored at -80°C in 50 mM Hepes, pH 7.4, approx 225 mM NaCl, 10% glycerol and protease cocktail. A 4 liter preparation of culture supernatant yielded 2 mg purified flagellin. In-gel tryptic digestion and protein identification by LC-MS ------------------------------------------------------------ Gels were fixed and stained (Bio-Safe Blue, BioRad). All of the following procedures were performed by the CCF Mass spectroscopy core facility. Excised gel bands were reduced (100 mM DTT), and alkylated (100 mM iodoacetamide). Proteins in the gel bands were digested with modified trypsin (Promega, 20 μg/mL) with an overnight incubation at 37°C. Tryptic peptides were extracted from the gel with 50% acetonitrile, 0.1% acetic acid, concentrated in a SpeedVac (Thermo Savant) to remove acetonitrile, and reconstituted to 20 uL with 0.1% acetic acid. Extracted peptides were subjected to reversed phase (50 uM ID packed with Phenomenex Jupiter C18, 6 cm capillary column) liquid chromatography (2%--70% solvent B; Solvent A, 50 mM acetic acid, aqueous, Solvent B acetonitrile), coupled to a Finnigan LCQ DECA ion trap mass spectrometer for peptide sequencing, as described \[[@B38]\]. Preparation of GST-IκBa1-54 and GST-cJUN1-79 kinase substrates -------------------------------------------------------------- IκBα amino acids 1 to 54 fused to GST or cJUN amino acids 1--79 fused to GST were prepared as previously described \[[@B37]-[@B39]\] and stored in kinase buffer (20 mM Hepes, pH 7.6, 10 MM MgCl2, 10 mM NaCl, 2 mM beta-glycerophosphate, 10 mM PNPP). Preparation of cells for microscopy ----------------------------------- HT29 cells for microscopic examination were grown in 6 well plates on sterile cover slips to a density of 50--75%. Cells were stimulated as described above. After stimulation, cover slips with HT29 cells were washed 2 times with ice cold PBS and fixed with 4% w/v formalin at room temperature for 20 minutes. Cells were washed 4 times with PBS prior to mounting for visualization of *Salmonella*invasion. Cover slips were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA), and cover slips sealed to slides. Cells for antibody staining were treated with absolute methanol for 20 minutes following formalin fixation, then washed 3 times with PBS supplemented with 0.1% BSA (PBSB) and used directly or stored in the cold after azide was added to 0.02%. For p65(RelA) localization, cells on coverslips were blocked for 1 h at 37°C with PBS supplemented with 1% BSA. The PBSB was removed, washed once with PBSB and coverslips were placed cell-side down onto 150 μl of p65 antibody (Zymed, South San Francisco, CA) diluted 1:1500 in PBSB on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 3 × 5 min with PBSB. Coverslips were then removed and placed cell-side down onto 150 μl of FITC-labeled donkey anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) (1:300 in PBSB) on a square of parafilm and placed in a humidified chamber at 37°C for 1.5 h. Coverslips were removed and placed cell-side up in 6-well dishes and washed 5 × 5 min with PBSB, removed and placed cell-side down onto slides mounted with Vectashield (Vector Laboratories, Burlingame, CA) with DAPI and then sealed. NF-κB localization was determined by indirect immunofluorescence. Samples were observed on a Leica DMR upright microscope (Leica Microsystems Inc., Heidelberg, Germany) at 400× with oil immersion and equipped with FITC and UV filters. Images were collected with a MicroMax RS camera (Princeton Instruments Inc., Princeton, NJ), and Image Pro plus, version 4.5, software (Media Cybermetics Inc., Carlsbad, CA). Color enhancements were performed with Image Pro plus software. Visible light plus color overlays for Fig. [1](#F1){ref-type="fig"} and Fig. [5B](#F5){ref-type="fig"} were performed with MetaMorph Software (Universal Imaging Corp., Downington, PA). Bacterial infection and cell stimulation ---------------------------------------- Mouse embryo fibroblasts (MEFs) or HT29 cells were grown in DMEM as above to a density of 90% prior to stimulation. All cells were washed with warm PBS and supplemented with DMEM without serum or antibiotics in preparation for stimulation. Cells were stimulated with; 10 ng/ml TNFα, 1 μg/ml flagellin unless specified otherwise, 20 μg/ml Anisomycin, 12.5 ng/ml PMA, or 10^8^*Salmonella*/ml at 37°C for desired times and extracts prepared as below. Cells harvested beyond one hour were washed with warm PBS and supplemented with warm DMEM, 2 mM glutamine, and 200 ug/ml gentamycin after 1 hour and returned to 37°C until extract preparation desired. Whole cell extract preparation ------------------------------ Cells were washed with ice-cold PBS and all subsequent steps carried out at 4°C or on ice. Cells were scraped from the dish in ice-cold PBS, and collected by centrifugation at 1000 × g for 1 minute. Cells were lysed by suspension in 50 mM Tris-HCl, pH 7.6, 400 mM NaCl, 25 mM beta-glycerol phosphate, 25 mM NaF, 10 mM PNPP, 10 % glycerol, 0.5 mM sodium orthovanadate, 0.5% nonidet-40 (NP-40), 5 mM benzamidine, 2.5 mM metabisulfite, 1 mM PMSF, 1 mM DTT and protease inhibitor cocktail as described \[[@B3]\]. Electromobility shift assays (EMSA) ----------------------------------- NF-κB DNA binding assays were carried out as previously described \[[@B3],[@B35],[@B38]\]. Anti-p65 antibody (Zymed, South San Francisco), anti-p50 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), and anti-STAT3 antibody (Santa Cruz) were used for EMSA supershifts. Invasion assay -------------- HT29 cells, 90--95% confluent in 35 mm round dishes, were prepared for stimulation as above and treated with a 1 ml suspension of Salmonella SJW1103 or SJW134 or left untreated in triplicate as above. After one hour, HT29 cells were washed 4× with warm PBS, supplemented with warm DMEM, 2 mM glutamine, and 200 μg/ml gentamycin, and incubated at 37°C for 4 hours. Cells were then harvested as above and lysed by suspension in 1 ml sterile distilled water. Ten-fold serial dilutions were prepared in PBS and 100 μl of each dilution was plated on LB agar plates and grown at 37°C for 20 hours. Colonies were counted and averaged. Kinase assays ------------- Whole cell extracts (250 μg) were supplemented with 150 μl of Buffer A (20 mM Hepes, pH 7.9, 20 mM beta-glycerophosphate, 10 mM NaF, 0.1 mM orthovanadate, 5 mM PNPP, 10 mM 2-mercaptoethanol, 0.5 mM PMSF, and protease inhibitor cocktail), and immuno precipitation kinase assays carried out as described \[[@B3]\] using either IKKα monoclonal antibody (PharMingen -- Becton Dickson), anti-JNK1 (Santa Cruz Biotechnologies, Santa Cruz, CA), or anti-hemagglutinin (HA) epitope antibody (Covence Antibodies, Princeton, NJ) as indicated. Protein G immunopellets were collected by centrifugation at 500 × g for 30 sec, washed 3 times with Buffer B (Buffer A plus 250 mM NaCl), and one time with Buffer C (Buffer A plus 50 mM NaCl and 10 mM MgCl~2~). Immunopellets were resuspended in 30 μl Kinase buffer with 0.1 mM orthovanadate, 50 μM \"cold\" ATP, 5 μCi γ-^32^P-ATP, 2 mM DTT, and 2 μg of soluble GST-IκBα1--54 or GST-cJUN1-79, and incubated at 30°C for 30 minutes. Reactions were stopped by the addition of 15 μl 4× SDS-PAGE loading buffer, heated at 95°C for 5 minutes, and resolved on 10% SDS-PAGE gels by standard procedures. Gels were rinsed, stained with Bio-Safe Coomassie (Bio-Rad) to visualize protein bands, rinsed, photographed then dried and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY) to detect substrate phosphorylation. Immunoblotting -------------- Protein samples (40 μg) were resolved by SDS-PAGE on a 10% acrylamide gels by standard procedures, and proteins transferred to PVDF membrane (Millipore) and probed with antibodies as described \[[@B3]\]. Membranes were washed 3× briefly with TBST, incubated with a 1:1000 dilution (1:800 for anti-TLR5) of the primary antibody in TBST, 1% non-fat milk for 1 hour, washed 3 × 5 min with TBST, and then incubated with a 1:2000 dilution of the appropriate HRP-conjugated secondary antibody in TBST, 0.5% non-fat milk for 1 hour. Primary antibodies used were: anti-IKKα/β (H-470, Santa Cruz), anti-JNK1, anti-ERK2 (K-23, Santa Cruz), anti-phospho-ERK (E-4, Santa Cruz), anti-p38MAPK (Cell Signaling Technologies, Beverly, MA), anti-phosopho-p38MAPK (Cell Signaling), anti-TLR5 (H-127, Santa Cruz), anti-muc1 (H-295, Santa Cruz) and anti-actin (C-11, Santa Cruz). Secondary antibodies used were: anti-mouse IgG HRP conjugate (Amersham-Pharmacia), anti-rabbit IgG HRP conjugate (Amersham-Pharmacia), anti-goat IgG-HRP conjugate (Santa Cruz). HRP activity was detected by ECL (Amersham-Pharmacia) as per manufacturers instructions, on Kodak X-OMAT AR film. Construction of dominant-negative TLRs -------------------------------------- All DN-TLRs were constructed using PCR. The universal 5\' primer consisted of a 5\'KPN I restriction site followed by sequences encoding the kozak sequence, translational start site, and preprotrypsin leader sequence of pCMV-1 (Sigma) that all the wild-type TLRs were initially cloned into. The 3\' anti-sense (AS) primers were human TLRgene-specific primers (sequences available upon request) that created a stop codon immediately after a conserved tryptophan in Box 9 of the TLR TIR homology domain according to Bazan \[[@B71]\], thus creating carboxy terminus deletions. The 5\' end of the AS primer contained a number of convenient restriction sites to allow directional cloning. PCR was performed with turbo-Pfu polymerase (Stratagene, La Jolla, CA) using standard procedures on individual wild-type TLR pCMV-1 plasmid DNAs (5 ng each, kind gifts of R. Medzhitov, Yale Univ. and R. Ulevitch, TSRI) \[[@B48],[@B49]\] with the 150 ng each of the universal 5\' sense primer and individual gene-specific TLR 3\' primers. PCR products were cleaned-up with PCR cleanup kit (Qiagen, Germany) digested with appropriate restriction enzymes, gel purified and then ligated into the mammalian expression vector pCDNA3.1 (Invitrogen). Positive clones were sequenced to verify the mutations and tested for expression in transient expression assays and detected on immunoblots by probing with anti-FLAG M2 monoclonal antibody (Sigma). All wild-type and DN-TLR alleles are amino terminus FLAG epitope-tagged. Transfections ------------- HT29 cells were transfected with Lipofectamine Plus (Invitrogen) as previously described \[[@B3]\]. In transfections monitoring reporter gene expression, transfections were performed at least three times in 6 well dishes in triplicate with the total DNA mass kept constant at 4 μg (2 μg effector plasmid DNA, 100 ng 2× NF-κB Luc reporter gene, 50 ng pRL-TK, a thymidine kinase promoter driven *Renilla*luciferase normalization reporter and 1.85 μg pCDNA3.1 plasmid DNA as bulk filler DNA) and fire-fly luciferase expression was normalized to *Renilla*luciferase expression using the dual-luciferase assay (Promega, Madison, WI). Fold inductions were calculated and values between experiments did not vary more than 15%, a representative experiment is presented. Transfection of 293T cells was performed with lipofectamine 2000 (Invitrogen) in 6-well dishes in triplicate as per the manufacture\'s protocol. TLR expression plasmids were added at 2 μg/well, and NF-κB and normalization control plasmids were as above with HT29 cells and pCDNA3.1 plasmid DNA as bulk filler DNA to a final DNA mass of 4 μg/well. Fold inductions were calculated and values between experiments (N of 3) did not vary more than 10%, a representative experiment is presented. Real Reverse Transcription and Real Time PCR (RT^2^PCR) ------------------------------------------------------- Cells (N = 3) were stimulated 3 hours at 37°C with TNFα or FliC or left untreated and harvested for total RNA isolation. Total cellular RNA was extracted from cells with Trizol reagent (Invitrogen) \[[@B3]\] and reverse transcribed with ReactionReady first strand cDNA synthesis kit (SuperArray Bioscience Corp., Fredrick, MD). RNA (2.5 ug per 20 ul reaction) was reverse transcribed using random primers and Moloney murine leukemia virus reverse transcriptase per manufacturer specified conditions. Controls without reverse transcriptase (minus RT) was also generated for each RNA sample. RT^2^PCR was performed with an iCycler (Bio-Rad) to quantify TLR1 through TLR10 mRNA, 18S rRNA, and GAPDH mRNA. RT^2^PCR (25 ul reaction volume) was performed with the appropriate primers (SuperArray) per manufacturers instructions in triplicate with HotStart Taq DNA polymerase (SuperArray) at 95°C for 15 min to activate Taq and amplified for 40 cycles (95°C, 30 sec, 55°C, 30 sec, 72°C, 30 sec). RT^2^PCR was performed on the minus RT controls with TLR5 primers to detect DNA contamination. Real-time PCR analysis was performed using SYBR-green (Perkin-Elmer) according to manufacture\'s instructions with the specific primer pairs indicated above and primer pairs for 18S ribosomal RNA as reference RNA (Classic 18S primer pairs -- Ambion Inc). Cycle time (Ct) was measured using the iCycler™ and its associated software (Bio-Rad). Relative transcript quantities were calculated by the ΔΔCt method using 18S ribosomal RNA as a reference amplified from samples using the Classic 18S primer pairs from Ambion, Inc (Austin, TX). Normalized samples were then expressed relative to the average ΔCt value for untreated controls to obtain relative fold-change in expression levels. Fold change in mRNA expression was expressed as 2^ΔΔCt^. ΔCt is the difference in threshold cycles for the TLR mRNAs and 18S rRNA. ΔΔCt is the difference between ΔCt non-simulated control and ΔCt stimulated sample. Values for fold-induction varied less than 5% among replicates. Abbreviations ============= The abbreviations used are: FBS, fetal bovine serum; IL-1, interlukin-1, SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; EMSA, electromobility shift assay; IB, immunoblot; KA, kinase assay; GST, glutathione S-transferase; PBS, phosphate-buffered saline; TNFα, tumor necrosis factor α; NF-κB, nuclear factor kappa B; IKK, Ikappa B kinase; IκB, Ikappa B; PCR, polymerase chain assay; RT-PCR, reverse transcription polymerase chain assay; Gu-HCl, guanidinium hydrochloride ; MAPK, mitogen activated protein kinase; SAPK, stress-activated protein kinase; ERK, extracellular regulated kinase; TLR, toll-like receptor; DN, dominant-negative; JNK, Jun N-terminal kinase; AP-1, activator protein-1; MEF, mouse embryo fibroblast; WCE, whole cell extract; IEC, intestinal epithelial cell; MCP1, macrophage chemoattractant protein 1; TTSS, type III secretion system; Sip, *Salmonella*invasion protein; PMA, phorbol 12-myristate 13 acetate; PNPP, para nitrophenyl phosphate; TK, thymidine kinase; BF, bright field; NP-40, nonidet-40; NRS, normal rabbit serum; IN, input; Ct, cycle time. Authors\' contributions ======================= TT and AD initiated the study and performed the majority of the experiments and contributed equally and were assisted by NK and JL. MD constructed a number of DN-TLRs and JD developed the study, provided funding support, oversaw the project and also constructed a number of mutant TLRs. Acknowledgements ================ We would like to thank Drs.E.Galyor (Compton Laboratory, Berkshire (UK)), Drs. R.M. Macnab (Yale University, New Haven, Conn), S. Mizel (Wake Forest University, Winston-Salem, NC), and M. Kagnoff (UCSD, La Jolla, CA) for bacterial strains. We would also like to thank S. Akira and O. Takeuchi (Osaka University, Osaka, JA) for the gift of MyD88 and TLR 2/4 double knockout cell lines. We also would like to thank S. Falkow (Stanford University, Stanford, CA), M. Karin (UCSD, La Jolla, CA), R. Medzhitov (Yale University, New Haven, Conn) and R. Ulevitch (TSRI, La Jolla, CA) and V. Dixit (then at Univ. of Michigan, Ann Arbor, MI) for gifts of plasmids. We would also like to thank B. Williams (CCF, Cleveland, OH) for thoughtful discussions and support on this project. This work was supported in part by grants from the National Institutes of Health, CA84406 (to J.A.D.) and the U.S. Army, DAMD 17-01-C-0065 (to B. Williams).

PubMed Central

2024-06-05T03:55:47.505168

2004-8-23

PMC516441

Background ========== Individuals with coronary disease (CAD) are at high risk for subsequent cardiovascular disease events and mortality\[[@B1]\]. Clinical trials have shown that this risk can be substantially reduced though the detection, treatment, and control of dyslipidemia\[[@B2],[@B3]\]. To that end, clinical guidelines have been established for the lipid management of CAD patients\[[@B4],[@B5]\]. Adherence to these guidelines is far from ideal\[[@B6],[@B7]\]. Substantial racial disparities in the diagnosis and management of dyslipidemia have been reported in the general population and among CAD patients \[[@B8]-[@B16]\]. Improved lipid management through the diagnosis of dyslipidemia has been a focus of quality improvement programs in the outpatient fee-for-service setting such as Medicare\'s Health Care Quality Improvement Program\[[@B17]\]. Lipid testing is used as an indicator of lipid management in the fee-for-service setting because testing is a service identifiable in insurance claims data. In contrast, assessment of treatment and goal attainment in the fee-for-service setting requires resource-intensive medical record review which is generally not performed for large national patient populations. This study describes outpatient lipid management for African Americans and Caucasians with CAD seen in medical practices throughout the United States. Data from medical records were examined for indicators of lipid management including lipid testing, lipid-lowering drug prescription, and goal attainment. Our objectives were to characterize lipid management across race-sex groups and evaluate the extent of disparities for the three components of lipid management: detection, treatment, and control. We then discuss the implications of our findings with respect to possible underlying causes and health policies for closing the gap in race-sex lipid management disparities. Methods ======= The Quality Assurance Program ----------------------------- The Quality Assurance Program (QAP) is a national program sponsored by Merck & Company conducted during the late 1990\'s to identify physician practice patterns and to promote evidence-based best practices for the medical management of patients with cardiovascular disease seen in the outpatient setting \[[@B18]\]. The QAP database provides abstracted medical record data collected in two distinct time periods and study populations nationwide. Our analyses were limited to the study population identified in the most recent period of data collection (QAP-II). Patients from QAP-II included in these analyses were seen at participating medical practices from January, 1995 through March, 1998. Medical records were reviewed by Access Medical Ltd (Arlington, VA) using a standardized electronic abstraction tool developed specifically for QAP-II. The QAP database contains data obtained from the medical record including race, sex, date of birth, medical history, and medical procedures. The most recent serum lipid testing results and the most recently recorded prescriptions for lipid-lowering drugs were also determined from the medical record. The medical record of each patient was reviewed only once. Patients were not followed over time. Patient and physician identifying information were not included in the QAP-II database to ensure confidentiality. QAP participant selection ------------------------- Physicians and medical practices throughout the United States with high numbers of prescriptions for medications used in the treatment of cardiovascular disease were invited to participate in the QAP. The specialties of participating physicians included cardiology, internal medicine, family medicine, and endocrinology. Patients with cardiovascular disease were randomly selected within each participating medical practice. Inclusion and exclusion criteria -------------------------------- Patients included in the QAP study were at least 21 years of age with CAD and/or heart failure and were seen at least twice in two years by the participating physician. Patients were excluded if the medical record indicated a terminal illness, history of a transplant or awaiting transplant, or deceased. Patients without medical record documentation of CAD were excluded from analysis. The presence of CAD was ascertained during analysis from abstracted medical record data based on medical history, *International Classification of Diseases, Ninth Revision, Clinical Modification*diagnosis codes (410--414), and cardiac procedures consistent with CAD (i.e., coronary artery bypass graft, angioplasty, and stent). Only patients with medical record documentation of African-American or Caucasian race were included. To reduce the influence of between-state variation in lipid management from states that contribute little information about African American populations, medical practices were excluded if they were located in states with fewer than 10 African-American patients in QAP-II Indicators of lipid management ------------------------------ Measures of detection (lipid testing), treatment (lipid-lowering drug prescription), and control (goal attainment) were the indicators of lipid management considered in this study. Low density lipoprotein cholesterol (LDL-C) testing was measured as the percentage of patients with at least one serum LDL-C value documented in the medical record. The use of lipid-lowering drugs (i.e., \"treated\" patients) was measured as the percentage of patients with medical record documentation of at least one prescription for a statin (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) or non-statin lipid drug (e.g., gemfibrozil). Goal attainment among those with documented LDL-C values was based on recommended guidelines for patients with coronary artery disease (LDL-C \< 100 mg/dL)\[[@B5]\]. Analysis -------- We conducted a cross-sectional analysis of abstracted medical record data obtained from QAP-II. Indicators of lipid management (i.e., LDL-C testing, lipid-lowering drug prescription, and LDL-C goal attainment rates) and potential confounding or explanatory variables were examined within strata of race and sex. Co-morbid conditions including diabetes mellitus, myocardial infarction, heart failure, and hypertension were identified from medical history and diagnosis codes. Logistic regression analyses were performed to evaluate the associations of race and sex with each of three dichotomous lipid management indicators while controlling for multiple confounding and explanatory variables. We accounted for correlations within medical practices using the generalized estimation equation (GEE) method\[[@B19]\]. This method was implemented in generalized linear models with PROC GENMOD of SAS Version 9 (SAS, Inc, Cary, North Carolina) for logistic regression with correlated data\[[@B20]\]. Separate models were run for each indicator of lipid management as the dependent variable. The entire study population was included for logistic regression analyses of LDL-C testing as the dependent variable. Regression analyses with lipid-lowering drug prescription as the dependent variable included only patients with LDL-C tests. Regression analyses for LDL-C goal attainment were limited to patients who received at least one LDL-C test and had a documented prescription for a lipid-lowering drug. The independent variables included in the regression models in addition to race and sex were age, medical history (diabetes mellitus, myocardial infarction, heart failure, hypertension), and geographic region of medical practice. Logistic models predicting lipid-lowering drug prescriptions included a term for serum LDL-C concentration in addition to the above variables. LDL-C concentration was included to control for the severity of hyperlipidemia as a factor in the physician\'s decision to treat with lipid-lowering drugs. Results ======= Population characteristics -------------------------- A total of 23,123 CAD patients with documented race and sex seen by 1,171 physicians at 238 medical practices in 23 states were included in the study. Of these patients, 1,046 were African American and 22,077 were Caucasian. African-American compared to Caucasian patients were more likely to be women (53% versus 36%). The average age for the study population was 69 years and ranged from 22 to 97 years. Within each race group, women were older than men on average and within each sex group Caucasians were older than African Americans (Table [1](#T1){ref-type="table"}). The prevalence of co-morbid conditions was high for all race-sex groups. Despite their younger ages, African Americans were more likely than Caucasians to have diabetes, heart failure, and hypertension. Almost half of African-American women and a third of African-American men had diabetes compared to about a quarter of the Caucasian population. About half of African-American men had heart failure and over three quarters of African-American women had hypertension. Consistent with the geographic distribution of race-specific populations, African-American patients were more likely seen in southern medical practices than elsewhere. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of patients by race and sex.\* ::: Characteristic African Americans Caucasians ------------------------------ ------------------- ------------- ------------- ------------- Age (mean ± SEM) 66.8 ± 0.5 62.7 ± 0.6 72.1 ± 0.1 67.1 ± 0.1 Medical History   Diabetes Mellitus 43 33 25 21   Myocardial Infarction 38 47 40 48   Heart Failure 42 48 39 34   Hypertension 78 70 63 52 Region   Northeast 27 25 34 31   Midwest 24 30 31 33   South 42 36 22 23   West 8 8 13 13 Serum Lipid Levels  LDL-C (mean ± SEM mg/dl)^†^ 131.3 ± 2.5 133.0 ± 2.5 124.8 ± 0.6 117.2 ± 0.3  LDL-C at goal (%)^†^ 19 18 25 31 \*Percent of race- and sex-specific total unless otherwise specified. ^†^For patients with documented tests having valid values ::: Lipid testing ------------- Within sex strata, the percent of patients with LDL-C tests was lower for African-Americans compared to Caucasians (Figure [1](#F1){ref-type="fig"}). Within race strata the percent of patients with LDL-C tests was lower for women than for men. The LDL-C testing rate for Caucasian men was over 1.4 times higher than that for African-American women and about 1.3 times higher than that for African-American men. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Lipid Management among CAD patients.**Lipid testing, treatment, and goal attainment rates for African-American and Caucasian women (W) and men (M). :::  ::: Lipid treatment --------------- Almost 60% of Caucasian men with LDL-C testing were prescribed lipid-lowering drugs (Figure [1](#F1){ref-type="fig"}). The proportion of tested Caucasian women receiving these drugs (55%) was similar to, but slightly lower than that for Caucasian men. Less than half of tested African-American men (47%) and women (46%) were prescribed lipid-lowering drugs. Goal attainment --------------- Including those prescribed and not prescribed lipid-lowering drugs (Table [1](#T1){ref-type="table"}), a quarter of Caucasian women and less than 20% of African-American men and women achieved the recommended LDL-C goal. But a higher proportion (31%) of Caucasian men achieved goal. The mean serum LDL-C concentration for Caucasian men (117 mg/dL) was lower than that for Caucasian women (125 mg/dL) and for African-Americans. Mean LDL-C concentrations were similar for African-American men (133 mg/dL) and women (131 mg/dL). Among patients tested and treated with lipid-lowering drugs about two-thirds or more failed to achieve goal (Figure [1](#F1){ref-type="fig"}). Tested and treated Caucasian men had the best goal attainment rates (35%). Tested and treated Caucasian women had lower levels of goal attainment than Caucasian men. Goal attainment for Caucasian men and for women of either race exceeded that of African-American men. Only about 1 of 5 African-American men prescribed lipid-lowering drugs achieved LDL-C goal. Logistic regression ------------------- Results from GEE logistic regression analyses controlling for age, co-morbid conditions, and geographic region (Table [2](#T2){ref-type="table"}) were consistent with results from the stratified analyses described above. Relative to Caucasians, African-American men and women were under-tested, under-treated, and less likely to be at goal. Relative to men, regardless of race, women were less likely to be tested. But if tested, women were as likely as men of the same race to receive prescriptions for lipid-lowering drugs. African-American women were the least likely to be tested (Odds Ratio, OR = 0.49), and if tested, African-American men were the least likely to be prescribed lipid drugs (OR = 0.59). Among those tested and treated, African-American men were least likely to be at goal (OR = 0.47). That is, among tested patients who were prescribed lipid-lowering drugs, the odds for goal attainment for Caucasian men were more than twice the odds for African-American men. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Lipid testing, pharmacologic treatment, and goal attainment among African-American men and women and Caucasian women relative to Caucasian men from logistic regression analyses.\* ::: African American Caucasian --------------------------------------- ------------------ ------------------ ------------------ ------ LDL-C tested (n = 23,104) 0.49 (0.37,0.64) 0.60 (0.47,0.77) 0.80 (0.74,0.86) 1.00 Lipid drug prescribed (n = 14,499)^†^ 0.62 (0.46,0.83) 0.59 (0.45,0.78) 1.04 (0.95,1.13) 1.00 LDL-C goal attainment (n = 8,336) ^‡^ 0.55 (0.36,0.82) 0.47 (0.30,0.74) 0.76 (0.68,0.85) 1.00 \*Odds ratio and 95% confidence intervals (95% CI) from logistic regression models accounting for within-practice correlations with GEE and controlling for race, sex, age, medical history (diabetes mellitus, myocardial infarction, heart failure, hypertension), and geographic region of medical practice. ^†^Regression model includes serum LDL-C concentration. ^‡^LDL-C goal attainment (\<100 mg/dL) among those with documented LDL-C values and treated with lipid-lowering drugs. ::: Discussion ========== Consistent with previous reports, our findings demonstrate that outpatient lipid management for CAD patients in the late 1990\'s had much room for improvement and that substantial race-sex disparities existed\[[@B8],[@B12],[@B16],[@B21]-[@B26]\]. African Americans experienced markedly lower levels of LDL-C testing than Caucasians and, as a result, they may benefit more than Caucasians from interventions to improve testing. Among those tested, African Americans were less likely to be treated and, if treated, they were less likely to be at goal compared to Caucasians. Marked lipid testing disparities by sex suggest a need for more aggressive testing in women. Much of the information needed to assess lipid management can be found only in the medical record. This information is generally not available to national and local public health programs attempting to implement policies promoting quality improvement in the many and diverse medical practices treating large patient populations. The most readily available data for patients seen in the fee-for-service setting is derived from administrative insurance claims for the reimbursement of costs associated with drugs and services. Insurance coverage for lipid-lowering drugs varies across plans. In contrast, lipid testing for CAD patients is a widely covered service that can be identified using electronic billing data without the need to review patient records in medical practices. For this reason, lipid testing is a focus of national efforts in the Medicare population to improve outpatient lipid management in the fee-for-service setting\[[@B17]\]. Our findings demonstrate that substantial disparities in treatment and goal attainment exist among CAD patients with lipid tests. This implies that current public health programs and policies designed to increase lipid testing alone will have limited impact on lipid management disparities. Substantial disparities in lipid treatment and control will likely persist in the absence of disparities in testing. Underlying causes of inadequate lipid management among CAD patients are multiple and likely vary by race and process of care (i.e., detection, treatment, and control). Factors that limit patient-physician encounters and continuity of care may partially account for racial disparities in lipid management. For example, African-American Medicare consumers with diabetes were more likely to receive outpatient care from emergency departments and had fewer physician visits per year than their Caucasian counterparts\[[@B12],[@B27]\]. The QAP data provide insufficient information to evaluate whether health care access and continuity explain lipid management disparities. A report regarding racial disparities in the use of prescription drugs from the Center for Studying Health System Change provides evidence of other factors explaining racial disparities in lipid management\[[@B11]\]. In this report, Medicare consumers 65 years of age and older were surveyed regarding their ability to obtain prescription drugs. African Americans were more than twice as likely as Caucasians to have not filled a prescription because they could not afford it. More than 16% of Medicare insured African Americans reported that they could not afford to fill at least one prescription in 2001. One-fifth of African Americans and 13% of Caucasians with low income could not afford to fill at least one prescription. During the time period of QAP-II, Medicare did not cover the cost of lipid-lowering drugs. Many Medicare consumers have supplemental insurance that assists with drug costs. In the Medicare population, African Americans were less likely than Caucasians to have supplemental insurance and more likely to be of low income\[[@B11]\]. Barriers to lipid management due to affordability may result in racial disparities if affordability differs by race. Patients who can not afford treatment may be less likely to aggressively pursue it with their physicians and may be less likely to comply with physician recommendations for testing and treatment. Secondary prevention of cardiovascular diseases among CAD patients with pharmacologic agents such as statins has been shown to be cost effective\[[@B28]\]. But drug costs that may exceed \$2,000 annually can be beyond the reach of low income and underinsured patients\[[@B29]\]. These costs are more likely a barrier for African Americans than for Caucasians and may contribute to lipid management disparities. The fact that African Americans are more likely to be of low income has greater implications than simply the inability to afford medications. Income is one of several indicators of socioeconomic status correlated with factors related to health including education\[[@B30]\]. Low education has been identified as a factor limiting a patient\'s personal involvement in lipid management\[[@B31]\]. There is evidence that African Americans are less knowledgeable about cholesterol compared to Caucasians\[[@B24]\]. African Americans may be less aware of the need for lipid management and less likely to pursue it with their physician. Achieving LDL-C goal is challenging for all races, but African Americans may require especially aggressive lipid management accompanied by an enhanced understanding of reasons for failure to achieve goal. A recent report concerning patients with CAD and/or diabetes seen at a Veterans Affairs Medical Center found that African Americans were less likely to achieve lipid goal than Caucasians when prescribed identical doses of the same lipid-lowering drug even though African Americans had more clinic visits and lipid tests\[[@B10]\]. The authors speculate that racial disparities in goal attainment may have occurred due to differences in compliance, lifestyle, and baseline LDL-C (higher for African Americans). In a study of LDL-C lowering with pravastatin in an African-American population, only 13% of patients with a LDL-C goal of 100 mg/dL actually achieved it. Incorrect drug regimen, inadequate lipid monitoring, and compliance problems were thought to have contributed to these goal attainment failures\[[@B32]\]. Additional studies are needed to investigate the underlying causes of lower goal attainment for African Americans receiving treatment for dyslipidemia. Physicians have indicated that oversight is a common reason for failure to adhere to lipid testing guidelines\[[@B33]\]. Whether oversight contributes to racial disparities in lipid management is not known, but oversight can be alleviated by system changes in the medical practice\[[@B34]\]. Awareness is growing that implementation of electronic health records is a necessary component of efforts to improve healthcare quality and prevent medical errors\[[@B35],[@B36]\]. The impact of electronic systems on disparities is an interesting area for future research. Lipid management is also influenced by physician attitudes about guidelines and drug effectiveness\[[@B37]\]. A better understanding is needed of physician attitudes and their relations with healthcare disparities. Physician-patient interactions are influenced by race and cultural factors related to race. African-American and Caucasian patients may differ with respect to cultural perceptions of health and disease and the ability of patients to influence or control their health outcomes\[[@B38]\]. African Americans may be less likely than Caucasians to trust their physicians and racial groups may view their relationship with physicians differently\[[@B39]\]. A survey of adults seen in a managed care setting revealed that African Americans viewed their visits with physicians as less participatory than did Caucasians, but they felt more participatory when seeing a physician of their own race\[[@B40]\]. The relatively poorer lipid management for African Americans compared to Caucasians may be partially explained by racial differences in the prevalence of co-morbid conditions. African Americans in the QAP were more likely than Caucasians to suffer from multiple chronic conditions including diabetes and heart failure. It has been shown that patients with diabetes compared to those without diabetes receive poorer lipid management and are less likely to be at goal\[[@B41]\]. An earlier report from the QAP has shown that patients with CAD and heart failure were less likely to receive lipid testing and cholesterol-lowering drugs than those without heart failure\[[@B42]\]. Co-morbid conditions may hinder lipid management in a number ways. These patients may be more likely to have contra-indications to lipid treatment and to present with acute life-threatening conditions that divert attention away from lipid management. In addition, it has been reported that patients with multiple chronic conditions are less likely to afford medications than those with one or fewer chronic conditions\[[@B11]\]. But even after controlling for several highly prevalent co-morbid conditions in logistic regression analyses, we find that significant lipid management disparities persist. There are a number of limitations in the QAP study design and its population that potentially pose a threat to internal and external validity. These limitations suggest that our findings may not reflect the experiences of the general population. The medical practices included in the QAP were restricted to those writing large numbers of prescriptions for cardiovascular disease drugs. It is difficult to evaluate the impact of this selection bias, but we suspect that medical practices included in the QAP represent the larger and more sophisticated providers of care. Thus, lipid management among QAP participants may be better than that found in the general population. Another limitation to our study is the lack of information characterizing medical practices, physicians, and patients with respect to factors related to race, sex, and lipid management. Because of the unavailability of this information, we could not identify underlying factors potentially explaining our findings. Lipid management data for the QAP patients seeing multiple physicians were unavailable from physicians not participating in QAP and this may have influenced results in an unpredictable manner. Considering the relatively small proportion of the study population identified as African American (\<5%), it is likely that African Americans in this study do not represent the national population. We have no specific information about QAP medical practices or their patient populations, but we speculate that African Americans in the QAP were likely receiving better care on average than their counterparts in the general population, especially those with little or no access to outpatient care. If this is true, then racial disparities in lipid management in the general population may be even greater that those suggested by our findings. The time frame for this study includes years 1995 through 1998. Despite changes in guidelines and therapies since that time, our findings remain relevant to current practices. They provide historic context and baseline measures of lipid management disparities necessary to evaluate trends. In addition, they direct attention to the persistent need to provide aggressive treatment to high risk populations with CAD, especially African Americans and women who continue to be underserved. Finally, they highlight the growing view that public health strategies in lipid management must shift focus from testing to treatment and goal attainment\[[@B43]\]. Clearly, a multi-pronged approach including all three elements of the process of care (i.e., detection, treatment, and control) is needed to improve lipid management for all race-sex groups, and particularly for African Americans. Although beyond the scope of this report, the application of conceptual models to the process of care may be useful in understanding how racial disparities arise at each step of the process. Furthermore, policies addressing health promotion and non-medical determinants of health such as socioeconomic status, community environment, and lifestyle choices need also be considered in confronting these disparities\[[@B44],[@B45]\]. Successful lipid management likely depends on a variety of processes that determine the provision of medical services including their availability, accessibility, and acceptability. The substantial racial disparities in lipid management among patients in contact with medical providers suggest that the effectiveness of medical services and patient characteristics play a prominent role in lipid management. Policies promoting appropriate delivery of care through system change as well as those ensuring equal access to care are required to eliminate lipid management disparities in the population of high-risk CAD patients\[[@B34],[@B46]\]. Conclusions =========== Our results suggest that policies and programs focusing solely on the elimination of lipid testing disparities can have only limited benefit in reducing the major disparity in lipid management. The elimination of lipid management disparities will require policies that view untested, untreated, and under-treated individuals as separate populations with unique challenges and solutions\[[@B43]\]. Disparities in testing are just one element in explaining overall disparities in lipid management and ultimately, in cardiovascular outcomes. Future research should address patient, physician, and health system factors that lead to lower rates of testing, treatment and goal attainment for African Americans. Disparities in treatment and goal attainment must be better understood and reflected in policy in order to improve the health of underserved populations through optimal lipid management. List of abbreviations ===================== CAD Coronary Artery Disease LDL-C Low-density lipoprotein cholesterol QAP Quality Assurance Program GEE Generalized Estimation Equation Competing interests =================== Analyses were funded by an unrestricted grant from Merck & Co., Inc. No other competing interests are declared. Authors\' contributions ======================= MM conceived of the study, provided analytic and statistical support, and was the leading contributing author. KF participated in the design of the study and provided analytic support and interpretation of findings. LCE provided critical technical review and major contributions to the discussion section. CS provided conceptual guidance, assistance with QAP project database, and interpretation of analytic findings. CA provided critical review, QAP project experience, and contributions to the presentation and interpretation of findings. RS provided critical review, QAP project experience, and contributions to discussion and interpretation of findings. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2261/4/15/prepub> Acknowledgements ================ The contributions of Dr. Lori Carter-Edwards were funded in part by grants from the Agency for Healthcare Research and Quality (R24HS13353) and the National Center on Minority Health and Health Disparities (R24MD000167).

PubMed Central

2024-06-05T03:55:47.511802

2004-8-18

PMC516442

Background ========== The medicinal leech, *Hirudo medicinalis*, has been used with increasing frequency during the past few decades for salvage of venous compromised pedicled flaps, microvascular free-tissue transfers and replantations. Although the therapeutic use of leeches in medicine dates back 50 BC; for centuries they were collected from various water supplies and utilised under septic conditions with the risk of wound infection and infestation. The supply of leeches was modernized by medicinal leech farm set up in the 1970s \[[@B1]\]. Today, leech therapy is indicated in plastic and reconstructive surgery, to relieve venous congestion and to improve the microrevascularization of flaps (Fig [1](#F1){ref-type="fig"},[2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"},[4](#F4){ref-type="fig"}) or replants with an increase in success rate by 60%--83%. However, in the presence of infection as a complication of medicinal use of leeches; the success rate for flap salvage may decrease to ≤ 30% \[[@B2]\]. *Aeromonas*is the most common microorganism in leech infections and may cause a wide spectrum of deseases such as cellulitis, ocular infections, arthritis, myocarditis, peritonitis, meningitis, bacteremia and sepsis \[[@B2]-[@B5]\]. In many countries, wild leeches are still provided from local markets and utilised in many plastic and reconstructive surgery clinics depending on antibiotic prophylaxies. In the first stage of the study, bacterial content of the transport fluid of leeches that were bought from local markets were studied, also oral and intestinal flora cultures and antibiograms of the identified microorganisms were performed subsequently. In the next stages hypochloric acid, a disinfectant, was tested on animals that were not bred in specific laboratories, to suppress the relevant microorganisms in order to avoid possible infections, without changing the life style and behavior of leeches in terms of sucking function. Methods ======= This study was approved by the Animal Care and Ethics Committee of our institution. Stage 1 ------- Ten leeches were obtained with their 100 cc original water from different stores. Their sizes varied between 6--10 cm in length and 0, 5 -- 1, 5 cm in width. They were transported in sterile boxes immersed in the water which the animals were kept at the market. For 2 days they were observed at room temperature. Specimens were taken and smears from transport fluids were prepared. All leeches were held with sterile gloves and prepared in sterile conditions. Specimens were obtained from their mouth (the smaller and more mobile pole) with the help of sterile cotton -- swab. After traction was applied between the mouth and posterior muscular organ, the animal was cleaned with alcoholic solution of povidone-iodine, as described by Hokelek et al. \[[@B6]\]. A longitudinal incision was made on the animal and crop or intestine was exposed (Figure [5](#F5){ref-type="fig"}). Using sterile cotton-swabs, smears were prepared from the intestinal material and they were planted in MacConkey and blood agars in 5 minutes. The growth was observed after 24--48 hours of incubation at 37°C. In addition to classical bacterial identification methods, Analytical Profile Index 32 GN (BioMérieux, France) kit was also used for identification. Further biochemical studies were made on microorganisms that were found to be *A. hydro/caviae*and *A. sobria*for definite identification. Since bacterial growth was observed in specimens taken from oral and intestinal flora or transport fluid of all animals except one ; antibiograms for aeromonas isolates were performed. Stage2 ------ 30 leeches from different stores were obtained and transported in different sterile boxes with their 100 cc original water as in stage 1. On the following day, samples were taken from transport fluids and oral region and planted in MacConkey and blood agar. Twenty five leeches, which had bacterial growth in either oral flora or transport water were used for the second stage. 100, 50, 25, 12.5, 6.25 *ppm*solutions were prepared by adding apropriate amounts of hypochloric acid into the transport fluids of 25 leeches seperately with five animals in each concentration group. After 10 minutes of contact with hypochloric acid solutions at 20°C, smears were taken from mouths and transport fluids of each leech under sterile conditions with cotton-swabs. Cotton-swabs were put into a neutralizing media (lecithinized agar media) because they could also contain disinfectant fluid which would prevent microorganism reproduction. Then the neutralized smears were planted into MacConkey and blood agar \[[@B7]\]. The leeches were then put into boxes containing distilled water and observed. Although the leeches which had been left in the 100, 50, 25 *ppm*solutions lost their muscular activity and died ;the leeches which were treated with 12.5 and 6.25 *ppm*hypochloric acid solutions were alive. Bacterial growth was not observed in either concentration group for the specimens taken from oral flora and transport fluid, however bacterial cultures were positive for in specimens taken from the intestine or crop. Of these microorganisms, bacterial colonies for *Aeromonas spp*were counted. To compare the changes in the number of *Aeromonas*colonies in leech intestinal flora induced with the application of hypochloric acid, the intestine of five new leeches were exposed as described for Stage 1. Based on the findings of Stage 2, a concentration of 12.5 ppm was chosen for Stage 3 studies. Stage 3 ------- 20 leeches were obtained with their 100 cc original water from different stores and equally divided into two groups. One group was treated with hypochloric acid while the others had no treatment at all. After taking specimens from the transport fluids and oral floras, for 10 leeches 12.5 *ppm*hypochloric acid solution was prepared with the transport fluids. After waiting for 10 min at 20°C temperature, smears were taken from the oral floras and fluids again. All leeches were put into boxes containing distilled water and watched for survival and blood suction function on sedated rats (ketamine 30 mg/kg and xylazine 10 mg/kg). After shaving dorsal region of 20 rats, 2 mm long skin incissions were done in order to produce bleeding. The leeches were applied to the bleeding areas atraumatically and the duration of suction was determined and compared statistically (ANOVA) between the leeches treated and not treated with hypochloric acid solution. Statistical significance was presumed at *p*\< 0.05. Results ======= Stage 1 ------- Bacterial growth was observed in specimens taken from the oral flora in 4 animals (40%), intestinal flora in 9 animals (90%) and transport media in 5 animals (50%). In one leech, no growth was observed in either oral, intestinal and transport fluid cultures. In all culture media, 4 different gram (-) bacterial growth (*aeromonas species*, *peudomonas species*, *acinetobacter species*, *sphingobacterium species*) was observed. In one media, Gram (+) growth was seen :*meticilline susceptible coagulase (-) staphylococcus*. Dominant microorganisms were *aeromonas species*, Table [1](#T1){ref-type="table"}. This was an expected result. The biochemical characterization and identification of *Aeromonas spp*. was investigated by using conventional methods and a commercial kit ID 32 GN ATB System (BioMérieux). In this study, with this system the results were obtained as *A. hydro/caviae*, *A. sobria*, *A. salmonicidia and A. spp*, with the latter nomenclature designating all other *Aeromonas species*. The most commonly encountered agents, *A. hydro/caviae*and *A. sobria*underwent further biochemical studies, as suggested by Abbott, et al.\[[@B4]\]. As a result, it was seen that *A. hydro/caviae*was in fact *A. hydrophila*, and *A. sobria*was in fact *A. veronii biovar sobria*(Table [2](#T2){ref-type="table"}) Antimicrobial susceptibility testing was performed by the disc diffusion method according to NCCLS (National Commity Clinical Laboratory Standart / American). The *Aeromonas spp*. were found to be susceptible to third generation cephalosporines, aminoglycosides and co-trimoxazole, and all aeromonas spp. were resistant to ampicillin-sulbactam and amoxycillin-clavulanic acid (Table [3](#T3){ref-type="table"}). Three *Aeoromonas spp*. were found to have inducable beta lactamase which meant that although they were susceptible, they might develop resistance to beta lactams other than carbapenems during treatment. Stage 2 ------- Fluid and oral flora cultures that were prepared before hypochloric acid application showed multiple microorganism growth, as it was in stage 1. After adding hypochloric acid there was no growth in transport fluid and oral media cultures in either concentration so that no identification and antibiogram study could be performed. After 10 minutes in hypochloric acid solution, leeches were taken to sterile containers which contained distilled water. The muscle activity of all leeches that had been treated in 100 *ppm*solution ended in 3 min, in 50 *ppm*ended in 7 min, in 25 *ppm*ended in 21 min and they died eventually. The ones which had been immersed in 12.5 *ppm*and 6.25 *ppm*solutions survived. The *Aeromonas*colonies in the intestinal flora of these last two groups were compared with the control animals\'. With respect to colony suppression, while the difference between the control group, 12.5 ppm group was significant, (p \< 0.05, F = 30.1, ANOVA) however the difference between control group and 6.25 ppm group was not significant. Stage 3 ------- All the leeches survived while hyperactivity followed by hypoactivity was seen in the group of leeches treated with hypochloric acid. In this group, two of the leeches never made suction but the other eight leeches sucked the bleeding area for 5--16 min (mean 12.2 ± 3.8). Non hypochloric acid treated group sucked for 9--22 min (mean 15.5 ± 4.4) and the difference between two groups was not significant (p \> 0.05, F = 2.6, ANOVA). In 70 % of cultures that were prepared from transport fluids of leeches and in 50% of cultures that were prepared from oral floras of leeches before hypochloric acid application, bacterial growth (mainly *Aeromonas spp*) were observed. After hypochloric acid application no growth was observed in any of the specimens taken from animals, therefore no identifications or antibiograms could be performed. Discussion ========== Many reports showed that venous compromise is a more common complication than arterial occlusion in free-tissue transfers. Although surgical correction is the first choice for management of venous compromised flaps ; in cases where surgical correction is not feasible or fails, nonsurgical procedures such as exanguination treatments can be used. Among them, medicinal leech therapy has become widely accepted to promote perfusion of venous compromised flaps and to relieve congestion. However there are reports in the literature concerning about infections specially with aeromonas bacteria after leech therapy calling attention for appropriate antibiotic prophylaxis \[[@B8],[@B9]\]. Many species of *Aeromonas*survive in water. These also can live in materials embedded in water, drainage tubes, fountains and containers for distilled water. Among other aeromonas species *A. hydrophilia*is the mostly isolated one from the intestines of leeches. Hemoglobin that is sucked by the leech is denaturated by *aeromonas*. One of the products, heme, is used by aeromonas and the other, globulin, is used by the leech\[[@B10]\]. This shows the endosymbiotic relationship of leeches and aeromonas. Many investigators isolated *aeromonas spp*as the dominant bacteria in infections related with leech usage \[[@B11]-[@B13]\]. *Aeromonas spp*. can cause infections with contamination through the bite point of the leech or on the macerated skin. The infections caused by these microorganisms can be minimized by easy measures. Leeches should not be handled with the forceps from the containers because these traumas can cause regurgitation of the leech into the wound and eventual contamination. Therefore, leeches should be taken with sterilized gloves without traumatisation of the animal. Same care should be taken while carrying the leech from the wound. When the leech is filled up, it puts itself off. If it is taken before this time, the risk of regurgitation is high. Low amounts of cigarette smoke or a heat source can be used in order to cause the leech to put itself off. During leech application, the routine surgical antibiotic prophylaxis must be changed because aeromonas spp. are resistant to first generation cephalosporins which are widely used at surgical wards. These are usually susceptible to third generation cephalosporins, aminoglycosides, tetracyclines and quinolones. Lineaweaver et al. recommended third generation cephalosporins for the prophylaxis of surgical procedures after which leeches were utilised \[[@B10],[@B12]\]. In countries, where special farms for medicinal leeches do not exist, animals obtained from markets should be studied for their floras and antibiotic susceptibilities. Eroğlu et al. investigated the floras and the antibiotic susceptibility of leeches in Black Sea region. Most commonly *A. hydrophila*, *Ochrobacter antropia*, *nonfermantating gram (-) rods*, *Acinetobacter Iwofii*and *A sobria*were isolated. All of the isolates were sensitive to ciprofloxacine, cefotaxime, ceftazidime, gentamycine and trimetoprim-sulfamethoxasole \[[@B14]\]. Likewise, we aimed to investigate the general flora of leeches we use occasionally and antibiotic susceptibility of these bacteria. Our results correlated with the other studies about this subject in the literature. The interesting observation in our study was there were 3 *Aeromonas hydrophylia*colonies which produced inducable betalactamase. In case of infections with these species, beta lactam antibiotics except carbapenems should not be used. In addition, we aimed to supress possible bacterial contamination of the transport media and oral floras of leeches but not intestinal flora because of the endosymbiotic relationship of the aeromonas and leech, with hypochloric acid solutions at disinfectant concentrations that does not affect the life style of leeches. Investigators have attempted to disinfect the guts of leeches before they are placed on patients by placing the ectoparasites in % 0.02 chlorhexidine for 15 seconds or in antibiotic solutions (tetracycline or cefoperazone solutions) for 12 hours, but these attempts were unsuccessful \[[@B2]\]. Mackay et al. incubated leeches in solutions of antibiotics to which *Aeromonas spp*. are sensitive for 12 hours but could not eradicate *aeromonas*from the intestines \[[@B11]\]. We preferred hypochloric acid which is an ideal disinfectant and has a wide antibacterial spectrum for desired purpose. Chlorine in the form of hypochloric acid exhibits rapid microbicidal activity by inhibiting of key enzymatic reactions within the cell and eventual protein denaturation. It has a rapid bactericidal effect and can dissolve in water. However, it has some disadvantages as it irritates mucosal membranes, has interactions with some chemicals and metals \[[@B15]\]. It was showed that bactericidal effect of hypochloric acid of 100 *ppm*concentrations on pseudomonas was achieved within 10 min exposure \[[@B7]\]. *Aeromonas spp*. cause similar nosocomial infections and has similar antibiotic susceptibility with *pseudomonas spp*. Therefore, in the second stage of our study, beginning with 100 *ppm*, we applied decreasing concentrations of hypochloric acid in transportation fluids, observed bacterial growth by taking specimens from transport material and oral flora of *hirudo medicinalis*. We tried to find the dilution ratio that was closest to 100 *ppm*, suppresing oral flora and transfer liquid and also allowing the leech to survive with normal function. As a result of the second stage of the study we found out that concentration to be 12.5 *ppm*. In the third stage, we observed that mean sucking duration of the hypochloric acid treated group was shorter than the other group but the difference was not significant (12.2 min & 15.5 min respectively). However hypochloric acid had successful disinfection effect for transport liquid and oral flora. In our opinion, such a decrease in duration of sucking function can be preferable to infection possibility. Since leeches are much cheaper than antibiotics, it is logical to use them once, after treating with hypochloric acid, and never utilise them again. We do not advise to use \"full-up\" leeches over and over again by putting them in hypertonic solutions to force them vomitting because if they regurgitate, the intestinal flora can readly contaminate the environment where the leeches are kept and cause infection eventually. Although the oral flora and transport enviroment studies do not really address the problem since excretory contamination is probably the major factor in leech infections ; we believe that to take any measure in order to decrease the infection risk during utilisation of ordinary leeches is valuable. Conclusions =========== We can comment that preparation of 12.5 *ppm*hypochloric acid solution with transport fluids of ordinary leeches obtained from the local market for 10 minutes and then taking the leeches gently from water before application can prevent possible infections caused by contamination from leech oral flora and transport medium. Competing interest ================== None declared. Author\'s contributions ======================= AA and SVK participated in design of the study, involved in the dissections of leeches. EH and ST participated in the design of the study and writing of the manuscript.HN, NG and BO involved in the antibiogram tests. SNK participated in design of the study. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/28/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Tissue defect on the dorsum of the hand of a male patient after a crush type injury. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Venous insufficiency of the posterior interosseous flap used for defect reconstruction. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Leech usage in the treatment of venous insufficiency. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Late follow up of the salvaged flap. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Intestinal exploration of Hirudo medicinalis and taking specimen with cotton swab. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Results of the cultures prepared from transport fluids, oral and intestinal smears of 10 leeches. ::: **Number of leech** **Source of the culture material** **Bacterial Growth** --------------------- ------------------------------------ -------------------------------------------------------------------------------- **1** oral flora no reproduction intestinal flora no reproduction transport fluid no reproduction **2** oral flora Aeromonas hydro/cavie, Aeromonas spp, Acinetobacter spp intestinal flora Aeromonas hydro/cavie, Aeromonas spp, Acinetobacter spp transport fluid Aeromonas hydro/cavie, Aeromonas spp, Acinetobacter spp **3** oral flora no reproduction intestinal flora Aeromonas hydro/cavie transport fluid no reproduction **4** oral flora no reproduction intestinal flora Aeromonas hydro/cavie transport fluid Aeromonas hydro/cavie, Aeromonas spp, Acinetobacter spp **5** oral flora no reproduction intestinal flora Aeromonas sobria transport fluid no reproduction **6** oral flora no reproduction intestinal flora Aeromonas sobria transport fluid Acinetobacter spp, methicillin sensitive-coagulase negative Staphylococcus spp **7** oral flora Aeromonas salmonicidia, Pseudomonas spp, Sphingobacterium spp intestinal flora Aeromonas hydro/cavie, Aeromonas sobria transport fluid Aeromonas hydro/cavie, Aeromonas salmonicidia **8** oral flora Aeromonas hydro/cavie intestinal flora Aeromonas sobria transport fluid Pseudomonas spp **9** oral flora Aeromonas hydro/cavie intestinal flora Aeromonas hydro/cavie transport fluid no reproduction **10** oral flora no reproduction intestinal flora Aeromonas hydro/cavie transport fluid no reproduction ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Definitive identification of A. hydro/caviae and A. sobria with biochemical tests. ::: **No** **2** **3** **4** **5** **6** **7** **8** **9** **10** -------------------------- ------- ------- ------- ------- ------- ------- ------- ------- -------- ------- ------- ------- ------- ------- ------- ----- **◆** **O** **I** **F** **I** **I** **F** **I** **I** **I** **F** **O** **I** **O** **I** **I** **Results of API 32 GN** h/c h/c h/c h/c h/c h/c s s h/c s h/c h/c s h/c h/c h/c **M** \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ **Esculin** \+ \+ \+ \+ \+ \+ \- \- \+ \- \+ \+ \- \+ \+ \+ **Catalase** \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ **Indole** \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ **VP** \+ \+ \+ \+ \+ \+ \+ \- \+ \+ \+ \+ \+ \+ \+ \+ **LDC** \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ **ODC** \- \- \- \- \- \- \- \- \- \- \- \- \- \- \- \- **DNase** \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ **Hem.** \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ **Glucose** \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ \+ **Lactose** \- \- \- \- \- \- \- \- \- \- \- \- \- \- \- \- **Urea** \- \- \- \- \- \- \- \- \- \- \- \- \- \- \- \- **Citrate** \+ \+ \+ \- \+ \+ \- \- \- \+ \- \+ \+ \+ \+ \- **Results** h h h h h h v v h v h h v h h h **Abbreviations :**No: Number of leech, ◆: Source of the culture material, API 32 GN: Analitycal Profile Index 32 GN (BioMérieux), O: Oral flora, I: Intestinal flora, F: Transport fluid, h/c: A. hydro/caviae, h: A. hydrophila, s: A. sobria, v: A. veronii biogroup sobria, M: Motility, VP: Voges-Proskauer, LDC: Lysine decarboxylase, ODC: Ornithine decarboxylase, Hem: Hemolysis, Lactose: Asit from Lactose. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### The antibiograms of aeromonas isolates from 9 different leeches. ::: **Leech** --------- ----------- ---- --- --- --- --- ---- ---- --- **AMP** R R R R R R R R R **SAM** R R R R R R R R R **AMC** R R R R R R R R R **CZ** R R R S S R R R S **CXM** S S S S S S S S S **FOX** S S S S S S S S S **CRO** S S S S S S S S S **SCF** S S S S S S S S S **FEP** S S S S S S S S S **GM** S S S S S S S S S **TOB** S S S S S S S S S **NET** S S S S S S S S S **AK** S S S S S S S S S **SXT** S S S S S S S S S **NOR** S S S S S S S S S **IPM** S S S S S S S S S **MEM** S S S S S S S S S **TZP** S S S S S S S S S **AZM** S S S S S S S S S **IBL** \+ \+ \+ **Abbreviations :**h:A. hydrophila, v:A. veronii biogroup sobria, R:Resistant, S:Sensitive AMP:Ampicillin, SAM:Sulbactam/ampicillin, AMC:Amoxicillin/clavulanate, CZ:Cefazolin, CXM:Cefuroxim FOX:Cefoxitin, CRO:Ceftriaxone, SCF:Sulbactam/cefoperazone, FEP:Cefepime, GM:Gentamicin, TOB:Tobramycin, NET:Netilmicin, AK:Amikacin, SXT:Trimethoprim/sulfamethoxazole, NOR:Norfloxasin, IPM:Imipenem, MEM:Meropenem, TZP:Piperacillin/tazobactam, AZM:Aztreonam, ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### The number of bacterial colonies before and after hypochloric acid applications of 12.5 *ppm*and 6.25 *ppm*concentrations and control group. ::: **No** ***Concentration of hypochloric acid*** **Transport fluid Colony count** **Mouth Colony count** **Gut Colony count** -------- ----------------------------------------- ---------------------------------- ------------------------ ---------------------- **1** **before** 12 16 Not studied ***12.5 ppm*after** No reproduction No reproduction 25 **2** **before** 23 14 Not studied ***12.5 ppm*after** No reproduction No reproduction 18 **3** **before** 30 34 Not studied ***12.5 ppm*after** No reproduction No reproduction 50 **4** **before** 25 16 Not studied ***12.5 ppm*after** No reproduction No reproduction 44 **5** **before** 18 No reproduction Not studied ***12.5 ppm*after** No reproduction No reproduction 30 **1** **before** No reproduction 20 Not studied ***6.25 ppm*after** No reproduction No reproduction 100 **2** **before** 27 7 Not studied ***6.25 ppm*after** No reproduction No reproduction 120 **3** **before** 20 No reproduction Not studied ***6.25 ppm*after** No reproduction No reproduction 72 **4** **before** No reproduction 8 Not studied ***6.25 ppm*after** No reproduction No reproduction 95 **5** **before** 15 12 Not studied ***6.25 ppm*after** No reproduction No reproduction 75 **1** **Control group** Not studied Not studied 100 **2** Not studied Not studied 150 **3** Not studied Not studied 120 **4** Not studied Not studied 105 **5** Not studied Not studied 140 :::

PubMed Central

2024-06-05T03:55:47.514439

2004-8-25

PMC516443

Background ========== Each year between 75.000 and 200.000 infant deaths are attributed to malaria infection in pregnancy globally, and between 200.000 and 500.000 pregnant women develop severe anaemia as a result of malaria in Sub-Saharan Africa (SSA) \[[@B1]\]. Pregnant compared to non-pregnant women are at an increased risk for malaria, and the severity of the clinical manifestations in the women and her foetus depends on the level of pre-pregnancy immunity \[[@B2]\]. While in areas of low malaria endemicity all pregnant women are equally susceptible to the consequences of malaria infection, in areas of high endemicity women appear to be most susceptible during their first pregnancy \[[@B3]\]. However, more recent publications point to significant susceptibility in primigravidae as well as in multigravidae \[[@B4]\]. Pregnancies in women living in malaria endemic regions are associated with a high frequency and density of *P. falciparum*parasitaemia, with high rates of maternal morbidity including fever and severe anaemia, with abortion and stillbirth, and with high rates of placental malaria and consequently low birth weight in newborns caused by both prematurity and intrauterine growth retardation \[[@B1],[@B3],[@B5]\]. In order to reduce malaria-related ill health, regular chemoprophylaxis has been recommended to all pregnant women living in malaria-endemic areas \[[@B6]\]. Most African countries, including Burkina Faso, include routine chemoprophylaxis in their official antenatal care programmes. However, in practice, coverage of chemoprophylaxis is limited due to low accessibility and quality of antenatal care (ANC) services as well as problems with compliance \[[@B7]\]. It has been estimated from a survey in four African countries that less than 20% of women use a prophylactic regimen close to the WHO recommendations \[[@B8]\]. While insecticide-treated bed nets and curtains (ITN) have been shown to substantially reduce malaria morbidity and mortality in children, results from initial trials on the efficacy of ITNs for malaria prevention in pregnancy produced conflicting evidence and were classified as inconclusive by the Cochrane Collaboration \[[@B3],[@B9]\]. However, with the publication of the findings from a major ITN trial in a holoendemic area of western Kenya, the use of ITN during pregnancy is getting more credibility \[[@B10]\]. In Burkina Faso, the official policy for malaria and anaemia prevention in pregnant women comprises of chloroquine and iron/folic acid supplementation respectively. However, in the rural areas of Burkina Faso little information exists on coverage with ANC and on compliance with preventive regimens. We assessed the coverage of antenatal care and investigated the knowledge and adoption of preventive practices with respect to malaria and anaemia in users and non-users of antenatal care. Methods ======= Study area ---------- The study took place in the rural part of the research zone of the *Centre de Recherche en Santé de Nouna*(CRSN) in Nouna Health District, northwestern Burkina Faso. The CRSN research zone consists of Nouna town and 41 of the surrounding villages with a total population of around 60.000 inhabitants. The Nouna area is a dry orchard savanna, populated mainly by subsistance farmers of different ethnic groups. Malaria is holoendemic but highly seasonal in this part of Westafrica \[[@B11]\]. Most malaria transmission takes place during or shortly after the rainy season which usually lasts from June until October \[[@B11]\]. Modern health services in the CRSN research zone are limited to four village-based health centres and the district hospital in Nouna town. As a consequence, malaria control is mainly based on home treatment with chloroquine, the official first-line treatment drug in Burkina Faso. Roughly half of all households in the area possess at least one untreated bed net and since 2000, ITN are distributed as part of an ongoing trial in young children \[[@B12],[@B13]\]. The official policy for malaria and anaemia prevention during pregnancy in Burkina Faso consists of a curative dose of 1 500 mg chloroquine during three days followed by a weekly dose of 300 mg chloroquine and a combined dose of daily 200 mg iron and 0.25 mg folic acid. This regimen should be followed from the first ANC visit until six weeks after delivery. Since the year 2002 it is the official policy in Burkina Faso to offer ANC services free of charge. This includes ANC card, physical examination and counselling, and malaria/anaemia prevention drugs. However, urine examination, gloves, and drugs for other concomitant diseases still have to be paid for. Urine examination and gloves usually cost 150--200 F CFA (1 Euro = 650 F CFA). Study design ------------ The study was cross-sectional and descriptive in nature, using both qualitative and quantitative methods for data collection. The study was implemented in May and June 2003. The research team comprised of the investigators and six trained local interviewers familiar with the common spoken local languages and French. The questionnaires were pre-tested before administration. The study took place in eight of the 41 villages of the CRSN study area. Villages were selected as follows: At a first stage, the four villages of the CRSN study area where a health centre exists were purposely selected. To account for the socio-demographic variability and geographical accessibility, in each of these health centre defined sub-areas, another village distant of at least 5 kilometres to the health centre was randomly selected. Qualitative research -------------------- We conducted six Focus Group Discussions (FGD). Two with pregnant women users of ANC, two with husbands of pregnant women users of ANC, and two with pregnant women non-users of ANC. Respective FGDs were held with groups of six to 12 participants from study villages with and without a health centre. Key informant interviews were conducted with four maternity health workers, seven traditional birth attendants and 29 women group leaders. The interviews assessed their knowledge, attitudes and practices about malaria and anaemia prevention in pregnancy. Quantitative research --------------------- The design of the quantitative survey instrument was informed by the results of the qualitative interviews. A structured questionnaire was administered to all women from the eight study villages who had delivered a life child during the last six months (n = 225). Information on births was available through the existing Demographic Surveillance System (DSS) in the study area \[[@B14]\]. The questions focussed on socio-demographic characteristics, obstetrical history, knowledge and practice of preventive measures against malaria and anaemia during pregnancy, factors influencing the utilisation of ANC services and on the compliance with chloroquine and iron/folic acid supplementation during pregnancy. The questionnaires were filled in by the interviewers who also cross-checked given answers with all available ANC cards (n = 156/225). On the ANC cards, the estimated age of pregnancy at first visit was reported through the fundal height in centimetres, as women were not able to recall their last menstruation period during ANC visits. These data were afterwards transformed into a specific scoring system. We used two definitions for malaria prophylaxis. A complete curative dose of 1 500 mg chloroquine followed by regularly weekly 300 mg doses afterwards (complete prophylaxis), and an incomplete regimen consisting of only 300 mg weekly doses (incomplete prophylaxis). A combined dose of daily 200 mg iron and 0.25 mg folic acid was defined as a complete prophylaxis. Statistical analysis -------------------- The data were entered in Microsoft Access 2000, cleaned, and then analysed with Epi Info 2000. Univariate analysis was done with chi-square test or Fisher\'s exact test to compare proportions for categorical variables. Results were considered to be significant when the 2-sided P value was \<0.05. Ethical aspects --------------- We received ethical approval from the institutional Ethical Committee at the Department of Tropical Hygiene in Heidelberg, Germany, and the local Ethical Committee in Nouna, Burkina Faso. Oral informed consent was obtained from all participants. Results ======= Study population ---------------- The characteristics of participants on the quantitative survey are shown in table [1](#T1){ref-type="table"}. The great majority of the survey women were married, illiterate, housewife/farmers and their ages range between 15 and 49 years. The distribution of ethnicity among survey women was as follows: 41% Bwaba, 39% Marka, 15% Mossi, 3% Samo and 2% others. The median number of pregnancies among survey women was 6 (range 1--13). There were no differences between the background characteristics of users compared to non-users of ANC services, except regarding distance to the nearest ANC services. When comparing women living at a distance ≤ 5 km with women living \>5 km, distance was significantly associated with ANC use (p \< 0.001). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Background characteristics of women interviewed ::: **ANC** -------------------- ---------- --------- ---------- **Age Group**  15--19 24 (15) 12 (18) 36 (16)  20--29 81 (51) 37 (56) 118 (52)  30--49 54 (34) 17 (26) 71 (32) **Education**  No Schooling 145 (91) 62 (94) 207 (92)  Primary Education 14 (9) 4 (6) 18 (8) **Parity**  1 26 (16) 8 (12) 34 (15)  2--3 46 (29) 17 (26) 63 (28)  4--13 87 (55) 41 (62) 128 (57) **Distance\*(Km)**  0--4 99 (62) 5 (8) 104 (46)  5--7 43 (27) 59 (89) 102 (45)  8--15 17 (11) 2 (3) 19 (8) \* Distance to the nearest health center in kilometers ::: ANC coverage ------------ In this study population, the minimal ANC coverage (defined as at least one ANC visit during pregnancy) was 159/225 (71%) and 63/225 (28%) if we consider the optimal frequency of at least three ANC visits (national goal). Minimal ANC coverage was 97/102 (95%) in villages with a health centre vs 62/123 (50%) in remote villages (p \< 0.001), while the optimal ANC coverage was 55/102 (54%) in villages with a health centre vs 8/123 (7%) in remote villages (p \< 0.001). Among ANC users, 27%, 40% and 33% of women visited ANC services one time, two times or more than two times respectively during their pregnancy. The first ANC visit of ANC users was in 14% during the first trimester, in 57% during the second trimester, and in 27% during the third trimester. Malaria and anaemia prevention knowledge and behaviour ------------------------------------------------------ Malaria and anaemia were considered as the most common diseases during pregnancy by the majority of the participants in the FGD, key informant interviews and survey women. In the Dioula language (lingua franca in the region) malaria is equivalent to \"Soumaya\", light to moderate anaemia to \"Djolidessé\" and severe anaemia to \"Djoliban\". Most women in the FGD were knowledgeable about the malaria prevention effect of chloroquine and the anaemia prevention effect of iron/folic acid. The knowledge of malaria and anaemia prevention measures by ANC users is given in table [2](#T2){ref-type="table"}. Regarding malaria, the majority of survey women stated that it can be prevented with chloroquine (white tablets), while only a minority mentioned mosquito nets or others measures. Regarding anaemia, iron/folic acid (red colour tablets or vitamins) supplementation was stated by the majority of survey women as being protective, while a much smaller percentage of women mentioned nutrition as an important factor. Stating chloroquine and mosquito nets as prevention measures against malaria and iron/folic acid against anaemia was significantly associated with ANC use (p \< 0.002; p \< 0.001; p \< 0.001). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Knowledge of preventive measures against malaria and anemia ::: **ANC** **All** ***P*Value** ------------------------ ---------- --------- -------------- ---------- **Malaria prevention**  Chloroquine 128 (66) 35 (59) 163 (65) \<0.001  Mosquito nets 40 (21) 2 (3) 42 (17) \< 0.001  Hygiene 6 (3) 3 (5) 9 (4) n.s.  Protective clothing 12 (6) 0 (0) 12 (5) \<0.05  Does not know 7 (4) 19 (32) 26 (10) \<0.001 **Anemia prevention**  Iron/Folic acid 129 (76) 16 (25) 145 (63) \<0.001  Adequate nutrition 29 (17) 8 (13) 37 (16) n.s.  Does not know 11 (7) 39 (62) 50 (22) \<0.001 ::: Table [3](#T3){ref-type="table"} shows data on self-reported use of malaria and anaemia prophylaxis with chloroquine and iron/folic acid in the population of ANC users together with respective data taken from their ANC cards. A correct prescription of chloroquine (complete prophylaxis) on ANC cards was seen in 60%, and a correct prescription of iron/folic acid (complete prophylaxis) was seen in 78%. In contrast, self-reported oral information during ANC visits on complete and incomplete prophylaxis regimens were lower and matched well with self-reported information on the chloroquine and iron/folic acid dosages taken. Most women reported being compliant with the oral information on chloroquine and iron/folic acid regimens from first ANC visit until delivery. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Prescription in ANC card of chloroquine prophylaxis and iron/folic acid supplemen-tation, self-reported ANC instructions and self-reported intake among ANC users (n = 159) ::: **Chloroquine (%)** **Iron/folic acid (%)** ------------------------------------------------------------- --------------------- ------------------------- **Prescription on ANC card**  Complete prophylaxis 95 (60) 124 (78)  Incomplete prophylaxis 29 (18) \-  Incorrect prescription 32 (20) 32 (20)  No prescription (no ANC card) 3 (2) 3 (2) **Instructions given at ANC visits (self-reported)**  Complete prophylaxis explained 46 (29) 111 (70)  Incomplete prophylaxis explained 65 (41) \-  Incorrect instructions given 41 (26) 41 (26)  No instructions given 7 (4) 7 (4) **Dosages taken (self-reported)**  Complete prophylaxis 46 (29) 149 (94)  Incomplete prophylaxis 67 (42) \-  Incorrect dose 43 (27) 8 (5)  No prophylaxis 3 (2) 2 (1) **Duration of chloroquine and iron/folic acid prophylaxis**  From 1^st^ANC until before delivery 20 (13) 20 (13)  From 1^st^ANC until delivery 115 (72) 124 (78)  From 1^st^ANC until after delivery 21 (13) 13 (8)  No prophylaxis 3 (2) 2 (1) ::: Factors influencing the use of ANC services ------------------------------------------- Of ANC users, the great majority reported to be satisfied with the quality of ANC services. Only 16% of ANC users were aware of the fact that ANC services have recently become free of charge, and 42% reported that they still had paid for services. Most (73%) ANC users had paid between zero and 200 F CFA, while 12% and 6% had paid between 200 and 1.500 and between 1.500 and 7.500 F CFA respectively. Apart from distance to the next health centre, lack of resources and ignorance were the most frequent stated reasons during qualitative and quantitative interviews why women did not attend ANC services. The majority of ANC non-users reported no specific prophylaxis during their pregnancy, but 6% took irregularly self medication for malaria/anaemia prevention. Moreover, 30% of ANC non-users had sought advice from traditional birth attendants (TBA). Typical statements in the FGDs with non-users of ANC services werecollected as follow: • *\"ANC is not free of charge, you must pay for ANC card and the medicine and it could be up to 500 CFA and for me I find it is expensive. Our husbands find it expensive too, that is why the majority of us can not attend ANC services\".* • *\"I don\'t know the advantages of ANC services, some of the pregnant women if they don\'t fell sick they wouldn\'t accept to attend ANC \".* • *\"There is lack of advices from health workers, if you go to the ANC for the 1*^st^*time, they give drugs, they don\'t tell you when you should come back and how many visits you should attend. That is how they do here\".* • *\"If you are not sick, you don\'t pay for drugs, what you pay for is the ANC card at 150 F CFA\....\"* • *\" But even the ANC card is free of charge, only the gloves cost 100 F CFA\....\"* • *\"I had not yet heard about the free ANC services \... Since I know now that ANC is free of charge I will attend the services.\"* • *\"We do not have money to go to the health centre it will be nice to have one in our village\".* Itching, vomiting and fatigue were the most frequently stated side effects of malaria/anaemia prevention drug regimens during interviews, sometimes leading to non-compliance. Most women also stated that bed nets are considered too expensive for their household. Discussion ========== The main findings of this study are (1) that coverage of antenatal care is far from complete, particularly in villages without a health centre, (2) that malaria- and anaemia-related knowledge and compliance with preventive measures is comparatively high with a wide gap between users and non-users of antenatal care and (3) that health services need to improve their response to the women\'s need for preventive care in pregnancy. Use and coverage of antenatal care ---------------------------------- In this community-based study from a rural area of Burkina Faso, we found an overall ANC coverage of 71%, which is however not representative given the used methodology. Most women had two ANC visits during their pregnancy, mainly during the second and third trimester. As we found ANC coverage to be much higher in villages with a health centre compared to villages quite distant from a health centre, and as we included similar numbers of health centre and non-health centre villages, our ANC coverage figure is likely to be an overestimate. The national demographic and health survey in Burkina Faso claims an ANC attendance (at least one ANC during one pregnancy) of 59% \[[@B15]\]. Although health workers from rural health centres in Burkina Faso are advised to do regularly outreach work in the villages of their respective catchment areas, in practice such visits are rare due to a number of reasons such as lack of transport. Our findings thus support the need for better access of rural SSA women to ANC services \[[@B16]\]. Other reasons given for non-use of ANC services included ignorance and lack of money. This confirms similar observations from other rural African areas \[[@B20]\]. Interestingly, the Burkinabé Government had recently changed its policy towards free ANC service provision. However, our findings show that this policy is rather confusing as some parts of ANC procedures are not included. Consequently it was not yet fully understood by the population. However, this change in policy was considered promising during our interviews, and it was also reassuring that most of the ANC users were satisfied with the quality of ANC services. Knowledge and compliance with preventive interventions related to malaria and anaemia ------------------------------------------------------------------------------------- Malaria and anaemia were seen as important disease entities during pregnancy in our interviews. Moreover, most women interviewed were quite knowledgeable about effective malaria/anaemia prevention measures. However, compared to ANC users ANC non-users were significantly less knowledgeable about malaria/anaemia prevention measures. The interpretation is not straight forward because more knowledgeable women may be more likely to attend antenatal care or increased knowledge may be the result of health education in anatenatal care; most likely both effects contribute to the observed gap between ANC users and ANC non-users. Responsiveness of health services --------------------------------- There is now a broad agreement on the need of new strategies for community participation on the implementation of effective malaria control activities, as well as for a better education of service providers of both the public and the private sector \[[@B17]\]. It was reassuring to find nearly all of the women with reported ANC use to have an ANC card in their house. Specific prescriptions were found on most cards, but in particular malaria prophylaxis prescriptions were often not complete. This explains why self-reported compliance with recommended prevention regimens was sub-optimal with regard to malaria. Such discrepancies have been observed already in a recent study in pregnant women of Nouna town \[[@B18]\]. However, self-reported compliance matched well with the reported oral instructions given by respective health workers. This points to the importance of correct oral information in rural areas with high prevalence of illiteracy \[[@B19]\]. The best model for effective and cost-effective malaria prevention during pregnancy in SSA still needs to be developed. Chloroquine has been the mainstay for malaria control in sub-Saharan Africa (SSA), but the emergence of chloroquine-resistant *Plasmodium falciparum*has put into question the efficacy of this well-known drug \[[@B21]\]. The first cases of in vitro and in vivo Chloroquine resistance in Burkina Faso were seen in 1983 and 1988, respectively, and reported clinical failure rates after use of Chloroquine for treatment of uncomplicated malaria in children were around 5% in the early 1990s and 10% during the most recently performed surveys \[[@B22]\]. Although this is considered still below the threshold of clinical failures considered to require a change of first-line treatment, it has recently been shown that chloroquine failed to prevent malaria in pregnant women of Burkina Faso \[[@B23]\]. Current alternatives include intermittent treatment with pyrimethamine-sulfadoxine and the use of ITNs. Sulphadoxine-pyrimethamine, given in 1--3 therapeutic dosages during the second and third trimester of pregnancy, has recently been demonstrated to be an effective and cost-effective schedule for malaria prevention \[[@B24]\]. Compared to chloroquine prophylaxis, this regimen has the advantage that it is given to women when they attend antenatal clinics, thus avoiding problems with compliance. ITNs are increasingly considered as an important tool in the prevention of malaria in young children and pregnant women, and the provision of ITN through ANC services has recently been proposed as a promising distribution channel \[[@B13],[@B25]\]. The findings of this study confirm the cost barrier to the private purchase of bed nets and ITNs and thus support the call for major subsidies if a high ITN coverage is going to be achieved \[[@B12],[@B13],[@B25]\]. Competing interests =================== None declared. Authors\'Contributions ====================== CM, AJ and OM designed the study. FS and BK were responsible for the conduct of the study in Burkina Faso. CM analysed the data. All authors contributed to the interpretation of the data, helped write the paper, and read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2393/4/18/prepub> Acknowledgements ================ We thank the staff of the CRSN and the population of the study villages for their support. The study was funded by the Deutsche Forschungsgemeinschaft (SFB 544, Control of Tropical Infectious Diseases). Caroline Miaffo was supported by a grant from the German Academic Exchange Service (DAAD).

PubMed Central

2024-06-05T03:55:47.517023

2004-8-27

PMC516444

Background ========== Besides brittle bones, all other clinical characteristics of osteogenesis imperfecta (OI) are variable, and even different members of the same family may present with a dissimilar degree of severity \[[@B1]\]. Severity appears to follow a continuum in the OI population, and it is therefore very difficult to establish a clinical prognostic classification in definite categories. This disease has received different names through history, including osteopsathyrosis, Vrolik\'s disease, fragilitas ossium, mollities ossium, Lobstein\'s disease, and Van der Hoeve syndrome \[[@B2]\]. In a first attempt to classify OI, in 1906 Looser divided the disease in two forms \[[@B3]\]: \"congenita\" (Vrolik) and \"tarda\" (Lobstein), depending on the severity of the presentation. In OI congenita, multiple fractures may occur *in-utero*, whereas in OI tarda, fractures happen at the time of birth or later. OI tarda has also been sub-divided in \"gravis\" and \"levis\" \[[@B3]\]. This classification is no longer tenable because it failed to encompass the obvious clinical variability apparent in this disorder. There has also been an attempt to classify OI according to radiological characteristics \[[@B4]\]. Some of the features suggested by the authors of this classification are not present until age 5 or 10 years, and children of early ages can not be classified using this scheme. There has also been suggested that OI could be classified OI according to severity \[[@B5],[@B6]\]. The most commonly used classification, initially proposed by Sillence in 1979, divides the patients in four types (I-IV) \[[@B7],[@B8]\]. In fact, it is now widely recognized that there may be more types of OI \[[@B9]-[@B11]\]. Some of the forms of congenital brittle bones have been considered OI and have been added as numbers V, VI and VII \[[@B8],[@B12]-[@B14]\], while others have been designated with eponyms (Cole-Carpenter syndrome \[[@B15]\], Bruck syndrome \[[@B16]\]), and others designated with clinical features (OI with denser areas in bones \[[@B17]\], OI with optic atrophy, retinopathy, and severe psychomotor retardation \[[@B18]\], OI with microcephaly, and cataracts \[[@B19]\], osteoporosis-pseudoglioma syndrome). The numeric classification (I-VII) is somewhat confusing, as the characteristics of each type overlap. Furthermore, there is no consensus about basic characteristics of the different types. For example, it is not clear if type I OI includes individuals with short stature, or if bone deformity rules out the diagnosis of Type I OI. Also, it is unclear if an individual with normal height can have type IV OI. Type II OI is defined as lethal, but there are cases of children with clinical characteristics of type II OI who have survived several years. Furthermore, the current classification does not allow for prognostic predictions, as individuals with type I OI may have numerous fractures and chronic pain in the course of their lives. This is part of the lack of a precise definition of OI. Thus, despite the fact that OI has been known for more than 200 years, there is no consensus about the definition for the disease. For some, it includes only those forms of congenital brittle bones secondary to mutations in the genes codifying for type I pro-collagen (COL1A1 and COL1A2). For others, it is a group of conditions with the common feature of congenital brittle bones. The example of the osteogenesis imperfecta-pseudoglioma syndrome illustrates this concept very clearly. This syndrome has been re-named as \"osteoporosis-pseudoglioma syndrome\" once the causal mutation has been identified to be in the LRP5 gene, elsewhere than the type I pro-collagen genes \[[@B20],[@B21]\]. Following this example, I propose to define osteogenesis imperfecta as syndromes resulting from mutations in either COL1A1 or COL1A2 genes, and to group all other syndromes with congenital brittle bones as \"syndromes resembling OI\" (SROI), pending the identification of their causal mutation. Here, a review of the different forms of syndromes with congenital brittle bones is presented (Table [1](#T1){ref-type="table"}). A working-group of international experts (like the Villefrance criteria in Ehlers-Danlos Syndrome) to clarify the issue of definition of congenital brittle bones syndromes and their classification is warranted. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### syndromes with congenital brittle bones ::: ---------------------------------------------------------------------------------------------- Osteogenesis Imperfecta  Mild OI with normal stature  Moderate OI with short stature  Severe OI  Lethal OI  Congenital brittle bones with dense areas in bones Syndromes resembling OI (SROI)  Congenital brittle bones with craniosynostosis and ocular proptosis  Congenital brittle bones with congenital joint contractures  Osteoporosis-pseudoglioma syndrome  Congenital brittle bones with optic atrophy, retinopathy and severe psychomotor retardation  Congenital brittle bones with microcephaly and cataracts  Congenital brittle bones with redundant callus  Congenital brittle bones with mineralization defect  Congenital brittle bones with rhizomelia ---------------------------------------------------------------------------------------------- ::: Discussion ========== Here, syndromes with congenital brittle bones are divided in osteogenesis imperfecta (those secondary to mutations in the type I pro-collagen genes) and syndromes resembling OI (those secondary to mutations other than the type I pro-collagen genes, identified or not) Osteogenesis imperfecta (types are ordered based on severity from mild to severe) ================================================================================= Type I OI --------- The most common mutation causing OI type I causes a reduction in the production of otherwise normal type I collagen secondary to the effect of a null allele mutation. Patients often have normal stature, and a slightly low stature does not preclude the diagnosis of type I OI. \"Type I OI\" is not a synonymous of \"mild OI\". Individuals may have few or no fractures, mostly during the first years of life or even at birth \[[@B22]\], or numerous fractures throughout their lives. They may have triangular face. They are fully ambulatory, and do not have bowing of the long bones, although vertebral fractures may be present. Most have blue sclera, but it can be white, or blue color may fade as the individual grows older. This condition is transmitted as an autosomal dominant trait. Despite the absence of fractures, bone density can be very low, with no relation with clinical severity, underscoring the relative lack of significance of bone density measurements assessing severity in patients with OI. In many instances bone density is normal during the first months of life, and individuals progressively fail to increase bone mineral density with age. In some cases the diagnosis is an incidental finding after a fracture \[[@B23]\]. Dentinogenesis imperfecta can be present even in very mild cases. Early hypoacusia \[[@B24],[@B25]\] and cardio-vascular problems, particularly aortic valvular disease \[[@B22]\] can be present in these subjects. Type IV OI (Moderate OI with short stature) ------------------------------------------- These individuals typically have short stature, bowing of long bones, and vertebral fractures. Scoliosis and joint laxity may be present. Patients with this form of OI are generally able to ambulate, but they may require aids for walking. Based in the presence of DI, moderate OI has been sub-divided in two forms, \"a\" and \"b\" \[[@B26]\]. These patients have white sclera. Precise diagnosis of this type of OI is often difficult, as the clinical characteristics are not clear in the literature, and different centers base the diagnosis on different criteria. Type III OI (Severe OI) ----------------------- These patients have triangular face, product of an enlarged head and under-development of the facial bones. They also have chest deformities, markedly short stature, and severe deformities of the long bones, vertebral fractures, and scoliosis. They are frequently wheelchair-bound, although some are able to walk with aids. Prenatal diagnosis is hard but sometimes possible using ultrasonography \[[@B27]\]. Long bones are severely bowed, and altered structure of the growth plates lead to a particular structural alteration of the metaphyses and epiphyses described a \"popcorn appearance\". Severe cases may have respiratory complications that can compromise survival. Type II OI ---------- In this form of OI, most newborns do not survive the perinatal period. Causes of death are malformations or hemorrhages of the central nervous system \[[@B28]\], extreme fragility of the ribs, or pulmonary hypoplasia \[[@B29]\]. The infants present with multiple intra-uterine fractures, including skull, long bones and vertebrae, beaded ribs, and severe deformity of the long bones \[[@B30]\]. Prenatal differential diagnosis between severe and lethal OI is usually not possible. Extremely severe cases can be dismembered during delivery \[[@B31]\]. The vast majority of cases are autosomal dominant new mutations \[[@B32]-[@B34]\]. It has been suggested that there may be different clinical forms of lethal OI \[[@B35]\]. Despite severity, a few patients have survived for several years. Congenital brittle bones with dense areas in bones -------------------------------------------------- Described in one infant \[[@B17]\] who died shortly after birth and presented with an OI phenotype that differed from the usual lethal form. The skeleton had regions of increased bone density, and this girl had dysmorphic facial features, including loss of mandibular angle, low set ears, soft skull, and large anterior and posterior fontanelles. Bilateral upper and lower limb contractures were present with multiple fractures in the long bones and ribs. The metaphysis of the long bones were dense in x-rays. The patient died after a few hours and histopathological studies identified extramedullary hematopoiesis in the liver, little lamellar bone formation, decreased number of osteoclasts, abnormally thickened bony trabeculae with retained cartilage in long bones, and diminished marrow spaces similar to those seen in dense bone diseases such as osteopetrosis and pycnodysostosis. Genetic testing showed that the child was heterozygous for a COL1A14321G → T transversion in exon 52 that changed a conserved aspartic acid to tyrosine (D1441Y). Abnormal proa1(I) chains were slow to assemble into dimers and trimers, and abnormal molecules were retained intracellularly for an extended period \[[@B17]\]. Because of this mutation, this form of congenital brittle bones should be included in the OI group. Syndromes resembling OI (SROI) ------------------------------ ### Congenital brittle bones with craniosynostosis and ocular proptosis (Cole-Carpenter syndrome) Two boys \[[@B15]\] and a girl \[[@B36]\] with this particular form of SROI have been described in the literature. Both boys were normal at birth, but after several months, they developed multiple metaphyseal fractures, associated with low bone density in the entire skeleton and craniosynostosis, hydrocephalus, ocular proptosis, and facial dysmorphism. One of the patients had also hypercalciuria. Neurological development is normal in this form of SROI. Both patients where wheelchair-bound at adult age, with very short stature, severe bone involvement and normal intellectual and neurological development \[[@B10]\]. No mutation has been identified as causing this syndrome. ### Congenital brittle bones with congenital joint contractures (Bruck syndrome) First described by Bruck et al in 1897 in an adult patient \[[@B16]\], in this form of SROI patients are born with brittle bones, leading to multiple fractures and joint contractures and pterygia (arthrogryposis multiplex congenita) due to dislocation of the radial head \[[@B37],[@B38]\]. Wormian bones are present, and inheritance appears to be recessive \[[@B39],[@B40]\]. In three patients that underwent pro-collagen mutation testing, it was not possible to demonstrate any mutations in the COL1A1 and COL1A2 genes \[[@B38]\]. The basic defect was mapped to locus 17p12 (18 cM interval), where a bone telopeptidyl hydroxylase is located \[[@B41]\]. The mutation leads to underhydroxilated lysine residues within the telopeptides of collagen type I, and therefore to aberrant crosslinking in bone, but not in cartilage or ligaments. ### Osteoporosis pseudoglioma syndrome \[[@B20],[@B21]\] This form of SROI was first described in three families in 1972 \[[@B42]\]. Other report described a South African family of Indian origin with the condition \[[@B43]\]. Inheritance is autosomal recessive. Individuals with Osteoporosis-pseudoglioma syndrome have mild to moderate OI with blindness due to hyperplasia of the vitreous, corneal opacity and secondary glaucoma. The ocular pathology may be secondary to failed regression of the primary vitreal vasculature during fetal growth \[[@B44]\]. The genetic defect has been mapped to chromosome region 11q12-13 \[[@B45]\]. The defect is specifically in the LRP5 gene that encodes for the low-density lipoprotein receptor-related protein 5 \[[@B44]\]. Treatment with pamidronate has shown promising results in this group of patients \[[@B46]\]. ### Other forms of SROI with ocular involvement Two other forms of SROI with ocular involvement have been described: one variant with optic atrophy, retinopathy, and severe psychomotor retardation \[[@B18]\], another with microcephaly and cataracts \[[@B19]\]. ### Congenital brittle bones with redundant callus formation Originally published as \"type V OI\" \[[@B12]\]. Patients with this syndrome develop hyperplastic calluses in long bones after a fracture or intramedullary rodding surgery \[[@B22]\]. These patients present with hard, painful and warm swellings over long bones which initially may suggest inflammation or osteosarcoma. After a rapid growth period, the size and shape of the callus may remain stable for many years \[[@B47]\]. Microscopically there is increased production of abnormal extracellular matrix, that is poorly organized and incompletely mineralized in the callus area \[[@B48]\]. Histological studies in bone outside the callus area showed that the bone lamellae are arranged in a mesh-like fashion, as opposed to a parallel arrangement in patients with OI \[[@B12]\]. A series of case reports of hyperplastic callus formation in patients with clinical characteristics compatible with OI can be found in the literature \[[@B3],[@B47],[@B49]-[@B54]\]. These large calluses may also be present in flat bones \[[@B55]\]. These patients may also have calcification of the interosseous membrane between the radius and ulna, determining a clinical sign, as affected individuals are unable to pronate and supinate the forearm. The radial head may be dislocated. Patients with this syndrome have white sclera and no DI. Mutations in the pro-collagen genes could not be identified so far. Inheritance appears to be autosomal dominant, with variable penetrance. ### Congenital brittle bones with mineralization defect Undistinguishable from moderate to severe OI on a clinical basis, this rare form of SROI \[[@B13]\], has been classified as \"Type VI OI\". It can only be diagnosed by bone biopsy, where a mineralization defect affecting the bone matrix and sparing growth cartilage is evident. These patients have no DI and no wormian bones. Despite the histological mineralization defect, there are no radiological signs of growth plate involvement. The pattern of inheritance is not clear, but the case of two siblings from healthy consanguineous parents has been described, suggesting gonadal mosaicism or a somatic recessive trait \[[@B13]\]. No mutations of COL1A1 and COL1A2 genes have been found in these patients, and collagen structure appears to be normal. This form of SROI shares several characteristics with fibrogenesis imperfecta ossium \[[@B56],[@B57]\]. ### Congenital brittle bones with rhizomelia A particular form of SROI with short humerus and recessive inheritance was described in a First Nations community of Quebec, and published as \"type VII OI\" \[[@B14]\]. The individuals affected have short humeri and femora. The phenotype is clinically moderate to severe. Fractures may be present at birth, and the condition progresses to early lower limb deformities, coxa vara and osteopenia. The bone morphology in congenital brittle bones with rhizomelia is not different than that of mild OI by histomorphometry. The genetic defect has been mapped to the short arm of chromosome 3 by linkage studies \[[@B58]\], where there are no genes that codify for type I pro collagen. Summary ======= A definition for osteogenesis imperfecta is proposed based on the presence of type I pro-collagen mutations. Inclusion of related syndromes with unidentified mutations should be considered as SROI until further defined. Different forms of syndromes with congenital brittle bones are reviewed. A debate about the definition of OI and a possible clinical and prognostic classification are warranted. List of abbreviations ===================== OI Osteogenesis Imperfecta SROI syndromes resembling osteogenesis imperfecta DI Dentinogenesis imperfecta Competing interests =================== None declared. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2431/4/16/prepub>

PubMed Central

2024-06-05T03:55:47.520336

2004-8-31

PMC516445

Background ========== About 8% of the Earth\'s crust is comprised of aluminium. This element is the most abundant metal naturally present in air, soil and water. Consequently, environmental exposure to aluminium is potentially possible. Its ingestion is unavoidable since aluminium compounds are added not only to most water supplies but also to many processed foods and medicines. Aluminium is a known neurotoxicant. It enters the brain, where it contributes to some neuro-degenerative diseases including dialysis encephalopathy, osteomalacia, osteodystrophy, in particular those related to dialysis treatment of uremic subjects \[[@B1]\]. Only a small amount of Al(III) ions in dialysis solutions may cause these disorders. Aluminium may contribute to Alzheimer\'s disease \[[@B2]\]. Aluminium is also able to give rise to toxicity in the bones and hematopoietic system in humans \[[@B3]\]. Positively charged aqua and hydroxy-monomeric forms have been found to be the most toxic species of aluminium to living organisms in the terrestrial and aquatic environments \[[@B4]\]. Generally, aluminium sulphate is used as a coagulant in the treatment of water to help the removal of suspended matter and highly coloured humic substances \[[@B5]\], thus reducing the dose of chlorine later required to ensure satisfactory microbiological quality. Hence, potable water often contains high aluminium levels of natural origin and/or from the water purification process \[[@B6]\]. The selective removal of aluminium ions have been extensively investigated by applying several techniques \[[@B7]-[@B9]\]. Among them, the use of specific polymeric adsorbents has been considered as one of the most promising techniques \[[@B10],[@B11]\]. Specific adsorbents consist of a ligand (e.g., reactive textile dye, ion-exchange functional groups or chelating agents) which interacts with the metal ions specifically, and a carrier solid matrix. There have been several separation approaches performed under magnetic field \[[@B12]\]. The most well known technique is the magnetically stabilized fluidized bed. Magnetically stabilized fluidized bed exhibits combination of the best characteristics of both packed and fluidized bed. These include the efficient fluid-solid mass transfer properties, elimination of particle mixing, low pressure drop, high feed-stream solid tolerances, good fluid-solid contact, elimination of clogging and continuous countercurrent operation \[[@B13]\]. Especially, when dealing with highly viscous mediums contact with the magnetic adsorbent in a magnetically stabilized fluidized bed is desirable because of high convective transport rates. Recently, there has been increased interest in the use of magnetic adsorbents in biomolecule coupling and nucleic acid purification \[[@B14]\]. Magnetic adsorbents can be produced using inorganic materials or polymers. High mechanical resistance, insolubility and excellent shelf life make inorganic materials ideal as adsorbent. The main disadvantage of inorganic supports is their limited functional groups for ligand coupling. Magnetic adsorbents can be porous or non-porous \[[@B15]\]. They are more commonly manufactured from polymers since they have a variety of surface functional groups which can be tailored to use in different applications \[[@B16]-[@B22]\]. In the present study, we attempted to use alizarin yellow-attached magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads as specific adsorbent for aluminium removal from aqueous solutions in a magnetically stabilized fluidized bed. Al(III) adsorption on the alizarin yellow-affinity beads from aqueous solutions containing different amounts of Al(III) ions and at different pHs is reported here. Finally, reuse of the dye-affinity beads is also discussed. Materials and methods ===================== Materials --------- 2-hydroxyethyl methacrylate (HEMA), was purchased from Sigma (St. Louis, MO, USA), and was purified by vacuum distillation under a nitrogen atmosphere. The comonomer, ethylene dimethacrylate (EDMA, Merck, Darmstadt, Germany) was used as the crosslinking agent. Magnetite particles (Fe~3~O~4~, diameter \< 1 μm) were obtained from Aldrich (USA). Alizarin yellow (3,4-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid, sodium salt mono-hydrate) was purchased from BDH (Poole, UK). All other chemicals were obtained from Merck as analytical grade. All water used in the adsorption experiments was purified using a Barnstead (Dubuque, IA) ROpure LP^®^reverse osmosis unit with a high flow cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 NANOpure^®^organic/colloid removal and ion exchange packed bed system. Preparation of magnetic PHEMA beads ----------------------------------- Details of the preparation and characterization of the mPHEMA beads were reported elsewhere \[[@B23]\]. The mPHEMA beads were prepared by suspension polymerization. A typical suspension copolymerization procedure of mPHEMA beads was performed as below: The dispersion medium was prepared by dissolving 200 mg of poly(vinyl alcohol) (PVA; molecular weight: 50.000) within 50 ml of distilled water. The desired amount of 2,2\'-azobisisobutyronitrile (AIBN) (0.06 g) was dissolved within the monomer phase 12.0/4.0/8.0 ml (EDMA/HEMA/toluene) with 1.0 g magnetite particles. This solution was then transferred into the dispersion medium placed in a magnetically stirred (at a constant stirring rate of 600 rpm) glass polymerization reactor (100 ml) which was in a thermostatic water bath. The reactor was flushed by bubbling nitrogen and then was sealed. The reactor temperature was kept at 65°C for 4 h. The temperature was then raised to 90°C and kept constant by a thermostated water bath during the polymerization time (2 h). After polymerization, the mPHEMA beads were separated from the polymerization medium. The residuals (e.g., unconverted monomer, initiator and other ingredients) were removed by a cleaning procedure. Briefly, beads were transferred into a reservoir, and washing solutions (i.e., a dilute HCI solution, and a water-ethanol mixture) were recirculated through the system which includes also an activated carbon column, to be assured that the magnetic beads were clean. Purity of the magnetic beads was followed by observing the change of optical densities of the samples (λ: 280 nm) taken from the liquid phase in the recirculation system, and also from the DSC thermograms of the magnetic beads obtained by using a differential scanning microcalorimeter (Mettler, Switzerland). Optical density of uncleaned magnetic beads was 2.63, but after the cleaning operation this value was reduced to zero. In addition, when the thermogram of uncleaned beads was recorded, it had a peak around 60°C. This peak might originate from AIBN, but after application of the cleaning procedure, no peak between 30--100°C was observed on the thermogram. The dry density of the magnetic beads was measured with pycnometer by dispersing the dry beads in ethanol. Alizarin yellow attachment -------------------------- Preparation and characterization of the alizarin yellow-attached mPHEMA beads were reported in our previous paper in detail \[[@B24]\]. In order to prepare the alizarin yellow-attached magnetic beads following procedure was applied. 5.0 g of dry magnetic beads was weighed and transferred into the SOCl~2~(Carlo Erba, Italy) (10 ml). This reaction medium was boiled in rotary evaporator for 6 h. Then, 2.5 g alizarin yellow was dissolved in absolute ethanol (30 ml). Alizarin yellow-attachment process was performed in ethanol solution for 24 h. At the end of this reaction period, the alizarin yellow-attached beads were removed by filtration and washed with ethanol, water and tetrahydrofuran several times until all the unbound dye molecules were removed. The dye attached beads were stored at 4°C with 0.02% sodium azide to prevent microbial growth. The leakage of the alizarin yellow from the dye-attached beads was investigated within the media at the selected pH in the range of 2.0--7.0. These media were the same which were used in the Al(III) adsorption experiments. The medium with the dye attached magnetic beads was stirred for 24 h at room temperature. Then, magnetic beads were separated from the medium, and the alizarin yellow concentration was measured in the liquid phase by spectrophotometry at 500 nm. Magnetically stabilized fluidized bed procedure ----------------------------------------------- Al(III) adsorption studies were carried out in a magnetically stabilized fluidized bed. Beads suspended in pure water were degassed under reduced pressure (by using water suction pump) and magnetically stabilized into a column (10 cm × 0.9 cm inside diameter) equipped with a water jacket for temperature control. The vertically oriented magnetic field was produced by passing DC current through two modified Helmholtz coils (1.5 cm diameter × 2.5 cm thick) spaced 4 cm apart. At a current of 1.6 A (50 W), each coil produced a magnetic field of 40 Gauss. Equilibration of the column was performed by passing four column volumes of phosphate buffer (pH: 7.4) before injection of the Al(III) solution. In a typical adsorption system, 50 ml of the aqueous Al(III) solution was passed through the column containing magnetic beads, by a peristaltic pump for 2 h. After loading, the column was washed with deionized water to wash out Al(III) impurities. The concentrations of the Al(III) ions in the aqueous phases after the desired treatment periods were measured by using a graphite furnace atomic absorption spectrophotometer (AAS 5EA, Carl Zeiss Technology, Zeiss Analytical Systems, Germany). Deuterium background correction was used. Pyrolitic graphite coated tubes were used for AAS measurements. The instrument response was periodically checked with known Al(III) solution standards. The experiments were performed in replicates of three and the samples were analyzed in replicates of three as well. For each set of data present, standard statistical methods were used to determine the mean values and standard deviations. Confidence intervals of 95% were calculated for each set of samples in order to determine the margin of error. In the first group of experiments, the flow rate of the aqueous solution (i.e., 50 ml of the solution with a Al(III) content of 50 mg/L) was changed between 0.5--3.0 mL/min. In the second group of experiments, Al(III) adsorption from aqueous solution was studied at different pH\'s (2.0--7.0). Adsorption isotherm was also obtained in the magnetically stabilized fluidized bed. Aqueous solutions containing different amount of Al(III) were used in these experiments. The changes in the Al(III) concentration with time was followed to obtain the adsorption curves. The amount of adsorbed Al(III) per dry magnetic beads was calculated by using the concentrations of the Al(III) in the initial solution and in the equilibrium. Desorption and repeated use --------------------------- In all cases adsorbed Al(III) ions were desorbed using 0.1 M HNO~3~solution. In a typical desorption experiment, 50 ml of the desorption agent was recirculated through the magnetically stabilized fluidized bed containing dye-affinity magnetic beads for 1 h. The concentrations of the Al(III) ions in the desorption medium were measured by using a graphite furnace atomic absorption spectrophotometer. The desorption ratio was calculated from the amount of Al(III) adsorbed on the magnetic beads and the final Al(III) concentration in the desorption medium. In order to test the reusability of the dye-affinity magnetic beads, Al(III) adsorption-desorption procedure was repeated ten times by using the same magnetically stabilized fluidized bed. Batch procedure --------------- Adsorption of Al(III) from aqueous solution was also investigated in batch experiments. Aqueous Al(III) solution (50 ml) was treated with the magnetic dye-affinity beads at room temperature, in the flasks agitated magnetically at an agitation speed of 600 rpm for 2 h. The suspension was brought to pH 5.0 by adding sodium hydroxide and nitric acid. The pH was maintained in a range of ± 0.1 units until equilibrium was attained. Polymer amount was kept constant at 100 mg per 50 ml. Al(III) determination was performed in water sample in an atomic absorption spectrophotometer coupled to a graphite furnace atomiser. Adsorption values (mg/g) were calculated as the difference in Al(III) ion concentration of the pre- and post adsorption solutions divided by the weight of dry magnetic affinity beads. Results and discussion ====================== Characteristics of mPHEMA beads ------------------------------- mPHEMA beads (in the size range of 80--120 μm) carrying alizarin yellow were prepared as a specific affinity adsorbent for removal of Al(III) from the water which was used for preparation of dialysis solution. mPHEMA beads used in this study were prepared and characterized in our earlier study \[[@B24]\]. The main criteria of selection of PHEMA is due to its mechanical strength and chemical stability. With the goal of testing the mechanical stability of the magnetic beads, a sample of these magnetic beads was treated in a ball mill for 60 min. Negligible percentage of the sample was broken. The dry density of the magnetic beads was measured as 1.27 g/cm^3^. The magnetic beads are crosslinked hydrogels. They do not dissolve in aqueous media, but do swell, depending on the degree of cross-linking and on the hydrophilicity of the matrix. The equilibrium swelling ratio (the ratio of the volumes of the microbeads before and after swelling) of the beads used in this study is 34%. The simple incorporation of water weaken the secondary bonds within the hydrogels. This enlarges the distance between the polymer chains and causes the uptake of water. It should be mentioned that the water uptake properties of the mPHEMA beads did not change after Alizarin Yellow attachment. After the attachment of the dye (i.e., alizarin yellow) the size of the swollen beads did not change, but the colour became dark yellow, which is a clear indication of the incorporation of the dye molecules in the structure of the mPHEMA microbeads. As shown in our previous paper, the dye molecules were attached to the mPHEMA beads by covalent bonding via hydroxyl groups \[[@B24]\]. The mPHEMA beads containing 208 μmol alizarin yellow/g polymer, which was the maximum amount of dye-attachment that we have reached, were used in this study. Alizarin Yellow release from the mPHEMA beads was also monitored continuously. There were no dye release in any of the adsorption and desorption media, which assured that the cleaning procedure used for removal of physically adsorbed alizarin yellow molecules from the mPHEMA beads was satisfactory. Column performance ------------------ The adsorption capacity at different flow-rates are given in Figure [1](#F1){ref-type="fig"}. The adsorption capacity decreased significantly from 17.2 mg/g to 6.9 mg/g polymer with the increase of the flow-rate from 0.5 ml/min to 3.0 ml/min. One of the explanation for such phenomenon would be a faster ligand-metal ion (i.e., alizarin yellow) dissociation rate compared to the association rate. Hence, the adsorbate (i.e., Al(III) ions) would pass through the magnetically stabilized column without adsorption at high flow-rate. Second explanation could be that the increased nonideal flow hydrodynamics of liquid phase and the solid phase for magnetically stabilized fluidized bed. These phenomena can be summarized by the increase of the axial dispersion coefficient in the axial dispersion model \[[@B25]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effect of flow-rate on Al(III) adsorption. Alizarin yellow loading: 208 μmol/g; Al(III) concentration: 50 mg/L; pH: 5.0; T: 25°C. :::  ::: Adsorption capacity ------------------- Figure [2](#F2){ref-type="fig"} shows the adsorption profile of Al(III) ions. The amount of Al(III) ions adsorbed per unit mass of the polymer (i.e. adsorption capacity) increased first with the initial concentration of Al(III) ions then reached a plateau value at about an initial Al(III) ions concentrations of 50 mg/L, which represents saturation of the active attachment sites (which are available for Al(III) ions) on the beads. The maximum adsorption capacity of Al(III) ions was of 647 μmol/g (17.5 mg/g). Unit mass of the mPHEMA beads carries 208 μmol alizarin yellow which was found by elemental analysis. From the mass-stoichiometry, it seems that one attached alizarin yellow molecule interacts with around three Al(III) ions. Since alizarin yellow has seven coordinating sites of a single sulphur and six oxygen atoms, it can form a ternary complex which is coordinated with water molecules at vacant coordination sites of metal-alizarin yellow complexes. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effects of Al(III) concentration on Al(III) adsorption. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; pH: 5.0; Adsorption time: 60 min; T: 25°C. :::  ::: It should be noted that the nonspecific adsorption (adsorption on plain mPHEMA beads) of Al(III) ions was relatively low (0.63 mg/g). mPHEMA beads do not contain ion exchange or chelating groups. Preferred coordination structure and preferred coordinating ligand atom may be utilized for this adsorption. Al(III) ions may interact with Oxygen atoms as the ligand. Diffusion of Al(III) ions into the swollen polymeric structure and retention in the pores may also contribute to this nonspecific Al(III) adsorption. Adsorption isotherms -------------------- An adsorption isotherm is used to characterize the interactions of each molecule with the adsorbent. In this case it provides a relationship between the concentration of the Al(III) ions in the solution and the amount of Al(III) ions adsorbed on the solid phase when the two phases are at equilibrium. The Langmuir adsorption model assumes that the species are adsorbed at a fixed number of well-defined sites, each of which is capable of holding only one molecule. These sites are also assumed to be energetically equivalent, and distant from each other so that there are no interactions between molecules adsorbed on adjacent sites. Adsorption isotherms were used to evaluate adsorption properties. The Langmuir adsorption isotherm is expressed by Equation 1. The corresponding transformations of the equilibrium data for Al(III) gave rise to a linear plot, indicating that the Langmuir model could be applied in these systems and described by the equation: Q = Q~max~. b . C~eq~/ (1 + bC~eq~)     (1) where Q is the adsorbed amount of Al(III) (mg/g), C~eq~is the equilibrium Al(III) concentration (mg/mL), b is the Langmuir constant (mL/mg) and, Q~max~is the maximum adsorption capacity (mg/g). This equation can be linearized so that C~eq~/Q = 1/(Q~max~. b) + C~eq~/Q~max~.     (2) The plot of C~eq~versus C~eq~/Q was employed to generate the intercept of 1/Q~max~.b and the slope of 1/Q~max~. The maximum adsorption capacity (Q~max~) data for the adsorption of Al(III) was obtained from the experimental data. The correlation coefficient (R^2^) was 0.989. The Langmuir adsorption model can be applied in this affinity adsorbent system. Maximum adsorption capacities determined using Langmuir isotherms show that dynamic capacity (25.3 mg/g) was somewhat higher than the batch capacity (12.6 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Effect of pH ------------ Metal ion adsorption onto specific adsorbents is pH dependent. In the absence of complexing agents, the hydrolysis and precipitation of the metal ions are affected by the concentration and form of soluble metal species. The solubility of metal ions is governed by hydroxide or carbonate concentration. Hydrolysis of metal ions becomes significant at approximately pH 7.5--8.5. Therefore, in the present study, we changed the pH range between 2.0--7.0. The effect of pH on the Al(III) adsorption of this alizarin yellow-attached mPHEMA beads is also shown in Figure [3](#F3){ref-type="fig"}. The magnetic mPHEMA beads exhibited a low affinity in acidic condition (pH \< 4.0), a somewhat higher affinity between pH 4.0 and 7.0. High adsorption capacities at around neutral pH values imply that Al(III) ions interact with dye molecules not only through the oxygen atoms by chelating, but also electrostatically through sulfonate groups, which are ionized at neutral pH. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effects of pH on Al(III) adsorption. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; Al(III) concentration: 50 mg/L; Adsorption time: 60 min; T: 25°C. :::  ::: Competitive adsorption ---------------------- Competitive adsorption of the metal ions from tap water in Ankara and dialysis water (reverse osmosis) was also investigated. The water containing different amounts of each metal ion was treated with dye beads in MSFB. Table [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"} show the adsorbed amounts for each metal ion. The adsorption capacity of the dye-attached mPHEMA beads for Cu(II) and Al(III) ions was higher than that for other ions. But it should also be noted that the extent of adsorption of each type of metal ion is strongly dependent upon their relative concentrations within the medium. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Aluminium removal from the tap water. ::: Metal Ion % Concentration of Metal Ions (ng/ml) Metal Ion Adsorption (μg/g) Adsorbed Metal Ions (%) ------------- ------------------------------------- ----------------------------- ------------------------- Al(III) 80.1 40.3 ± 0.1 98.9 Fe(III) 32.1 10.8 ± 0.2 54.3 Cu(II) 145.3 28.3 ± 0.2 35.1 Cd(II) 0.05 nd \- Pb(II) 0.03 nd \- Zn(II) 20.4 1.5 ± 0.1 28.6 Adsorption Conditions: Flow-rate: 0.5 ml/min; pH: 5.0; T: 25°C, nd: not determined. Each experiment was repeated three times. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Aluminium removal from dialysis water. ::: Metal Ion % Concentration of Metal Ions (ng/ml) Metal Ion Adsorption (μg/g) Adsorbed Metal Ions (%) ------------- ------------------------------------- ----------------------------- ------------------------- Al(III) 18.96 9.46 ± 0.1 99.7 Fe(III) 0.05 \- \- Cu(II) 0.82 0.16 ± 0.01 40.0 Zn(II) 1.26 0.44 ± 0.01 69.8 Adsorption Conditions: Flow-rate: 0.5 ml/min; pH: 5.0; T: 25°C. Each experiment was repeated three times. ::: The World Health Organization (WHO) and the European Community (EC) guide values for Al(III) ions for tap water is 200 ng/ml \[[@B26],[@B27]\]. Al(III) concentrations both in tap water and dialysis water are below this value. It should be noted that polymer treatment (i.e, adsorption) significantly decreases the metal content and these purified waters can be used safely especially for the preparation of dialysis solutions. Magnetic dye-affinity beads exhibits the following metal ion affinity sequence: Al(III) \> Cu(II) \> Fe(III) \> Zn(II). Desorption and repeated use --------------------------- Desorption ratios were very high (up to 97.6%) with the eluant system and under conditions used. When HNO~3~is used as a desorption agent, the coordination spheres of chelated Al(III) ions are disrupted and subsequently Al(III) ions are released from the solid surface into the desorption medium. Therefore, we conclude that HNO~3~is a suitable desorption agent for the dye adsorbents, and allows their repeated use. In order to show the reusability of the dye-attached mPHEMA beads, adsorption-desorption cycle was repeated ten times by using the same sample of affinity adsorbent. As can be seen from Figure [4](#F4){ref-type="fig"}, adsorption capacities did not noticeable change during the repeated adsorption-desorption cycles. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Repeated use of dye-attached mPHEMA beads. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; Al(III) concentration: 50 mg/L; Adsorption time: 60 min; pH: 5.0; T: 25°C. :::  ::: Comparison of magnetically stabilized fluidized bed and batch system -------------------------------------------------------------------- As can be seen in Figure [5](#F5){ref-type="fig"}, maximum Al(III) adsorption from aqueous solution is 11.8 mg/g for batch system and 17.5 mg/g for MSFB system. These results indicated that the adsorption capacity obtained in MSFB system is considerably higher than obtained in batch sytstem. This means, in equilibrium binding experiments, maximum capacity was 38.8% lower as compared to the value obtained in MSFB. This result could be explained in two ways. (i) The dye ligand-Al(III) dissociation rate in the batch system is higher than the association rate in the MSFB system. (ii) Alizarin yellow ligand is found both on the surface and in the pores of the magnetic beads. In the presence of flow, the Al(III) solution is forced from the surface into the pores thus eliminating the surface diffusion. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Comparison of MSFB and batch system. Alizarin yellow loading: 208 μmol/g; Flow-rate: 0.5 ml/min; Al(III) concentration: 50 mg/L; Adsorption time: 60 min; pH: 5.0; T: 25°C. :::  ::: Conclusions =========== The medical relevance of aluminium has stimulated the development of cost and time effective separation techniques including polymeric carriers. Magnetic adsorbents have several potential advantages over conventional adsorbents \[[@B28]-[@B32]\]. The magnetically stabilized columns require faster processing times and high flow-rates with a much lower operating pressure than a packed bed column. In this study, mPHEMA beads, in the size fraction of 80--120 μm, were produced by a dispersion polymerization of EGDMA and HEMA in the presence of magnetite particles. These novel magnetic beads were then successfully attached with reactive dye-ligand, namely alizarin yellow. The highest dye loading was 208 μmol/g. Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 4.0. Al(III) adsorption onto the mPHEMA beads was negligible (0.63 mg/g). Higher adsorption values (up to 17.5 mg/g) were observed using alizarin yellow attached mPHEMA beads for the adsorption of Al(III) ions from aqueous solutions. Al(III) ions could be repeatedly adsorbed and desorbed without significant losses in their adsorption capacities.

PubMed Central

2024-06-05T03:55:47.521890

2004-8-26

PMC516446

Background ========== Harm reduction programs operate with the assumption that some people who engage in high-risk behaviors are unwilling or unable to abstain. Using a \"low-threshold approach,\" they do not require that clients abstain from drug use in order to gain access to services, nor expect adherence to one service to be eligible for another. Rather than having abstinence goals set for them, clients in such programs take part in a goal-setting process, an approach that has been shown to correlate consistently with retention and success \[[@B1]-[@B3]\]. Providers help clients make connections among their complex attitudes, behaviors, and the change they are trying to pursue as a result of an interactive process -- not a dogmatic format. Behavior change is regarded as incremental and based on the premise that people are more likely to initiate and maintain behavior changes if they have the power both to shape behavioral goals and enact them. Research on harm-reduction/syringe-exchange programs has been limited largely to demonstrating their success with reducing the transmission of HIV/AIDS among drug users \[[@B4]-[@B11]\]. While this is an important accomplishment, little is known about their impact in assisting drug users in making changes in life conditions, circumstances, and quality of life. This is partially because few efforts have been made to establish appropriate measures of client and program progress in these areas. The traditional field of drug treatment has generated many assessment tools including the Addiction Severity Index used extensively for treatment planning and outcome evaluation \[[@B12]\]. This tool, and others like it, such as the Chemical Dependency Assessment Profile (CDAP) \[[@B13]\] and the assessment forms created at the Institute of Behavioral Research at Texas Christian University (TCU/DATAR), generate severity ratings that are subjective ratings of the client\'s need for treatment derived by the clinician. The ASI interview asks questions related to domains or \"problem areas\" in substance abusing patients that have been determined by clinicians, not the patients themselves. Thus, despite the formally established validity and reliability of the tool, and others like it, the measures are developed from the perspective of the clinician and researcher and are designed to generate information that is consistent with their view of the world, not the world of drug users. Given the tenets of harm reduction in which drug users participate in their own goal setting, such tools lack cultural sensitivity and relevance. Denning\'s work on harm reduction psychotherapy (2000) is considerably more grounded in the life circumstances of drug users. Her Multidisciplinary Assessment Profile (MAP), a baseline assessment tool to use with chemical dependency clients, however, was not designed to generate information about what drug users consider to be realistic goals and progress towards these goals. If service providers are to guide an effective interactive process of goal setting, it is important that they understand the parameters of realistic, incremental behavior change from the perspective of the client. Since the development of the ASI, there has been growing movement to acknowledge the value of participatory research in which the \"subjects\" of research become directly involved with shaping the research agenda and designing data collection tools. Such an approach empowers the community participating in the research so that members are not objects acted upon but rather partners in an endeavor to improve their circumstances. This approach increases the cultural appropriateness of the way the research is conducted, the potential validity and reliability of the data that are generated, and the utility of the results. In order to advance the study of harm reduction programs and our understanding of how drug users define their own progress, we conducted participatory research to develop outcomes of harm reduction programming. The goal of the research was to involve program clients in a process that would generate valid measures that are 1) culturally relevant to the way they see the world and live their lives; 2) incremental -- i.e., capable of measuring small changes, and 3) hierarchical -- i.e., capable of showing how clients improve over time. This article summarizes information on the research methods used and the outcomes that were generated. Methods ======= The research study was conducted in two phases. In the first phase, drug users participated in a group process using nominal group technique (NGT) \[[@B15]\] to develop the outcome measures. In the second phase, other drug users participated in focus group interviews to reflect on the measures developed and their validity for the drug-using population. Below, the sample and methods of the two phases are described in more detail. Sample ------ Study participants were recruited from the New York Harm Reduction Educators, Inc. program, which has delivered comprehensive services to over 40,000 enrollees at six sites located in East Harlem and the Bronx, New York. To involve recipients in the study, clients were recruited by program staff and given a \$10 incentive for participation. The study was advertised at the main program site, and a stratified convenience sample of approximately 120 clients was recruited for phase one of the study, and 120 for phase two, with some duplicate count of clients who participated in both phases of research. The sample was stratified by neighborhood, by time in the harm-reduction program, and by whether participants took part in the syringe exchange only or accessed a fuller range of services. The demographics of the sample closely represented the larger program and included 26 % African American, 50% Latino, and 24% white; 72% male and 28% female; 17% ≤ 29 years of age; 36% between 30--39, and 46% ≥ 40 years of age. Methodology ----------- In the first phase of the research, a facilitator independent of the program used NGT with 10 groups of approximately 12 individuals per group. This group process provides a structure for small group meetings so that client participation is maximized and judgments effectively pooled. The technique was especially helpful in establishing priorities in that it neutralized differences in status and verbal dominance among participants. Before the present study, NGT was used successfully with clients in the program to identify areas of life functioning that people like themselves (i.e., drug users) deemed to be the most important and meaningful in their lives. The top 10 \"life\" areas were money (income); housing; food (nutrition); family relations; self-improvement; connectedness to services/benefits/programs; dealing with negative feelings (mental health); health problems (physical health); and legal and drug use problems. To generate items in a scale for every outcome listed above, 12 people were recruited for a group that used several NGT process steps. First, the facilitator asked the group to contemplate a question related to the outcome of interest. For example, \"What ways do people in your circumstance make money?\" was asked to generate the outcome of source of income; \"What ways or places do people in your circumstance get something good to eat?\" was asked to generate the outcome of source of nutrition; and \"What are the types of places that people in your circumstance live?\" was asked to generate the outcome of housing. Next, the members of the group brainstormed their answer(s) to the question posed to them and the facilitator recorded these answers on a large chart. The facilitator continued to call upon the members until all ideas were recorded. Next, all group members received a packet of 10 index cards numbered 1 to 10. The facilitator engaged the group in ranking the ideas according to every individual\'s order of preference. This step started with the facilitator asking individuals to take out the index card marked \#1. She then asked every group member to select from the list that idea that he/she considered the best (i.e., the best way to make money, the best way/place to get something good to eat, the best place to live, etc.) and write it down on card \#1. She then asked everyone to take out card \#10 and select the idea that he/she considered the worst (i.e., the worst way to make money, the worst way/place to get something good to eat, the worst place to live, etc.) and write it down on card \#10. Finally, she led them through a similar ranking process with cards \#2 -- \#5, used by the individuals to record their \"next best\" preferences and cards \#6 -- \#9 to record their \"next worst\" preferences. This group work resulted in 10 scaled outcomes. In the second part of the research, 10 focus group interviews were conducted to allow more of the target population to reflect on the validity of the measures and further explore the meanings of the scaled items based on the lives of drug users. In most cases, a completely different group of drug users who had participated in the NGT process for a certain outcome were selected to participate in the focus group related to that same outcome. Data Analysis ------------- The analysis of the data from each NGT group for every outcome was done by first eliminating all ideas that received no votes. The remaining data were then analyzed by: 1) determining the total score for all remaining ideas (the individual score of each idea was based on the card number on which it was recorded -- i.e., 1--10 -- and the total was the sum of individual scores); 2) determining the mean score for every idea (dividing the total score for an idea by the total number of votes -- i.e., cards received for the idea; 3) rank-ordering the ideas by mean score; and 4) grouping the 40 or more individual ideas with similar mean scores by larger concepts so that every outcome had about 12 hierarchical, scaled items from best to worst, with mean scores from low to high. Results ======= The results of the preliminary research are displayed in Table 1 (see [Additional file: 1](#S1){ref-type="supplementary-material"}) showing every individual outcome and the hierarchical scale of items that measure incremental change from better to worse. \"Better\" items in every outcome, near the top of the scale, were those items that had received a mean score of 5 or better while \"worse\" items, near the bottom of the scale, were those items that received a mean score of 6 or higher. A summary of results for every outcome is presented below. Money (income) -------------- The outcome \'money (income)\' includes a hierarchical scale of 11 items measuring better to worse \'ways of making money\'. According to the clients, better ways of making money were from entitlements; a legitimate job; from family (i.e., through marriage, inheritance); or borrowing from others. Worse ways of making money were from hustling (i.e., conning or informing to police); stealing (i.e.,\"boosting\" -- stealing); drug trade (e.g., selling, holding, transporting); panhandling; more serious criminal activity (i.e., credit card fraud, robbery, hitman); sex work; and selling blood or body parts. When other program clients reviewed this outcome scale, they felt it reflected their lives overall. One exception was that many felt that \"job\" (employment, peddling, odd jobs, volunteer) should be at the top of the scale rather than \"entitlements.\" Some saw entitlements as \"an easy way to make money -- \"it\'s a way of survival,\" -- while having a job offered more independence and feelings of self-esteem. Other changes suggested by individuals were that \"stealing,\" \"drug trade\" and \"hustling, police informant\" should be further down on the scale while \"panhandling\" should be further up. Strong negative feelings were expressed about being a police informant;\" \"Where I come from, when out on the street\..., you don\'t inform the police! Police will lie to you and use you, then throw you back to the dogs and you\'re dead.\" Focus group participants spoke about what money meant to them and what life was like when one made money via drug trade, sex work or selling blood or body parts. Like most people, they felt having money offered a sense of freedom and independence. In addition, many felt that having it contributed to self-esteem and allowed you to help others you cared for: \"It lets you help a family member who needs help\"; \"It helps support my spouse\... and give some money to my son when I can.\" Regarding money making from drug trade, study participants felt, overall, that it was unstable and risky. It\'s like addiction. It\'s an adrenaline pump \... that keeps you in it for so long. You do it to keep your habit going\... If you sell what you use, it\'s not good. You wind up doing all the drugs, then you have to run for your life from the dealer. It\'s an unstable life\...and the consequences are great if you get caught. You go to jail, get clean, come back out, and start all over again. It\'s a never-ending cycle\.... Eventually your luck runs out and you either go to jail or get killed. Sex work was also described in negative terms: That\'s the last thing you want to have to do, man or woman. Selling your body is the worst thing you could do for yourself. You can get HIV, hepatitis C, or even get killed. You don\'t know who this person is that you are engaging in this sex at with. He could be a serial killer. Selling blood and body parts were described as both limited and, again, risky: Now you are put on a computer and once they have a record of your blood test, you can\'t do it because they can screen you from sight to sight\... If you sell a body part, they might not wait for you to die to get it, they might hunt you down and kill you. Housing ------- The outcome \'housing\' is made up of a hierarchical scale of 11 items measuring the better to worse places to live. Based on client input, better places to live were a house or apartment that you rented or owned; a friend\'s home; a drug program; a family member\'s home; housing provided through a social program; institutionalized housing, such as a shelter or hospital; and homelessness that was considered \"least severe (e.g., sleeping on the subways or at a bus station). Clients considered worse places to live to be jail, homelessness considered to represent an intermediate level of severity (sleeping in cars, bathrooms, hallways, abandoned buildings) or the most severe level (in tunnels, caves, sewers, parks, on a roof). When other program clients reviewed this outcome scale they agreed, overall, that it reflected the reality of their lives. A few suggested minor changes in the scale could be made such as moving \"jail\" as a housing option to the very end of the scale because, as one said, \"I don\'t want my freedom taken away -- it\'s degrading and lowers your self-esteem being subject to a strip search at any time.\" A few others suggested putting \"apt/room that you rent or own\" before \"friend\'s home.\" Most felt that it was better to have your own place than to live with family or friends because \"it makes you feel independent, feel human -- it\'s an accomplishment.\" They also offered some insight on what it was like being a drug user and living with a family member vs. a friend. A family member would let you get away with more than a friend would. With a friend, you would have to be on time, with all of your part of the rent money, food money, and clean up after yourself. With family you might dib and dab a little with the rent money or get out of doing some things around the house. However, when you\'re on drugs, going to your family is not good because they can give you the boot. Other \"windows\" of insight into how drug users deal with housing came in participants\' discussion of being in a drug program, institutionalized housing (shelters, hotels, hospitals, SROs), and living on the street. Participants thought that drug programs offered an important option for housing but also saw them as a last resort: They give you structure and are stable -- you can get food, clothes and confidence. Last resort when you have no place to stay and have no money\... It\'s the place to go if you want to change your life around. Some program clients saw institutionalized housing as a crutch, while others discussed the advantages and disadvantages of various types of institutionalized housing: I became \"shelterized\" after being in a shelter for several years. I got locked into a routine where you don\'t want to take care of yourself because the basics are provided for you. Life was sweet, too sweet -- I had no responsibility. Some hotels, depending on where they are located, have a high level of theft with no security. Hospitals will take you in depending on the weather and your state of mind. The homeless quarters and the psyc ward are often connected. Sometimes you have to fake it, like you need psychiatric care, if you want to get off the street for a while. If you act like you are going to harm yourself, they give you a bed fast. SROs are like apartments but they have rules\... Some don\'t allow company but others do. You have to sign visitors in and out at the desk. Tier 1 and 2 housing is for families. But it\'s only temporary -- 30 to 60 days. Then you have to pack up everything and move somewhere else. Section 8 housing has a lot of limitations. You have to be a family that is homeless living in a shelter, a victim of domestic violence, or in the witness-protection program. You can\'t have any felonies on your record. Some of section 8 is only available if you are HIV positive. Generally, clients felt that living on the street was a last resort but an option that could work: If you bum everything out by not following rules, stealing, looking for fights or taking drugs, then the street will become your home\... But you can make the street work for you if you know how to survive. I did. You pick your area and take a claim for it. I had a half car that was my roof\... I even evicted some people from my neighborhood because they didn\'t act right. We had our own rules. Food (nutrition) ---------------- The outcome \'food (nutrition)\' is made up a hierarchical scale of 11 items measuring the better to worse \'ways/places to get something good to eat\'. According to the clients, better ways/places to get something good to eat were to cook food yourself; from friends or family; from a supermarket; from place that gave out free food (from soup kitchen, shelter, pantry, social gathering); buying food (with food stamps or from money earned); or from a restaurant. Clients considered worse ways/places of getting food were from begging or stealing; from institutions (hospital, jail), from trying to provide for yourself (hunting, fishing); and, lastly, from the garbage. After other program clients reviewed the developed scale, they generally agreed that it reflected their lives. The exceptions were that some felt that \"buying food\" should be placed higher in the scale, preferably at the top. The rationale was that before you could cook food yourself, you needed to buy it. In addition, others felt that \"food from facilities\" and \"providing your own food\" should be placed before stealing food in that stealing food involved risk and possible repercussions. Study participants spoke about what they considered \"something good to eat.\" They most often mentioned that food needed to be tasteful, although not necessarily nutritious. The feelings that they associated with getting something good to eat were \"feeling good,\" \"wanting to act civil,\" and \"wanting to treat people better.\" Feelings associated with not getting something good to eat were \"feeling cheated\" and \"developing an instant attitude.\" They also discussed why \"cooking food yourself\" was higher on the scale than \"going to a restaurant\" or having others prepare the food for you. The importance of self-sufficiency emerged when participants spoke about the value of \"cooking it the way you want it\" and the feeling of comfort that came from \"doing it yourself\" and being able to \"be at home with my girl and be able to afford a full-course meal.\" Participants had much to say about the topic of \"free food\" and the better/worse places of getting. Although \"the price was right,\" and they were all aware of \"street sheets\" listing several places to go for free food, they also spoke about traveling long distances, waiting on long lines, walking up several flights of stairs, and having to have a referral and register with a program to get food. The factors that affected their decision about where to get food were the attitudes of the staff, the quality of food offered (i.e., brand names were preferred over generic, U.S.D.A. -- grade foods), and whether the program also offered other needed services (e.g., some pantries offered services like showers and laundry facilities). Some participants contended that some program staff in pantries \"pick through the groceries and bag up the best stuff for themselves and friends.\" Other revealing insights that drug users had about food were that they did not feel it was ever necessary to steal or beg for food: \"There are plenty of places to get food. Anyone you see stealing food or begging is doing it for a profit, to be able to purchase something else.\" A number of participants referred to \"dumpster divers\" (people who eat street or building garbage) as people who were mentally ill and took great risk of eating contaminated food. Most felt more comfortable eating leftover, pre-wrapped food from fast-food restaurants than resorting to street dumpsters. Family relations ---------------- The outcome family relations includes a hierarchical scale of 15 items measuring the better to worse \'types of family relations\'. At the top of the scale clients considered better ways of relating to family to include loving your family; taking part in special family gatherings; having positive communication (open, honest, tolerant); interacting directly with members (playing games, picnics, talking about family history); arguing; showing support and respect, spending high quality time; and engaging in passive contact (movies, TV, reading the Bible). Clients felt the worse ways of relating to family were showing a lack of respect between members (being stigmatized/disrespected for who you are, talking about drugs around children); members\' having negative attitudes toward one another (envy, judgment, alienation); conflicting lifestyles; engaging in abusive relations (incest, sexual abuse, violence); having difficulties with financial support; abandoning a family member; or being deceitful (stealing, gossiping, lying). Upon reflecting on the scaled outcome later, other program clients generally agreed that the scale reflected their lives, with a few exceptions. Several felt that \"arguing,\" in the middle of the scale, should be placed further down on the scale as they saw it as a way of relating that often leads to abuse. In addition, there was some disagreement on the order of the items considered the worse types of family relations. Some clients felt that \"abusive relations\" should be listed last. These individuals spoke painfully about how abusive relations in their childhood had damaged them throughout life, and others spoke about how exposure of the abuse within their family had created lasting division between members. Client input on the items \"love,\" \"respect,\" and \"negative attitudes\" illuminates the meaning of these terms in the lives of drug users. \"Love\" was seen as a building block and foundation for family relations. It was equated with respect among family members, with one client asserting that \"Love for my family may mean not spending time with them so that I do not expose them to my drug use.\" Clients felt that indicators of family respect were listening, letting people have their say, giving people the benefit of the doubt, and living life your way without interfering with others. Clients reflected on the negative attitudes they had experienced around family members. Along with outwardly judgmental remarks, clients also experienced a great deal of nonverbal behavior that they interpreted as negative attitudes. Examples included when they walked into the room where family members were conversing, and people suddenly stopped speaking or hid their purses. Overall, clients felt that a family member\'s drug use should not necessarily engender negative attitudes among other family members and that their families needed to learn more about understanding the harm reduction approach. Connection to services, programs and benefits --------------------------------------------- The outcome \'connection to services, programs and benefits\' consists of a hierarchical scale of 12 items measuring the better to worse \'types of services/ programs/benefits available to drug users\'. Clients felt better types of services to connect with were those related to housing; HIV/AIDS assistance; mental health; drug treatment; entitlements (i.e., public assistance, SSI, and social services); and harm reduction (outreach, needle exchange, condoms). They considered the less preferred (i.e., worse) available services to be those in mainstream institutions (churches, library, legal services); \"getting-connected\" services (escort services, resource directories); support services (12 step, women\'s groups); family-prevention services (parenting skills, domestic violence services); stress reduction (acupuncture, field trips); and work (WEP) programs. Later, when other clients reviewed the developed outcome, most felt that it overall reflected their lives. The one change that a sizable number of clients called for was to put harm reduction services farther up on the scale. The value they placed on this type of service was shown in a number of comments: Harm reduction has taught us a lot about taking care of yourself physically, mentally and emotionally. If you are using drugs, it teaches you how to use drugs safely and in a safe environment. If you want to stop using, there are places to go to get the help you need. If you are out on the street hustling, selling your body it teaches you about using protection. Harm reduction is very important because it taught me a lot about how to take care of myself, manage my drug use, use my needles properly, and reduce my stress. In addition, certain individuals, based on their circumstances, made other suggested changes. One client who disclosed himself as HIV sero-positive said that \"AIDS-related services\" was the best service on the list for him. Another client remarked that \"drug treatment\" would need to be listed before housing since you are required to be drug-free to get housing. Still another felt that all the listed services were important \"because they can assist me in preparing for my future.\" The clients discussed why \"support services\" (12-step programs, advocacy groups) were fairly far down on the scale. Overall, they felt this was because participation in these programs was dependent on giving up drugs, which some people are not ready to do. They also felt some people do not agree with the philosophy of the programs nor are they ready to be in a group environment. Clients also spoke at length about why the \"WEP (Work Related) program\" was listed last on the scale. Although they thought it might benefit people who have no skills, they felt, overall, that the program was degrading. For those who may have skills, it\'s kind of degrading in a way because you are working for a check that you are receiving from public assistance. For a single person you might get \$68.00 every 2 weeks and you are doing the same kind of work that the people earning above minimum wage are getting. You can be working in the Parks department, cleaning people\'s toilets or picking up paper in the street for the sanitation dept. Some of it can be real degrading and discouraging. Self-improvement ---------------- The outcome \'self-improvement\' consists of a hierarchical scale of 12 items measuring the better to worse \'ways of improving yourself\'. Study participants felt that better ways of improving yourself were having a better relationship with yourself (self-love, respect) and with others; getting and staying clean from drugs; being spiritual; taking part in self-help groups (12-step programs, support groups); working or developing work skills; and engaging in stress-reduction activities. They considered less preferred or \"worse\" ways of improving yourself to be helping others; taking care of yourself (i.e., going to dentist, taking medications, dieting, going to gym); being more responsible (i.e., living on a budget, accomplishing goals); behaving yourself (stop lying, stay out of trouble); and having a hobby (i.e., art work, boating, fishing, hunting). After a different group of program clients had reviewed this outcome, most felt that it adequately reflected their lives. A few individuals suggested that \"caring for self\" and \"being more responsible\" (i.e., items \#9 and \#10) should be listed further up on the scale. In addition, one individual felt that \"becoming more spiritual\" should be first on the scale, \"because if you have God in your life, everything else will fall into place.\" Program clients were asked about the meaning of \"self-improvement,\" \"self-respect,\" \"relating to others\" and other items as they appeared on the scale. Concerning self-improvement, clients often thought of the topic as one that involved personal goal attainment. Setting goals that are positive and reaching them. Then setting another and reaching it, one step at a time\.... Setting up a network that will help me to build a foundation of positive aspects in my life that I can follow. The clients described self-respect as requiring self-esteem, as being linked with showing respect for others and with how you physically appear to others, and as dependent on managing your drug use. If you have self-esteem and care for yourself\..., respect will come. By you respecting yourself and wanting to be treated a certain way, you know you have to respect others to get it back in return. If I looked better, I would feel better about myself. A lot of time when people are drugging, they get caught up in a lot of things and before they know it, they have done some things that have cost them their self-respect, so getting it back is important to be able to get on with your life. In clients\' discussion of the meaning of \"relating better to others,\" several indicators emerged such as honest communication; holding an intelligent conversation about yourself; being comfortable relating your feeling to others; and listening. Their thoughts on \"getting/staying clean\" (item \#3) demonstrated the challenges they face and the degree to which their lives must change to stay clean. It was a hard process for me because I would always fool myself that it wasn\'t the right time\.... You can\'t do it for someone else, it has to be for you. It took me becoming homeless to decide that I had to make some changes in my life. Now that I have a new apartment, I want to keep it. My budget won\'t allow me to get high and keep my rent paid. Once I got out (of jail), my body was clean but my mind was still dirty. Mentally I still wanted to do drugs\... I had to leave people, places and things alone because I feel powerless over the influence of others. Being around positive people and getting the support of groups helped me stay clean. Clients also provided rich detail on what they meant by \"behaving myself\" (item \#11), including this response: It\'s the whole package. Your attitude, the way you talk, the language you use. When you start to change your life for the better, everything changes. You don\'t use a lot of 4-letter words. You want to socialize with different people in a different atmosphere. Not getting high where you work at; being more responsible. Finally, the clients were asked why \"working/developing work skills\" (item \#6) was as far down on the scale as it was. Most acknowledged that this was a goal that many drug users are not yet ready to achieve, given their difficulty functioning in an environment that they are not used to. If you are coming into work and you are in this other world where you are not sick (to others), but you are not well either, it is hard to function. You have to have a functional mind that is able to concentrate on work\...and for a lot of people, they are not there yet. Alternatively, their discussion suggested that volunteering was a better way to approach the world of work: \"I started volunteering here at NYHRE, and I intend to go to computer school so I can get a better job.\" Mental health ------------- The outcome \'mental health\' consists of a hierarchical scale of 13 items measuring the better to worse \'ways of handling negative feelings\'. Study participants felt that better ways of handling negative feelings were getting informal support; (from friends, support groups), spiritual help (going to church, praying); or professional help (from a doctor, counselor); working; engaging in diversions (interacting with children; going to ball game or the beach, singing), or in stress reduction (smoking, massage, sex) and physical activities (exercise, cooking, sports). They considered worse ways of dealing with negative feelings to be engaging in violence against self (suicide, bulimia, anorexia); outward violence (hurting others, breaking things); bringing negative feelings into social relations (into marriage, when visiting someone in jail); withdrawing (\"isolating\"); and engaging in illicit activity (working the streets, using drugs, gambling). When another group of program clients reviewed the scaled outcome, they saw the relevance of all the items and agreed on the general order of the items in the scale. A few clients suggested some minor adjustments in the scale, however. For example, a few felt that \"professional help\" (item \#3) should be considered the best way of handling negative feelings, rather than \"get support\" (from friends, support groups). For the most part, however, the majority of the clients agreed that getting support from friends and support groups was more functional for people in their circumstance than going to a professional because of issues of availability. As one person put it, \"The drug man never sleeps\" and people involved in this culture need easy access to those who can help them with their negative feelings. When you are out in the street drinking and drugging, there is something going on at every corner 24 hours of the day. Support from friends and groups are available to you on those off hours when \"professional help\" is not. This was also the rationale for few clients as to why \"spiritual help\" should be placed before professional help -- you can pray at any time. Other individual clients felt that \"social relationships\" should be further up on the scale because peers and loved ones were often the most understanding. You need communication with someone that understands you and is willing to put up with your shortcomings. Problems do arise if one gets high and the other does not, but you can usually work this out. Things get tough sometimes but she helps to keep the balance in the relationship. My spouse helped me with my addiction. In regard to how drug users resorted to abuse in deal with negative feelings, the clients often referred to circumstances involving drugs. When verbal abuse did not work, they often resorted to physical abuse. A spouse will be abused when you want your drugs and you don\'t have the money. You know that she has the money but she won\'t give it to you. When selling drugs and someone comes to you with short money, even if it is only \$1, he might get his butt whipped. Dealing with health problems ---------------------------- The outcome \'dealing with health problems\' consists of a hierarchical scale of 12 items measuring the better to worse \'ways of handling health problems\'. The clients in the study considered better ways of handling health problems to be using home remedies (external and internal cleansing, praying); stress reduction; drug treatment/therapy; \"clean living\" (i.e., reduced drug use, taking meds, stopping smoking); seeing the doctor; and getting health screenings. They felt less preferred (i.e., worse) ways of dealing with health problems were maintaining a good diet, getting health education information, exercising, using alternative therapies (i.e., fasting, herbs, psychic readings, witch doctors), exhibiting negative emotions (depression, denial, suicide, anger); and using illicit drugs. After another group of program clients reviewed this outcome scale, they generally felt that it reflected their lives, with a few exceptions. Several felt that \"see a doctor,\" \"educate yourself\" and \"alternative therapies\" should be higher on the scale. Most clients felt that \"home remedies\" should stay at the top of the scale because \"they work the best.\" When they spoke about their experiences with doctors, it often was not positive. The waiting is horrible. As an inpatient, you could die before you see a doctor. Once you are identified as an addict, whether on methadone or still using drugs, you\'re discriminated against. Sometimes I am too leery to go to see a doctor. I may wait for someone else to go to the doctor first and then get their opinion. When asked about the health problems they encountered, client usually mentioned serious conditions (cancer, STDs, HIV, pneumonia), indicating that ailments were not a health program unless they had become serious. Regarding drug treatment, clients saw it as a positive way to deal with health problems, with certain parameters. \"Drug treatment is not going to help you if you are not ready to stop using\... It won\'t help you unless you have a follow-up plan like a support network at a church, family or groups, and being around positive people.\" Clients also discussed how other items on the scale were related to their drug use. Several felt that \"using illegal drugs\" should be at the very bottom of the scale, but opinions about this varied based on level of drug use. Clients knew that drugs could eventually bring about bad health but were often so out of touch with their feelings while doing drugs that they thought they were healthy: When I was on a constant run (doing drugs), I didn\'t get sick. Thought I had a wonder drug. Didn\'t feel anything; drugs preserved me. I didn\'t get headaches, toothaches or colds. If I was sick, the drugs controlled my inner body, I couldn\'t feel a thing. They felt that the item on the scale \"educate yourself about health\" was especially important for drug users who are often controlled by their substance: No one used to take vitamins because your drug controlled your mind. You couldn\'t eat properly because you had to get high first. Education about my health has helped me make some changes. Before I didn\'t go to a doctor. Now I make an effort to go on a regular basis. Dealing with drug use problems ------------------------------ The outcome \'dealing with drug-use problems\' consists of a hierarchical scale of 17 items measuring the better to worse \'ways of handling drug-use problems\'. Study participants felt that better ways to handle drug-use problems were to admit the problem (and make amends with family); engage in religious activity (go to church, pray); get social support (from support groups, asking for help, making new positive friends); go into drug treatment; quit using drugs; get professional help (therapy, education about drugs, medications); stay distracted (keep busy, play with kids); and avoid the drug culture (avoid places that trigger drug use, drug paraphernalia). Clients considered the less helpful (i.e., worse) ways of handling drug-use problems were to follow a treatment plan (go to the hospital, take and not sell medications), get family support or spiritual guidance (from 12-step programs, minister); be in jail; be honest with yourself (reflect on past behaviors and pain associated with use); be deceitful (lie, manipulate others); engage in illegal activity (i.e., deal drugs, steal, prostitution); \"isolate\"; and continue to binge. After reviewing the developed outcome, another group of program clients generally agreed that the scale reflected their lives on the better to worse ways of handling drug-use problems. Several people commented that the items that were near the bottom of list, or the worse ways of handling drug use, were not ways of handling the problem but were, in fact, the kinds of things that went on when your drug use was out of control. They described in graphic terms what this looked like: To be focused every minute of every day on just getting the next bag of dope. My life is non-functional\... I am a zombie. Wake up in the morning, get dressed and head straight for the corner to hustle up enough money to get that bag of dope. Binging is like being on a mission. You go all the way out until everything is gone\.... It can be one hour, a day or longer. It is when you have used all your resources and there is no more to be had. There is no one left for you to use or manipulate. They also talked about why it was important to handle problems with drug use. One person admitted, \"Your drug use is like a marriage, something you live with for life,\" and several clients talked about what their life looked like when they were able to handle their drug-use problems. I need to have something with structure in my life to keep going\... so you can function better\... go forward\... handle you apartment, raise your kids, keep yourself clean\... stay out of jail and live a longer life. My everyday life is my life now. The clients made several other insightful remarks about various items in this outcome. They commented on how \"praying\" helped them to function: \"Praying helps me get things straight in my head.\" \"It makes me strong and gives me more confidence,\"; \"Praying makes me more humble\"; and \"When I pray I feel more positive in my thinking.\" Regarding the item \"follow a treatment plan,\" some people felt it was farther down on the scale because of the coercive aspect they associated with it: \"Sometimes following a treatment plan is what you have to do because you have to see your parole officer every week, so you are forced to do it.\" In describing their experience in jail, many felt it did not help with problems with their drug use because it is very easy to get drugs in jail. The clients did feel, however, that it was something to be avoided at all costs: What you experience in jail makes you never want to go through that again. It takes your freedom away. It changed me. Now I don\'t even steal a Hershey bar. Dealing with legal problems --------------------------- The outcome \'dealing with legal problems\' consists of a hierarchical scale of 11 items measuring the better to worse \'ways of handling legal problems\'. The participants in the study considered better ways to handle legal problems were to pay, go see, and speak with a legal professional; address the problem yourself (go to law library, represent yourself, write to the judge); speak to a non-legal person (employer, counselor, parole officer, case manager); respect the law (by serving time, making court appearances); and learn from mistakes. The clients in the study considered worse ways of dealing with legal problems to be disrespecting the law (breaking the law, not respecting authority); facing the consequences of one\'s actions (serve time in prison or drug program, give up parental rights); avoiding legal responsibility (run from parole, leave the state, not show up in court, jump bail, ignore bench warrants); and relying on support from friends. When a different group of program clients reviewed the outcome they agreed overall with the order of the items in the scale. They offered rich detail on specific items on the scale and insight into how drug users experience legal problems. Drug users confront a wide variety of legal problems, including being arrested for various drug-related charges; police harassment related to petty crimes like loitering or suspicion of a crime; legal problems related to one\'s children and the Bureau of Child Welfare; and taking part in a hearing to qualify for SSI. Clients talked about their experience with legal professionals and items near the top of the scale. Many agreed that it was best when you could pay for an attorney, or, as one client put it, \"Money talks and bullshit walks.\" However, they also realized that the steps in dealing with a professional first involved seeing and talking to one to find out the fee for services. Clients had varied experiences with professional attorneys, with several agreeing that legal aid lawyers were most helpful. I prefer legal aid lawyers because they work from the heart and not by what you put in their pockets. In housing I had a legal aid lawyer who helped me in a very positive way. A private, paid lawyer to help me keep my kids did not do what he was supposed to do. It was an SSI case and I had to pay and I got very little help or feedback from the lawyer at all. As with other outcomes that have been reviewed, the participants in the study spoke favorably about trying to solve the problem themselves (item \#4 on the scale). It is good to do everything you can to help yourself first before you pay a legal professional or seek out their help. You might be homeless, out there on the streets\...and ready to come in and get your life together\.... You need to investigate how to clean up any legal problems that may be lingering. Sometimes friends may have gone through a similar experience and can tell you some of the steps they took to avoid jail or paying fines. Clients talked about how they \"learned from legal mistakes\" (item \#7) and what \"disrespect the law\" (item \#8) meant to them. Learning from legal mistakes often involved experiencing the consequences when the police caught up with you: I use to smoke my pipe out on the street and didn\'t care about the cops or anybody\...When I saw the cops, I would run and hide and thought I got away from them. But when I came out of hiding, they were waiting for me and I got arrested. Clients associated several different acts with \"disrespecting the law\" and often spoke of \"testing\" the authorities by jumping the subway turnstile, going to the bathroom in the street, jay-walking, and cursing out the cops. Another interesting insight into the lives of drug users was how the clients felt that the law did not understand their ability to manage their drug use and lead a responsible life. Overall, they knew that when their drug use was out of control, they realized their respect for the law was \"the farthest thing from your mind.\" One woman described how during a chaotic period she became suicidal and her children were taken away by the authorities. However, after she had effectively managed her drug use for some time and had sought out legal representation, even then she had not been allowed access to her children for the past six years. Discussion ========== The methodology used in the study contributed to the field of harm reduction and how to work more effectively with drug users. Meaningful outcomes for active drug users cannot be accurately measured in \"either/or\" terms (i.e., drug use vs. abstinence) or reflect a yardstick of achievement that is not culturally based in the lives of program clients. In that harm reduction programs work with drug users \"where they are\" and strive for incremental change that can be realistically accomplished, the results of the study represent the field\'s first attempt to establish relevant, culturally sensitive outcomes for measuring client and program success. Rather than using measures/standards developed by researchers in a different cultural world, the generated outcomes seek to more closely represent the lives of the population we are trying to understand and serve. The ethics and benefits of this participatory research/evaluation approach have been acknowledged by many \[[@B16]-[@B19]\]. This said, it should be noted that that the major limitation of the research was that it was done with a representative sample of only one harm reduction program in an urban area. And, although the sample included a variety of program clients whose drug use was characterized on a continuum from stable to chaotic, the external validity of the measures may be questionable for use with harm reduction programs having a different client population. The methodology is one that can be used by other harm reduction programs to help them identify ranked goals in each of the life areas for their particular population. In addition to its methodological contribution, the study provided some important insights into drug users\' lives and values, and an increased understanding of how this population envisions a better quality of life. Based on a number of items in the scaled outcomes, results showed that drug users often cited traditional measures in defining their life progress. Examples of this include having a legitimate job as a positive measure of source of income; being homeless and sleeping on the street as a measure of an undesirable housing situation; having an open and honest relationship with one\'s family as a measure of positive family relations; avoiding legal responsibility (i.e., jumping bail, running from parole) as a measure of an ineffective way of dealing with legal problems; and getting/staying clean of drugs as a positive measure of how to deal with drug use. It is generally felt that drug users\' low ratings of certain activities as solutions to their problems (i.e., crime, prostitution) does not reflect a belief that these activities are inherently bad or immoral. Rather, these ratings reflect drug users\' pragmatic nature and recognition that these activities can be impractical and dangerous. Other findings from the qualitative research were somewhat counterintuitive to those outside the drug-use culture and reflect the realities of poverty, racism, social isolation, past trauma, and discrimination faced by individuals in this stigmatized population. For example, the study sample considered the very best way of making money was through entitlements (i.e., welfare, disability). This finding reflects the fact that since drug users struggle for day-to-day survival and often have a criminal record, many do not have the confidence, skills or opportunities to make a legitimate living in the dominant culture. It also reflects the reality that getting needed resources via entitlements is relatively easy and dependable for drug users while getting pay from a part-time job can be problematic when an employer in the drug trade can disappear on pay day. The stark realities of the drug user world were apparent in the fact that sex work and selling blood or body parts were ways that some individuals survived. Findings related to the housing and nutrition outcomes also provided a window into the lives of many drug users. For example, being in jail was considered a more preferred housing arrangement than many forms of being homeless; eating out of the garbage -- \"dumpster diving\" -- was considered the worst way to try to get something good to eat. Interestingly, when it came to family relations, violence among family members was considered less problematic than being deceitful (stealing, gossip, lies) and may partially reflect the culture\'s shared value of openly expressing feelings. In regard to the outcome of connectedness to services and programs, it was not that surprising that the sample valued those services that addressed their day-to-day survival needs (entitlement benefits and programs related to housing, AIDS, and drug treatment), more than those that addressed less immediate needs (prevention, stress reduction, nutrition, and employment services). Certain consistencies across the outcomes and items in the scales shed light on other values of drug users and the barriers they face. Overall, the sample indicated that the most preferred way of living was one in which they could try to work things out for themselves and remain independent of the dominant culture. This was seen in such examples as \"having your own place to live\"; \"cooking food yourself as a way of getting good food\"; \"developing a better relationship with self as a way of improving yourself\"; \"praying and getting support from friends in order to deal with negative feelings\"; \"relying on home remedies to address health problems\"; \"admitting you have a problem and making amends as a way of handling problems with drug use\"; and \"going to a law library and doing research as a way of dealing with legal problems.\" These findings are not surprising, considering the fact that drug users often experience stigma and discrimination when they try to rely on the traditional service delivery system and, as a result, remain isolated and marginalized. It is also a finding that challenges the dominant view of drug users as lazy, dependent, and not wanting to change. Implications for the field ========================== This preliminary research needs to be expanded in order to develop more valid and reliable outcome measures for the field of harm reduction. Other harm reduction programs are encouraged to conduct similar research with drug users in their locales and share their results. In addition, to validate that the outcomes are not simply research artifacts but reflective of the target population, field research can be conducted. For example, \"hanging out\" with the homeless and watching them as they spend the day looking for the best place to be homeless would help validate the outcome on housing. With measures that are science-based and evaluation results that can demonstrate the effectiveness of this approach to working with drug users, harm reduction programs will stand a better chance of receiving funding from potential donors. In addition to their use, these measures for program outcome evaluation can also benefit the clinical component of programs. Case managers could collaboratively use the methodology in sessions with individual clients to develop relevant and realistic treatment plans. Future plans for behavior change developed in one-on-one sessions and using user-generated measures of progress have more potential for achievement than plans that do not consider how clients define progress. Conclusion ========== Clearly drug users have a set of workable solutions for meeting their own survival needs. Results of the present study show that they can often view institutions in the existing culture as irrelevant when addressing day-to-day living problems. The scales and rankings of many of the socially approved ways of solving life\'s problems show that just as there is an underground economy, there is a whole underground subculture where those marginalized from the mainstream have developed a culture with its own set of relevant structures, informal relationships, and home-grown recipes for addressing life\'s challenges. These study findings convey an understanding of drug users as people who are interested in positive change in all areas of their lives, and that by empowering them in a process to identify their own goals, they may be more motivated and engaged in the program. The findings also provide some initial empirical evidence that challenges traditional ways of measuring drug use based solely on quantity and frequency. Results suggest that more appropriate measures may be the extent to which the drug users organize their lives around drug use, the extent to which drug use is integrated into their lives, and the extent to which drug use negatively impacts other aspects of their lives. They also suggest that harm reduction and other programs serving active drug users and other marginalized people should not rely on institutionalized, provider-defined solutions to the problems in living faced by their clients. Rather, drug users should be assisted with problem solving by being encouraged to consult their own set of culturally shared solutions. Competing interests =================== None declared. Authors\' Contributions ======================= T.R. conceived the study, designed the research, and provided input on the manuscript. S.R. coordinated the study, provided oversight on data collection, analyzed the data and wrote the manuscript. Both authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional file 1 Outcomes of Harm Reduction Programming to Measure Incremental Change from Better to Worse. This is a tabular form of all categories of behavior discussed in the paper. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The authors wish to acknowledge several individuals who contributed to the research. First, we thank the clients of the NYHRE harm reduction program who took part in the research. Their patience and commitment to the group process was commendable and their honesty and directness appreciated. Thanks also go to two people at NYHRE who logistically set up of the research: Martha Hornsby, former Deputy Director and Eddie Rivera, Project Coordinator. We also appreciate the work of AED staff and consultants: Kathryne Leak and Sarah Anderson who conducted the groups with clients, Stacy Silverstein who assisted with the development of focus group protocol, Amy Richie who analyzed the data, Noemi Corujo who formatted the manuscript and Elayne Archer who edited it. Finally, we would like to thank Edith Springer and Ernie Drucker, PhD for their encouragement and input on this research.

PubMed Central

2024-06-05T03:55:47.524644

2004-8-26

PMC516447

Background ========== Medical care aims not only to improve health status but also to respond to patient needs and wishes and to ensure their satisfaction with care \[[@B1]\]. Likewise, conducting surveys to measure satisfaction with psychometrically validated questionnaires entails assessment of the quality of care organization and procedures \[[@B2]\]. Patient judgement on medical care also contributes to medical outcome. In the case of ambulatory care, it has been clearly shown that satisfied patients are more likely to cooperate with treatment, to maintain a continuing relationship with a practitioner \[[@B3]\] and thus enjoy a better medical prognosis \[[@B4]\]. From a conceptual point of view, the construct of patient satisfaction as been defined by Ware as an \"attempt to capture the personal evaluation of care that cannot be known by observing care directly\" and to consider opinion of patients as a multidimensional subjective indicator of quality of care \[[@B5]\]. The model most commonly, though implicitly, used in satisfaction work is the discrepancy model (degree of fulfillment of expectation is related to satisfaction level) giving to patient expectations a central role \[[@B6]\]. This model, according to Sitzia \" implies that concentrating upon areas of expressed dissatisfaction is more valuable than obtaining consistency of expressed satisfaction\" \[[@B4],[@B7]\]. In France, measuring satisfaction has been mandatory since 1996 and several questionnaires have been developed to evaluate inpatient care \[[@B8]-[@B12]\]. Most existing outpatient satisfaction questionnaires have been developed to assess primary care practice, especially general practice \[[@B13]-[@B20]\]. However, it could be hypothesized that content of questionnaires evaluating primary care physician may be different from that of questionnaires exploring hospital consultation with a specialist because of differences in patient expectations. So it could be assumed that dimensions that are very important in the case of primary care like human qualities of the physician and medical information could have a lesser importance in case of hospital consultation, while technical competency could have a more important place \[[@B21]-[@B23]\]. Few questionnaires have been developed for hospital consultations. Of these, some were specific to one type of consultation like oncology \[[@B24]\], rheumatology \[[@B25]\] or diabetes clinics \[[@B21]\], while others were non-generic questionnaires \[[@B14]\]. There is one French-language questionnaire on satisfaction with outpatient hospital care, however this questionnaire was developed from an \"expert\" viewpoint \[[@B26]\]. Hence the decision to construct a complementary \"patient-oriented\" questionnaire implicating potential respondents in the generation and selection of items. Even if health care organization differs across countries, the role of the hospital in most countries is very similar and it could be expected that the questionnaire developed in France could be used in other countries with a public health system, in particular European countries. The main objective was to develop, according to psychometric standards, a generic outpatient satisfaction questionnaire that could be used to compare hospital outpatient departments one with another or the same department over time. The questionnaire needed to be brief, understandable and easy to complete for outpatients aged18 years or older in medicine, surgery and psychiatric hospital consultations. It was designed to be self-administered. The French final version is being adapted in English, German, Italian, Spanish and Hebrew. The secondary objective was to define administration procedures in routine study that minimize non-response bias. Three situations were tested: i) questionnaire issued and completed at hospital, immediately after consultation; ii) questionnaire mailed and completed at home before any reminder; iii) questionnaire mailed and completed at home only after reminder(s). The groups were compared for satisfaction. Overview of the questionnaire development ----------------------------------------- It comprised 2 phases. First, a qualitative phase for item generation and construction of a first version of the questionnaire (41-item version). Secondly, a quantitative phase comprising 2 steps. A first validation phase that provided a shortened version of the questionnaire (27-item version). Second, a replication validation phase to corroborate results from the previous steps. Finally a very short-form version (9-item) was constructed. All versions are presented in the Appendix (see additional file 1). A steering committee supervised the questionnaire development procedure, comprising methodologists, hospital practitioners and persons from patient associations defending health care user rights. All analyses were performed using SAS software (version 8). Method ====== Qualitative phase of item generation ------------------------------------ A psychologist conducted 25 individual semi-structured interviews with recent outpatients, using the critical analysis incident technique \[[@B27]\]. Subjects were asked to detail specific events they had experienced and situations associated with neutral, pleasant or unpleasant emotions that had influenced their opinion on consultation. An interview guide constructed according to the chronological order of a consultation was used. The interviews were pursued until new ideas were exhausted. Patients expressing ideas that were too general or those talking about non-personal experiences were interrupted in order to refocus on a particular personal experience. Each interview lasted 30 minutes on average. All the different wordings of a given idea were recorded. Interviews were transcribed and items were generated from the verbatim statements (n = 105 items). A literature review was carried out on validated satisfaction questionnaires \[[@B5],[@B13]-[@B20],[@B23]-[@B26],[@B28]-[@B30]\]. This yielded a preliminary list of areas of satisfaction with consultation. Items found in the literature but not in the interviews were collated (n = 26). This procedure also identified other factors related to outpatient satisfaction with consultation (patient and physician profiles), relevant for inclusion in the questionnaire. The aim was to select the variables linked to satisfaction, independent from place of consultation (department), for the final questionnaire. These variables constitute background adjustment factors needed to avoid bias in comparing departments one with another or the same department over time (age of the patient for example) \[[@B31]\]. A list of satisfaction items (n = 131) was constructed classified into the following domains: administrative procedures, appointment making, receptionist and nurses, waiting time, facilities, duration and privacy of the consultation with the doctor, human relationships with the doctor, information provided by the doctor and shared decision-making, doctor\'s technical competence, coordination and continuity of care, and global satisfaction. The source of items (interview v. literature) was indicated. Using the Delphi technique \[[@B32]\], the steering committee and six patients (members of the National League against Cancer) selected items within each domain (n = 60). The number of items to be chosen was proportional to the number of items proposed in each domain. The list of items was submitted as often as necessary to obtain a consensus of at least 80% among the raters. A focus group (one two-hour meeting) coordinated by two of the authors (IG, SV) including two patient association representatives and three patients, with previous individual access to the list of items, checked acceptability of item wording and exhaustiveness of the list. A pilot study was conducted on 55 outpatients from different outpatient departments using a preliminary questionnaire comprising the selected items, to check comprehensibility and acceptability of items and response patterns. Confusing items were removed, rewritten or replaced. The list of the items extracted from this qualitative phase is shown on the appendix. Questionnaire ------------- The questionnaire obtained from the qualitative phase and tested in the first study comprised 41 negatively and positively worded satisfaction items (Appendix \[see Additional File 1\]). The traditional approach was chosen, in which the item is structured as a statement of opinion. A Likert five-point response balanced scale was chosen (in French : \'yes certainly\', \'yes probably\', \'neither yes nor no\', \'probably not, \'certainly not\') because it seems to be the best format \[[@B5],[@B33]\] and the most often used \[[@B5],[@B13],[@B14],[@B17]-[@B19],[@B24],[@B28],[@B29]\]. A \'does not apply\' category was provided for 19 items relating to situations not universally relevant. Each item was scored from 0 to 4, 4 indicating greatest of satisfaction. Non-response and \'does not apply\' categories were considered as missing data. Patients were asked to answer for their last consultation in the department. Several other items on general satisfaction were also included in this questionnaire: one overall satisfaction item, using a seven-point scale (from \'not at all\' to \'completely\' satisfied) and two items on intended behavior (to recommend, to consult again), using a four-point scale (\'yes certainly\', \'yes probably\', \'probably not\', \'certainly not\') and one open-ended question. These items were included to test concurrent validity. The questionnaire also comprised data on sociodemographic profile, medical status, visit background and characteristics. and an overall satisfaction with life (using a 7 point scale, from \'not at all\' to \'completely\' satisfied). This last variable was included because of the relationship between affective disposition and the expression of satisfaction \[[@B34],[@B35]\] and because of the relationship between satisfaction with life and satisfaction with care \[[@B36]\]. Samples and studies design of the quantitative phase ---------------------------------------------------- ### First study (first validation phase) To select items, a first study was conducted in 2001--2002 in 10 wards of 3 short-stay public teaching hospitals of Paris area (Paul Brousse, Bichat and George Pompidou European hospitals). Data was collected in 7 medical departments (internal medicine, rheumatology, 2 cardiology, dermatology, infectious disease, and oncology) and 3 surgical outpatient departments (urology, orthopedic, surgical gynecology). All consecutive eligible ambulatory patients over 18 years in scheduled consultation with a physician were included, to obtain approximately 100 subjects per department. Patients hospitalized before or immediately after the consultation were excluded. Research assistants approached outpatients immediately after consultation and invited them to participate. Outpatients were randomized prior to being approached. Outpatients randomized in group 1 completed the questionnaire alone immediately after consultation and left it in a box. Patients of group 2 received the questionnaire by mail at home for completion. They were asked to complete and return it by post in a prepaid envelope carrying a neutral address. Non-respondents were sent up to 3 more questionnaires at one-week intervals. To assess reliability over time a sample of 38 respondents from the second group was sent a second questionnaire to return completed, without any reminder. Finally of the 1548 outpatients approached, 70.9% agreed to participate (n = 1097) and 65.1% completed the questionnaire (n = 1007). Response rates were 57.0% in group 1 and 73.7% in group 2 (40.2% before any reminder, 63 % after one reminder, 69.7% after two and 73.7% after three). Reasons for non-participation were refusal or lack of time (12.9% of the overall sample), language barrier (8.5%), inability for medical reasons (7.2%), other reason (0.6%) and agreement but no return of the questionnaire after 3 reminders (5.8% of the overall sample and 12.0% of group 2). Compared to respondents, the non-respondent group comprised older subjects (60.2% v. 52.6% aged over 50 years, p \< 0.001), more foreigners (12.5% v. 29.1%, p \< 0.001) and more patients consulting for the first time in the department (28.0% v. 22.4%, p = 0.02). Response rates also differed according to the department (p \< 0.001) and the hospital (p \< 0.001). ### Second study (replication phase) To confirm the results of the previous study, a second study was conducted in the year 2002 in two departments (internal medicine and infectious disease) in one short-stay public teaching hospital. All consecutive outpatients of 18 years and over (not hospitalized immediately after consultation) were included to obtain 100 participants per department. The questionnaires were posted with a prepaid envelope. One reminder was sent 10 days after the first mailing to non-respondents. Participation rate was 33.9% before reminder and 56.5% after (n = 248). Results ======= First validation phase ---------------------- ### Item selection A first selection of items was made from descriptive response distribution for each item. The criteria used to guide item selection/deletion were: high rates of non-response and \'not applicable\' response (≥ 20%) except for items where high rates in this response category were expected, ceiling and floor effects (≥ 50%), and unacceptable test-retest reliability (weighted kappa coefficient\<0.60). Pragmatic considerations also tempered selection: interest of the item in itself, number of items covering the same domain, redundancy. Results showed that the proportion of missing responses per item was low. As predictable, for the two items relating to accessibility of the service in case of emergency (items 5 and 6, Appendix \[see Additional File 1\]) the number of \'does not apply\' responses was high (30.7% and 45.0%). A ceiling effect was observed for all items (from 54.4% to 79.6%), except for those on facilities and waiting time (items 10 to 13). Test-retest reliability was good for 20 items (weighted kappa ≥ 0.7 for 10 items and from 0.6 to 0.69 for 10 items). For 5 items, the coefficient ranged from 0.45 to 0.56. The item on doctors\' warnings on side effects of treatment (item 22) had a very low weighted kappa (k = 0.17). At this stage 12 items were discarded. Item 22 was retained for its clinical relevance (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Item description and scaling properties of the questionnaires extracted from the validation phase (26 item version) and from the replication phase ::: --------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------- --------------------- --------------------------- --------------------- ------------------------------ --------------------- ------------------------ -------------- --------------- **Intermediate questionnaire 26-items retained at the and of the first validation phase (first study, n = 1007)** **Final questionnaire 27-item questionnaire tested at the replication phase (second study, n = 248)** Title of the scales Consultation with the doctor Appointment making Reception Waiting time & facilities Overall scale Consultation with the doctor Appoint-ment making Reception & facilities Waiting time Overall scale [Items properties]{.underline} \# of items in the scale 13 6 3 4 26 13 6 5 3 27 \# of questionnaires with at least 1/2 of items completed ^(1)^ 996 931 1004 1001 1003 235 248 247 244 248 \# of items with \'non response\' ≥ 20% 0 0 0 0 0 0 0 0 0 0 \# of item with \'does not apply\' response ≥ 20% 0 2 0 0 2 1 0 0 0 1 \# of items with ceiling effect ≥ 50% (≥ 60%) 13 (12) 6 (4) 3 (2) 0 (0) 22 (18) 12 (4) 1 (1) 2 (0) 0 (0) 15 (5) \# of items floor effect ≥ 50% 0 0 0 0 0 0 0 0 0 range of Weighted kappa (\# of items with kappa ≥ 0.60) 0.14--0.83 (10) 0.46--0.77 (3) 0.45--0.78 (1) 0.68--0.82 (4) 0.14--0.82 (18) \- \- \- \- [Scaling properties]{.underline} Mean score (± sd) 85.1 (17.2) 83.2 (19.9) 88.0 (14.5) 69.6 (24.9) 82.7 (13.7) 84.1 (17.2) 80.6 (18.5) 75.3 (18.3) 61.3 (19.6) 78.9 (15.3) Ceiling / floor effect (%) 26.2 / 0.10 32.2 / 0.2 38.5 / 0.1 16.2 / 1.1 4.2 / 0.1 25.8 / 0.4 24.7 / 0.4 13.7 / 0.5 19.0 / 4.9 4.4 / 0.4 Skewness value /SE -3.00 -2.09 -3.5 -0.86 -2.67 -0.98 -0.83 -0.58 -0.20 -0.76 Range of interscale correlations 0.33--0.35 0.34--0.37 0.35--0.40 0.33--0.40 \- 0.46--0.51 0.51--0.59 0.42--0.49 0.42--0.53 \- \# of items with own scale correlation ≥ 0.40 ^(3)^ 12 6 2 4 \- 13 6 5 3 \- \# of items with own scale ^(3)^correlation greater than with other scale 13 6 3 4 \- 13 6 4 3 \- Cronbach alpha coefficient 0.85 0.82 0.69 0.77 0.88 0.94 0.87 0.79 0.89 0.94 Intraclass coefficient \[IC95%\] 0.69 \[0.49--0.83\] 0.84 \[0.71--0.91\] 0.86 \[0.75--0.92\] 0.83 \[0.71--0.91\] 0.90 \[0.81--0.94\] \- \- \- \- --------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------- --------------------- --------------------------- --------------------- ------------------------------ --------------------- ------------------------ -------------- --------------- \(1) Including non response and \'does not apply\' response\' (2) From the final principal component factor analysis (3) Corrected for overlap ::: ### Factorial structure The 29 items retained were entered into principal-components factor analysis (PCFA) with \'varimax\' rotation and the 26 items with substantial loading ≥ 0.40) on only one factor were retained (Appendix \[see Additional File 1\]). Another PCFA was computed on the 26 remaining items to determine the structure of the instrument. The screeplot revealed a predominant eigenvalue with nevertheless a four-dimensional structure (the following eigenvalues showed a smooth decrease). Hence the proposal is to consider a four-dimension structure with the possibility of an overall score. One dimension grouped the 13 items relating to consultation with the physician. The second dimension grouped the 6 variables relating to appointment-making. The third and fourth related respectively to waiting time or facilities (4 items) and reception (3 items). None of the 26 items loaded on more than one factor. Only item 26 (\'doctor in touch with attending physician\') had a borderline loading (0.37), but it was kept because coordination of care in hospital care is important. The stability of the 4 factors was ascertained with PCFA on subgroups and with \'oblique\' rotation (male v. female and surgery v. medicine). ### Scale properties Scores for each scale were based on the standardized sum of the items, giving a range from 0 (low satisfaction) to 100 (high satisfaction). Scores were computed when at least half the items in a scale were completed. Because of a ceiling effect, mean scale scores are relatively high except for the \'waiting time and facilities\' scale (Table [1](#T1){ref-type="table"}). Interscale correlations were good for the four scales. One item had a borderline correlation with its own scale (r = 0.37 for item 7 \'the consultation room was clearly sign-posted\') and one item had a low correlation (r = 0.33 for item 26 \'doctor in touch with attending physician\'). All items had a higher correlation to their hypothesized scales than to other scales. Reliability was good, meeting both Cronbach alpha and intraclass correlation coefficient requirements (Table [1](#T1){ref-type="table"}). A very strong association between the overall scale, intended behaviors, comments and global satisfaction question was noted, suggesting good concurrent validity (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Association between overall satisfaction scale, intended behaviors and global satisfaction item from the first validation study (n = 1007) and replication study (n = 248) ::: **First validation phase First study (n = 1007)** **Replication phase Second study (n = 248)** -------------------------------------- --------------------------------------------------- ---------------------------------------------- --------------- ---- ------------- --------- Overall satisfaction item ^(3)^ 1 (*not at all satisfied*) 13 58.0 (21.6) 2 12 68.7 (17.7) 3 18 56.0 (13.5) \<0.001 ^(4)^ \- na \- 4 39 59.4 (14.0) 5 125 72.1 (11.5) 6 285 80.3 (10.0) 7 (*completely satisfied*) 496 90.6 (7.6) To recommend to relatives or friends certainly not 13 52.7 (18.2) \<0.001 ^(4)^ \- na \- probably not 30 57.3 (16.7) yes probably 272 75.4 (13.1) yes certainly 665 87.6 (9.5) To consult again certainly not 10 58.7 (24.2) \<0.001 ^(4)^ \- na \- probably not 20 57.3 (17.9) yes probably 213 72.5 (14.5) yes certainly 756 86.6 (10.3) To consult again 9 Do not agree \- na \- 46 57.8 (14.4) \<0.001 agree 18 64.2 (12.4) Fully agree 1 84.3 (11.7 Content of the open-ended question negative comment 303 76.1 (14.7) 85 72.1 (14.9) mixed comment 110 78.4 (13.5) \<0.001 8 81.0 (12.4) \<0.001 no comment 442 85.4 (12.1) 91 82.0 (15.8) positive comment 152 91.2 (8.2) 42 85.5 (11.1) na: non available (1) Overall satisfaction score (26 items scale extracted from the first study) (2) Overall satisfaction score (27 items final scale extracted from the second study) (3) 7-point scale from 1 \'not at all satisfied\' to 7 \'completely satisfied\' (4) ANOVA test regrouping the responses 1,2 and 3 ::: Replication phase ----------------- ### Questionnaire tested (see appendix) A modified version of the questionnaire was constructed at the end of the previous step. To avoid the ceiling effect highlighted in the previous stage, responses choices were modified (using the pattern \'fully agree\', \'agree\', \'moderately agree, \'not really agree\', \'not agree at all\'). One satisfaction item on waiting time was added and one item on the facility was reworded to improve the chance of revealing a \'waiting time\' subscale and a \'reception-facilities\' subscale) and because reliability of the \'reception\' subscale was borderline. Patient demographic variables identified at the previous stage as having a relationship with satisfaction scores, one item on intended behavior and an open-ended comment field were also added to the questionnaire. ### Final psychometric properties of the final 27-item version questionnaire The number of items with ceiling effect decreased. Item completion rates were good (Table [1](#T1){ref-type="table"}). PCFA was performed on the 27 items. The screeplot highlighted the same internal structure. The \'varimax\' rotation revealed that two dimensions were identical to those identified in the first study (\'consultation with the doctor\' and \'contact-appointment\') (Table [4](#T4){ref-type="table"}). The two others were slightly altered: the three items on \'waiting time\' were isolated from items about \'facilities\' that grouped themselves with the \'reception\' factor. All items had a good loading on their own factor. Item 9 (\'pleasantness and availability of receptionist\') was the only item with secondary loading on another component. It was kept because it was the only item on human qualities of non medical staff which were cited very often by patients in the qualitative phase (Table [1](#T1){ref-type="table"}). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Principal components factor analysis (varimax rotation) computed with the final 27-items version of the questionnaire (second study, n = 248) ::: **Factor 1** **Factor 2** **Factor 3** **Factor 4** ---- --------------------------------------------------------------------------------------- -------------- -------------- -------------- -------------- 1 easy to make an appointment by phone 0.07 **0.49** 0.12 0.39 2 Pleasantness of staff answering the phone 0.24 **0.56** 0.30 0.19 3 Acceptable time lapse to obtain appointment 0.19 **0.81** 0.22 0.18 4 Possibility of obtaining an appointment on convenient day and hour 0.20 **0.77** 0.20 0.14 5 Contacting someone in the facility on the phone for help or advice in case of problem 0.30 **0.68** 0.18 0.08 6 In an emergency, getting a quick appointment in the facility 0.19 **0.77** 0.24 0.11 7 Inside the hospital the consultation room was clearly sign-posted\' 0.17 0.15 **0.67** \- 0.16 8 Administrative procedures (completing papers and paying) fast and easy 0.15 0.25 **0.61** 0.16 9 Pleasantness and availability of receptionist 0.20 ***0.44*** **0.59** 0.23 10 Waiting room pleasant 0.11 0.15 **0.78** 0.26 11 Premises clean 0.17 0.22 **0.66** 0.18 12 Saw the doctor at the appointed time 0.24 0.18 0.14 **0.84** 13 Waiting time acceptable 0.25 0.29 0.10 **0.85** 14 Information on how long to plan for 0.23 0.16 0.20 **0.78** 15 The doctor was welcoming **0.69** 0.09 0.38 0.08 16 Took an interest in me not just my medical problem **0.72** 0.06 0.15 0.18 17 spent adequate time with me **0.82** 0.14 0.21 0.07 18 Examined me carefully **0.78** 0.04 0.21 0.07 19 Explained what he/she was doing during the consultation **0.78** 0.15 0.17 0.12 20 Wanted to know if I had pain **0.75** 0.13 0.17 0.15 21 Asked if I was taking medication for other health problems **0.72** 0.07 0.08 0.09 22 Warned me about possible side effects of treatment (operation, drugs)- **0.68** 0.30 0.01 0.19 23 Took my opinion into account **0.79** 0.20 0.02 0.10 24 Explained decisions **0.76** 0.25 0.18 0.20 25 I got the information I wanted **0.78** 0.23 0.12 0.17 26 he/she is in touch with my GP **0.57** 0.20 0.02 0.15 27 Agree with doctor\'s instruction **0.49** 0.32 0.04 \- 0.06 ::: For item-scale correlations, item 9 also correlated to these two scales (\'reception-facilities\' factor and \'contact-appointment\'). It was decided to attribute it to the factor that maximized internal consistency (\'reception-facilities\' scale). All items met the requirement of being highly correlated to their own scale, all interscale correlations were satisfying, as well as internal consistency (Table [1](#T1){ref-type="table"}). The scale overall was significantly associated with comments and intended behaviors (Table [2](#T2){ref-type="table"}). Construction of a unidimensional 9-item form -------------------------------------------- As the factorial analysis of both the first validation and replication phases revealed a predominant factor that could be split into four underlying dimensions, it was decided to construct a unidimensional form of the questionnaire, that could produce an overall global outcome that could be very useful in case of evaluation study. Within each dimension, one third of the items were selected according to two criteria: items without \'not applicable\' response choice, and items having strong loading on the principal component in PCFA. Thus 9 items were selected, 4 items from the \'consultation with the doctor\' scale, 2 from the \'contact-appointment\' scale, 2 from \'reception-facilities\' and 1 from \'waiting time\' (Appendix \[see Additional File 1\]). Final PCFA on these 9 items showed scale unidimensionality. Item loading on this factor ranged from 0.56 to 0.78. Item-scale correlation corrected for overlap ranged from 0.47 to 0.65. Internal consistency was good (Cronbach α = 0.86). Effect of mode of questionnaire administration on estimation of patient satisfaction ------------------------------------------------------------------------------------ First study showed that compared to the satisfaction score obtained with completion at home, mean scores for all hospital-completed satisfaction scales were very significantly higher. In the group that completed the questionnaire at home, comparison between respondents before any reminder and respondents after reminder(s) showed no difference in satisfaction scores, whatever the scale considered (Figure [1](#F1){ref-type="fig"} -- Satisfaction scores according to the place of completion and time of answering \[before v. after reminder\]). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Satisfaction scores according to the place of completion and time of answering (before v. after reminder) (first study, n = 1007). :::  ::: Differences between departments ------------------------------- A multiple linear regression showed that differences between departments were highly significant, even if patient characteristics that influenced patients\' satisfaction were taken into account (i.e. age, satisfaction with life and previous consultation). Satisfaction scores ranged from 79.3 to 91.7 for \'consultation with the doctor\' scale, from 72.8 to 94.2 for \'appointment making\' scale, from 83.4 to 91.3 for \'reception\' scale, from 57.3 to 80.5 for \'waiting time-facilities\' and from 77.8 to 89.3 for the overall scale). Older age, good satisfaction with life and numerous previous consultations in the department were all associated with high levels of satisfaction, independently from the department (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Association between demographic, medical, outpatient consultation characteristics considered as explanatory variables and overall satisfaction score as dependant variable (1) (linear regression analysis from the first study) ::: **DF** **F-value** **P-value** ------------------------------------------------------------------------------------------------------------------------------------------------------ -------- ------------- ------------- **Demographic profile** Age (quantitative variable) 1 7.75 **0.006** Matrimonial status (Married or living with partner v. single v. divorced, separated, or widowed) 2 0.93 0.42 Working status (employed v. student v. unemployed v. Retired v. prolonged sick leave v. other) 5 0.79 0.56 Level of education (university yes v. no) 1 1.72 0.19 **Overall satisfaction with life**(quantitative variable) 1 51.1 **0.001** **Modes of care provision** Outpatient department (n = 10) 9 4.3 **0.001** \# of consultations in the department (first v. 2 to 3 v. 4 to 5 v. more then 5) 3 2.92 **0.03** At least one hospitalization in the ward 1 0.73 0.39 **Medical profile** Duration of the health problem justifying the consultation (less than 6 month v. 6 month and more) 1 FGF 0.22 Severe medical problem (\'yes definitely\' v. \'yes rather\' v. \'neither yes nor no\', v. \'not really\', v. \'definitely not\' v. \'do not know\') 5 1.19 0.31 Comorbidity (yes v. no) 1 1.97 0.16 Perceived health status, compared to persons of same age (better v. similar v. worse) 2 0.49 0.61 829 observations used in the analysis, r^2^= 0.21 (1) First short version of the satisfaction questionnaire List of other variables not entered into the model because of non significance in the bivariate analysis (p \> 0.1): gender, nationality, gender of the physician, motive of the consultation (physical, psychological or mixed) and prescription of test or medication and having a general practitioner. ::: Discussion ========== Psychometric properties of the scale ------------------------------------ The 27-item and 9-item versions of the questionnaire developed here appear sufficiently concise, valid and reliable to provide a non-biased subjective evaluation of outpatient viewpoint on the quality of care and services in hospital consultations. The questionnaire demonstrated very good internal consistency and good reliability over time. The construction strategy presented here follows most of the recommendations for \"good practice\" in validation of measurement tools of patient satisfaction with care \[[@B7]\]. Questionnaire content comprises culture-specific features, but overall remains consistent with various north American and European studies \[[@B21],[@B23],[@B26],[@B37]\]. The predominant role given to patients in the early development stages, the literature review and the implication of various experts ensure good content, construct and face validity. This first qualitative step, often insufficiently detailed and structured in satisfaction questionnaire construction, is indeed crucial \[[@B38]\]. The quantitative phase (i.e. first validation with replication) used not only statistical and psychometric results to reach decisions, but also the \"intrinsic\" and \"clinical\" relevance of items. This is a very important point. First, because satisfaction studies aim not only to measure quality from user viewpoint, but also to highlight practical elements that can be modified to improve quality. Second, questionnaires that are perceived to have content validity are needed to generate interest in results among health professionals and provide incentive for changes in approach to their jobs. Third, the tendency of health professionals to develop \"home-made\" questionnaires and their reluctance to use validated questionnaires developed elsewhere can be countered if questionnaire items are perceived as relevant. Dimensions of the questionnaire ------------------------------- Each dimension comprises items exploring both technical aspects of care (i.e. equipment, competence, accessibility, continuity, compliance, pain management, waiting and consultation time\...) and interpersonal aspects of care (i.e. information, decision sharing, attitude\...). These aspects are both predictors of patient opinion on care and services \[[@B22],[@B23],[@B37]\] because implementation of appropriate technical medical strategies is necessary, but not sufficient, to achieve desired outcome. Good management of the human is needed because, as Donabedian remarks, \"the interpersonal process is the vehicle by which technical care is implemented and on which its success depends\" \[[@B1]\]. According to this author, technical and interpersonal performances are the first circle around the \"bull\'s eye\" of the \"quality of care\" target. The most important dimension explaining outpatient opinion of hospital quality is the actual consultation with the physician, representing half the items in the tool. This is consistent with other generic patient questionnaires on satisfaction with ambulatory care, also comprising a majority of items related to the medical intervention \[[@B17],[@B19],[@B21],[@B26],[@B29],[@B37]\]. No independent subscales regarding specific aspects of the patient-physician encounter (i.e. communication, professional competence, interpersonal skills\...) were identified here. They have been regularly identified by authors developing GP satisfaction questionnaires \[[@B5],[@B17],[@B29],[@B37],[@B39],[@B40]\]. This could be explained by the fact that, as hypothesized, expectations of outpatients with respect to hospital care differs from expectations from primary care. Possibly patients have different needs and expectations according to the type of consultation, hospital specialists generating more mixed expectations because the specific technical competence of hospital specialists predominates and patients have greater difficulty in dichotomizing doctors\' skills into \"affective \" and \"technical\' dimensions \[[@B23],[@B41],[@B42]\], whereas \"affective\" qualities have a predominant role in primary care \[[@B15],[@B16],[@B43],[@B44]\]. This is corroborated by the fact that generic questionnaires designed to evaluate hospital care (inpatient or outpatient) most often do not identify such human versus technical dimensions \[[@B8],[@B10],[@B26],[@B45],[@B46]\]. The three other dimensions (\'contact-appointments\', \'reception-facilities\' and \'waiting time\') are all related to organizational non-medical aspects of care. These dimensions are classically identified in other generic questionnaires \[[@B17],[@B18],[@B21],[@B23],[@B28],[@B29],[@B40],[@B42]\]. Comparison of the two factorial structures shows stability for all dimensions except \'reception-facilities\' and \'waiting time\'. From a strictly psychometric viewpoint, these two dimensions, both exploring events occurring just care quality, these two dimensions can pinpoint independent improvement measures, and calculating two different scores may improve the probability of highlighting the impact of such measures. Differences between departments and role of background factors -------------------------------------------------------------- It was shown that satisfaction scores were strongly related to consultation department, regardless of outpatient, physician and care-provision characteristics. These results suggest that this measure is more sensitive to levels of department performance than to patient profile or to modes of consultation, as shown elsewhere \[[@B47]\]. Therefore it is important that each department should identify its weak points to implement specific targeted actions to improve care quality. As in numerous studies, it was observed that older patients have a higher opinion of care provided than others \[[@B7],[@B23],[@B48]\]. For several authors, this contributes to construct validity of satisfaction questionnaires \[[@B41]\]. The same was observed for patients with multiple contacts with a department \[[@B10]\]. This could be explained by a better match between expectations and experience for multiple consultants, dissatisfaction during first contact leading patients to consult elsewhere. The strong relationship between overall satisfaction with life and opinion on care expresses the influence of the individual affective disposition trait (i.e. general tendency of an individual to be optimistic or pessimistic) which influences job satisfaction, a concept very close to patient satisfaction with care \[[@B49]\]. Other studies have found relationships between satisfaction and variables strongly associated with perception of overall quality of life, like mental health status and health-related quality of life \[[@B12],[@B24]\]. The influence of these three background factors suggests the need to adjust patient satisfaction scores on these three variables (i.e. patient age, number of contacts and satisfaction with life) when comparing performances between departments or measuring performance over time within departments \[[@B31]\]. Impact of data collection method -------------------------------- For patients completing the questionnaire immediately after consultation in the hospital, satisfaction estimates were higher than in case of home completion, in spite of procedures to preserve anonymity and confidentiality at hospital. Little data exists on the impact of place of completion for self-administered questionnaires on satisfaction with consultation: two studies conclude that patients express less satisfaction when the questionnaire is completed at home rather than in the medical facility \[[@B13],[@B50]\] and one concluded that there was no difference according to data collection methods, but lacked power because of small sample size \[[@B51]\]. This could be interpreted as an over-estimation of patient satisfaction in case of completion in the facility, patients being more prone to express their real opinion when they have more time to consider the consultation and are safely back home \[[@B13]\]. Moreover response rates in the hospital completion group were relatively low (57%) expressing both refusal to participate or inability to respond, and reluctance to answer a satisfaction questionnaire immediately after consultation because of long waiting time beforehand, or because a relative, an ambulance or a taxi is waiting to take the patient home. It could be concluded that completion at home may be better than immediately after consultation. In the present study, no difference was observed between respondents without reminder and respondents only after reminder(s). This result is in agreement with other studies assessing inpatient satisfaction \[[@B9],[@B52]\]. It could be concluded that reminders are not necessary to produce non-biased data. Limitations =========== This work entails several limitations. First, overall response rates only reached 65% despite reminders sent to patients receiving mailed questionnaires at home. However, unlike other studies, the response rates calculated did not exclude patients unable to respond for medical reason (i.e. who were very ill or did not understand French) and homeless patients giving an invalid address (shelter\...). Second, non-respondents differed from respondents regarding two background factors influencing satisfaction levels, with over-sampling of less-satisfied subjects in the respondent group (young patients and first consultants). There are also differences in participation rates between departments that could lead to over-estimating real differences between departments, because the more satisfied outpatients within each department may have been excluded. Third, validation is a continuous process and further studies are required to confirm these first results. The experimental nature of these studies may have induced bias in questionnaire responses. So there is a need to replicate findings using confirmatory statistical methods (IRT or structural equation model for example) using the data from non experimental, routine studies. Conclusion ========== Good estimation of patient opinion on hospital consultation can be obtained with these two questionnaires. When comparing performances between departments or the same department over time scores need to be adjusted on the three variables that influence satisfaction independently from department (patient age, previous consultation in the department and overall satisfaction with life score). Mail-back completion at home of the questionnaire seemed preferable to completion in the consultation facility immediately after the consultation. Reminders are not necessary to produce non-biased data. Authors\' contribution ====================== IG -- initiation of the research, supervision of the project and drafting the manuscript; SV -- coordination of the 2 studies, participation in the interpretation of the results and revision of the draft paper; CDS -- performing statistical analyses; PD and PR- participation in the conception, design and coordination of the research; BF -- participation in the interpretation of the results, supervision of the statistical analysis and revision of the draft paper. Acknowledgments =============== The authors would like to thank the members of the steering committee (Prof. P. Auquier, Mr JP Escande, Mr C Gilioli, Mr T Greacen, Prof. F Guillemin, Dr J Labarère, Prof. J Lellouch, Dr A Leplège, Dr C Pourin), the National League against Cancer, the participating departments of Paul Brousse hospital (Prof. C Jasmin, Prof. D Vittecoq), Georges Pompidou hospital (Prof. B Augereau, Prof. JN Fiessinger, Prof. JP Lemerle, Prof. PF Plouin) and Bichat-Claude Bernard hospital (Prof. B Crickx, Prof. L Boccon-Gibod, Prof. C Lejeunne, Prof. O Meyer, Prof. R Taurelle, Prof. A Vahanian). This study was funded by a 2000 national PHRC grant (Programme Hospitalier de Recherche Clinique) from the French Health Ministry. Approval was given by the CNIL (Commission national Informatique et Libertés).

PubMed Central

2024-06-05T03:55:47.530655

2004-8-4

PMC516448

Background ========== Sir William Osler (1849--1919), one of the most distinguished physicians of the nineteenth and early twentieth century, recognised that only some doctors are happy in their professional lives: \"To each one of you the practice of medicine will be very much as you make it -- to one a worry, a care, a perpetual annoyance; to another, a daily joy and a life of as much happiness and usefulness as can well fall to the lot of man.\"\[[@B1]\] The modern medical workplace is a complex environment, and doctors respond differently to it, some finding it stimulating and exciting, whereas others become stressed and burned out from the heavy workload. The medical workplace also provides an environment where new skills are continually being learned, both as a result of medical knowledge evolving and because a doctor\'s work changes, in part due to career development and progression through different jobs. In an important study, Delva *et al*\[[@B2]\] used earlier research \[[@B3],[@B4]\] to develop two separate instruments for studying how doctors work, the Approach to Work Questionnaire (AWQ) and the Workplace Climate Questionnaire (WCQ). In Canadian physicians \[[@B2],[@B5]\] the AWQ showed three separate factors, which were called *Surface-Rational*, *Surface-Disorganised*, and *Deep*(see table [1](#T1){ref-type="table"}). These approaches related to different methods and motivations for continuing medical education. Those with a deep approach preferred independent and problem-based learning and motivation was internal. Surface-rational and surface-disorganised approaches were primarily driven by external motivation, with the preferred mode of continuing education learning being independent for the surface-rational, and in consultations for the surface-disorganised. The WCQ showed three dimensions, called *Choice-Independence, Supportive-Receptive*, and *Workload*(see table [1](#T1){ref-type="table"}), which correlated with the AWQ. Doctors reporting Choice-Independence and Supportive-Receptive work environments had a Deeper approach, whereas those describing an environment dominated by Workload tended to be more Surface-Disorganised. Some doctors are unhappy with their work, which can manifest as stress (usually assessed by the General Health Questionnaire) or burnout, which has three separate components of emotional exhaustion, depersonalisation and reduced personal accomplishment (see table [2](#T2){ref-type="table"}). Greater stress and burnout in doctors are related to the personality trait of neuroticism or \'negative affectivity\' \[[@B6]\]. The AWQ and WCQ provide a snapshot of a doctor\'s learning environment and approach to work at one particular time, as also do measures of stress and burnout. A key question, as Deary *et al*recognised \[[@B6]\] when considering stress, is the extent to which different approaches to work and the climate of the workplace are consequences *of the workplace*or *of the doctor*. At first sight it might seem that the workplace itself has to be the primary force driving both workplace learning and workplace climate. However, it is also possible that approaches to learning and work mainly depend upon pre-existing differences among doctors, differences that may already have manifested earlier in the doctors\' careers. The AWQ bears a strong formal similarity to the surface, deep and strategic study habits and learning styles identified by the Study Process Questionnaire (SPQ), which assesses the motivations and approaches used by students in higher education (see table [3](#T3){ref-type="table"}). The similarity is not accidental since the AWQ was developed by adapting items from Entwistle and Ramsden\'s Approaches to Study Inventory \[[@B7]\], which has a similar factor structure to that of the Study Process Questionnaire \[[@B8]\]. It is therefore expected that there may be significant continuities across approaches to study and approaches to work. In this paper we describe a large cohort of UK doctors, typically aged 29 or 30 at the time of the study, who have been qualified for five or six years, who are practising as SHOs or SpRs in hospital or are in general practice, and who previously had been studied when aged 17 or 18 at application to medical school in autumn 1990 \[[@B9]\], in their final year at medical school \[[@B10]\] and as PRHOs \[[@B11]\]. The main interest here will be in the extent to which a doctor\'s present approaches to work and their workplace climate, as well as their stress and burnout, relate to earlier measures of study habits and personality at application to medical school and subsequently. Method ====== Participants ------------ In the autumn of 1990 a questionnaire was sent to all individuals with European Community postal addresses who had applied to any of the five UK medical schools taking part in the study \[[@B9]\]; they represented about 70% of all applicants and acceptances for medical school in that year. The response rate was 93%. Students who were accepted for entry in 1991, 1992 or 1993 were followed up in their final year at medical school (1995--1998), when the response rate was 56%, and at the end of their PRHO year (1996--1999), when the response rate was 58%. In 2002 a tracing exercise searched the *Medical Register*and *Medical Directory*from 1995 to 2002 to find the addresses of as many doctors as possible who were in the original survey, and who were known not to have died, left medical school during basic medical sciences, or otherwise to be no longer in the survey. For study design see figure [1](#F1){ref-type="fig"}. Questionnaire ------------- Questionnaires were sent to all individuals with current or recent GMC addresses. The questionnaire consisted of a single folded A3 sheet of paper (4 A4 sides). Included in the present questionnaire (described in the results as \'2002\') were the 12-item General Health Questionnaire (GHQ) \[[@B12]\]; an abbreviated version of the Maslach Burnout Inventory (aMBI), which has three sub-scales, Emotional Exhaustion, Depersonalisation, and Personal Accomplishment \[[@B13],[@B14]\]; a three-item scale modelled on the aMBI, which assesses Happiness with a Medical Career \[[@B15]\]; an abbreviated version of the Study Process Questionnaire, which has three sub-scales of Surface, Strategic and Deep learning \[[@B16],[@B17]\]; an abbreviated questionnaire assessing the \'Big Five\' personality dimensions of Neuroticism, Extraversion, Openness to Experience, Agreeableness and Conscientiousness \[[@B15],[@B18]\]; and abbreviated versions of the Approach to Work Questionnaire (aAWQ) and the Workplace Climate Questionnaire (aWCQ) \[[@B19]\], each of which has three sub-scales, and for which a detailed description is provided in the **Supplementary Information** (see [Additional file: 1](#S1){ref-type="supplementary-material"}) \[[@B2]\]. The GHQ, aMBI and personality questionnaire had also been administered previously in the PRHO survey, and the SPQ had been administered in the Applicant and Final year surveys. Procedure --------- Questionnaires, along with a postage-paid return envelope, were posted at the beginning of December 2002. Two reminders were sent to non-respondents. Although the official closing date was 25^th^March 2003, a few questionnaires were returned up until the end of August 2003. Statistical analysis used SPSS version 10.5, and structural equation modelling used LISREL 8.52. Results ======= The tracing exercise looked for 2,912 individuals thought to have completed basic medical sciences and entered a clinical course. Eighty-nine had never been on the UK Medical Register, and either had failed finals, had never registered, or had emigrated. Of 2,823 individuals who were traced, 2,754 doctors were on the 2002 Register, 7 returned to the Register during 2002, and 64 were on an earlier Register. Of 2,823 questionnaires sent, 176 were returned by the Post Office as undeliverable, 10 doctors were travelling and hence uncontactable, and2 had died. Of the remaining 2,635 doctors, 1,668 returned questionnaires, giving a response rate of 63.3%. There was no evidence of response bias (see **Supplementary Information** see [Additional file: 1](#S1){ref-type="supplementary-material"}). Respondents ----------- The mean age of respondents on 1^st^December 2002 was 30.4 years (SD 1.86, range 28.3 -- 49.2). There was substantial variation in the scores on the aAWQ and the aWCQ, and the factor structures of the aAWQ and aWCQ were similar to those reported elsewhere \[[@B19]\] (see **Supplementary Information** see [Additional file: 1](#S1){ref-type="supplementary-material"}). There was also substantial variation on the measures of stress, burnout and satisfaction with medicine as a career, with 21.3% of doctors (345/1617) reporting GHQ scores of 4 or more, the conventional level of \'caseness\'. Approaches to work and learning were correlated with climate in the workplace, and as in the Delva *et al*study, the highest correlations were for a surface-disorganised approach correlating with high workload, and a deep approach correlating with a supportive-receptive environment and with choice-independence (table [4](#T4){ref-type="table"}). Approaches to work ------------------ Table [4](#T4){ref-type="table"} shows correlation of the stress measures with approaches to work and study habits. The largest correlations were of a surface-rational approach with a strategic learning style, and a deep approach to work with a deep learning style. In each case the correlations were not only highly significant when study habits were measured in the final year at medical school, six or seven years earlier, but were also very significantly correlated with study habits measured at selection, twelve years earlier. Correlations of approaches to work and stress, burnout and satisfaction with medicine were generally small, and generally were only with measures taken in 2002, and not with measures taken as a PRHO, five or six years earlier. The sole exception was that a surface-disorganised approach correlated with high stress as measured by the GHQ, both in 2002 and with stress when the doctors were PRHOs. Workplace climate ----------------- Table [5](#T5){ref-type="table"} shows correlations between the workplace climate and study habits, stress, burnout and satisfaction with medicine. In contrast to the associations with approaches to work, the workplace climate showed only small correlations with study habits, but showed strong correlations with stress, burnout and satisfaction with medicine. In particular, high stress in the PRHO year showed very significant correlations with measures in 2002 of a perceived high workload, a less supportive-receptive environment, and less choice-independence. In addition, emotional exhaustion both in 2002 and in the PRHO year were related to a high perceived workload in 2002. Personality ----------- Table [6](#T6){ref-type="table"} shows the correlations of approaches to work and workplace climate with the \'Big Five\' measures of personality, measured both in 2002 and also measured five to six years previously when the doctors were PRHOs. The surface-disordered approach to work is associated with high neuroticism and low conscientiousness, the PRHO correlations also being highly significant in each case. Neuroticism, both in 2002 and as a PRHO, is also associated with a perceived high workload (although in contrast to its prediction of a surface-disordered approach, conscientiousness is not a significant correlate of workload). The deep approach to work and learning is associated with being extravert and with greater openness to experience, and again the measures taken six years earlier are predictive. Finally a supportive-receptive work climate is associated with greater reported agreeableness, both in 2002 and six years earlier as a PRHO. There were no substantial correlations between personality and the surface-rational approach to work or choice-independence in work climate. Multiple regressions -------------------- Tables [4](#T4){ref-type="table"} to [6](#T6){ref-type="table"} show a large number of correlations, which are not always straightforward to interpret, both because they are numerous and because many variables are themselves inter-correlated. Multiple regression was used to clarify the relationships (for technical details see **Supplementary Information** see [Additional file: 1](#S1){ref-type="supplementary-material"}). Each individual measure of the aAWQ and aWCQ was regressed on the measures of study habits at application (n = 3) and in the final year (n = 3), of stress and burnout during the PRHO year (n = 4) and in 2002 (n = 4), and of personality in the PRHO year (n = 5) and in 2002 (n = 5). Alpha for entry was set at p \< 0.0001 in view of the large sample size and the number of independent variables. The variables that were significant are shown in tables [4](#T4){ref-type="table"},[5](#T5){ref-type="table"} and [6](#T6){ref-type="table"} and [3](#T3){ref-type="table"} in italics. Of particular interest are variables that show not only show significant contemporaneous correlations but also significant correlations when measured five or more years previously. A surface-disorganised approach to work is predicted by surface learning in medical school and by higher neuroticism scores and lower conscientiousness (see tables [4](#T4){ref-type="table"} and [6](#T6){ref-type="table"}). The surface-rational approach to work is predicted by strategic learning in medical school, and by less openness to experience and higher conscientiousness. The deep approach to work is predicted by a deep approach to learning at medical school, by greater extraversion, by greater openness to experience, and by lower emotional exhaustion. A workplace climate dominated by a high workload is predicted by higher stress and emotional exhaustion measures five years earlier, and by lower openness to experience (see tables [5](#T5){ref-type="table"} and [6](#T6){ref-type="table"}). A supportive-receptive workplace is predicted by lower stress and depersonalisation, and a higher sense of personal accomplishment when measured previously, and by a more agreeable personality. Choice-independence in the work environment is predicted only by lower previous measures of stress. Stress, burnout and satisfaction with medicine ---------------------------------------------- Although in the previous analyses, stress and burnout have been used as predictors of approaches to work and workplace climate, they are also important outcome measures in their own right. Table [7](#T7){ref-type="table"} shows the correlations of the five \'stress-related measures\' (GHQ, the three burnout measures and satisfaction) with measures of learning style and personality, in each case measured on two separate occasions. Personality correlates with each of the measures, as do study habits. Because of the complex inter-correlations between the dependent variables, multiple regression was used, as before, to find the most important relationships (for technical details see **Supplementary Information** see [Additional file: 1](#S1){ref-type="supplementary-material"}). Doctors who are most stressed showed higher levels of neuroticism, both currently and previously, and those reporting most emotional exhaustion also had higher neuroticism levels, as well as being more introvert. High levels of depersonalisation related to lower levels of agreeableness. A greater sense of personal accomplishment related to previous deep approaches to study and learning, as well as to being more extravert. Overall satisfaction with medicine as a career related to lower levels of neuroticism. Path analysis ------------- The complex relationships described by the various correlations are best analysed and described by means of path analysis or causal modelling \[[@B20]\], which analyses the entire set of correlations between variables, using plausible assumptions about causality and removing non-significant paths. The path diagram, which was analysed using LISREL 8.52 \[[@B21]\], is shown in figure [2](#F2){ref-type="fig"}. Measures to the left can causally influence measures to their right. Based on the time-lagged correlations reported previously, we assumed that stress causes different approaches to work, and we also assumed that approaches to work cause differences in workplace climate rather than vice-versa. (Nevertheless, we acknowledge that the causation may well be reciprocal, as suggested by the originators of the scale \[[@B2],[@B19]\]; further longitudinal data will be required to test that hypothesis). Study habits are temporally and causally prior to stress, approaches to work and workplace climate. Personality, being a trait, was prior to all other measures. For technical details see the **Supplementary Information** (see [Additional file: 1](#S1){ref-type="supplementary-material"}). Although several of our variables are measured at different time points, we have chosen not to present a model in which each variable has been included on each occasion that it is measured, as the resulting diagram becomes unmanageably complex. Although the path diagram in figure [2](#F2){ref-type="fig"} is complex at first sight, the paths are readily interpretable. The diagram divides into two broad sections, with the measures of learning style and approach to work at the bottom, and stress at the top. Here we have simplified the model by omitting the closely correlated measures of burnout, and only including paths with t-values greater than 3.6. Estimates of all the paths are available in the **Supplementary Information** (see [Additional file: 1](#S1){ref-type="supplementary-material"}). Stress in our model is caused by personality differences, being greatest in those having high neuroticism scores, low extraversion scores, and low conscientiousness scores. It is unrelated to learning style. Learning styles at medical school relate to different personality measures, in particular showing no relationship to neuroticism. Deep learning is highest in extraverts who are open to experience, whereas strategic learning is highest in highly conscientious individuals with low openness to experience. Surface learning style is higher in introverts who are low in openness to experience. These findings are similar to those of others \[[@B22]\]. Approaches to work are mainly but not entirely driven by learning styles. A deep approach to work occurs in extraverts who are open to experience and have a deep learning style. The surface-rational and surface-disorganised approaches to work are both greater in those with a surface learning style. However, a surface-disorganised approach occurs in individuals with higher neuroticism scores, in those with lower conscientiousness scores, and in those who have been stressed, whereas the surface-rational approach to work occurs in strategic learners and in those who are low in openness to experience. Workplace climate has a range of influences. High perceived workload occurs in those with a surface-disorganised approach to work, who have been stressed and are more neurotic. In contrast, choice-independence and a supportive-receptive environment both occur in individuals who have not previously been stressed, the choice-independence approach occurring in those with a deep approach to work, whereas the supportive-receptive approach occurs in those who have higher scores on the personality trait of agreeableness. Discussion ========== Many doctors at the age of 30 are unhappy in their jobs, and a fifth of our sample reached the conventional GHQ criterion of psychiatric \'caseness\'. In contrast, many doctors reported high levels of personal accomplishment, choice and independence in their work environment, satisfaction with medicine as a career, and intellectual and emotional satisfaction from their work. That is not new; Sir William Osler in 1905 contrasted doctors \"whose stability of character and devotion to duty make one proud of our profession\" with those who find it difficult to keep \"the flame alive, smothered as it is apt to be by the dust and ashes of the daily routine\" \[[@B1]\]. In 2001, Richard Smith asked \"Why are doctors so unhappy?\" and concluded that \"The most obvious cause of doctors\' unhappiness is that they feel overworked and undersupported\" \[[@B23]\]. Certainly many doctors in our study report a high workload and a work climate that is neither supportive nor receptive, and those doctors also report more stress, burnout and dissatisfaction with medicine as a career. It is tempting therefore to conclude, as did an article in a special edition of *BMJ Careers*devoted to \"Doctors\' Wellbeing\", that excessive workload and absence of support are directly caused by poor working conditions: \"the way in which the NHS is run generates stress for members of the workforce every day\" \[[@B24]\]. However, such an interpretation is not straightforward in general \[[@B25]\]. It is particularly difficult for the doctors in our study because the study is longitudinal, and workload and lack of support correlate *with stress and burnout reported five or six years earlier*, when the doctors were PRHOs and carrying out entirely different jobs. High perceived workload and poor support are therefore determined as much by doctors themselves as by specific working conditions. That view was expressed in another article in the special edition of *BMJ Careers*: \"A critical element contributing to the stress that many conscientious doctors experience is internal\...\" \[[@B26]\]. A similar conclusion was reached in a previous study of ours when these doctors were PRHOs, and multi-level modelling showed that stress is not a characteristic of jobs but of doctors, different doctors working in the same job being no more similar in their stress and burnout than different doctors in different jobs \[[@B11]\]. If differences in reported workload are partly explained by differences among doctors, what in turn explains those differences? Doctors reporting a high workload also have what Delva *et al*\[[@B2]\] describe as a *surface-disorganised*approach to work, which in turn is correlated with being a surface learner at application to medical school, a dozen years previously. Surface-disorganised doctors are also high on the personality trait of neuroticism and low on the trait of conscientiousness; and again those correlations are with measures taken six years earlier when the doctors were PRHOs. Doctors reporting a work climate low in support were lower on the personality scale of agreeableness in the measures collected when they were PRHOs. Some doctors may be stressed and burned out, but what predicts those others who are happy in their work? Doctors reporting high satisfaction with medicine as a career have a deep approach to work, and that approach is more common in those who also had a deep learning style when they applied to medical school. Satisfaction with medicine also relates directly to the personality traits of greater extraversion and lower neuroticism, and the deep approach to work correlates with greater extraversion and more openness to experience. Doctors who describe their colleagues as receptive and supportive score more highly on the personality trait of agreeableness; and as in many other correlations reported here, that correlation is stable across time -- those who are more agreeable at the age of 24 have a more receptive and supportive work environment when aged 30. An overview of our findings is that approaches to work are predicted by earlier measures of study habits and learning styles, whereas perceived work climate, and its pathologies such as stress and burnout, are predicted mainly by personality. Although unfortunately our study did not measure personality during selection, the high stability of the Big Five measures across the life-span \[[@B27]-[@B29]\] (and across our two measures six years apart), as well as their heritable component \[[@B30]\], means that we have little doubt that personality at selection would also have been predictive, particularly given that a similar pattern of correlations was found in a different cohort of doctors in mid-career \[[@B15]\]. Other studies on very different groups of students have also found, like us, that both strategic and deep learning correlate with conscientiousness, and that deep learning also correlates with extraversion and openness to experience \[[@B22],[@B31]\]. Our study has, for various reasons, not looked at academic performance in relation to study habits, learning styles and personality, although previous work of ours has found clear correlations between learning styles and examination performance \[[@B32]\]. In contrast we have not found any correlation of undergraduate or postgraduate academic achievement with personality \[[@B15]\], and although some studies have found correlations of conscientiousness with academic achievement \[[@B33]\], this does seem to vary according to the learning context \[[@B34],[@B35]\]. Although we will be looking at this question again in more detail in a further analysis, it does seem probable that personality mostly has an indirect effect upon academic achievement via approaches to learning \[[@B31],[@B36]\]. If, as William Wordsworth said, \"the child is father to the man\", then the seeds of subsequent job satisfaction and dissatisfaction in doctors may be visible in the personality, motivations and learning styles of medical school applicants. This argument may provide some justification for using such measures in selection, particularly given the general association of job performance and satisfaction with personality \[[@B37]\] and motivation \[[@B38]\], and learning styles with personality \[[@B22]\] . However, just as genes are not destiny, so neither personality nor learning style is destiny. Nurture interacts with nature \[[@B39]\], the environment building upon the genes, and the genes using what is provided by the environment; the poetic complement to William Wordsworth is therefore Alexander Pope, who said, \"This education forms the common mind: Just as the twig is bent, the tree\'s inclined.\" Extreme introverts can, with sufficient insight, preparation and appropriate training become effective public speakers, less conscientious individuals can learn to be more organised and efficient, and those who are more neurotic can transcend their anxieties (and indeed neuroticism may be beneficial if sublimated into a professional concern for detail in critical situations, rather than merely being undifferentiated personal anxiety). . Formal education, particularly effective formal education \[[@B40]\], can also alter study habits and learning styles, which are less fixed and \'trait-like\' than personality measures \[[@B17]\]. Intercalated degrees increase deep and strategic learning and decrease surface learning at medical school \[[@B41]\], making it likely that they also encourage surface-rational and deep approaches to work. Deep and strategic learning also relate to the clinical experience gained by medical students \[[@B32]\], making it possible that greater patient involvement during undergraduate clinical training, rather than mere reliance on textbook learning to pass exams, a characteristic of surface learners, will also reduce surface-disorganised approaches to work. Conclusions =========== Longitudinal data suggest that personality and learning style are not merely *correlates*of approaches to work, workplace climate, stress, burnout and satisfaction with a medical career, but are *causes*, events later in time being predicted by events earlier in time \[[@B35]\]. Doctors with greater stress and emotional exhaustion, who were less satisfied with medicine as a career, had higher neuroticism scores and were more likely to be surface-disorganised. Lower conscientiousness on the personality measure also predicted greater stress. Extraverts reported more personal accomplishment and were more satisfied with medicine. The personality measure of agreeableness predicted a more supportive-receptive work environment. These results imply that differences in approach to work and workplace climate in our study result from differences among doctors themselves, as much as they do from differences in working conditions. Competing interests =================== None declared. Authors\' contributions ======================= The cohort study was designed by ICMcM. The present follow-up was designed by ICMcM and EP, who also prepared the questionnaire. AK was responsible for day-to-day running of the study, and for data entry and cleaning. ICMcM was primarily responsible for data analysis and for writing the first draft of the paper. ICMcM, EP and AK were all involved in preparing the final draft of the paper. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1741-7015/2/29/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional file 1 ::: ::: {.caption} ###### Click here for file ::: Acknowledgments =============== We thank the many doctors who have participated in this study over the past twelve years. We are also grateful to the three reviewers for their very helpful suggestions. This follow-up of the 1991 cohort was funded by the London Deanery. The survey of applicants was funded by the Leverhulme Trust and the Department of Health, follow-ups during medical school were funded by the Nuffield Foundation and the Department of Health, and the PRHO follow-up was funded by North Thames Postgraduate Medical and Dental Education. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Diagram showing the timing of the various stages of the survey. Note that not all applicants entered in October 1991 (despite in most cases having applied to do so), and subsequent stages of the study therefore took place at different times. Some entrants also took a year longer because of taking an intercalated degree (represented by the diagonal arrow from entrants in Oct 1991 to Final year in 1997), and a few other students delayed for other reasons. All doctors were surveyed at the end of their PRHO year, whenever that had occurred. The cohort was finally brought back into step as a single cohort with the 2002 follow-up when all doctors were studied at the same time, irrespective of the time at which they qualified. Questionnaires were given out at all the boxes shown (with the exception of a questionnaire to Entrants, who are shown merely to make the flow clearer). It should also be noted that there was a follow-up of a subset of the students in their third academic year, which is not shown here because the data are not discussed in this study. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Path diagram showing the relationships among the measures of personality, learning style, stress, approaches to work, and workplace climate. The width of arrows is proportional to the strength of an effect, which is shown alongside each line as a path (beta) coefficient. Negative effects are shown as red, dashed lines. For details of the statistical method and a fuller model incorporating all links, see **Supplementary Information**. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Dimensions of the AWQ and WCQ \[2\]. ::: **Approaches to work questionnaire** -------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ *Surface disorganised:* Feeling overwhelmed by work. For example, being unsure what is needed to complete a task, finding it difficult to organise time effectively, reading things without really understanding them. *Surface rational:* Preference for order, detail, and routine. For example likes to know precisely what is expected, puts of a lot of effort into memorising important facts when learning something new. *Deep approach:* Integrative approach that leads to personal understanding. For example, tries to relate new ideas to situations where they might apply. **Workplace climate questionnaire** *Choice-independence:* Perception of control over what one does and how one does it. *Supportive-receptive:* Perception that help is available in the workplace and colleagues are understanding. *Workload:* Perception of heavy workload and having to cope alone. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The three separate components of burnout \[42\]. *Note*: burnout on the MBI is indicated by higher scores on the emotional exhaustion and depersonalisation scales, and lower scores on the scale of personal accomplishment. ::: ---------------------------- -------------------------------------------------------------------------------------------------------------------------------- *Emotional exhaustion:* Reduced energy and job enthusiasm; emotional and cognitive distancing from the job. *Depersonalisation:* Cynicism; lack of engagement and distancing from patients; treatment of patients as inanimate, unfeeling objects. *Personal accomplishment:* A sense of efficacy and effectiveness; of involvement, commitment and engagement; of capacity to innovate, change and improve. ---------------------------- -------------------------------------------------------------------------------------------------------------------------------- ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Differences in motivation and process of the surface, deep and strategic approaches to learning assessed in the Study Process Questionnaire \[41\]. ::: **Motivation** **Process** --------------- --------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------ **Surface** Completion of the course Fear of failure Rote learning of facts and ideas Focussing on task components in isolation Little real interest in content **Deep** Interest in the subject Vocational relevance Personal understanding Relate ideas to evidence Integration of material across courses Identifying general principles **Strategic** Achieving high grades Competing with others To be successful Use techniques that achieve highest grades Level of understanding Patchy and variable ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Pearson correlation (Significance; N) of background measures with the Approaches to Work Questionnaire. Correlations significant at p \< 0.05 are in bold, and those with an absolute value of greater than 0.2 are underlined. Variables that are significant in the multiple regression (see text) are indicated in italics. ::: Time of measurement aAWQ Surface-Disorganised aAWQ Surface-Rational aAWQ Deep ---------------------------------------- --------------------- ----------------------------------------------- ------------------------------------------------- ----------------------------------------------- aWCQ Workload 2002 [**0.259**]{.underline} (p \< .001; N = 1644) 0.045 (p = .071, N = 1642) **-0.068**(p = .006, N = 1638) aWCQ Supportive-Receptive 2002 **-0.112**(p \< .001; N = 1636) **0.064**(P = .009; N = 1633) **0.102**(P \< .001; N = 1630) aWCQ Choice-Independence 2002 **-0.125**(P \< .001; N = 1647) -0.001 (P = .976; N = 1645) [**0.209**]{.underline} (P \< .001; N = 1641) SPQ Surface Learning Application ***0.119***(P \< .001; N = 1591) **0.105**(P \< .001; N = 1589) **-0.081**(P \< .001; N = 1584) Final Year **0.105**(P = .001; N = 999) **0.101**(P = .001; N = 1001) **-0.109**(P = .001; N = 996) SPQ Strategic Learning Application **-0.101**(P \< .001; N = 1591) [***0.203***]{.underline} (P \< .001; N = 1589) **0.052**(P = .038; N = 1584) Final Year **-0.157**(P \< .001; N = 999) [**0.215**]{.underline} (P \< .001; N = 1001) **0.149**(P \< .001; N = 996) SPQ Deep Learning Application **-0.078**(P = .002; N = 1591) **0.067**(P = .007; N = 1589) ***0.192***(P \< .001; N = 1584) Final Year **-0.131**(P \< .001; N = 999) **0.152**(P \< .001; N = 1001) [**0.240**]{.underline} (P \< .001; N = 996) General Health Questionnaire PRHO **0.169**(P \< .001; N = 974) 0.010 (P = .761; N = 974) **-0.079**(P = .014; N = 972) 2002 [**0.294**]{.underline} (P \< .001; N = 1611) 0.018 (P = .471; N = 1608) **-0.119**(P \< .001; N = 1604) aMBQ -- Emotional exhaustion PRHO 0.062 (P = .103; N = 705) -0.011 (P = .777; N = 704) -0.042 (P = .267; N = 704) 2002 [**0.223**]{.underline} (P \< .001; N = 1609) 0.007 (P = .782; N = 1607) ***-0.157***(P \< .001; N = 1602) aMBQ -- Depersonalisation PRHO -0.003 (P = .939; N = 661) -0.033 (P = .400; N = 658) -0.017 (P = .672; N = 659) 2002 [**0.224**]{.underline} (P \< .001; N = 1598) **-0.071**(P = .005; N = 1596) **-0.151**(P \< .001; N = 1590) aMBQ -- Personal accomplishment PRHO **-0.137**(P \< .001; N = 938) -0.032 (P = .335; N = 937) **0.156**(P \< .001; N = 936) 2002 0.024 (P = .335; N = 1604) -0.013 (P = .610; N = 1602) **0.095**(P \< .001; N = 1596) Satisfaction with medicine as a career 2002 **-0.162**(P \< .001; N = 1620) 0.037 (P = .135; N = 1617) [**0.211**]{.underline} (P \< .001; N = 1612) ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Pearson correlation (Significance; N) of background measures with the Workplace Climate Questionnaire. Correlations significant at p \< 0.05 are in bold, and those with an absolute value of greater than 0.2 are underlined. Variables that are significant in the multiple regression (see text) are in italics. ::: Time of measurement aWCQ Workload aWCQ Supportive-Receptive aWCQ Choice-Independence ---------------------------------------- --------------------- ------------------------------------------------- -------------------------------------------------- -------------------------------------------------- SPQ Surface Learning Application 0.022 (P = .389; N = 1587) 0.042 (P = .093; N = 1578) 0.004 (P = .874; N = 1591 Final Year 0.037 (P = .240; N = 999) -0.034 (P = .287; N = 998) 0.054 (P = .090; N = 1000) SPQ Strategic Learning Application 0.032 (P = .196; N = 1591) 0.043 (P = .090; N = 1578) **0.082**(P = .001; N = 1591) Final Year **0.063**(P = .048; N = 999) 0.020 (P = .520; N = 998) 0.036 (P = .255; N = 1000) SPQ Deep Learning Application 0.008 (P = .738; N = 1587) 0.029 (P = .251; N = 1578) 0.049 (P = .051; N = 1591) Final Year -0.032 (P = .306; N = 999) **0.071**(P = .026; N = 998) **0.065**(P = .041; N = 1000) General Health Questionnaire PRHO **0.183**(P \< .001; N = 973) **-0.134**(P \< .001; N = 970) **-0.110**(P = .001; N = 972) 2002 [***0.354***]{.underline} (P \< .001; N = 1606) [***-0.239***]{.underline} (P \< .001; N = 1597) [***-0.259***]{.underline} (P \< .001; N = 1609) aMBQ -- Emotional exhaustion PRHO [**0.211**]{.underline} (P \< .001; N = 703) -0.056 (P = .136; N = 701) -0.035 (P = .348; N = 703) 2002 [***0.400***]{.underline} (P \< .001; N = 1607) **-0.164**(P \< .001; N = 1607) [**-0.220**]{.underline} (P \< .001; N = 1609) aMBQ -- Depersonalisation PRHO 0.057 (P = .145; N = 658) **-0.109**(P = .005; N = 656) -0.020 (P = .609; N = 658) 2002 [**0.216**]{.underline} (P \< .001; N = 1595) [***-0.222***]{.underline} (P \< .001; N = 1594) **-0.165**(P \< .001; N = 1597) aMBQ -- Personal accomplishment PRHO -0.028 (P = .393; N = 937) **0.110**(P \< .001; N = 933) 0.063 (P = .055; N = 936) 2002 **0.163**(P \< .001; N = 1601) ***0.143***(P \< .001; N = 1600) 0.044 (P = .079; N = 1603) Satisfaction with medicine as a career 2002 [**-0.256**]{.underline} (P \< .001; N = 1617) [**0.317**]{.underline} (P \< .001; N = 1616) [**0.294**]{.underline} (P \< .001; N = 1620) ::: ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Pearson correlation (Significance; N) of Approach to Work Questionnaire and the Workplace Climate Questionnaire with personality. Correlations significant at p \< 0.05 are in bold, and those with an absolute value of greater than 0.2 are underlined. Variables that are significant in the multiple regression (see text) are in shaded boxes. ::: Time of measurement aAWQ Surface-Disorganised aAWQ Surface-Rational aAWQ Deep aWCQ Workload aWCQ Supportive-Receptive aWCQ Choice-Independence ------------------------ --------------------- -------------------------------------------------- ----------------------------------- ------------------------------------------------- ----------------------------------------------- --------------------------------- --------------------------------- Neuroticism PRHO **0.172**(P \< .001; N = 993) 0.021 (P = .510; N = 993) -0.050 (P = .113; N = 992) **0.111**(P \< .001; N = 992) -0.055 (P = .086; N = 989) -0.017 (P = .592; N = 991) 2002 [***0.335***]{.underline} (P \< .001; N = 1645) 0.021 (P = .405; N = 1643) **-0.111**(P \< .001; N = 1638) [**0.307**]{.underline} (P \< .001; N = 1642) **-0.180**(P \< .001; N = 1632) **-0.168**(P \< .001; N = 1644) Extraversion PRHO **-0.113**(P \< .001; N = 991) 0.056 (P = .079; N = 991) 0**.174**(P \< .001; N = 989) **-0.090**(P = .004; N = 990) **-0.087**(P = .006; N = 987) 0.049 (P = .124; N = 989) 2002 **0.173**(P \< .001; N = 1649) 0.037 (P = .133; N = 1647) [***0.288***]{.underline} (P \< .001; N = 1643) **-0.164**(P \< .001; N = 1647) **0.147**(P \< .001; N = 1637) **0.119**(P \< .001; N = 1649) Openness to experience PRHO -0.011 (P = .729; N = 975) **-0.077**(P = .016; N = 975) [**0.263**]{.underline} (P \< .001; N = 973) -0.037 (P = .250; N = 974) 0.036 (P = .259; N = 971) 0.033 (P = .297; N = 973) 2002 **-0.063**(P = .010; N = 1646) ***-0.108***(P \< .001; N = 1644) [***0.346***]{.underline} (P \< .001; N = 1639) ***-0.096***(P \< .001; N = 1643) 0.038 (P = .129; N = 1633) **0.080**(P \< .001; N = 1645) Agreeableness PRHO -0.034 (P = .285; N = 991) 0.048 (P = .130; N = 991) 0.038 (P = .235; N = 989) -0.024 (P = .455; N = 990) ***0.182***(P \< .001; N = 987) 0.026 (P = .421; N = 989) 2002 **-0.108**(P \< .001; N = 1650) 0.096 (P \< .001; N = 1648) 0.038 (P = .121; N = 1643) **-0.057**(P = .021; N = 1647) **0.153**(P \< .001; N = 1638) 0.017 (P = .498; N = 1650) Conscientiousness PRHO [***-0.353***]{.underline} (P \< .001; N = 991) 0.124 (P \< .001; N = 991) **0.084**(P = .008; N = 989) 0.023 (P = .467; N = 991) 0.057 (P = .074; N = 987) 0.056 (P = 077; N = 990) 2002 [***-0.477***]{.underline} (P \< .001; N = 1645) *0.126*(P \< .001; N = 1643) **0.136**(P \< .001; N = 1638) **-0.088**(P \< .001; N = 1641) **0.093**(P \< .001; N = 1632) **0.092**(P \< .001; N = 1644) ::: ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Pearson correlation (Significance; N) of stress, burnout and satisfaction with medicine as a career in 2002 with study habits at application to medical school and in th final year of medical school, and with personality in the PRHO year and in 2002. Correlations significant at p \< 0.05 are in bold, and those with an absolute value of greater than 0.2 are underlined. Variables that are significant in the multiple regression (see text) are in shown in italics. ::: Time of measurement Stress (GHQ) (2002) Emotional exhaustion (aMBI) (2002) Depersonalisation (aMBI) Personal accomplishment (aMBI) Satisfaction with medicine as a career ------------------------ --------------------- ------------------------------------------------- ------------------------------------------------- ------------------------------------------------- --------------------------------- ------------------------------------------------- SPQ Surface Learning Application 0.036 (P = .150; N = 1555) **0.105**(P \< .001;N = 1552) **0.068**(P = .008;N = 1542) -0.003 (P = .895;N = 1546) -0.033 (P = .186;N = 1563) Final Year 0.008 (P = .792;N = 984) **0.090**(P = .005;N = 983) **0.094**(P = .003;N = 976) -0.036 (P = .266;N = 980) -0.023 (P = .471;N = 989) SPQ Strategic Learning Application -0.011 (P = .671; N = 1555) 0.017 (P = .513;N = 1552) **-0.073**(P = .004;N = 1542) 0.021 (P = .416;N = 1546) 0.046 (P = .068;N = 1563) Final Year -0.014 (P = .655;N = 984) -0.052 (P = .102;N = 983) **-0.108**(P = .001;N = 976) 0.007 (P = .819;N = 980) **0.104**(P = .001;N = 989) SPQ Deep Learning Application 0.041 (P = .102; N = 1555) -0.001 (P = .974;N = 1552) **-0.055**(P = .031;N = 1542) ***0.123***(P \< .001;N = 1546) 0.042 (P = .101;N = 1563) Final Year -0.022 (P = .498;N = 984) **-0.105**(P = .001;N = 983) **-0.071**(P = .027;N = 976) **0.120**(P \< .001;N = 980) **0.134**(P \< .001;N = 989) Neuroticism PRHO **0.192**(P \< .001;N = 972) **0.233**(P \< .001;N = 972) **0.103**(P = .001;N = 972) 0.036 (P = .255;N = 975) **-0.188**(P \< .001;N = 981) 2002 [***0.461***]{.underline} (P \< .001; N = 1610) [***0.378***]{.underline} (P \< .001;N = 1607) [**0.235**]{.underline} (P \< .001;N = 1596) **0.090**(P \< .001;N = 1603) [***-0.314***]{.underline} (P \< .001;N = 1618) Extraversion PRHO **-0.111**(P = .001;N = 970) **-0.190**(P \< .001;N = 970) **-0.137**(P \< .001;N = 969) **0.077**(P = .016;N = 972) **0.228**(P \< .001;N = 979) 2002 [**-0.243**]{.underline} (P \< .001; N = 1614) [***-0.262***]{.underline} (P \< .001;N = 1611) **-0.171**(P \< .001;N = 1599) ***0.152***(P \< .001;N = 1606) [**0.307**]{.underline} (P \< .001;N = 1621) Openness to experience PRHO 0.012 (P = .721;N = 956) 0.017 (P = .604;N = 955) 0.009 (P = .777;N = 954) **0.096**(P = .003;N = 957) 0.042 (P = .194;N = 964) 2002 -0.046 (P = .066; N = 1611) 0.010 (P = .677;N = 1607) -0.028 (P = .262;N = 1596) **0.127**(P \< .001;N = 1603) **0.066**(P = .008;N = 1618) Agreeableness PRHO -0.028 (P = .376;N = 970) -0.062 (P = .055;N = 970) **-0.240**(P \< .001;N = 969) **0.091**(P = .005;N = 972) **0.101**(P = .002;N = 979) 2002 **-0.080**(P = .001; N = 1615) **-0.094**(P \< .001;N = 1611) [***-0.322***]{.underline} (P \< .001;N = 1600) **0.082**(P = .001;N = 1607) **0.135**(P \< .001;N = 1622) Conscientiousness PRHO -0.045 (P = .165;N = 971) **-0.088**(P = .006;N = 970) **-0.121**(P \< .001;N = 969) 0.010 (P = .767;N = 972) 0.045 (P = .160;N = 979) 2002 ***-0.196***(P \< .001; N = 1610) **-0.129**(P \< .001;N = 1606) **-0.165**(P \< .001;N = 1595) 0.031 (P = .209;N = 1602) **0.162**(P \< .001;N = 1617) :::

PubMed Central

2024-06-05T03:55:47.537265

2004-8-18

PMC516449

Dynamics of HIV infection and selection of equilibrium states ============================================================= Reports on the high turnover rates of HIV and its preferred target, CD4+ lymphocytes, during the latent phase of HIV infection \[[@B1]-[@B3]\] have established the virus as a prime suspect for direct demolition of the immune system. These clinical findings have also stimulated further efforts at modeling \[[@B4],[@B5]\], and quantitative experimental observation \[[@B6]\]. Major journals have a preference for experimental or clinical data, and the results of mathematical modelling have not reached the broader AIDS research community. For example, the most interesting result of a simple dynamic model published several years ago \[[@B7]\], namely the existence of multiple equilibrium states, one of which is virus-free, has not been discussed in any of the recent publications on HIV response to anti-viral drugs. For a general medical audience it would be desirable to describe the basic features of the dynamics of HIV infection without recourse to any mathematical formulations. Dynamics implies change over time and the behavior of a dynamic system is defined by stating how the system variables affect each other during a unit of time. In the simplest model there are three system variables: (i) the number of uninfected lymphocytes, (ii) the number of infected lymphocytes and (iii) the number of free virions. The system equations describe how these populations interact. For HIV/CD4+, some of these interactions are understood and generally accepted; others are more speculative, and are subject to further study. However, even with different assumptions about these lesser known aspects, the most interesting result is little affected because it derives from the fact that the rate of infection, i.e. the number of newly infected cells in a unit of time, is proportional to the *product*of the number of uninfected cells and the number of free virions. This makes the resulting equations non-linear, and when the question of equilibrium is addressed, which is done by setting all rates (changes with time) equal to zero, there are two distinct solutions. One of these is free of virus, i.e. the number of virions (and infected cells) is equal to zero, whilst the other equilibrium state has non-zero values for all three populations and thus corresponds to a chronic infection. Which one of the two equilibrium states the system attains depends on the values of the system parameters. The most natural parameter to consider for switching the states is the difference between the rates at which uninfected cells are dying and proliferating. If this parameter, the net extinction rate of healthy lymphocytes, is increased above a critical value, the virus-free equilibrium is selected. This selection (bifurcation) is driven by the conditions of stability; the chronic infection state becomes unstable, i.e. any disturbance takes the system out of it, whilst the virus-free state becomes stable. Once the net extinction rate exceeds the critical value, the system finds its way out of infection. It just so happens that the amount by which the extinction rate needs to be changed, and this based on our current best estimates of other values, is quite modest -- several percent of the total lymphocyte population needs to be removed daily. Depletion of lymphocytes as a therapy for AIDS, based on a population dynamic model, has been advocated by de Boer and Boucher \[[@B8]\]. They proposed that using a suitable immunosuppressant or CD4-killing drug in combination with an anti-viral therapy may eliminate the infection. This author has arrived at the same result independently using a population dynamics model (three populations, as described above), but also using an expanded model that includes the immune response and, in particular, Tat protein-induced apoptosis \[[@B9]\]. The intervention by lymphocyte depletion will work as predicted by modelling only if other parameters of the system remain substantially unaffected. This is an unlikely outcome with immunosuppressive drugs. Results from limited attempts to use them in HIV-positive patients \[[@B10]-[@B12]\] are interesting, but not very encouraging. In fact, the observed rise in CD4+ counts runs *contrary*to the expected effect of depletion. Activation of latent CD4+ by OKT3 and IL-2 with intention to purge the virus has also been attempted \[[@B13]\], but the outcome was a surprisingly prolonged depletion of CD4+ with little effect on the virus. Our knowledge of the immune system interactions seems inadequate to provide satisfactory explanations for such a response. As a further illustration of how complex different interventions with biological modifiers can be, treatments with depleting CD4 monoclonal antibody showed a preferential loss of naive T cells, but did not affect IFN-gamma secreting cells \[[@B14]\], providing a clue as to why such depletions did not meet expectations in treating autoimmune diseases. Depletion of lymphocytes from the lymphatic circulation ======================================================= The prediction of the theoretical model calls for the removal of 5 to 10 percent of the total lymphocyte pool per day. The critical value is subject to uncertain estimates of some parameters, and it does differ between the simple, three-parameter and the five-parameter, expanded model. Perhaps the best approach would be to begin depletion, monitor the response by the viral RNA, and then adjust the depletion rate. All of this suggests that some means of physical removal would be best suited. Extracorporeal blood cell separation is a possibility, but the estimate of several hours that the patient would have to spend on the machine daily for several weeks to months, is very discouraging. However, filtration of the thoracic duct lymph, where lymphocytes are present in high concentration, seems almost ideal. The technique of duct drainage for lymphocyte depletion was developed in the sixties and the seventies in order to reduce the risk of organ rejection \[[@B15]-[@B21]\]. It has found fairly broad acceptance in renal transplantation \[[@B22]-[@B25]\] where the patients would typically be treated for four weeks prior to receiving a transplant. With improved techniques of tissue matching and better immunosuppressive drugs, the thoracic duct drainage lost its appeal in transplant surgery, but it remains an interesting approach to treatments of autoimmune conditions such as rheumatoid arthritis (RA) \[[@B26]-[@B28]\]. Improvements in the biocompatibilty of implants could ostensibly even extend the impressive performance of access devices that have remained potent for hundreds of days \[[@B29]\]. The number of lymphocytes removed from RA patients by thoracic duct filtration \[[@B29]\] is in the range of modelling predictions for elimination of the virus (on the order of ten billion per day at the start of the treatment). An alternative to removal of lymphocytes by duct drainage or filtration is their diversion from the lymphatic system into the gastro-intestinal tract, which has been demonstrated in experimental animals \[[@B30]-[@B32]\]. Cells are killed while the precious protein is recycled, avoiding the problem of protein loss by drainage. There is no evidence that HIV could survive gastric passage. The drawback of such a procedure would be in the difficulty of controlling the number of lymphocytes removed. This may not be such a serious limitation, provided the critical value is exceeded. The rate of lymphocyte removal then simply determines the duration of the treatment and the reduction in the number of lymphocytes the patient will experience. This, of course, is an issue that needs careful consideration. Depletion of lymphocytes will cause a transient reduction of their pool (with counts predicted to drop to a few hundred CD4 lymphocytes/microliter), and thus affect the general immune competence. The intervention by depletion should be done as early as possible when the rates of removal necessary are lower and the total pool is less reduced. Concluding remarks ================== Unfortunately, theoretical predictions of system dynamics are not very encouraging for the prospects of HIV vaccines. In principle, the vaccination primes the system for a faster, stronger response, including proliferation of the responding lymphocytes. As the same cells are targets for the virus, the system moves away from the stability condition for the virus-free equilibrium. Apoptosis of uninfected CD4+ lymphocytes in HIV infection is an appropriate response (for the host), albeit insufficient, since the cause of apoptosis is Tat protein produced by only the infected cells themselves. This prevents the system from eliminating the virus because the apoptotic signal *weakens*along with the infection. This suggests a possibility for a pharmacological intervention based on Tat protein that could sustain the apoptotic signal without introducing molecular modifiers with potentially broader effects. If vaccinations were to work, upon entry of the pathogen, they should *provoke*apoptosis of lymphocytes, not their proliferation. Until such discoveries are made, and to test perhaps their ultimate potential, a simple surgical intervention to allow for removal of lymphocytes merits further investigation. Dangers posed to the patient would be significant, both due to morbidity of the procedure itself, and the expected, but difficult to precisely predict cumulative effects on immunocompetence. An attractive aspect of using physical means of depletion is the possibility to terminate the treatment instantly and completely, as soon as any major deviations from the expected response would arise, indicating a failure of the model and alarming for unexpected risks. Competing interests =================== None declared.

PubMed Central

2024-06-05T03:55:47.541848

2004-8-31

PMC516450

Introduction ============ With relatively few genes compared to many other virus families, the retroviridae have evolved over the millenia to maximise the functions of their RNA genome. The genome serves as a versatile template from which various proteins can be translated by the use of splicing and by translational flexibility using scanning, IRES and frameshifting. It is also an RNA molecule capable of interacting with itself, and cellular and viral proteins. By these means, from an RNA around 7 -- 12 kilobases long, the retroviridae have evolved to infect a wide range of species and cell types. A unique characteristic of retroviral genomes is the fact that they are dimeric. The reasons for this are as yet unclear, and are discussed below. In brief, it is thought that the diploid genome allows template switching during reverse transcription and may be linked to recombination in some viruses. It may also play a role in translation of proteins and packaging of the RNA. Much of the work on the nature, structure(s), and role of the dimer linkage has been based on Human Immunodeficiency Virus Type 1, and this has been recently reviewed (\[[@B1]\] and Russell *et al*this issue \[[@B2]\]). Whether or not HIV-1 is a representative model for other retroviruses is open to debate. However, there have been important contributions from investigators studying other retroviruses. They have shown both similarities with the HIV-1 motifs, and also, importantly, differences. The fact that distinct RNA structures are used by different retroviruses to perform the same purpose, namely to link their two RNA molecules, tells us something very important. For these viruses, whatever organism or cell they are infecting it has been advantageous to evolve to do so with a double complement of genome in their virion particles. However, diploidy may be used to benefit the virus in a number of ways and for different viruses the priorities may vary. This review will attempt to draw on several examples from viruses other than HIV-1, whilst of necessity drawing comparisons with the latter. The dimeric genome ================== Retroviruses were discovered at the beginning of the 20th century \[[@B3],[@B4]\]. The unique nature of their genome was first discovered in the 1960s \[[@B5],[@B6]\] but the actual dimeric genomes were elucidated, and visualised by electron microscopy, a decade later \[[@B7],[@B8]\]. Bender and colleagues extracted the RNA from several different retroviruses and examined it by electron microscopy under denaturing conditions. The RNA appeared to be joined at a discrete point, termed the dimer linkage site (DLS). Using bromodU to label the RNA at one end, they were able to show that the molecules were joined at their 5\' ends \[[@B9],[@B10]\]. Under less stringent conditions the genomes can be demonstrated to interact along their lengths \[[@B11]\] and it is this that probably contributes to confusing reports on the exact location of the primary DLS in different viruses. RNA dimerisation in the primate lentiviruses, predominantly HIV-1, has subsequently been extensively studied \[[@B1]\], yet little has been published on this process in the non-primate lentiviruses. Early studies of rapid harvest virions of the prototype lentivirus, Maedi Visna virus (MVV), identified viral RNA with a Svedberg coefficient of 35S immediately post-budding, which increased with time to 70S. It is possible that weakly interacting dimers formed during RNA encapsidation may have been denatured during purification, however these observations are supportive of a progression from monomeric to dimeric RNA associated with viral maturation \[[@B12]\]. Since 1990 \[[@B13]\] it has been possible to study *in vitro*the RNA elements involved in the dimer linkage first observed by EM. It was shown that RNA transcripts comprising sequences from the 5\' end of the viral genome would migrate as two species of RNA when subjected to electrophoresis. By this means many subsequent studies were able to focus on isolating the elements and structures involved in dimerisation, and to investigate the role of the viral structural proteins in this process. Multiple functions for the dimeric genome? ========================================== As yet investigators have not been able to agree on a distinct role for the dimer linkage. The fact that it is conserved amongst the retroviridae does not guarantee that its role will be the same in all retroviral families. The following section of the review will endeavour to explore some of the proposed roles, and examine the evidence from different retroviruses. The dimeric linkage and recombination ===================================== Several studies have demonstrated that, in HIV-1 and MLV, the dimer linkage serves as a \"hotspot\" for recombination \[[@B14],[@B15]\]. It is an obvious hypothesis, that in viruses which are known for their hypervariability, there exists the capacity to jump from one RNA molecule to another. Researchers have compared dimerising to non-dimerising controls, and the frequency and distribution of template switching. Templates containing the dimerisation site had a 4-fold higher transfer efficiency than the non-dimerising control \[[@B14]\]. This result implies that recombination would occur preferentially at the site where the RNA molecules were in close proximity. In the case of HIV-1, whilst it has been shown that template switching is facilitated by template homology \[[@B16]\], it has also been demonstrated that recombination can occur between viruses of different subtypes which might have different dimer initiation sequences (DIS) \[[@B17]\]. Bearing in mind the fact that the genome is linked at other sites besides the DIS \[[@B11]\], it seems probable that other hot spots for recombination exist. Interestingly, it has been suggested that the nucleocapsid protein (NC) promotes or stimulates the strand transfer reaction. As will be discussed below, NC and the precursor Gag protein both bind the RNA close to the DIS in HIV-1. In addition, there is evidence that the presence of a dimer in the virus particle facilitates the first strand-transfer reaction of reverse transcription \[[@B18]\]. Work in our laboratory has shown that the Maedi Visna Virus DIS is centred on a helix terminating in a GACG tetraloop between positions 281 and 300 in the viral genome; a region which is highly conserved between the ovine and caprine lentiviruses (Monie, personal communication, see Figure [3d](#F3){ref-type="fig"}). Intriguingly, this structure shows homology with structural motifs in the *Alpha-*and *Gammaretroviruses*, but not with DIS regions identified in the primate lentiviruses. Within the *Alpha*- and *Gammaretroviruses*GACG tetraloops are involved in the packaging of viral RNA \[[@B19],[@B20]\] and whilst not a component of the core M-MLV DIS motif \[[@B21]\], they may contribute to the process of dimerisation and the stability of the resultant dimer \[[@B22]\]. Importantly, it is possible to form heterodimers between transcripts from these viruses containing the GACG tetraloops and between MVV and M-MLV (personal observations). This raises parallels with recent studies of the dimerisation of murine leukaemia viruses and Harvey Sarcoma virus in which GACG tetraloops were found to regulate inter-species RNA heterodimerisation \[[@B23]\], whilst other linkage elements were postulated to mediate homodimerisation. Recombination, and the genomic variability it confers cannot be the sole function of the dimeric genome, since retroviruses with highly conserved genomes and little sequence variability such as HTLV-1 \[[@B24]\] are also dimeric. Translation and packaging? ========================== Another possible role is that of the dimer linkage acting as a switch, its presence permitting or restricting the packaging of RNA. In HIV-2 two regions were originally suggested as dimer initiation sites, one analogous to the palindromic sequence identified as the principal DIS in HIV-1 (termed SL1), one close to the PBS \[[@B25]-[@B27]\]. Recently, a region upstream of SL1 (also called the DIS, see Figure [1a](#F1){ref-type="fig"}) was identified as being critical for packaging \[[@B28]\]. An extensive deletion analysis of the 5\' leader of HIV-2 was carried out, and removal of nucleotides 380--404 (HIV~ROD~), termed the DM region, rendered the virus severely packaging deficient. The mutation had been designed based on the mfold \[[@B29],[@B30]\] prediction, that removal of these sequences would disrupt the SL1 structure and hence dimerisation (Figure [1b](#F1){ref-type="fig"}). *In vitro*studies using RNA transcripts comprising the leader region with and without the DM deletion, reveal that it does, indeed, render the viral RNA monomeric (personal observations). Using antisense oligonucleotides, another group have demonstrated that this region may, in fact, play a role in the dimerisation process itself \[[@B31]\]. By free energy minimisation this region is predicted to be unstructured, so it is not clear how the RNAs would interact with one another. In addition, whilst the SL1/DIS sequence is conserved amongst HIV-2 and SIV sequences in the database, that within the DM region is less so, and the substitutions which exist would affect the auto-complementarity of the sequence. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Structure of the HIV-2 leader region. 1a. Secondary structure model of the HIV-2 leader region based on mfold predictions. Indicated are the putative dimer linkage sites (in red). Also highlighted is the DM region defined as being critical for packaging \[28\], in blue). 1b. The effect of the DM deletion on the SL1/DIS stem loop. The stem is truncated and the internal bulge altered in approximately half the predicted structures. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Structure of the key elements involved in HIV-1 RNA dimerisation. 2a. Secondary structure model of the packaging signal of HIV-1~Lai~(\[64\] \[65\]), containing the principal DLS. 2b Proposed sequence of the RNA dimerisation process in HIV-1~Lai~. The initial kissing hairpin interaction (including loop B) followed by formation of the extended duplex (\[1\]). 2c. Loop B, one of the critical elements in the dimer interaction. The flexibility of this internal loop allows the duplex to form (\[44\]). :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Dimer linkages of the retroviridae (excluding the lentiviruses). 3a. Loose and tight dimers (\[51\]). 3b. Imperfect repeats (\[66\]). 3c. Palindromes (\[38\]). 3d. GACG loops (\[23\]). 3e. CAG tri-loops (see Figure 4). :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Proposed tertiary structure of the HTLV-1 dimer linkage. 4a Stereoview of 3D molecular modelling of a potential structure of the HTLV-1 DIS from nucleotide A730 to A744 using JUMNA (\[61\]). 4b. Close up of the terminal loop. Bases are coloured as follows: adenine, grey; cytosine, yellow; guanine, orange; and uracil, cyan. :::  ::: One of the key differences between HIV-1 and HIV-2 replication is their modes of packaging \[[@B32]\]. Whilst the Gag protein of the former captures the genomic RNA *in trans*, the latter uses predominantly a *cis*mechanism. One might postulate therefore, that, if retroviruses must package a dimeric genome, it is critical in the case of HIV-2 that the genome is dimeric before interacting with the Gag polyprotein. Hence, the effect of mutations in the DM region may be to render the RNA monomeric and thus to severely impair packaging. It is attractive to speculate that the reason packaging itself is not affected by DIS mutations to the same degree in HIV-1 \[[@B33]\] is this difference in protein:RNA interaction. If the RNAs can interact at points other than the principal DIS over time, then perhaps the *trans*mechanism is less dependent on a high affinity dimer linkage? Particle maturation and viral infectivity ========================================= A recurring observation amongst investigators is the fact that mutation or deletion of dimer linkage sites causes viral infectivity to be decreased \[[@B33]\]. One explanation for this might be that a dimeric genome is a prerequisite for maturation of virus particles. Certainly, immature HIV-1 particles are non-infectious, and viruses with their DLS mutated have been demonstrated to form only immature particles \[[@B34]\]. The DLS of Human T Cell Lymphotropic Virus Type 1 (HTLV-1) was identified as a 14-nucleotide sequence just downstream of the splice donor \[[@B35]\]. Removal of this region from the leader sequence rendered the RNA monomeric *in vitro*\[[@B24],[@B36]\]. When this deletion was introduced into the wildtype genome sequence, the only viral replication defect that was observed, following transfection and subsequent infection, was that of impaired infectivity \[[@B37]\]. Likewise, a similar effect was observed when the DLS of Human Foamy Virus was mutated \[[@B38]\]. Parent *et al*showed that if the RNA of Rous Sarcoma Virus (RSV) was engineered so that it was monomeric, the virus was non-infectious \[[@B39]\]. Interestingly, this group suggested that it might be a difference in localisation of structural proteins and RNA affecting subsequent dimer formation and viral infectivity \[[@B40]\]. This is an area that has not been explored to any extent. Also working with RSV, Bieth and colleagues found that, in an *in vitro*system, dimer formation appeared to inhibit synthesis of the Gag polyprotein precursor \[[@B41]\]. Structure of the dimer linkage ============================== Undoubtedly the best defined dimerisation structure is that involved in the dimer linkage of HIV-1. The discovery of the sequences involved, the subsequent description of the RNA:RNA interaction, and the elucidation of the tertiary interaction are described elsewhere \[[@B1]\]. The initial interaction between the two RNAs appears to be a kissing loop interaction (similar to that seen in the regulation of plasmid replication, \[[@B42]\]) followed by annealing of the two RNAs into an extended duplex (Figure [2b](#F2){ref-type="fig"}). The sequences contained within the palindrome are remarkably conserved. Using an *in vitro*selection system it has been possible to demonstrate that the DIS has evolved to satisfy both constraints for optimal dimerisation affinity, and the potential to homodimerise \[[@B43]\]. The dimer linkage is found at the terminal end of Stem Loop 1 (SL1) within the packaging signal region of HIV-1 (Figure [2a](#F2){ref-type="fig"}). The tertiary structure of the whole SL1 RNA has been determined \[[@B44],[@B45]\] and the structures have helped to determine exactly how the RNAs interact with one another. A number of elements appear to be critical for the dimer interaction: flanking purines and central nucleotides in the palindromic sequence \[[@B46]\] and loop B \[[@B47]-[@B49]\]. The tertiary structure of the latter has been described (Figure [2c](#F2){ref-type="fig"}), and there is some debate as to how flexible this internal loop might be. However, work by Borer *et al*, examining the interaction of NC with elements of the packaging signal, of which loop B is one, showed that, in fact, both structures might exist, the flexible one allowing NC binding at high affinity \[[@B50]\]. There are similar linkages in other retroviruses. The Avian Leukosis Viruses also interact firstly in a kissing hairpin manner, and then form an extended duplex (Figure [3a](#F3){ref-type="fig"}, \[[@B51]\]). Palindromes remain a theme throughout many of the viruses investigated to-date. As already mentioned, the DIS of HIV-2 is less well defined than that of HIV-1. Whilst there is a palindromic sequence at the top of a stem loop structure that closely resembles the HIV-1 DIS (see Figure [1](#F1){ref-type="fig"}), there are other regions which have also been demonstrated to be important for dimer formation \[[@B25],[@B26]\]. Other viruses with palindromic sequences as their DLS include HFV (Figure [3c](#F3){ref-type="fig"}) and MoMLV. In the case of MLV there are other sequences and structures which may play a role in dimer formation, including the GACG tetraloops mentioned previously \[[@B52]\]. The tertiary structure of this stem loop is the only proposed dimer linkage element yet to be determined in a retrovirus other than HIV-1 (\[[@B53]\]). RSV and VL30, also have imperfect repeat sequences in their dimer linkages \[[@B54],[@B55]\] (Figure [3b](#F3){ref-type="fig"}). Recent work by Monie and colleagues \[[@B36]\] describes the potential tertiary structure of the HTLV-1 dimer linkage, capped by a novel CAG tri-loop (Figure [3e](#F3){ref-type="fig"} and Figure [4](#F4){ref-type="fig"}). This tri-loop is formed by an unusual C:synG base pair closing the loop. Other similar loops have been described, in the domain IIId terminal loop of the hepatitis C virus internal ribosomal entry site (IRES) \[[@B56]\] and in stem loops required for initiation of transcription within the Bromoviridae \[[@B57]\]. Although sequence heterogeneity between HTLV-1 isolates is rare, distinct mutations identifying individual strains can be identified. Of 101 HTLV-1 sequences identified from the EMBL database, 90 showed sequence homology with HTLV-1~CH~, the strain used in the study. The other 11 sequences comprised three different variants. Eight contained a deletion of C736 (see Figure [4](#F4){ref-type="fig"}), two possessed the substitution A737G, and one possessed the substitution C733U. The substitution mutants have minimal impact on regional secondary structure, while the deletion may induce formation of a CAGG tetraloop. Interestingly, the A737G mutation possesses homology with 150 deposited HTLV-II sequences, suggesting a conservation of the DIS between HTLV-I and -II. Conclusions =========== The retroviral RNA genome structure does not stay static during the course of transcription, translation and ultimately packaging. Various investigators have suggested that this constantly changing RNA structure plays an intimate role in the viral replication \[[@B58]-[@B61]\]. It seems possible that linkage of the two RNA molecules constituting the genome is integral to the changes in RNA structure. As described in the article above, the dimer also acts as a mechanism for promoting recombination; may be a signal for packaging to occur; may be an inhibitory signal; may direct processes to occur in specific cellular compartments; and lastly, may be capable of interacting with cellular proteins. *In vivo*data has revealed just how important an intact dimmer linkage may be to a retrovirus. For instance, there are intriguing differences in the effect of dimer mutations on viral infectivity depending on the cell type being infected \[[@B62]\]. What the significance of this might be in the context of a viral infection is, as yet, unclear. The importance of the dimer linkage is perhaps most clearly exemplified by the observation that a patient infected with a viral isolate having a defective DLS, had a low viral load. The subsequent switch in the predominant virus to that with a competent DLS coincided with a rise in viral load \[[@B63]\]. One can speculate that, at least in the case of HIV-1, only those viruses with a whole, optimised dimer linkage are capable of efficient infectivity. For the purposes of examining the role of retroviral RNA dimer sequences in the context of animal models, the non-human retroviruses, including the non-primate lentiviruses will be of great importance. To sum up, retroviral dimeric genomes are linked by a variety of RNA structures, including kissing hairpins, GACG tetraloops and unusual CAG-tri loops. The differences in these interactions, and when or where they occur, may reflect different demands upon this unique feature, and highlight the elasticity of the RNA genome. Competing interests =================== None declared. Acknowledgements ================ Many thanks to Andrew Lever for advice on, and editing of the manuscript. Particular thanks also go to Tom Monie for Figure [4](#F4){ref-type="fig"}, allowing description of unpublished work, and critical reading of the manuscript. In addition, thanks to Martin Zaccharias and Jose Gallego for contributions to the other figures, Paul Digard and Ian Brierley for critique.

PubMed Central

2024-06-05T03:55:47.542634

2004-8-18

PMC516451

Introduction ============ The dimeric feature of the retroviral RNA genome was identified almost forty years ago. However, as with many topics in retrovirology, interest in this area was heightened with the realization that the causative agent of AIDS was a retrovirus. Since then, RNA and protein sequences involved in genome dimerization have been identified for a number of retroviruses, and the dimeric nature of the retroviral genome is known to be important for various critical events in the viral life cycle. These include reverse transcription and recombination, as well as genome encapsidation. To date, a number of informative reviews have been published on retroviral RNA dimerization \[[@B1]-[@B3]\], genome packaging \[[@B3]-[@B7]\], and the role of nucleocapsid (NC) protein in these activities \[[@B8],[@B9]\]. More recently, a comprehensive review was published that summarized the contributions of *in vitro*analysis to the identification of retroviral dimerization signals, and provided an overview of the HIV-1 5\' untranslated region (UTR) structure with reference to a number of proposed models \[[@B10]\]. Another, in this issue of *Retrovirology*, focuses on the different roles of different dimer linkage structures amongst various retroviruses \[[@B11]\]. In this review, we will focus on results from *in vivo*studies that provide insights into the relationship between retroviral RNA dimerization and packaging, and the biological relevance of these activities to viral replication. Retroviral RNA dimerization =========================== The first evidence for the existence of a dimerized RNA genome came in 1967 when it was shown that viral RNA from each of Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV), murine leukemia virus (MLV), and mouse mammary tumor virus (MTV) displayed sedimentation constants between 64S and 74S in sucrose gradients \[[@B12]\]. Since these sedimentation constants and corresponding molecular weights were much larger than those of most other known viral RNAs, the structure of these RNA genomes became a matter of great interest. Experiments showing that the 62S RSV RNA species could be converted to a 36S species by heat treatment suggested a disaggregation of the 62S RNA into smaller RNAs, and implied that the fast-sedimenting (62S) RSV RNA was actually an aggregate of smaller (36S) RNAs \[[@B13]\]. The first real understanding of this putative aggregate RNA structure came in 1975 when RNA from the endogenous feline retrovirus, RD-114, was visualized by electron microscopy (EM), and it was apparent that the 52S RNA molecule existed as an extended single strand that contained a central Y- or T-shaped secondary structure \[[@B14]\]. It appeared that this 52S molecule actually consisted of two half-size molecules, joined together by the Y- or T-shaped structure, which was termed rabbit ears (RE). It was later shown that the RNA had a poly(A) sequence at each of the two free ends. More importantly, this indicated that nucleotides involved in this RE, or dimer linkage structure (DLS), resided in the 5\' region of the RNA genome \[[@B15]\]. Similar structures were also reported for numerous other type C RNA viruses \[[@B16]-[@B21]\]. The absolute conservation of a DLS among retroviruses was strong evidence that the dimerization process must be critical to the retroviral life cycle. With the discovery that the causative agent of AIDS was also a retrovirus, inhibition of RNA dimerization was proposed as a possible therapy for HIV, and HIV-1 RNA dimerization became an intensely studied topic. Both *in vivo*and *in vitro*approaches have been used to study retroviral RNA dimerization. The *in vivo*approach is that whereby RNA is isolated from virions produced in tissue culture and then analyzed by native Northern blotting \[[@B22]\]. The other method involves synthesis of short segments of viral RNA *in vitro*, and then studying the ability of these fragments to form dimers. The HIV-1 DLS was originally identified when it was shown that an *in vitro*-transcribed fragment of HIV-1 RNA could form two major bands on a native gel after incubation at 37°C for 15 min \[[@B23]\]. The lower band had the expected size of the RNA fragment, while the upper band corresponded to a dimer. *In vivo*evidence for a role of the NC protein in the dimerization process was already available \[[@B24]\], and this study also showed that NC could bind to viral RNA and increase the rate of dimerization of the RNA fragments in these *in vitro*dimerization assays \[[@B25]\]. It was subsequently reported that an RNA fragment representing nt 1--311 of HIV-1 RNA (Mal strain; a chimera of subtypes A and D) could not only form dimers, but that RNAs containing these first 311 nt could dimerize 10 times faster than RNA sequences at positions 311--415 that were previously shown to be sufficient for HIV-1 RNA dimerization \[[@B25]\]. Based on these results, the authors concluded that sequences upstream of the splice donor site are involved in the dimerization process, and proposed that sequences in this region somehow hastened the reaction. The key nucleotides involved in this RNA dimerization event make up a palindromic sequence, 274-GUGCAC-279, between the PBS and the major splice donor \[[@B26]\], and RNA sequences on both sides of this palindrome can form a stem-loop structure with the palindrome in the hairpin loop. Deletion of this stem-loop motif (nt 265--287) completely abolished dimerization of the 1--615 HIV-1 RNA fragment *in vitro*. The palindromic region was termed the dimerization initiation site (DIS) and it was proposed that this structural element could be exploited for targeted antiviral therapy by antisense oligonucleotides \[[@B26]\]. These findings were later confirmed when a 19 nt sequence upstream of the 5\' major SD was shown to be part of the HIV-1 RNA dimerization domain (Lai strain; subtype B) \[[@B27]\], and it was found that *in vitro*dimerization of a 224--402 nt RNA fragment was completely blocked by an antisense oligonucleotide that targeted the palindrome \[[@B28]\]. This led to a \"loop-loop kissing complex\" \[[@B29]\] or \"kissing-loop model\" \[[@B27]\] of HIV-1 RNA dimerization, in which the 6 nt palindromes on each of the two monomeric RNA molecules interact through Watson-Crick base-pairing. Purine residues flanking the palindrome were later shown to be intricately involved in this initial interaction \[[@B30],[@B31]\] which is believed to shift the equilibrium toward the formation of dimers, allowing the stems to melt and anneal to their complementary sequences on the other RNA molecule, thus forming the stable extended duplex (Fig. [1](#F1){ref-type="fig"}). This model fits with the idea that immature virions contain a less stable dimer involving only base-pairing of the palindromes, but that the mature virions contain a more stable structure, the extended duplex. Subsequent phylogenetic analysis of over 50 HIV-1, HIV-2, and simian immunodeficiency virus (SIV) nucleotide sequences showed an absolute conservation of a predictable structure similar to the DIS, with the hallmark of the HIV-1 DIS motif being a 6 nt palindrome consisting of either a GCGCGC or a GUGCAC sequence \[[@B32],[@B33]\]. Similar kissing-loop models have also been proposed for a number of other retroviruses \[[@B34]-[@B41]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **HIV-1 5\' RNA Structural Elements.**Illustration of a working model of the HIV-1 5\' UTR showing the various stem-loop structures important for virus replication. These are the TAR element, the poly(A) hairpin, the U5-PBS complex, and stem-loops 1--4 containing the DIS, the major splice donor, the major packaging signal, and the *gag*start codon, respectively. Nucleotides and numbering correspond to the HIV-1 HXB2 sequence. (Adapted from Clever *et al*. \[73\] and Berkhout and van Wamel \[136\]) :::  ::: Despite ample *in vitro*evidence supporting the above model of dimer maturation, it was not yet known where or when the RNA dimer was actually formed *in vivo*. However, native Northern blotting analysis of RNA from two Moloney murine leukemia virus (MuLV) protease-negative (PR^-^) mutants displayed dimers that migrated more slowly, and showed lower melting temperatures, than that of wild-type \[[@B42]\]. It was therefore concluded that PR function is required for RNA maturation in MuLV. Similar experiments with a related virus also suggested that the RNA maturation event required an intact, unsubstituted Cys array within the NC domain \[[@B42]\]. On the basis of these results, a maturation pathway was proposed for MuLV in which Gag polyprotein molecules assemble into a nascent virion containing an immature dimer. The particle would then be released from the cell, and once Gag is cleaved by PR, NC would act on the immature dimer, converting it to the mature form. Evidence for the role of NC in this dimer maturation process came when *in vitro*analysis showed that NC could convert the less thermostable dimers to a more stable conformation \[[@B43]\]. Similar results were obtained by others showing that HIV-1 NC could activate dimerization of a 77--402 nt fragment of HIV-1 Lai RNA, as well as convert an unstable dimer, corresponding to the kissing complex, to a stable one \[[@B44]\]. Taken together, these thermostability conversions seem to resemble the RNA maturations reported *in vivo*, and, in agreement with earlier proposals \[[@B24]\], strongly suggest that NC is responsible for the dimer maturation depicted in Fig. [1](#F1){ref-type="fig"}. Subsequent *in vivo*analysis of a panel of HIV-1 NC mutants showed that Cys-Ser substitution of amino acid residues within the second zinc finger decreased genomic RNA dimerization to the same extent as disruption of the DIS \[[@B45]\]. This finding confirmed the involvement of NC in the dimerization process, and suggests that the kissing-loop model also applies to the *in vivo*situation. HIV-1 RNA packaging =================== Why a class of viruses would evolve to have such a unique genomic structure is not entirely clear, but it is speculated that the availability of two copies of the genome would be advantageous for recombination during the complex reverse transcription process, that is key to the retroviral life cycle \[[@B46]\]. Indeed, the dimeric nature of the genome is thought to be responsible for a high rate of recombination during infection \[[@B47]-[@B50]\]. Given that most dimerization signals overlap with known packaging elements, it was naturally assumed that it is the RNA dimer that is specifically recognized for packaging in the case of retroviruses, and that this dimeric feature ensures proper packaging of two copies of genomic RNA. A number of studies have attempted to address this question of a link between dimerization and packaging, but let us first review several aspects of the HIV-1 RNA packaging process. The first studies aimed at identifying the HIV-1 RNA packaging signal found that deletion of RNA sequences between the major splice donor (SD) and the *gag*coding region (i.e. SL3 and adjacent sequences in Fig. [2](#F2){ref-type="fig"}) decreased the levels of genomic RNA packaged into virions \[[@B51]-[@B53]\]. Since these sequences were downstream of the major 5\' SD, and therefore would not be found in any spliced viral RNA species, it was plausible that this region could be responsible for the selective packaging of genomic RNAs. Analysis of the putative ψ locus from a variety of retroviruses showed that these sequences had the ability to direct the selective encapsidation of heterologous RNAs to which they had been linked artificially \[[@B54]-[@B61]\]. In HIV-1, such autonomous packaging signals were mapped to the regions extending 30--40 nt immediately upstream and downstream of the *gag*start codon \[[@B62]\]; however, subsequent studies showed that RNA sequences upstream of the 5\' SD site also affected RNA packaging \[[@B63]\]. It was also known that retroviral encapsidation required trans-acting amino acid sequences in the Gag protein \[[@B51],[@B64]-[@B68]\], and several groups reported that HIV-1 Gag and NC exhibit specific binding affinity for the HIV-1 ψ site *in vitro*\[[@B23],[@B69]-[@B72]\]. These findings, combined with chemical and RNase accessibility mapping, as well as computerized sequence analysis, led to the generation of a model for the HIV-1 ψ site that comprised four independent stem-loops \[[@B73]\] (SL1-4 in Fig. [2](#F2){ref-type="fig"}). Three of these hypothetical stem-loop structures were each shown to serve as independent Gag binding sites, and were proposed to contribute individually to overall packaging efficiency. SL1, SL3, and SL4 were later shown to be critical for packaging specificity *in vivo*\[[@B74],[@B75]\]. Subsequent *in vitro*analysis from another group demonstrated that the major packaging signal is an extended bulged stem-loop whose RNA conformation is altered upon interaction with Gag \[[@B76]\]. However, more recent work indicates that SL2 and SL3 display much higher affinities for NC than SL1 and SL4 *in vitro*\[[@B77],[@B78]\]. Based on these findings, a model has been proposed to represent the initial complex formed between the NC domains of assembling Gag molecules and the dimeric ψ region \[[@B79]\]. In this model, SL1 is shown to form an RNA duplex between the two stands, while SL4, instead of directly binding to Gag, contributes additional RNA-RNA interactions that stabilize the tertiary structure of the ψ element. The RNA conformation resulting from this folding pattern is thought to expose SL2 and SL3 for high-affinity binding to Gag. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **The Kissing-Loop Model of HIV-1 RNA Dimerization.**HIV-1 RNA dimerization is initiated by a Watson-Crick base-pairing interaction between two palindromes in the loops of SL1 on two monomeric genomic RNAs. This interaction forms the loose unstable kissing-loop complex. Coincident with virus particle maturation, this unstable dimer is rearranged to form a more stable extended duplex that involves a mechanism whereby the base-pairs in the stems melt and then re-anneal to their complementary sequences on the opposite strand. Nucleotides and numbering correspond to the HIV-1 HXB2 sequence. (Adapted from Skripkin *et al.*\[26\] and Laughrea and Jetté \[27\]) :::  ::: Despite the clear results obtained from simplified *in vitro*studies such as those mentioned above, the SL1-4 region alone is not sufficient to target RNA into HIV-1 virions *in vivo*\[[@B80]\], and the minimal region required to confer autonomous packaging activity actually maps to a larger region covering the first 350--400 nt of the genome, including ≈ 240 nt upstream of SL1 \[[@B81]-[@B84]\]. In agreement with these studies, mutations that alter the stability of the poly(A) hairpin stem region, or delete the upper part of the hairpin, severely inhibited HIV-1 replication \[[@B85]\]. And, these deficits in replication correlated with reduced RNA packaging levels in virions, suggesting that the formation of the poly(A) hairpin is necessary for normal packaging of viral genomes. Subsequent research confirmed the importance of the poly(A) hairpin in the RNA packaging process \[[@B86]\], and it was shown that similar disruption of base-pairing in the stem of the TAR element also caused profound defects in packaging \[[@B81],[@B86]\]. Finally, deletion analyses of RNA sequences between the poly(A) hairpin and SL1 suggested that unspecified sequences within the U5-PBS region also contribute to HIV-1 RNA packaging \[[@B83],[@B86]\]. Our group later showed that GU-rich sequences in the lower stems of the poly(A) hairpin and the U5-PBS complex contribute to both dimerization and packaging \[[@B87]\]. In summary, all of the seven predicted stem-loop structures in the HIV-1 5\' UTR (Fig. [2](#F2){ref-type="fig"}) are known to be important for genome encapsidation, and all of these RNA structural elements have also been assigned other functions in various steps of the viral life cycle, e.g. the role of SL1 in the initiation of dimerization. The existence of such overlapping functions for these RNA structures raises the possibility that some of these functions, such as dimerization and packaging, might be linked. The evidence for and against the existence of such a link in HIV-1 will be the main focus of the remainder of this review. Is dimerization a prerequisite for packaging? ============================================= One of the first electron microscopy studies of a retroviral DLS in 1976 proposed that this region \"could have some role in packaging the RNA in the virus\" \[[@B16]\]. This raised the question of a possible link between dimerization and packaging that is still debated. The answer to this question has significance in our basic understanding of the retroviral life cycle and may also have implications for therapy, since many groups are actively studying these two activities as potential drug targets. Clues from *in vitro*studies ---------------------------- Early reports on *in vitro*dimerization of HIV-1 RNA showed that the DLS localized to a stretch of genomic RNA downstream of the 5\' SD (nt 311--415) \[[@B23],[@B88]\], and it was noted that this dimerization domain encompassed a previously identified packaging element that had also been shown to bind NC \[[@B51]-[@B53]\]. This dependence of HIV-1 RNA dimerization on *cis*elements required for packaging was immediately interpreted to mean that retroviral RNA dimerization, activated by either NC or Gag precursors, should direct genomic RNA into the virion, implying that dimerization might be a prerequisite for packaging. Since HIV-1, MuLV, and RSV all contain elements involved in dimerization that were also required for packaging \[[@B23],[@B89],[@B90]\], it was proposed that dimerization might function as a molecular switch that negatively regulates translation and positively regulates encapsidation \[[@B88]\]. The existence of a DLS downstream of the major splice donor would seemingly supply a convenient mechanism whereby only genome length RNA would be able to dimerize and subsequently become encapsidated into the virion. However, evidence questioning such a dimerization-mediated mechanism of genomic RNA packaging came from studies showing that sequences upstream of the SD site had even greater dimerization capabilities than those located downstream \[[@B25]-[@B27]\]. The involvement of such sequences (e.g. the DIS, SL1) in the dimerization process questioned the link between dimerization and packaging, because these sequences are also found in all HIV-1 spliced viral RNAs. Observations from *in vivo*studies ---------------------------------- Early *in vivo*studies analyzing the structure of virion-associated RNA from rapid-harvest avian retroviruses showed that viral RNA appeared to be a mixture of monomers and dimers \[[@B91]-[@B93]\]. Similar results had also been reported with PR \[[@B94],[@B95]\] and NC \[[@B24],[@B94],[@B96],[@B97]\] mutants, which argued against the notion that dimerization is a prerequisite for packaging. However, analysis of rapid-harvest virus in MuLV showed that genomic RNA was already in the form of a dimer shortly after budding, albeit as a less stable, physically different RNA dimer than that present in mature virions \[[@B42]\]. Based on these observations it was proposed that MuLV particles never package monomeric RNAs, but rather that the dimeric RNA structure might be integral to the packaging signal that is recognized by Gag during assembly. It was also speculated that the previously reported presence of monomers in viral RNA preparations had resulted from the physical dissociation of fragile unstable dimers during RNA preparation. Similar experiments performed on PR^-^mutants of HIV-1 showed that substantial amounts of monomeric RNA could be detected \[[@B98]\]. Since PR^-^dimers were shown to be less stable than wild-type dimers, it was assumed that dimers were preferentially packaged in PR^-^particles, but that some fragile dimeric structures had dissociated during RNA preparation. Based on these *in vivo*results with both MuLV and HIV-1, it was concluded that dimerization is a prerequisite for packaging and should be considered to be a general feature of retrovirus assembly. Further insights into this topic can be obtained by examination of results from a number of studies aimed at understanding the role of the DIS in HIV-1 replication. One such study, in which DIS loop palindrome sequences were mutated, found that mutation of the palindrome to shorter or longer versions of GC stretches did not have major effects on viral RNA dimerization; however, partial RNA packaging defects were observed that also corresponded to diminutions in viral replication \[[@B33]\]. Based on these data, it was proposed that these DIS loop mutants might have experienced a partial dimerization defect that caused inefficient packaging \[[@B33]\]. In a similar study, mutation of the palindrome, as well as deletion of the upper stem-loop of SL1 caused drastic reductions in viral infectivity and decreases in both dimerization and packaging of HIV-1 genomic RNA \[[@B32]\]. In an attempt to explain how these mutations could affect both activities, a model was proposed in which Gag does not specifically recognize the dimerized genome but rather initially interacts with one molecule of genomic RNA that happens to be linked (dimerized) to a second such molecule. Then, during packaging, Gag would effectively bind to two genomic RNA molecules at once. Hence, defects in dimerization would result in subsequent packaging defects. Based on these data, it was also concluded that the encapsidation and dimerization processes are coupled to some extent. Although several groups had attempted to delineate the relationship between dimerization and packaging, the fact remains that the RNA signals that are important for both of these activities overlap in most retroviral genomes; this makes it difficult to interpret the results of mutagenesis studies. In an attempt to generate viruses that would be expected to display selective defects in dimerization or packaging, one group designed a panel of constructs containing mutations in SL1, SL3, or both \[[@B99]\]. Results from this study showed that deletion of either SL1 alone, or SL3 plus adjacent flanking sequences, reduced genomic packaging, while deletion of SL1 and SL3 simultaneously caused an even further reduction. With respect to dimerization, complete deletion of SL1, or even disruption of the base-pairing in the upper stem, resulted in elevated levels of monomer-sized RNA species on native Northern blots, again confirming the importance of this region for the *in vivo*HIV-1 RNA dimerization process. Yet, these mutant genomes could still be packaged, suggesting that HIV-1 RNAs need not be dimers for this to happen. Thus, the authors concluded that dimerization is not a prerequisite for packaging but rather serves an independent function in the retroviral life cycle. In the above-cited article, the effects of SL3 mutations on dimerization were not studied, but our group later showed that viruses containing even minor substitutions in or around SL3 could have significant effects on both dimerization and packaging \[[@B100],[@B101]\]. In summary, the *in vivo*studies described above commonly observed that mutations in 5\' RNA sequences affected both dimerization and packaging, presumably due to the close proximity of the RNA dimerization and packaging signals. Can monomers be packaged? ------------------------- In an attempt to separate the dimerization and packaging functions, and to characterize the DIS-DLS region without altering packaging activity, one group generated mutant constructs carrying a duplication of approximately 1000 nt from the HIV-1 5\' region (termed E/DLS) including the encapsidation signal and the DIS-DLS \[[@B102]\]. They found that the presence of an ectopic E/DLS near the 3\' region of the genome resulted in the appearance of monomeric RNA in virus particles, suggesting that monomers can be packaged and that dimerization of HIV-1 genomic RNA is not required for packaging. However, they also found that two intact E/DLS regions had to be present on the same RNA molecule in order for packaging of monomers to occur. Therefore, it was assumed that these monomers had been generated from an intramolecular interaction between the two E/DLS regions. If we assume that such an intramolecular interaction between two DLS structures would occur on a single RNA molecule, however, might such a structure then not also appear as a dimer to a Gag protein that was attempting to package it? Although these data were interpreted to mean that dimerization is not required for packaging, they also suggest that some structure that is generated by the interaction of the two E/DLS regions might be recognized by Gag in order to facilitate packaging. In the context of wild-type genomic RNA containing only one E/DLS region, such a structure might then only be generated by an intermolecular interaction between two RNA molecules, i.e. a dimer. Hence, these results also imply that dimerization might be required for proper packaging. In a follow-up study, the same group created mutant HIV-1 particles that contained only monomeric RNAs, and concluded that these mutants demonstrated the complete separation of encapsidation from physical dimerization of retroviral RNA \[[@B103]\]. However, they also reported that these viruses packaged only monomers, and that packaging efficiencies were approximately half those of wild-type, implying that dimerization is the sole mechanism to ensure the packaging of two copies of viral genomic RNA into each virus particle. In addition, the packaged monomers might have originally been weak dimers that dissociated during extraction and analysis, as has been pointed out in previous reports \[[@B42],[@B102]\]. However, the above results do raise the issue of packaging specificity in mutant viruses. We and others have shown that, in COS cells, HIV-1 can incorporate significant amounts of spliced viral RNA when proper packaging of full-length viral genomic RNA is reduced \[[@B99],[@B104]\]. During assembly, Gag will always successfully package some RNA, and it is important to know the degree of specificity with which monomers versus dimers are packaged. If monomer-packaging mutants concomitantly package high levels of spliced viral RNA, then it is likely that packaging specificity may have been compromised by the existence of an extra E/DLS, and that the packaging of the monomers was non-specific. However, a lack of spliced viral RNA in these virions would indicate that the monomers were packaged with a high degree of specificity, and would have implications as to whether or not Gag initially recognizes viral genomic RNA in a dimeric versus a monomeric state. None of the viruses engineered to package only monomers were able to efficiently establish a new round of infection, suggesting that dimerization is required for replication if not for packaging. It is difficult to predict what other effects the addition of large segments of highly structured RNA might have on the viral life cycle. Another group reported similar phenotypes in the context of an HIV-1 mutant that was designed to have altered Gag/Gag-Pol ratios \[[@B105]\]. Analysis of virion-derived genomic RNA from these viruses showed an increase in packaging of monomers, demonstrating that stable RNA dimers are not required for encapsidation of HIV-1 genomic RNA. Interestingly, these viruses also showed drastically reduced infectivity. Insights from forced evolution studies -------------------------------------- We have also been studying the HIV-1 5\' UTR and its putative interactions with Gag, and how these interactions affect dimerization and packaging activities. The DIS is known to be important for viral replication \[[@B32],[@B33],[@B63],[@B99],[@B106]-[@B109]\], reverse transcription \[[@B47],[@B48],[@B107],[@B109]\], RNA dimerization \[[@B32],[@B99],[@B106],[@B109]-[@B111]\], and packaging \[[@B32],[@B33],[@B74],[@B99],[@B107],[@B108],[@B110]\], as well as packaging specificity \[[@B99]\]. However, despite the obvious importance of this stem-loop structure, work from our group has shown that defective viral replication caused by deletions in the DIS can be largely corrected by a series of compensatory point mutations identified in matrix, capsid, p2, and NC \[[@B112]-[@B114]\]. These findings imply that the RNA sequences comprising the DIS interact in some way with these domains of Gag, and that when the RNA sequences are mutated, the virus will acquire adaptive mutations that potentially restore putative RNA-protein interactions over long-term culture. Since the originally deleted RNA sequences were in the DIS, we had naturally assumed that the major defect of these mutants would relate to RNA dimerization, and that compensatory mutations had arisen to correct defective RNA dimerization activity. To our surprise, this was not the case. Although our mutants did indeed yield reduced levels of dimerized genomic RNA in virus particles, the compensatory mutations in Gag that restored replication capacity \[[@B112]-[@B114]\] did not correct dimerization defects \[[@B109]\]. Rather, compensatory mutations apparently resulted in increased overall levels of viral genomic RNA that were packaged into virus particles, irrespective of impaired RNA dimerization. Similar effects on packaging were observed in the context of compensatory mutations identified during long-term culture of viruses containing mutations outside the DIS, such as the poly(A) hairpin and the U5-PBS complex \[[@B87]\], and between the PBS and SL1 \[[@B115]\]. These findings again question the link between dimerization and packaging, since our compensatory point mutations were able to increase RNA packaging levels without correcting dimerization. One possibility is that the revertant viruses somehow gained the ability to package wild-type levels of RNA without correcting dimerization defects, i.e. they packaged more monomers. However, we also cannot rule out the possibility that our point mutations in Gag may have restored weak dimerization properties to the mutated RNAs, and that the latter dimers dissociated during extraction and analysis. In a follow-up study, we created two other DIS deletions and combined them with various combinations of the previously identified compensatory point mutations. We showed that these mutant viruses, ΔLoop (lacking the loop region of SL1) and ΔDIS (lacking the complete SL1) displayed defects in replication, RNA dimerization, and packaging. Once more, all of these but dimerization were largely corrected by the compensatory point mutations in Gag \[[@B104]\]. Even a virus that lacked the DIS, e.g. ΔDIS, and which never showed any signs of viral growth in tissue culture, was able to replicate to significant extent when it also possessed the compensatory mutations. The mechanism(s) whereby these compensatory point mutations functioned to restore replication had eluded us for some time. Recently, however, we employed an RNase protection assay to discriminate between genomic and spliced viral RNA packaged into virus particles. Our results showed that all of our 5\' UTR mutant viruses aberrantly packaged increased levels of spliced viral RNA compared to wild-type virions. More importantly, however, the effect of one of our compensatory point mutations (i.e. MP2; a Thr-\>Ile substitution at position 12 of the SP1 spacer peptide in Gag) was to exclude spliced viral RNA from being packaged into mutant virions \[[@B104]\]. Surprisingly, this single point mutation was also able to restore significant levels of virus replication to our ΔDIS mutant virus, which had been noninfectious in both T cell lines and blood mononuclear cells. Previous work had suggested that the packaging of spliced viral RNA is a mechanism used by packaging mutants to fill the space that would normally be occupied by genomic RNA \[[@B99]\]. Were this the case, then the MP2-mediated exclusion of spliced viral RNA from the virus particle should have been accompanied by increased packaging of genomic RNA. In the absence of MP2, the mutant particles contained lower levels of genomic RNA and higher levels of spliced viral RNA packaged than wild-type. In contrast, the presence of MP2 led to the exclusion of spliced viral RNA, but had no effect on packaging of genomic RNA. In the context of dimerization and packaging in the mutated viruses, it is possible that spliced viral RNAs, which do contain some RNA elements involved in RNA dimerization, including the DIS, might form heterodimers with molecules of genomic RNA. These putative heterodimers might be packageable, but it is unlikely that virions containing such genomes would be able to replicate, e.g. the noninfectious ΔDIS mutant. However, in the presence of MP2, the modified Gag protein might in some way block the formation of such an RNA heterodimer, thereby increasing the probability that dimers form between two genomic RNA molecules, resulting in partially restored levels of virus replication. Since these genomic RNA molecules are already mutated in dimerization signals, these weaker dimers would probably appear on a gel as monomers. In such a model, MP2 would act to restore dimerization, resulting in increased replication capacity, suggesting that dimerization is required for proper packaging to ensure that a particle is infectious. Unfortunately, this is virtually impossible to prove with current *in vitro*and *in vivo*protocols. New approaches to study dimerization and packaging within the cell will hopefully allow new hypotheses to be tested. The packaging of spliced viral RNA and/or the exclusion of such RNA species raises the question of whether the viral RNA sequence, or possibly the RNA structure, is important in proper assembly and/or structural integrity of the virus particle itself. Evidence in support of this possibility comes from studies on the binding of NC, in the context of full-length Gag, to viral genomic RNA. This might concentrate Gag proteins onto one or more RNA molecules, thereby facilitating Gag-Gag multimerization in a template-driven manner. Hence, viral genomic RNA would be a structural element, or scaffold, on which the virion can assemble \[[@B116]\]. Other reports have shown that viral RNA can affect particle morphogenesis \[[@B116]-[@B119]\] and structural stability \[[@B120],[@B121]\], although the mechanisms involved are unclear. If RNA structure, or even the dimeric versus monomeric state of the RNA, truly does play a role in virion assembly and/or stability, this might also explain the apparent detection of monomeric RNA in the HIV-1 mutants mentioned above. For example, the duplication of large E/DLS sequences would undoubtedly have altered the overall structure of viral RNA, which might have resulted in the formation of unstable virus particles \[[@B102],[@B103]\]. Degradation of such particles could have indirectly caused the dissociation of dimers that would then appear as monomers on a gel. The fact that these viruses were all noninfectious may also have been due to the formation of unstable virus particles. Consistent with this concept, we found by electron microscopy that HIV-1 mutants lacking DIS stem sequences displayed an increased proportion of immature virus particles \[[@B114]\]. This might mean that either the RNA structure, or the lack of a properly formed dimer, resulted in the production of virus particles with abnormal morphology. Since RNA can affect Gag cleavage, it is possible that mutations in the RNA might have also compromised the cleavage of Gag precursor proteins, which may subsequently have affected particle maturation \[[@B122]\]. We believe that proper RNA dimerization may be a prerequisite for efficient virion assembly and structural stability. As stated, the link between dimerization and packaging is a subject of ongoing debate \[[@B32],[@B33],[@B42],[@B98],[@B99],[@B102],[@B103],[@B109],[@B110]\], but we and others view dimerization as a prerequisite for packaging. Genomic RNA can be packaged as monomers \[[@B99],[@B102],[@B103],[@B105],[@B109]\], or alternatively as weak dimers that appear as monomers on gels, but mutant viruses that exhibit dimerization defects generally do not grow as well as wild-type viruses. The fact that our ΔDIS-MP2 virus can replicate in tissue culture, despite being severely compromised in genome dimerization, is evidence that efficient dimerization is not required for packaging or replication. In the absence of an authentic DIS, other sequences that affect dimerization may form a weak dimer that allows RNA to be recognized and adequately packaged \[[@B87],[@B100],[@B101]\]. The contribution of the DIS might then be to significantly increase the efficiency of the dimerization process, resulting in more efficient packaging and replication. In conclusion, we agree with opinions expressed by others that the generation of virus particles able to package monomeric genomes is possible, but that dimerization is likely to be a prerequisite for the production of infectious viral progeny \[[@B10]\]. The DIS as a therapeutic target? ================================ It is clear that virus replication capacity is significantly affected whenever dimerization and/or packaging are compromised, suggesting that these activities can be exploited as anti-HIV drug targets. Indeed, the DIS was first proposed to be a potential therapeutic target at least 10 years ago, and antisense molecules were directed at this region of viral RNA \[[@B26],[@B123]\], without practical outcome. Other approaches directly target the HIV-1 kissing-loop complex, which resembles the eubacterial 16S ribosomal aminoacyl-tRNA site, i.e. the target of aminoglycoside antibiotics such as paramycin and neomycin \[[@B124]\], both of which specifically bind to the kissing-loop complex. Drugs based on antibiotics with high affinity and specificity for the DIS may be a worthwhile approach, although efficacy might be compromised by the fact that HIV can replicate in the face of mutations that decrease genomic dimerization by more than 50% \[[@B104]\]. RNA interference (RNAi) is a novel mechanism that regulates gene expression in which small interfering RNAs direct the targeted degradation of RNA in a sequence-specific manner (reviewed in Lee and Rossi \[[@B125]\]). Although RNAi is a powerful tool, it is not yet clear whether its therapeutic potential will materialize. This not-withstanding, several reports show that specific degradation of HIV-1 RNA is possible in infected cells \[[@B125]\], and reductions of p24 levels by as much as 4 logs have been achieved using RNAi directed against HIV-1 *tat*and *rev*\[[@B126]\]. DNA vectors are currently being engineered that will allow for long-term production of siRNAs for use against chronic diseases, such as HIV-1. The DIS might also be a good candidate for sequence-specific targeting of HIV by RNAi therapy since it is highly conserved among naturally occurring virus isolates, and, due to its position upstream of the major splice donor, is contained in all HIV-1 RNA transcripts, both spliced and unspliced. Effective DIS-directed degradation of HIV RNA should confer the same viral phenotype as observed with our ΔDIS mutant, which never showed signs of virus replication in either permissive T cell lines or blood mononuclear cells \[[@B104]\]. One concern with use of RNAi is how accessible certain RNA sequences might be. For example, complex secondary structures might cause some sequences to be buried and therefore inaccessible to the siRNA. However, this would not be a concern with DIS-directed RNAi, since the DIS contains a 6 nt palindromic sequence that is believed to initiate the dimerization process by binding to an identical sequence on another molecule of genomic RNA. If two 6 nt stretches of RNA can find each other on two 9200 nt strands of highly structured RNA, they should also be accessible to siRNAs. Recently, the practicality of RNAi-based therapies against HIV-1 was called into question when it was shown that HIV-1 was able to escape the antiviral pressure of RNAi by generating substitutions or even deletions within RNAi target sequences \[[@B127],[@B128]\]. This again highlights the versatility and plasticity of the HIV-1 genome. However, in these studies, the RNAi target sequences were located within the *tat*and *nef*genes, and the mutations that were generated blocked the effects of the RNAi without conferring any major detriment to virus replication. In contrast, RNAi may be more useful if targeted to more critical RNA elements within the genome, such as the DIS or the Ψ region, since any escape mutations that occur might result in viruses with severely impaired replication ability. All of these DIS-directed strategies rely on specifically targeting the viral RNA itself, which might not be practical given our inadequate knowledge of the overall structure of the HIV-1 5\' region. The fact that RNA sequences such as SL1 and SL3 are known to form relevant RNA-protein interactions raises the possibility that the protein component of these interactions might also provide potential targets for anti-HIV therapy. Such approaches are currently being explored in research aimed at designing inhibitors of the TAR-Tat RNA-protein interaction \[[@B129]\]. Similar approaches might also be developed to target RNA-protein interactions involving SL1 or SL3 and Gag. Future directions ================= Current HIV combination therapies have demonstrated that a multi-targeted approach against the virus results in the greatest degree of suppression of virus replication. Therefore, the identification of novel targets for anti-HIV therapy could significantly improve HIV treatment strategies. HIV-1 RNA dimerization is clearly a critical event that could be exploited as a target once its complete mechanism is elucidated. It is pleasing to see that a number of laboratories that have actively researched RNA dimerization and packaging are now moving beyond conventional *in vitro*and *in vivo*approaches toward more biologically relevant methods. One group has taken chemical modification protocols commonly used for *in vitro*RNA analysis, and adapted them for use in virus-producing cells. Hence, structural analysis of viral RNA, that would previously be carried out only *in vitro*on short fragments of artificially transcribed RNA, can now be performed on *in vivo*-generated HIV-1 genomic RNA (J.-C. Paillart and R. Marquet, personal communication, and \[[@B130]\]). This method also allows comparisons of cellular and virion-derived HIV-1 RNA and represents a middle ground between classic *in vitro*and *in vivo*approaches. The goal of this work is to provide insight on the true structure of the HIV-1 leader, and on which RNA substructures are involved in dimerization. Preliminary data suggest that viral RNA may already be dimerized in the cytoplasm (J-C. Paillart and R. Marquet, unpublished data). This method might also have application in regard to *in vivo*foot-printing that could allow the study of RNA-protein interactions in the context of virus-producing cells. The structure of the viral RNA that exists in the cell has long been a topic of interest, and recent data suggest that different RNA sequences might be involved in higher order intrastrand structures that favor the dimerization of the two RNA molecules. Such a model has been proposed \[[@B131]\], and is supported by numerous *in vitro*dimerization studies conducted on HIV-1, HIV-2, and SIV RNA \[[@B41],[@B131]-[@B133]\]. The model proposes that the HIV-1 5\' UTR can form two alternating conformations, termed the long-distance interaction (LDI) and the branched multiple hairpin (BMH) structures. The LDI conformation is believed to exist when the RNA is in a monomer form, and is thought to form a long extended base-paired structure with almost all of the proposed stem-loop sequences buried. This structure is thought to be favored during certain steps of the life cycle, such as translation. In this model, NC has been shown to bind the LDI structure to induce a switch to the BMH structure \[[@B131]\], in which the DIS and ψ would then be exposed in a manner able to mediate dimerization and packaging. Such a \'riboswitch\' is an attractive hypothesis, especially since similar mechanisms have recently been proposed to account for previously unexplained results in the field of gene regulation \[[@B134]\]. Although there is currently little *in vivo*evidence directly supporting such a model in the case of retroviruses, the results of previous mutagenesis studies from several laboratories correlate with those that would be predicted from the riboswitch model, both concerning RNA packaging and RNA dimerization status \[[@B135]\]. In regard to dimerization being a prerequisite for packaging, it would also be interesting to test whether an HIV-1 RNA molecule in the LDI conformation can be packaged. Since the BMH conformation is believed to mediate dimerization, one would assume that the LDI structures would not be packageable if dimerization is truly a packaging prerequisite. Others have developed a fluorescence resonance energy transfer (FRET)-based system to allow visualization of RNA-Gag interactions within cells (A.M. Lever and co-workers, unpublished data). Such a system might provide insight into the timing of genome selection and packaging. It will also be interesting to determine whether this system can be adapted to pinpoint how retroviral RNA dimerization takes place within cells, and whether dimerization indeed occurs before RNA is selected for packaging. Competing interests =================== None declared. Author\'s contributions ======================= RSR gathered the information discussed in this review, and was primary author of the manuscript. CL and MAW carefully read the manuscript and offered insightful suggestions for its revision. All authors read and approved the final version. Acknowledgements ================ The authors wish to acknowledge past and present members of the Liang and Wainberg laboratories, for continued contribution to this field. We apologize to those researchers whose work has not been cited due to publication restraints. RSR is the recipient of a Doctoral Research Award from the Canadian Institutes of Health Research (CIHR). CL is a Chercheur-Boursier of the Fonds de la Recherche en Sante du Quebec (FRSQ) and a New Investigator of the CIHR. Research in our labs has been supported by grants from the CIHR, the FRSQ, and the Canadian Foundation for Innovation. We are also grateful to Diane and Aldo Bensadoun for support of our research program.

PubMed Central

2024-06-05T03:55:47.544680

2004-9-2

PMC516581

Background ========== Severe acute maternal morbidity (SAMM), also known as \"near miss\", is defined as \"A very ill pregnant or recently delivered woman who would have died had it not been that luck and good care was on her side\" \[[@B1],[@B2]\]. This concept is relatively new in maternal care, but is increasingly becoming important in areas with low maternal mortality ratios or where the geographic area is small \[[@B3],[@B4]\]. The use of data collected on SAMM has been shown to be a mechanism for identifying health system failures or priorities in maternal health care more rapidly than maternal deaths \[[@B5]\]. It has the advantage of events still being rare enough not to overload clinicians and data capturing personnel within a facility. Its routine use as an indicator, however, is limited due to the lack of uniform criteria of identification of the cases. This study was undertaken to systematically review all available studies on SAMM with a view to establishing the global prevalence and examining the usefulness as a maternal health indicator. Methods ======= This study is a part of a bigger systematic review undertaken by the UNDP/UNFPA/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP), Department of Reproductive Health and Research at the World Health Organization (WHO) to obtain prevalence/incidence data on maternal mortality and a range of morbidities including SAMM. The methodology of the systematic review followed an a priori protocol and involved an extensive search of all relevant published/unpublished data from 1997 to 2002. The methodology of the systematic review and the search strategy have been described elsewhere \[[@B6]\]. In brief, we searched 10 electronic databases, WHO regional databases, internet and reference lists, contacted experts in the field, and hand-searched relevant articles in the WHO Library. Criteria for inclusion of studies in the review were: inclusion of data relevant to pre-defined conditions, specified dates for data collection period, including data from 1990 onwards, sample size \>200 and a clear description of methodology. A data extraction instrument was used to extract data from included studies. This instrument includes 48 items distributed in five modules three of which were relevant to this analysis. Modules were designed to collect information on (i) the general study level characteristics such as design, population, setting, (ii) prevalence/incidence of maternal morbid conditions, and (iii) quality assessment of morbidity reports. Reporting of definitions and of the procedures used for identification of cases for morbidities were part of quality assessment. We did not assign quality scores to articles, but preferred to present available information on variables regarded as quality components (including reporting of definitions, case-identification criteria, characteristics of setting and participants). Nearly 65 000 reports were screened initially by titles and/or abstracts of which more than 4500 were retrieved for full-text evaluation. More than 2500 of these were included in the review. Data extracted were entered into a specifically constructed database and tabulated using SAS software. A small number of the articles in the review report on SAMM, near miss or a similar definition such as severe morbidity, critically ill obstetric patient. Although we included other articles reporting on individual severe morbidities within their own category of conditions (e.g. severe hypertension within hypertensive disorders of pregnancy), this particular article is concerned with those papers which define a separate entity of SAMM or similar, or those which report on the most recognised end-points for SAMM (i.e. emergency hysterectomy and obstetric admissions to intensive care units). We later conducted an updated search of MEDLINE and Popline using the keywords \'near miss morbidity\' and \'severe maternal morbidity\' to find out about any recent publications that could be of importance, but were not included in the database due to the end date of the original search (2002) for the bigger systematic review. The date of this complementary search was June 2004. We describe below the included studies with an emphasis on the different definitions used and criteria for identification of the cases. Results ======= A total of 30 reports of SAMM are included in the systematic review (twenty-seven identified by the original search and three by the complementary search). Study designs are mainly cross-sectional and most of them are conducted in tertiary care hospitals (see [Additional file 1](#S1){ref-type="supplementary-material"}). Most of the studies describe the characteristics of the setting and participants as well as reporting definitions and procedures for identification of the cases. There are essentially two types of definition of severe acute maternal morbidity; one describing what the authors meant by a near miss; and the other describing a response to an event such as hysterectomy or admission to intensive care unit (ICU). Fourteen studies define a specific SAMM or near-miss \'condition\' \[[@B1],[@B2],[@B7]-[@B18]\] while nine consider admissions to ICU as near-miss cases \[[@B19]-[@B27]\] and the remainder report on emergency hysterectomies \[[@B28]-[@B34]\]. In the majority of articles there is an intuitive agreement on what a near miss means -- a woman who almost died but survived. Identification of cases, however, is complex and varies widely across studies. We listed the articles in [Additional file 1](#S1){ref-type="supplementary-material"} in three categories according to criteria used to classify patients as being near miss; disease-specific (specified criteria for common conditions, e.g. pre-eclampsia, haemorrhage); management-specific (specified criteria related to response to disease, e.g. hysterectomy or admission to ICU); and organ-system dysfunction/failure based (specified criteria for dysfunction or failure related to each organ system). One study reports the proportion of the admissions to ICU separately as well as the total number of SAMM cases \[[@B8]\]. We included this report under the section of organ-system based identification criteria. Individual prevalence rates for SAMM vary between 0.80% -- 8.23% in the first category of studies that use disease-specific criteria. Case-finding criteria differ significantly within this category as well. In addition, some of the studies with similar criteria use mainly clinician\'s evaluation for identification \[[@B15]\], while others have established threshold levels for the degree of severity of the conditions of concern \[[@B7],[@B18]\]. This is probably due to contextual factors such as the availability of facilities with sufficient diagnostic tools. The range is 0.38% -- 1.09% in the group of reports that use organ-system based criteria and include unselected women. Rates are lower (0.01% -- 2.99%) and variation is lesser in the category of studies using management-based criteria. An expected finding is the difference between resource-poor and more advantageous settings in the prevalence of SAMM. In resource-poor settings, 4--8% of pregnant women who deliver in the hospitals will experience SAMM when case-identification criteria are based on specific diseases. This rate is around 1% when the organ failure is considered. In more developed country settings, the rates are around 1% with disease-specific and 0.4% with organ-system based criteria, respectively. The results also suggest that the use of organ-system based criteria is more specific in identifying the real SAMM cases. Discussion ========== Due to the wide variation in identification of the cases, it is not possible to pool data and make a summary estimate for SAMM. Because of the variation in case identifications in the three categories of identification criteria as well as variation within each category (e.g. for disease-specific criteria -- the use of physician\'s evaluation versus technology requiring tests) it is difficult to make comparisons as well. Nevertheless, it is evident that the prevalence of SAMM is higher in studies conducted in less developed country settings. Rates seem to be higher also in studies that use disease-specific criteria as compared to those using organ-system based criteria for similar settings. This finding suggests less specificity of disease-specific criteria in identifying real SAMM cases. Although less specific, the use of disease-specific criteria has some advantages; it is easy to interpret, cases can be identified retrospectively, and the quality of care for that particular disease can be assessed \[[@B18],[@B35]\]. However, the approach concentrates on certain diseases, and thus, other problems such as pulmonary embolus, which is an important cause of maternal death in developed countries could be ignored \[[@B36]\]. In addition, definition of conditions may not always be straightforward. For example, the same threshold for severe haemorrhage could have different consequences in women with normal haemoglobin levels or those with severe anaemia. Furthermore, although detailed objective criteria are established for case identification in developed country settings \[[@B18]\], the limited availability of resources in less developed settings may not permit this level of detail. Therefore, identification of cases is likely to be less accurate when the diagnosis depends on clinical estimates \[[@B2],[@B15]\]. Use of management-specific criteria is advantageous in that it is simple to identify the cases, but it depends on many other variables such as the availability of ICU beds, the facilities in an ICU, or differing views about and indications for hysterectomy. Also, the approach does not include all SAMM cases. One study reporting SAMM according to organ-system based criteria and admissions to ICU separately shows that admissions to ICU represent only one third of all SAMM cases \[[@B8]\]. Use of organ-system based criteria allows for identifying all severe morbidities and then investigating the primary cause, thus does not discard any particular condition. It is the most reproducible across similar areas and criteria can be defined according to resources available. High technology requiring laboratory and haemodynamic investigations can be avoided. However, it is the most labour-intensive for identifying cases, hence criteria for inclusion as near miss must be strict. Bias can be introduced if data collection is incomplete and prevalence can be underestimated. Two approaches are used as potential methods of assessing the care SAMM cases receive. \"Mortality Index-MI\" is defined as the ratio of maternal deaths among the SAMM cases to the sum of maternal deaths and SAMM cases \[[@B35],[@B37]\]. It represents the proportion of women who presents with a SAMM and subsequently dies \[[@B37]\]. Another approach is to calculate the ratio of SAMM to mortality \[[@B8],[@B18]\]. We attempted to calculate the ratio of SAMM to mortality for all studies included under the categories of disease-specific or organ-system based case identification criteria (see [Additional file 1](#S1){ref-type="supplementary-material"}). We did not calculate MI because it was not clear from some reports whether reported maternal deaths were identified as SAMM or not prior to death. It is clearly illustrated in the studies that more SAMM cases are likely to die in resource-poor settings than in more developed country settings. For example, the studies conducted in Niger, Benin and Malaysia give the morbidity to mortality ratio as 11--12 \[[@B10],[@B15],[@B17]\] while this is 117--223 in studies conducted in Europe \[[@B11],[@B18]\] in the category where disease-specific criteria are used. The same applies to the category of organ-system based criteria; morbidity: mortality ratio is 5--8 in South Africa \[[@B1],[@B14],[@B15]\] and 49 in Scotland \[[@B8]\]. These findings suggest that an indicator that relates SAMM to maternal deaths could be a useful method in assessing the care SAMM cases receive. However, the definitions and identification of cases should be standardised at least for similar settings and the indicator needs to be clearly defined. Conclusion ========== Considering all complexities in definition and case-identification of SAMM, it is necessary that studies clearly describe their identification criteria for the cases. There is a clear need to set criteria to identify SAMM cases. Use of organ-system based criteria seems to be a more useful approach in identifying cases as variation in defining criteria can be avoided, particularly for similar settings. It would then be easier to establish summary estimates for SAMM prevalence which could serve as a measure of maternal health and quality of care indicator. Competing interests =================== None declared. Authors\' contributions ======================= AMG and LS coordinated the conduct of the systematic review of maternal morbidity and mortality. RCP and LS outlined the manuscript. LS reviewed the studies and wrote the initial draft. AMG and RCP substantially improved the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional file 1 Table describing important variables of all studies included in the systematic review ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank Dr Paul FA Van Look for his thorough review of the manuscript and valuable inputs.

PubMed Central

2024-06-05T03:55:47.549648

2004-8-17

PMC516767

Background ========== In the classical cell cycle, the nuclear DNA contents vary only within the range of 2C and 4C, where C is the haploid DNA content per nucleus. When mitotic DNA replication in somatic cells is not followed by cell division (a process called endoreduplication), variation of cellular ploidy levels (designated as somatic polyploidy or endopolyploidy) can result \[[@B1]\]. Endopolyploidy is considered to be developmentally regulated \[[@B2]\] and has been described in several plant species including maize \[[@B3],[@B4]\], sunflower \[[@B5]\], tomato \[[@B6]\], *Arabidopsis*\[[@B7]\] and brassicas \[[@B8],[@B9]\]. Presence of endopolyploidy as a result of endoreduplication is also a common feature of insects and mammals \[[@B10],[@B11]\]. In orchids, endoreduplication has been described in the raphid crystal idioblasts of *Vanilla*\[[@B12]\] and in parenchyma cells of *Vanda*seedlings \[[@B13],[@B14]\]. The family Orchidaceae has an estimated 17,000 to 35,000 species, making it the largest and an important family of the flowering plants \[[@B15]\]. Many of the members of the orchid family are commercially valuable, and are micropropagated \[[@B16]\]. The explant sources used for orchid micropropagation include inflorescence, leaves, floral buds and roots \[[@B16],[@B17]\]. However, very little is known about the ploidy variation in different explant tissues of the orchid plants during different developmental periods and at the stage when they are used as explants for micropropagation. Increased knowledge of the degree of endopolyploidy in the explant tissue source will be highly valuable for the maintenance of the original ploidy level in culture \[[@B8]\]. In this paper, systemic variation of cellular ploidy and DNA content in different tissues of *Spathoglottis plicata*, a common tropical terrestrial orchid species, from field-grown plants and *in vitro*cultures was investigated. Results ======= Ploidy level of nuclei isolated from leaves and roots of greenhouse-grown plants -------------------------------------------------------------------------------- Flow cytometry analysis of nuclear preparations from entire young leaves of 1--3 cm in length revealed that there were three peaks of fluorescence corresponding to 2C, 4C and 8C DNA content of somatic cells (Fig. [1A](#F1){ref-type="fig"}). About 50% of the nuclei were found to have 2C DNA content, 25% were 4C and 15% were found to have 8C DNA content (Fig. [1G](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Nuclear DNA content and distribution of endopolyploid nuclei in vegetative tissues of the greenhouse-grown plants: A. Young leaves, B. Basal part of the mature leaves, C. Middle part of the mature leaves, D. Tip of the mature leaves, E. Root tips, F. Root segments with root hairs. The Y-axis presents the number of nuclei (events); the X-axis presents 3-decade log value of relative DNA content (PMT4). G. The population of endopolyploid nuclei in tissues A-F. :::  ::: More detailed analysis was done on mature leaves of 48 cm in length. Tissues taken from different regions of the mature leaves showed that the pattern of ploidy levels was different at different regions. For tissues taken from the basal (petiolar end) of the leaves, there were three fluorescence peaks corresponding to 2C, 4C and 8C nuclear DNA content (Fig. [1B](#F1){ref-type="fig"}). However, no 8C peak was observed from nuclei preparations taken from tissues of the middle (Fig. [1C](#F1){ref-type="fig"}) and tip (Fig. [1D](#F1){ref-type="fig"}) regions of the same leaf; only 2C (35--40% of cell populations) and 4C (60--65% of cell population) nuclei were identified (Figs. [1C,1D,1G](#F1){ref-type="fig"}). In the young root tips, 2C, 4C and 8C peaks were observed (Fig. [1E](#F1){ref-type="fig"}) and the distribution skewed toward 2C population, which accounted for more than 60% of the nuclei population analyzed (Fig. [1G](#F1){ref-type="fig"}). The percentage of nuclei population with 4C and 8C DNA content in the root tip was relatively small and accounted for only about 10% each (Figs. [1E,1G](#F1){ref-type="fig"}). Cells from root segments taken at least 2 cm away from the tips were all 2C (Fig. [1F](#F1){ref-type="fig"}). Ploidy level of nuclei isolated from floral tissues --------------------------------------------------- Preparations from floral pedicels (Fig. [2A](#F2){ref-type="fig"}), columns (Fig. [2B](#F2){ref-type="fig"}) of freshly open flowers, and growing flower stems (Fig. [2F](#F2){ref-type="fig"}) revealed that there were 2C, 4C and 8C nuclei. The proportions of 2C and 4C nuclei ranged from 40--50% (Fig. [2H](#F2){ref-type="fig"}), and only about 8 -- 10% of nuclei were found to be 8C (Fig. [2H](#F2){ref-type="fig"}). In pedicels of un-open flower (Fig. [2G](#F2){ref-type="fig"}), there was a 16C peak in addition to 2C, 4C and 8C peaks. The majority of nuclei were in 4C (28%) and 8C (46%) peaks. The 2C and 16C peaks each had less than 10% of the total nuclei (Fig. [2H](#F2){ref-type="fig"}). Nuclei isolated from the sepals (Fig. [2C](#F2){ref-type="fig"}), petals (Fig. [2D](#F2){ref-type="fig"}) and ovary tissues (Fig. [2E](#F2){ref-type="fig"}) were all 2C. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Nuclear DNA content and distribution of endopolyploid nuclei in floral tissues of the greenhouse-grown plants: A. Pedicels, B. Columns, C. Sepals, D. Petals, E. Ovary tissues, F. Growing flower stems, G. pedicels of un-open flowers. The Y-axis presents the number of nuclei (events); the X-axis presents 3-decade log value of relative DNA content (PMT4). H. The population of endopolyploid nuclei in tissues A-G. :::  ::: Ploidy level of cells from *in vitro*cultures --------------------------------------------- Protocorms of *S. plicata*were found to have 2C, 4C and 8C nuclei (Fig. [3A](#F3){ref-type="fig"}) with majority (over 70%) of them with 2C DNA content (Fig. [3E](#F3){ref-type="fig"}). In the young leaves of plantlets, majority (70%) of the nuclei isolated were 4C, and about 20% of were 2C nuclei and the rest 8C (Figs. [3B,3E](#F3){ref-type="fig"}). In the root tips of cultures, there were about 40% each of 2C and 8C nuclei, and the proportion of 4C nuclei was only about 10% (Figs. [3C,3E](#F3){ref-type="fig"}). Nuclei taken from root tissues at the region with root hairs were all 2C (Figs. [3D,3E](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Nuclear DNA content and distribution of endopolyploid nuclei in vegetative tissues of the tissue-cultured plants: A. Protocorms, B. Young leaves, C. Root tips, D. Root segments with root hairs. The Y-axis presents the number of nuclei (events); the X-axis presents 3-decade log value of relative DNA content (PMT4). E. The population of endopolyploid nuclei in tissues A-D. :::  ::: Discussion ========== As references to the DNA content of gametic nucleus of individuals, DNA \'C\' values have been estimated in several thousand animal and plant species \[[@B18]\]. For angiosperms, information on \'C\' values is used in a wide range of biological fields \[[@B19]\]. The 1C DNA values in angiosperm plants differ approximately 1000 folds, ranging from 0.13 pg in *Arabidopsis thaliana*to 127.4 pg in *Fritillaria assyriaca*\[[@B18]\]. The DNA content per genome is usually considered to be constant between cells in an individual, and relatively constant between individuals of the same species \[[@B18]\]. However, in some plant species, intraplant ploidy variations were reported, and this implied that the nuclear DNA content in these species is not static and hence a great amount of variation occurs \[[@B8]\]. For example, a survey of *Arabidopsis thaliana*revealed endopolyploidy in hair trichomes, leaf epidermal cells, root tip cells, and cells in the hypocotyls \[[@B7],[@B20]\], but not in the inflorescence \[[@B7]\]. In some cell types, the extent of endoreduplication appears to be intrinsically controlled by the differentiation programme, but environmental influences such as light can also affect endoreduplication \[[@B21]\]. The patterns of endopolyploidy may be affected by plant growth conditions in some plants. For example, leaves of *in vitro*grown tomato and potato plants were found to have lower level of endopolyploidy than leaves of plants grown in the greenhouse \[[@B22],[@B23]\]. However, in *S. plicata*, patterns of endopolyploidy were found to be similar in both tissue-cultured plants and greenhouse-grown plants. Endoreduplication was found to occur in actively growing tissues with of *S. plicata*such as young leaves (1--3 cm in length, newly initiated) and root tips from greenhouse-grown plants. Similarly, endopolyploid cells were found in protocorms, young leaves and root tips from *S. plicata*seedlings in tissue culture. The common feature for protocorms, young leaves of 1--3 cm and root tips are that they are young and active in cell division and growth. In other orchids such as *Dendrobium*, endopolyploidy was found in root tips and newly expanded young leaves \[[@B24]\]. In the root segment with root hairs of *S. plicata*, endopolyploidy was neither found in tissue-cultured plants nor it was detected in greenhouse-grown plants. These results imply that the presence of the endopolyploidy during *S. plicata*development is an intrinsic programme, and it is not much affected by the growth condition. Besides root tips and newly developing young leaves, endopolyploidy was observed in mature leaves in a few *Dendrobium*species and cultivars \[[@B24]\]. Endopolyploidy was also detected in mature leaves of *S. plicata.*Furthermore, when the tip, middle and basal parts of the mature leaf were examined, different patterns of ploidy levels were obtained. Endopolyploidy was found only in leaf base part of mature leaves. A mature leaf represents a continuous developmental system, with the young, less green meristem cells at the basal petiolar end and the older, photosynthetically active cells at the tip \[[@B25]\]. Previous research shown that in cucumber and succulent plants with small genome, the level of endoreduplication does not increase once an organ is fully developed \[[@B26],[@B27]\]. The tips and middle parts of mature leaves in *S. plicata*are fully developed. Endoreduplication in these tissues is unlikely since it would lead to further cell expansion. The pattern of DNA ploidy variation within the mature leaf is closely associated with the developmental status. The mechanism that resulted in endopolyploidy, however, remains unclear. In *S. plicata*, endopolyploidy was present in some floral tissues such as columns, growing flower stems and pedicels of both un-open and freshly open flowers. However, other floral tissues like sepals, petals and ovary tissues were found to have only 2C nuclei. In the growing un-open flower pedicels, the highest ploidy level even reached 16C. In cabbage, endopolyploidy was reported in cabbage flowers \[[@B8]\], and detailed patterns of endopolyploidy were found in various developmental stages of petals \[[@B9]\]. In cabbage petals, differentiation of expanding cells was characterized by endoreduplication \[[@B9]\]. In the proximal part of the cabbage petal, differentiation was accompanied with endoreduplication and cell enlargement. By contrast, no endopolyploid nucleus was found in the distal part of the lamina in the mature cabbage petal \[[@B9]\]. This study suggested that the developmental program of the cabbage petals might induce the initiation of endoreduplication \[[@B9]\]. In *Arabidopsis*, endopolyploidy was found in hypocotyls, cotyledonary leaves, rosette leaves, stems of bolting plants and floral leaves, but was not found in inflorescences \[[@B7]\]. Given the small size of columns within *Arabidopsis*floral buds, and the small population of endopolyploid nuclei found in columns and pedicels of *S. plicata*in this study, the minute population of the endopolyploid nuclei could easily be neglected when the whole floral buds were used for sampling. In *S. plicata*, it was found that the average size of nuclei was larger in columns and pedicels that have a measurable amount of endopolyploid cells than in other flora tissues without endopolyploidy (unpublished results). Similarly, a correlation was found between cell size and ploidy levels during cabbage petal development \[[@B9]\]. In *Dendrobium*, the post-pollination physiological changes were found to be different between floral tissues such as columns, ovary tissues, sepals and petals \[[@B28]\]. Edgar and Orr-Weaver \[[@B10]\] suggested that as endoreduplication is often found in large cells or cells with high metabolic activity, it might be a common strategy for cell growth without division. Further evidence was found in legumes where cell differentiation to a specialized function as pod wall tissues was accompanied by endoreduplication, and higher ploidy levels coincided with maximum pod growth \[[@B29]\]. During tomato fruit development, the pericarp tissue of young green fruit did not have higher ploidy (usually within 2C and 4C), but most of the cells in pericarp became endopolyploid (up to 256C) as the fruit developed further \[[@B6]\]. In tobacco single cell culture, endoreduplication was associated with plant growth regulators. When auxin was applied alone, endoreduplication was induced and the DNA content kept pace with the increment of cell volume. When both auxin and cytokinin were supplied subsequently, the cells divided first as amitosis leading to DNA endoreduplication, then followed by normal mitosis cell cycles \[[@B30]\]. Gibberellin and ethylene were found to play important roles in the endoreduplication of *Arabidopsis*hypocotyls \[[@B31]\]. In cabbage, mammals, *Drosophila melanogaster*and some small genome plants like *Arabidopsis*, it is thought that endoreduplication is developmentally regulated \[[@B8],[@B10],[@B32]\]. The systemic endopolyploidy revealed within different tissues of *S. plicata*raises the question of its possible implications. In tobacco, it was reported that the morphogenetic response of the tissues culture was related to the nuclear DNA content variation within stem explants of different ages \[[@B25]\]. In *Oncidium*Gower Ramsey, a hybrid orchid, only root tips, cut surfaces of stem segments and young leaves were able to form callus in tissue culture. Other explants such as old leaves and the roots without meristem tips could not form any callus \[[@B33]\]. Molecular data showed that the nuclear DNA modulation was closely related to the acquisition of embryogenic competence in cultured carrot hypocotyls \[[@B34]\]. In various tissues in cabbage plants, the number of endocycles was tissue-specific and was characteristic of the developmental stage \[[@B8],[@B9],[@B32]\]. These studies suggested that pattern of endopolyploidy may represent the characteristic of the developmental and physiological properties of the tissue. The role of endoreduplication in plant development is still not well understood. The presence of endopolyploidy was proposed to be associated with several factors, such as taxonomic position of a species, life cycle, genome size, and organ type \[[@B35]\]. Recent investigation of 16 plant species suggested that endopolyploidization might provide a mechanism to facilitate plant growth \[[@B35]\]. Endoreduplication benefits fast growth in several ways. In polyploid cells, the increased gene dosages may enhance the transcriptional and metabolic activities. In addition, several processes are eliminated in the endoreduplication cycle such as the reorganization of the cytoskeleton and condensation of the chromosomes, and that might allow faster growth \[[@B36]\]. Ploidy level also plays a role in controlling the size of the cells, the organs or the whole plant \[[@B6],[@B9],[@B37]\]. One of the common features of plant development is the uneven enlargement of plant cells coupled to somatic endoreduplication, which indicates that the enlargement of plant cells might be the consequence of the increased genome size \[[@B9],[@B37]\]. This research may also have an impact on the orchid industry. Orchidaceae is the largest family of the flowering plants, and many of its members are commercially hybridized \[[@B38]\]. Clonal propagation is a common and essential practice for multiplication of hybrid orchids because the genotypes of the hybrids are usually heterozygous \[[@B39]\]. Many tissues have been used as explants for micropropagation including inflorescence, leaves, floral buds and roots \[[@B16],[@B40],[@B41]\]. Somaclonal variation is undesirable, and it is a major problem encountered in commercial micropropagation of orchids if true-to-type plants are required \[[@B39],[@B42]\]. The mechanism of the somaclonal variation is poorly understood \[[@B42]\]. Polyploidy is considered as a possible cause for somaclonal variation in tissue cultures \[[@B43]\], but how polyploidy is generated during tissue culture is unclear \[[@B39],[@B42]\]. The presence of systemic endopolyploidy and DNA content variation within different tissues of *S. plicata*as revealed in this study suggests that endopolyploidy and DNA content variation in explants might be a cause for somaclonal variation in tissue culture derived orchid plantlets. Thus, the pre-knowledge about the ploidy variation in different explant tissues is valuable for clonal propagation or for deliberate induction of variants in culture. Further systemic investigation of the relationship between somaclonal variation and type and endopolyploid level of source explants will provide indepth knowledge for micropropagation of orchids. Conclusions =========== Systemic variation of cellular endopolyploidy in different tissues during growth and development of *Spathoglottis plicata*from field-grown plants and *in vitro*cultures was developmentally regulated. Pattern of endopolyploidy is a character of the developmental and physiological properties of the tissue. This finding provides useful information for understanding of the plant development and for industrial propagation of orchids. Methods ======= Plant materials --------------- *Spathoglottis plicata*L. is a common tropical terrestrial orchid. The plants were grown in pots and placed in the greenhouse at 28 ± 4°C without artificial lighting. The following materials were taken for analysis: a) young leaves (1--3 cm in length), b) mature leaf (48 cm in length), c) root tips (2 cm including the tip), d) root segments from region with root hairs (2 cm away from the root tip), e) newly opened flowers, f) growing flower stems and g) developing floral pedicels of un-open flower (4--6 days before flowering). Seedpods were surface-sterilized for 20 min in 20% (v/v) Clorox™ solution and subsequently rinsed 3 times with autoclaved water. Seeds were germinated aseptically in 9 cm diameter petri dishes containing 25 ml Knudson C orchid medium (Duchefa, Netherlands) with 2% (w/v) sucrose and 0.8% (w/v) agar. All cultures were incubated at 25 ± 2°C under a 16 h photoperiod (light intensity: 54 μm^-1^m^-2^s^-1^). The following materials from *in vitro*cultures were examined: a) protocorms (6 weeks after germination), b) young leaves (1--2 cm in length) from seedlings, c) root tips (1 cm segments from tips), d) root segments with root hairs (at least 1 cm away from the root tips). Preparation of nuclei and flow cytometry analysis of nuclear DNA content ------------------------------------------------------------------------ Extraction of nuclei and staining of DNA were performed according to the method of Arumuganathan and Earle \[[@B44],[@B45]\] with some modifications. All preparations were done on ice. Tissues (about 0.3 -- 1.0 g) were sliced with razor blades into strips of less than 1 mm in 1 ml extraction solution (1 mM MgSO~4~, 5 mM KCl, 0.5 mM HEPES, 1 mg/ml dithiothreitol, 2.5 mg/ml Triton X-100, pH 8.0) and extracted for 45 minutes. After filtering through a 45 μm Falcon cell strainer, 100 μl of propidium iodide (1 mg/ml) and 2.5 μl of 500 μg/ml DNase-free RNase (Boehringer Mannheim, Indianapolis, IN) were added to each sample followed by 30 min incubation at 37°C. A Coulter EPICS^®^Elite ESP with 15 mW 488 nm Cyonics Argon air-cooled laser flow cytometer was used to measure the relative fluorescence of nuclei. For each sample, at least 10,000 nuclei were analyzed. Data were analyzed with WinMDI27B software (Joseph Trotter™). Abbreviations ============= C, DNA content of the unreplicated haploid chromosome complement PI, propidium iodide PMT4, photo multiplier tube Authors\' contributions ======================= MY carried out experiments in the project. CSL and MY prepared the manuscript. Acknowledgement =============== The authors thank M. S. Choy for his help on flow cytometry.

PubMed Central

2024-06-05T03:55:47.551229

2004-9-1

PMC516768

Background ========== Two mtDNA macro-haplogroups (M and N) that arose from the African haplogroup L3 encompass virtually all mtDNAs outside Africa \[[@B1]-[@B4]\]. The phylogenetic node N (including R) has spread its branches all over Eurasia, in contrast to haplogroup M, which is found in Eastern Eurasia but is virtually absent in Europe. The numerous branches of N are, however, generally segregated to either the eastern (e.g. A, B \[[@B5]\], Y \[[@B6]\], R9 \[[@B7]\] or western (e.g. N1 \[[@B8],[@B9]\], N2 (comprising of W and its sister-clade identified by \[[@B10]\]), TJ, HV, U \[[@B11]\]) Eurasian-specific pools. The majority of Indian mtDNAs belong to macro-haplogroup M \[[@B8],[@B12]-[@B21]\]. While the topology of the M sub-haplogroups that are common in mainland East Asia (M7, M8 (including C, Z), M9 (including E), D, G \[[@B7],[@B22],[@B23]\]) and in Africa (M1 \[[@B24]\]) is established in detail, the internal haplogroup structure of M in India has remained largely undefined. We have previously demonstrated that transitions at nps. 477G, 1780, 8502 and 16319 designate Indian-specific haplogroup M2, the most frequent M clade in India \[[@B15]\]. Another Indian-specific M clade supported by HVS-I variation as well as coding region markers, is M6 \[[@B15]\]. Haplogroups M3, M4 and M5 have been discriminated preliminarily by their characteristic HVS-I mutations \[[@B19]\], but since their defining positions, 16126, 16311, and 16129, respectively, are phylogenetically unstable \[[@B25],[@B26]\], it is unlikely that the proposed haplogroups are monophyletic. Most numerous sub-groups of macro-haplogroup N in India are the Indian-specific variants of the phylogenetic node R including haplogroups R5, R6, U2(a, b, c) \[[@B8],[@B13],[@B27]\]. The overwhelming majority of the Iranian mtDNAs have been shown to lie in the West Eurasian domain of the global human mtDNA pool \[[@B27],[@B28]\]. Here we focus on the analysis of mtDNA lineages that are shared between Indians and Iranians and bear signals of pre-Holocene expansion in the region. India congregates four linguistic domains (Indo-European, Dravidic, Austro-Asiatic and Tibeto-Burman) that occupy non-random spheres of the geographic distribution of its populations. The majority of the recent studies based on mtDNA variation have, in contrast to some \[[@B21]\], provided evidence that linguistic groups of India do not represent genetically homogeneous units and are not, therefore, traceable to different immigration waves from distinct sources \[[@B8],[@B13],[@B19]\]. The complexity that arises in defining populations and groups of populations in India based on genetic and cultural criteria has been recently demonstrated in South Indian tribal and caste populations. The combined data from mtDNA, Y-chromosome and autosomal genes indicated that the tribes and castes derive largely from the same genetic heritage of Late Pleistocene southern and southwestern Asians, and have received limited gene flow from external sources since the Holocene \[[@B15]\]. Similar results were obtained by Cordaux et al. \[[@B29]\], who demonstrated that caste and tribal groups exhibit similar levels of molecular variance. However, genetic distances indicated that the Tibeto-Burman speaking tribal populations (from eastern India) were more closely related to East Asians than to other Indians \[[@B29]\]. This is consistent with an earlier suggestion placing the origin of these tribal groups east of India -- in Tibet and Myanmar \[[@B30]\]. In this study, we have analyzed the mtDNA variation in a sample of 796 Indians and 436 Iranians (Table [1](#T1){ref-type="table"}), and combined the results with previously published data from the same geographic area. We also compared the mtDNA variation in India and Iran with that of Europe, China, and Thailand. The overall aim was to improve our understanding of the origins and composition of the Indian and Iranian gene pools and to determine the nature and the extent of gene flow between these regions. Through the analyses of the genetic variation of extant Southwest and South Asian populations we took an endeavor to envisage the exodus of anatomically modern humans from Africa. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the Indian and Iranian population samples whose mtDNA variation has been determined in the course of this study. ::: **INDIA** --------------- ------- ---------------- -------------------------------- ----- ----- ---------------- ---------------- West Bengal Caste Indo-European Mixed caste people Ben 50 80 Million Uttar Pradesh Tribe Indo-European Bhoksa Bho 5 32,000 Kerala Caste Indo-European Mixed caste people from Cochin Co 55 600,000 Kerala -^a^ Indo-European Cochin Jews CoJ 45 5,000 (in Israel) Gujarat Caste Indo-European Mixed caste people Guj 53 50 Million Himachal Tribe Tibeto-Burman Kanet Kan 37 33,000 Maharashtra Caste Indo-European Konkanastha Brahmin Kon 58 N/A West Bengal Caste Indo-European Kurmi Kur 55 N/A West Bengal Tribe Austro-Asiatic Lodha Lod 56 59,000 Sri Lanka Caste Dravidic Moor Mo 50 3.4 Million Maharashtra -^a^ Indo-European Parsi Par 55 76,000 Punjab Caste Indo-European Mixed caste people Pun 109 24 Million Rajasthan Caste Indo-European Rajput Raj 35 5 Million (in Rajasthan) Sri Lanka Caste Indo-European Sinhalese Sin 82 14.6 Million Uttar Pradesh Tribe Indo-European Tharu Tha 26 96,000 (in India) Uttar Pradesh Caste Indo-European Uttar Pradesh Brahmin UPb 25 2.5 Million total 796 **IRAN** northwest 226 southwest 138 northeast 30 southeast 6 central 36 total Indo-European Iranian IR 436 ca. 68 Million ^a^For some analyzes these populations where grouped with the caste populations. (see Table 1 in the Supplementary Material for the complete set of studied populations including those whose mtDNA variation was previously published) ::: Results and Discussion ====================== Geographic distribution of macro-clades M and N in India -------------------------------------------------------- We found haplogroup M ubiquitous at almost 58% among the caste, and 72% among the tribal populations (Table [2](#T2){ref-type="table"}), which is largely consistent with previous reports \[[@B8],[@B12]-[@B21]\]. Our results indicate that the frequency distribution of haplogroup M varies across different Indian regions by a significant cline towards the south and the east (see Figure [4](#F4){ref-type="fig"} for Spatial Autocorrelation Analysis (SAA) p \< 0.05). The variation among caste populations climbs from approximately 40% in Gujarat and (Indian-) Punjab to 65% in the southern states, and peaks at over 70% in West Bengal (Table [2](#T2){ref-type="table"}). We observed a similar geographic pattern among tribal populations, where the frequency varied from just over 50% in the northern states of Punjab and Himachal Pradesh, increased to 70%--80% in the southern states, and peaked at 86% in West Bengal (Table 8, see [Additional file 3](#S3){ref-type="supplementary-material"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Spatial Autocorrelation Analyses Correlograms of different haplogroups and haplogroup groupings frequencies in South Asia.**In the case of haplogroup M3a some datasets had to be excluded because lack of resolution (see footenote for Table 3. for detailes). There was no significant cline in the frequencies of haplogroup R2. In the case of haplogroups M6a and M6b only the potential cline along the Bay of Bengal is investigated. Therefor the number of distance classes is reduced to three. :::  ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Geographic, linguistic and socio-cultural distribution of major Indian-specific mtDNA haplogroups ::: HAPLOGROUP FREQUENCY (95% CR FOR PROPORTION) -------------------------------------------------------------------- ---------------------------------------------- -------------------- ------------------- ------------------ ----------------- ------------------ ------------------- ----------------- ------------------- ----------------- ----------------- ------------------- ------------------- ------------------- ------------------- India 2544^g^ 65 (63.1--66.8) 6.3 (5.4--7.3) 2.2 (1.7--2.9) 0.6 (.4--1) 2.9 (2.3--3.6) 2.1 (1.6--2.8) 0.9 (.6--1.4) 11.4 (10.2--12.7) 2.2 (1.7--2.8) 1.3 (.9--1.8) 5.3 (4.5--6.2) 3.4 (2.7--4.2) 10.5 (9.4--11.8) 12.1 (10.9--13.4) Iran 436 5.3 (3.6--7.8) 0 (0--.8) 0.5 (.1--1.6) 0 (0--.8) 0 (0--.8) 0.2 (.1--1.3) 0 (0--.8) 3.2 (1.9--5.3) 0.9 (.4--2.3) 0 (0--.8) 0.2 (.1--1.3) 13.3 (10.4--16.8) 3.9 (2.5--6.2) 90.8 (87.7--93.2) **Socio-cultural affiliation (Indian data only)** Caste population 1204 57.8 (54.9--60.5) 5.1 (4--6.5) 4.2 (3.2--5.5) 1.1 (.6--1.8) 3 (2.2--4.1) 1.4 (.9--2.2) 1.4 (.9--2.2) 14.1 (12.3--16.2) 3.3 (2.5--4.5) 1.7 (1.1--2.5) 7.8 (6.4--9.5) 4.8 (3.7--6.2) 4 (3--5.2) 17.1 (15.1--19.3) Tribal population 1332 71.5 (69--73.8) 7.5 (6.2--9) 0.5 (.2--1) 0.2 (0--.5) 2.7 (2--3.7) 2.7 (2--3.7) 0.5 (.2--1) 8.9 (7.5--10.6) 1.2 (.7--1.9) 0.9 (.5--1.6) 3 (2.2--4.1) 2 (1.4--2.9) 16.5 (14.6--18.6) 7.5 (6.2--9) Brahmins & Kshatriyas 313 60.1 (54.5--65.3) 1.9 (.9--4.1) 5.1 (3.2--8.1) 1 (.3--2.8) 0.6 (.2--2.3) 1 (.3--2.8) 2.2 (1.1--4.5) 14.7 (11.2--19.1) 4.5 (2.7--7.4) 1.3 (.5--3.2) 6.4 (4.2--9.7) 6.1 (3.9--9.3) 3.5 (2--6.2) 17.6 (13.8--22.2) Other Castes^h^ 517 61.5 (57.2--65.6) 6.2 (4.4--8.6) 4.6 (3.1--6.8) 1.2 (.5--2.5) 2.9 (1.8--4.7) 2.1 (1.2--3.8) 0.8 (.3--2) 12.6 (10--15.7) 2.3 (1.3--4) 1.5 (.8--3) 7.4 (5.4--9.9) 3.9 (2.5--5.9) 1.7 (.1--1.4) 16.6 (1.2--3.8) **Language groups of India** Indo-European 963 57.5 (54.4--60.6) 3.8 (2.8--5.3) 4.5 (3.3--6) 1.0 (.6--1.9) 2.7 (1.9--3.9) 1.1 (.6--2) 1.6 (1--2.6) 7.8 (6.3--9.7) 2.5 (1.7--3.7) 1.6 (1--2.6) 7.1 (5.6--8.9) 6.9 (5.4--8.6) 4.0 (3--5.5) 19.9 (17.5--22.6) Dravidic 1063 70.0 (67.2--72.7) 10.8 (9.1--12.8) 1.2 (.7--2.1) 0.5 (.2--1.1) 4.0 (2.9--5.3) 1.4 (.9--2.3) 0.5 (.2--1.1) 8.9 (7.4--10.8) 2.7 (1.9--3.9) 1.4 (.9--2.3) 5.9 (4.7--7.5) 1.2 (.7--2.1) 3.0 (2.1--4.2) 8.8 (7.3--10.7) Tibeto-Burman 249 59.8 (53.6--65.7) 0 (0--1.5) 0.4 (.1--2.2) 0 (0--1.5) 1.6 (.7--4) 1.2 (.4--3.5) 1.2 (.4--3.5) 0.8 (.2--2.9) 1.2 (.4--3.5) 0 (0--1.5) 0.8 (.2--2.9) 2.0 (.9--4.6) 61.4 (55.3--67.3) 3.2 (1.7--6.2) Austro-Asiatic (Mundari) 90 86.7 (78.1--92.2) 2.2 (.7--7.7) 0 (0--4) 0 (0--4) 0 (0--4) 26.7 (18.6--36.7) 0 (0--4) 4.4 (1.8--10.9) 0 (0--4) 1.1 (.3--6) 1.1 (.3--6) 1.1 (.3--6) 0 (0--4) 1.1 (.3--6) Indo-European ^i^ 250 66.4 (60.3--72) 8.0 (5.3--12) 0.4 (.1--2.2) 0.8 (.2--2.8) 4.8 (2.8--8.2) 1.6 (.6--4) 0.4 (.1--2.2) 8.8 (5.9--13) 2.0 (.9--4.6) 1.6 (.6--4) 6.4 (4--10.1) 4.0 (2.2--7.2) 1.2 (.4--3.5) 13.6 (9.9--18.4) Indo-European ^j^ 713 54.4 (50.7--58) 2.4 (1.5--3.8) 5.9 (4.4--7.9) 1.1 (.6--2.2) 2.0 (1.2--3.3) 1.0 (.5--2) 2.0 (1.2--3.3) 7.4 (5.7--9.6) 2.7 (1.7--4.1) 1.5 (.9--2.7) 7.3 (5.6--9.4) 7.9 (6.1--10.1) 5.0 (3.7--6.9) 22.2 (19.3--25.4) **Indian States and Bangladesh (excluding tribal populations)**^k^ Andhra Pradesh 245 65.3 (59.1--71.0) 7.7 (5--11.8) 2.8 (1.4--5.7) 1.6 (.6--4.1) 3.3 (1.7--6.4) 4.1 (2.3--7.4) 0 (0--1.5) 18 (13.7--23.2) 4.1 (2.3--7.4) 1.2 (.4--3.5) 5.3 (3.2--8.9) 2.4 (1.1--5.2) 1.2 (.4--3.5) 8.9 (6--13.2) Bangladesh 30 66.67 (48.6--80.8) 9.96 (3.61--25.7) 0 (.08--11.2) 0 (.08--11.2) 3.29 (.78--16.6) 3.29 (.78--16.6) 0 (.08--11.2) 6.67 (2.04--21.4) 0 (.08--11.2) 0 (.08--11.2) 6.67 (2.04--21.4) 3.29 (.78--16.6) 9.96 (3.61--25.7) 6.67 (2.04--21.4) Gujarat 57 40.4 (28.6--53.4) 5.3 (1.9--14.4) 7 (2.9--16.7) 1.8 (.4--9.2) 1.8 (.4--9.2) 1.8 (.4--9.2) 0 (0--6.2) 17.5 (9.9--29.4) 5.3 (1.9--14.4) 1.8 (.4--9.2) 1.8 (.4--9.2) 17.5 (9.9--29.4) 5.3 (1.9--14.4) 33.3 (22.5--46.3) Karnataka 47^h^ 55.3 (41.2--68.6) 2.1 (.5--11.1) 2.1 (.5--11.1) 0 (.1--7.4) 0 (.1--7.4) 0 (.1--7.4) 0 (.1--7.4) 19.1 (10.5--32.6) 6.4 (2.3--17.2) 0 (.1--7.4) 10.6 (4.7--22.7) 6.4 (2.3--17.2) 0 (.1--7.4) 14.9 (7.5--27.8) Kashmir 19 26.3 (11.9--49.1) 0 (.1--16.8) 0 (.1--16.8) 5.3 (1.2--24.9) 10.5 (3.2--31.7) 0 (.1--16.8) 0 (.1--16.8) 10.5 (3.2--31.7) 0 (.1--16.8) 5.3 (1.2--24.9) 10.5 (3.2--31.7) 5.3 (1.2--24.9) 21.1 (8.7--43.7) 31.6 (15.4--54.3) Kerala 100 54 (44.2--63.5) 4 (1.6--9.8) 0 (0--3.6) 0 (0--3.6) 0 (0--3.6) 0 (0--3.6) 5 (2.2--11.2) 25 (17.6--34.3) 9 (4.9--16.2) 1 (.2--5.4) 7 (3.5--13.8) 1 (.2--5.4) 4 (1.6--9.8) 11 (6.3--18.7) Maharashtra 117 65 (55.9--73) 3.4 (1.4--8.5) 11.1 (6.6--18.1) 0.9 (.2--4.6) 0 (0--3.1) 0 (0--3.1) 5.1 (2.4--10.7) 6.8 (3.5--12.9) 0.9 (.2--4.6) 0.9 (.2--4.6) 4.3 (1.9--9.6) 4.3 (1.9--9.6) 5.1 (2.4--10.7) 23.1 (16.4--31.5) Punjab 150 36 (28.8--44) 0.7 (.2--3.6) 4.7 (2.3--9.3) 1.3 (.4--4.7) 0.7 (.2--3.6) 0 (0--2.4) 2.7 (1.1--6.6) 11.3 (7.2--17.4) 0.7 (.2--3.6) 0.7 (.2--3.6) 7.3 (4.2--12.7) 14 (9.4--20.5) 5.3 (2.8--10.2) 42.7 (35--50.7) Rajasthan 36 69.4 (53--82) 0 (.1--9.5) 13.9 (6.2--28.8) 0 (.1--9.5) 0 (.1--9.5) 5.6 (1.7--18.2) 0 (.1--9.5) 22.2 (11.8--38.2) 5.6 (1.7--18.2) 2.8 (.7--14.2) 2.8 (.7--14.2) 5.6 (1.7--18.2) 0 (.1--9.5) 2.8 (.7--14.2) Sri Lanka 132 57.6 (49--65.7) 6.8 (3.7--12.5) 1.5 (.5--5.3) 1.5 (.5--5.3) 1.5 (.5--5.3) 0.8 (.2--4.1) 0 (0--2.7) 12.9 (8.2--19.7) 0.8 (.2--4.1) 3 (1.2--7.5) 12.1 (7.6--18.8) 2.3 (.8--6.5) 2.3 (.8--6.5) 15.2 (10--22.3) Tamil Nadu 51 64.7 (50.9--76.4) 5.9 (2.1--15.9) 3.9 (1.2--13.2) 2 (.5--10.3) 2 (.5--10.3) 2 (.5--10.3) 0 (0--6.8) 11.8 (5.6--23.4) 0 (0--6.8) 3.9 (1.2--13.2) 2 (.5--10.3) 3.9 (1.2--13.2) 23.5 (14--36.8) 7.8 (3.2--18.5) Uttar Pradesh 98 52 (42.2--61.7) 1 (.2--5.5) 8.2 (4.2--15.3) 1 (.2--5.5) 0 (0--3.7) 2 (.6--7.1) 1 (.2--5.5) 11.2 (6.4--19) 3.1 (1.1--8.6) 2 (.6--7.1) 15.3 (9.5--23.8) 3.1 (1.1--8.6) 4.1 (1.7--10) 13.3 (8--21.4) West Bengal 106 71.7 (62.5--79.4) 3.8 (1.5--9.3) 1.9 (.6--6.6) 0 (0--3.4) 5.7 (2.7--11.8) 0 (0--3.4) 0.9 (.2--5.1) 12.3 (7.3--19.9) 6.6 (3.3--13) 2.8 (1--8) 6.6 (3.3--13) 0.9 (.2--5.1) 0.9 (.2--5.1) 7.5 (3.9--14.2) ^a^a See Tables 8--13 (additional files 3--7, 9) for frequencies of other haplogroups including breakdown of R2, U7 and W b Includes all haplogroup M sub-haplogroups c Includes sub-haplogroups R2, R5, R6 and R10 d Composite of haplogroups U2a, U2b and U2c \[27\] e Composite of haplogroups A, B, D, G, M7, M8 (including C and Z), M9 (including E), M10, N9 (including Y), R9 (including F) f Composite of haplogroups HV, TJ, X, N1, N2 and U (except U2i) g Caste and tribal populations plus 8 Siddi \[49\] who belong to neither groupings h Datasets where caste affiliation was not determined (undefined/mixed caste population), Scheduled Caste populations (e.g. the Mukri, see text for explanation) and data from \[3\] (dataset of 27 haplogroup M samples) are excluded. i Indo-European speakers of southern India (Tamil Nadu, Kerala, Karnataka, Andhra Pradesh and Sri Lanka) j Indo-European speakers of northern India (States other than listed in the previous footnote) k The sum of the presented caste populations sample sizes is different from that given in the third row of the current table because of the addition of the Bangladeshi (n = 30) and extraction of the Mukri (n = 42), Orissa (n = 2) and Bihar (n = 2) data. ::: With the exception of the diverse set of largely Indian-specific R lineages, the most frequent mtDNA haplogroup in India that derives from the phylogenetic node N is haplogroup W \[[@B13]\]. The frequency peak of haplogroup W is 5% in the northwestern states -- Gujarat, Punjab and Kashmir. Elsewhere in India its frequency is very low (from 0 to 0.9%) (Table [2](#T2){ref-type="table"}) forming a significant spatial cline (Figure [4](#F4){ref-type="fig"}). At 15% among the caste and 8% among the tribal populations haplogroup U is the most frequent sub-clade of R in India (Table 12, see [Additional file 7](#S7){ref-type="supplementary-material"}). Approximately one half of the U mtDNAs in India belong to the Indian-specific branches of haplogroup U2 (U2i: U2a, U2b and U2c) \[[@B13],[@B27]\] (Table [2](#T2){ref-type="table"}). They are present throughout India without a clear geographical cline (Figure [2](#F2){ref-type="fig"}, panel U2i, SAA p \> 0.05). However, the spread of another subset of U, haplogroup U7 \[[@B13]\], is similar to that of haplogroup W, peaking at 12% and 9% in Gujarat and Punjab, respectively (Table 11, see [Additional file 6](#S6){ref-type="supplementary-material"}). The frequency of U7 is also high in neighboring Pakistan (6%) and particularly in Iran (9%) (Table 9, see [Additional file 4](#S4){ref-type="supplementary-material"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The spatial distribution of Indian-specific mtDNA haplogroups (U2, M4a, M18 and M25) and their sub-haplogroups. In the case of haplogroup M25 some datasets had to be excluded because the discrimination between M\* and the respective subgroup was not possible on the basis of the HVS-I data alone (see footnote of Table 3). For other details see legend to Figure 1. :::  ::: MtDNA haplogroups in Iran ------------------------- Over 90% of the mtDNAs found in Iran belong to haplogroups HV, TJ, U, N1, N2 and X, commonly found in West Eurasia (Table [2](#T2){ref-type="table"}). In contrast to Europe, where H is predominant among the mtDNA haplogroups, in Iran the frequency of haplogroup U (29%) is higher than that of haplogroup H (17%) (Table 9, see [Additional file 4](#S4){ref-type="supplementary-material"}). This difference accounts, at least partly for the presence in Iran of U sub-groups, such as U7 (9.4%), that are virtually absent in Europe. Compared to India, haplogroup M frequency in Iran is marginally low (5.3%) and there are no distinguished Iranian-specific sub-clades of haplogroup M. All Iranian haplogroup M lineages can be seen as derived from other regional variants of the haplogroup: eleven show affiliation to haplogroup M lineages found in India, twelve in East and Central Asia (D, G, and M8) and one in northeast Africa (M1). Indian-specific (R5 and Indian-specific M and U2 variants) and East Asian-specific (A, B and East Asian-specific M subgroups) mtDNAs, both, make up less than 4% of the Iranian mtDNA pool. We used Turkey (88.8 ± 4.0%) as the third parental population for evaluating the relative proportions of admixture from India (2.2 ± 1.7%) and China (9.1 ± 4.1%) into Iran. Therefore we can conclude that historic gene flow from India to Iran has been very limited. The package of the most ancient mtDNA haplogroups in India ---------------------------------------------------------- Approximately one tenth of the Indian haplogroup M mtDNAs fall into its major sub-clade M2, which is defined by the motif 477G-1780-8502-16319 \[[@B15]\]. M2 can be further subdivided into haplogroups M2a (transitions at nps 5252 and 8369) and M2b \[[@B15]\]. Haplogroup M2 and its two major sub-clades reveal coalescence times of 50 to 70 thousand years (Table [3](#T3){ref-type="table"}). Due to the increased frequency towards the southern part of India (Figure [1](#F1){ref-type="fig"}, panel M2, SAA p \< 0.05 Figure [4](#F4){ref-type="fig"}), M2 is significantly (p \< 0.05) more frequent among the Dravidic speakers than among the Indo-European speakers who are spread mostly in the northern regions of India (Table [2](#T2){ref-type="table"}). It is more plausible that geography rather than linguistics is behind this pattern, because the frequency of M2 amongst the Indo-European speaking populations in southern India is significantly higher than that in the north, while there is no significant difference between Dravidic and Indo-European speaking populations from the same geographic region (Table [2](#T2){ref-type="table"}). It is also notable that the frequency of M2 among the Brahmins and the Kshatriyas of Andhra Pradesh (CR 3.3 -- 19.2%) is not significantly (p \> 0.05) different from that among the other castes or the tribal populations of the region (CR: 5--12.9%, 11.2--18.3%, respectively). On the other hand, none of the 159 Brahmins and Kshatriyas from the northern states of India (Punjab, Rajasthan, Uttar Pradesh and West Bengal) belong to M2 while the frequency reaches nearly 3% (CR: 1.6--4.6%) among the other castes and tribal populations of the region. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Indian-specific sub-clades of mtDNA haplogroups M and R. ::: DIAGNOSTIC CODING REGION MARKERS ANCESTRAL HVS-I MOTIF COALESCENCE (years) ρ/nδ^2a^ COALESCENCE ^b^(years) ρ/nδ^2a^ n^c^ Proportion (95% CR) ------- ---------------------------------- ------------------------- --------------------- ---------- ------------------------ ---------- ------ --------------------- M2 477G-1780-8502 16223--16319 70,600 ± 21,000 0.02 70,100 ± 20,700 0.04 166 6.1 (5.3--7.1)  M2a 5252--8369 16223-16319-16270 48,300 ± 20,100 0.03 46,700 ± 22,800 0.06 79 2.9 (2.3--3.6)  M2b 16223-16319-16274 54,800 ± 25,000 0.02 57,600 ± 22,300 0.06 87 3.2 (2.6--3.9) M3a 4580 16126--16223 17,300 ± 7,400 0.10 17,300 ± 7,600 0.11 62 3.0^f^(2.4 -- 3.8) M4a 6620--7859 16223--16311 19,200 ± 9,000 0.23 19,100 ± 9,000 0.25 21 0.8 (0.5--1.2) M6 3537 16223-16231-16362 33,000 ± 13,900 0.04 30,000 ± 13,600 0.08 79 2.9 (2.3--3.6)  M6a 16223-16231-16356-16362 19,100 ± 7,600 0.17 15,700 ± 8,100 ^d^ 0.54 37 1.4 (1.0--1.9)  M6b 5585 16188-16223-16231-16362 6,000 ± 2,100 0.73 12,100 ± 4,500 0,82 37 1.4 (1.0--1.9) M18 16223-16318T 9,400 ± 3,200 0.31 17,100 ± 4,700 0.61 58 2.1 (1.7--2.7) M25 15928 16223--16304 19,400 ± 7,200 0.30 22,300 ± 8,600 0.32 25 1.0^g^(0.7 -- 1.5) R2^e^ 4216 16071 40,400 ± 14,300 0.45 52,500 ± 21,700 0.5 8 0.3 (0.2--0.6) R5 8594 16266--16304 66,100 ± 22,000 0.04 69,800 ± 24,800 0.05 58 2.1 (1.7--2.7) R6 -12282 AluI 16129--16362 30,000 ± 11,000 0.14 30,300 ± 11,600 0.19 35 1.3 (0.9--1.8) Total 2719 ^a^Efficiency of the sample for coalescence calculation (Saillard et al. 2000) ^b^Calculated without the tribal populations ^c^In the Indian subcontinent (a total of 2719 mtDNAs) ^d^Excluding also the scheduled cast of Mukri ^e^R2 is spread also in the Near East, Central Asia and Volga basin ^f^Excluding the datasets where M3\* / M3a discrimination was impossible: 246 subjects from Andhra Pradesh (Bamshad et al. 1998); 382 Pushtoon, Koragas, Naga, Yerava, Pardhi, Paniya Adi, Andh, Apatani and Soligas (Cordaux et al. 2003); 35 Kota and Kurumba (Roychoudhury et al. 2001) ^g^Excluding the datasets where M\* / M25 discrimination was impossible: 246 subjects from Andhra Pradesh (Bamshad et al. 1998); 39 Thoti and 29 subjects from Bangladesh (Cordaux et al. 2003); 12 Muria (Roychoudhury et al. 2001) ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **The spatial distribution of Indian-specific mtDNA haplogroups (M2, M6 and M3) and their sub-haplogroups.**Maps of South and Southwest Asia depicting the spatial frequency distribution of various Indian-specific mtDNA haplogroups. For India, the tribal populations were excluded (see \'Methods\' for explanation). The published (full reference in Table 6, see Additional file 1) and new data were averaged to the resolution of states in India, geographic regions in Iran and provinces in China and Thailand. Numbers adjacent to the data points indicate sample sizes. In the case of haplogroup M3a some datasets had to be excluded because the discrimination between M\* and the respective subgroup was not possible on the basis of the HVS-I data alone (see footnote of Table 3). :::  ::: We found that R5, which is defined by transitions at nps 8594 \[[@B27]\], 16266 and 16304, is the second most frequent sub-clade of R in India after haplogroup U. The coalescence age estimate for R5 was similar to that of M2 (Table [3](#T3){ref-type="table"}), whereas individual boughs within the R5 limb showed expansions from ca. 20,000 ybp to ca. 50,000 ybp (Figure 12, see [Additional file 8](#S8){ref-type="supplementary-material"}). Our data indicate that this diverse and ancient haplogroup is present over most of India (Figure [3](#F3){ref-type="fig"}, panel R5). Though absent among the Austro- Asiatic tribal groups, R5 is spread across the other language boundaries. In contrast to M2, R5 is more frequent among the caste (CR: 2.5--4.5%) than among the tribal populations (CR: 0.7--1.9%) (Table [2](#T2){ref-type="table"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The spatial distribution of Indian-specific mtDNA haplogroups (R5 and R6) and West Eurasian-specific U7, W and R2 in South and southwest Asia. For other details see legend to Figure 1. :::  ::: Together with the Indian-specific sub-clades of haplogroup U2 \[[@B13],[@B27]\], haplogroups M2 and R5 can be discriminated as a package of Indian-specific mtDNA clades harboring extremely deep coalescence times (around 50,000 -- 70,000 ybp). Together they constitute nearly 15% of the Indian mtDNAs. Importantly, these haplogroups are virtually absent elsewhere in Eurasia \[[@B13],[@B15]\], this study\]. Because most of Indian varieties of haplogroup M are still unclassified (M\*), this package is likely to be extended when more mtDNA coding region information will become available for the M\* lineages in India. The geographic distribution of the M2, R5 and U2i package seems to be rather uniform in the context of the Indian-specific maternal lineages (SAA p \> 0.05). When excluding the mtDNAs that are likely to have arrived more recently from West or East Eurasia, the share of the package among the caste populations in northern and southern India is roughly similar (CR: 16.1--24.5% and 19.5--26.0 %, respectively). However, in accordance with expectations from the individual haplogroup distributions, the tribal groups speaking Austro-Asiatic and Tibeto-Burman languages are characterized by considerably lower values (CR: 3.9--14.1% and 3.8--15.1%, respectively). The quest for finding the origin of haplogroup M and a plausible scenario for the peopling of Eurasia ----------------------------------------------------------------------------------------------------- Based on the high frequency and diversity of haplogroup M in India and elsewhere in Asia, some authors have suggested (*versus*\[[@B3]\]) that M may have arisen in Southwest Asia \[[@B16],[@B17],[@B31]\]. Finding M1 or a lineage ancestral to M1 in India, could help to explain the presence of M1 in Africa as a result of a back migration from India. Yet, to date this has not been achieved \[[@B15]\], this study). Therefore, one cannot rule out the still most parsimonious scenario that haplogroup M arose in East Africa \[[@B3]\]. Furthermore, the lack of L3 lineages other than M and N (indeed, L3M and L3N) in India is more consistent with the African launch of haplogroup M. On the other hand, one also observes that: i) M1 is the only variant of haplogroup M found in Africa; ii) M1 has a fairly restricted phylogeography in Africa, barely penetrating into sub-Saharan populations, being found predominantly in association with the Afro-Asiatic linguistic phylum -- a finding that appears to be inconsistent with the distribution of sub-clades of haplogroups L3 and L2 that have similar time depths. That, plus the presence of M1 without accompanying L lineages in the Caucasus \[[@B32]\] and \[our unpublished data\], leaves the question about the origin of haplogroup M still open. In contrast to haplogroup M, ancient sub-clades of haplogroup N are spread both east and west of India as well as within India itself. Several migration scenarios involving multiple \"out of Africa\" events punctuated by space, time or both, could be invoked to explain the phylogeography of these mtDNA haplogroups. Yet, using the parsimony criterion it can be argued that only a single early migration that brought ancestral lineages, M and N (with the latter having already given rise to R), to South Asia could account for the extant mtDNA phylogeography in Eurasia \[[@B15]\]. The finding of several largely South and West Asian-specific sub-clades (H, L, R2, and F\*) of the major Eurasian Y chromosomal haplogroups F and K also supports this scenario \[[@B15]\]. From South and West Asia the colonization would have sprung both east and west as region-specific mtDNA and Y chromosomal sub-clades appeared both in West- and East-Eurasia as well as in India itself (Figure [5](#F5){ref-type="fig"}). However, not all the West-Eurasian Y-chromosomal founder haplogroups are present in India. Haplogroup E, for example, was possibly carried to Europe and Western Asia later via the Levantine corridor \[[@B33]\]. Similarly, a Late Upper Palaeolithic origin and spread of mtDNA haplogroup X from Northeast Africa and Middle East has been suggested lately \[[@B34]\]. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Peopling of Eurasia.** Map of Eurasia and northeastern Africa depicting the peopling of Eurasia as inferred from the extant mtDNA phylogeny. The bold black arrow indicates the possible \"coastal\" route of colonization of Eurasia by anatomically modern humans (ca. 60,000 -- 80,000 ybp.). This \"Southern Coastal Route\" is suggested by the phylogeography of mtDNA haplogroup M, the virtual absence of which in the Near East and Southwest Asia undermines the likelihood of the initial colonization of Eurasia taking a route north around the Red Sea. Therefore, the initial split between West and East Eurasian mtDNAs is postulated between the Indus Valley and Southwest Asia. Spheres depict expansion zones where, after the initial (coastal) peopling of the continent, local branches of the mtDNA tree (haplogroups given in the spheres) arose (ca. 40,000 -- 60,000 ybp), and from where they where further carried into the interior of the continent (thinner black arrows). Admixture between the expansion zones has been surprisingly limited ever since. We note that while there is no obvious need to introduce the \"northern route\" -- from northeast Africa over Sinai to the Near East -- to explain the initial colonization of Eurasia, the spread of some mtDNA and Y-chromosomal haplogroups implies that the \"northern\" passage might have been used in a later period \[33, 34\]. :::  ::: The improved structure of autochthonous Indian mtDNA clades ----------------------------------------------------------- Nearly a third of Indian mtDNAs belonging to haplogroup M could be assigned to its existing boughs and limbs with the current knowledge of the mtDNA coding region polymorphisms (Table 9, see [Additional file 4](#S4){ref-type="supplementary-material"}). It is likely that the unclassified Indian M\* and R\* mtDNAs are also to a large extent autochthonous because neither the East nor West Eurasian mtDNA pools include such lineages at notable frequencies. Haplogroup M6 (Figure [6](#F6){ref-type="fig"}) is primarily found in the Indus Valley and on the western shores of the Bay of Bengal where its sub-clades M6a and M6b are concentrated towards the southwest and the northeast, respectively (Figure [1](#F1){ref-type="fig"}, panel M6, M6b cline is significant SAA p \< 0.05, Figure [4](#F4){ref-type="fig"}). The highest frequencies of M6a and M6b were found amongst the Mukri scheduled caste from Karnataka (17%) and in Kashmir (10%), respectively. The Mukri form an endogamous group of no more than 10,000 individuals, who dwell on an area less than 2000 km^2^\[[@B35]\]. That, together with the observation that all the sixteen M6 sequences found among the Mukri belong to a single haplotype, suggests that genetic drift has played a major role in the demographic history of the Mukri. The statistical significance of the high M6 frequency in Kashmir is undermined by the small sample size (19 individuals), which results in the very wide error margins for the frequency estimate (CR: 3.2--31.7%). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Network of HVS-I haplotypes belonging to haplogroup M6.** Circle areas are proportional to haplotype frequencies. Variant bases of the HVS-I are numbered as in (Anderson et al. 1981) minus 16,000 and shown along links between haplotypes. Character changes are specified only for transversions. Underlined samples are those in which the diagnostic coding region markers (3537 and 5585) were assayed by either RFLP analysis (M6: -3537 AluI; M6b: -5584 AluI) or direct sequencing. Sample codes are as in Table 6 (see Additional file 1). Coalescence estimates marked with an asterisk are calculated excluding tribal populations (see Materials and Methods for explanation). The coalescence estimate marked with two asterisks is calculated without the data on tribal and scheduled caste (the Mukri) populations (see text for details). :::  ::: Different geographic distributions characterize the sub-clades of M3 and M4 that we define by mtDNA coding region markers (Table [3](#T3){ref-type="table"}, Figures [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"}). Both M3a and M4a show time depths around 20,000 ybp. However, while M4a is sparsely spread in most of India with no obvious geographical cline (Figure [2](#F2){ref-type="fig"}, panel M4a, SAA p \> 0.05), the spread of M3a is concentrated into northwestern India (Figure [1](#F1){ref-type="fig"}, panel M3a, SAA p \< 0.05), suggesting that the region may have been the ancestral source. The frequency of M3a is at its highest amongst the Parsees of Mumbai (22%). Given the low M3a diversity amongst the Parsees -- the twelve M3a mtDNAs fall into the two most common haplotypes (Figure [7](#F7){ref-type="fig"}) -- the high frequency is likely a result of admixture and subsequent founder events. On the other hand, it is intriguing that, despite its low frequency, M3a penetrates into central and southwestern Iran (Figure [1](#F1){ref-type="fig"}, panel M3a) -- the historic origin of the Zoroastrian Parsees. In addition to the Parsees we found M3a at high frequencies amongst the Brahmins of Uttar Pradesh (16%) and the Rajputs of Rajasthan (14%) (Table 10, see [Additional file 5](#S5){ref-type="supplementary-material"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **A network of haplogroup M3a haplotypes.** Underlined samples are those in which the diagnostic coding region marker (4580) of M3a was assayed by either RFLP analysis (-4577 NlaIII) or direct sequencing. For other details, see the legend to Figure 6. :::  ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Network of HVS-I haplotypes belonging to haplogroup M4a.** Underlined samples are those in which the diagnostic coding region marker (6620) of M4a was assayed by either RFLP analysis (+6618 MboI) or direct sequencing. For other details, see the legend to Figure 6. :::  ::: Awaiting further information from complete mtDNA sequences, we defined haplogroup M18 by using the transversion at np 16318. This star-like cluster (Figure [9](#F9){ref-type="fig"}) is spread at low frequencies across India, with the exceptions of the very north and the coast of the Arabian Sea (Figure [2](#F2){ref-type="fig"}, panel M18). The high incidence (33%) of the M18 nodal haplotype among the Austro-Asiatic speaking Lodha of West Bengal (Table 10, see [Additional file 5](#S5){ref-type="supplementary-material"}) suggests a possible founder effect in this population. This explains the nearly two-fold difference between the coalescence estimates for this cluster calculated with and without the tribal data (Table [3](#T3){ref-type="table"}). ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Network of HVS-I haplotypes belonging to haplogroup M18.** For other details, see the legend to Figure 6 :::  ::: The G to A transition at np 15928 has been spotted on different branches (e. g. haplogroups T and M) of the mtDNA phylogeny \[[@B3],[@B36]\]. Quintana-Murci and colleagues observed this transition within haplogroup M in combination with the HVS-I motif 16048-16129-16223-16390 \[[@B27]\]. None of the mtDNAs in our study which harbor -- or stem from -- the 16048-16129-16223 motif were positive for the 15928 transition, suggesting an additional occurrence. In addition, we recorded this transition associated with three other HVS-I motifs on the background of haplogroup M (M8-Z: 16185-16223-16260-16298; M\*: 16223 and M\*: 16086-16223-16335). These occurrences cannot be monophyletic for obvious reasons. Yet, when combined with the transition at np 16304, G15928A roots a star-like subclade of haplogroup M that we tentatively named M25 (Figure [10](#F10){ref-type="fig"}). In this case, monophylecity is the most parsimonious assumption. This haplogroup is moderately frequent in Kerala and Maharashtra but rather infrequent elsewhere in India (Figure [2](#F2){ref-type="fig"}, panel M25). ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **Network of HVS-I haplotypes belonging to haplogroup M25.**Underlined samples are those in which the diagnostic coding region marker (15928) of M25 was assayed by either RFLP analysis (-15925 MspI) or direct sequencing. For other details, see the legend to Figure 6. :::  ::: Coalescence estimates for these Indian-specific mtDNA haplogroups (M3a, M4a, M6, M25 and R6) fall largely between 20,000 and 30,000 ybp. These estimates overlap with those of many West Eurasian-specific (e.g. H, HV, preHV, U3, U4, K, X \[[@B9],[@B34]\]) and East Eurasian-specific (A, F2, D4, M7c1, M7a1, M8a \[[@B7],[@B22]\]) mtDNA clades, suggesting a rather synchronic worldwide demographic expansion event in the late Pleistocene, during an interglacial period preceding the LGM. Several Indian-specific mtDNA clades demonstrate a similar spread-pattern in southern India. We found haplogroups M4a, M6a and M18 in southeastern Tamil Nadu and Andhra Pradesh while they were absent from neighboring Karnataka and Kerala (Figure [1](#F1){ref-type="fig"} panel M6a and Figure [2](#F2){ref-type="fig"} panels M4a and M18). One possible explanation is that admixture has been facilitated along the coastlines of the Arabian Sea and the Bay of Bengal. On the other hand, because the absolute frequencies of these haplogroups are rather low, it cannot be ruled out that an increase of sample sizes would disrupt the observed spread-pattern. Were the Austro-Asiatic speaking tribal people the earliest inhabitants of India? --------------------------------------------------------------------------------- By calculating nucleotide diversities and expansion times (using the method from \[[@B37]\]) for different linguistic groups of India, some previous studies on mtDNA variation have distinguished the Austro-Asiatic speaking tribal groups as the carriers of the genetic legacy of the earliest settlers of the subcontinent \[[@B17],[@B38]\]. However, because the linguistic groups of India do not cluster into distinct branches of the Indian mtDNA tree \[[@B13],[@B15],[@B19]\], this study\], calculating the beginning of expansion for those groupings is problematic and likely controversial as well. Recently, Basu et al. (2003) supported the conclusions of \[[@B17],[@B38]\] by reporting that the frequency of the ancient haplogroup M2 among the Austro-Asiatic tribal populations is as high as 19%, and that they lack the slightly younger haplogroup M4 (the likely paraphyletic mother-clade of M4a). The authors have regarded the HVS-I transition at np 16319 as sufficient in defining haplogroup M2. This assumption, however, might lead to an overestimation of M2 frequency and age. Indeed, the 16319 transition has arisen several times on the background of other Indian haplogroup M lineages (Table 7, see [Additional file 2](#S2){ref-type="supplementary-material"}), more specifically, in mtDNAs lacking the coding region markers that define M2 \[[@B15]\]. Although two out of the four M2 sequences reported by Basu et al. (2003) among the Lodha, Mundas and Santals (HVS-I sequences originally published by \[[@B17]\]) do harbor the characteristic M2a HVS-I motif (16223-16270-16274-16319-16352), without information from the coding region it is not clear whether the other two sequences (HVS-I motifs: 16092-16179-16223-16289-16294-16319 and 16147G-16172-16223-16319) represent novel M2 sub-clades (because these sequences cannot be affiliated with M2a or M2b) or derive from two independent branches of haplogroup M where 16319 transition has arisen recurrently. HVS-I motif 16147G-16172-16223, for example, is commonly associated with haplogroup N1a. Since sequence data on the five M2s among the Austro-Asiatic speaking tribe Ho, reported by Basu et al. (2003), have not been made available in the publication, we cannot rely on their haplogroup classification. Thus, we are left with one Munda and one Santal mtDNA belonging to haplogroup M2. They make up just 5% of the Austro-Asiatic tribal sample of 37 subjects (excluding the ten Ho). Interestingly, we found no instances of haplogroup M2 among the 56 Lodhas analyzed in this study. Consequently, when excluding the recurrences of the 16319 transition on the background of other sequence motifs, the frequency of M2 among the Austro-Asiatic speaking tribal groups from West Bengal in the combined dataset (Table 7, see [Additional file 2](#S2){ref-type="supplementary-material"}) is significantly reduced to about 2%. The corrected value is comparable to the M2 frequency (\>3%) in tribal populations speaking Indo-European languages of Punjab and Uttar Pradesh, but is significantly lower than its frequency (\>14%) among the Dravidic-speaking tribal groups of Andhra Pradesh (Table 8, see [Additional file 3](#S3){ref-type="supplementary-material"}). Language families present today in India, such as Indo-European, Dravidic and Austro-Asiatic, are all much younger than the majority of indigenous mtDNA lineages found among their present-day speakers at high frequencies (see [Additional file 9](#S9){ref-type="supplementary-material"}). It would make it highly speculative to infer, from the extant mtDNA pools of their speakers, whether one of the listed above linguistically defined group in India should be considered more \"autochthonous\" than any other in respect of its presence in the subcontinent. Additionally, we note that some recent linguistic and archaeological evidence place the spread of the Austro-Asiatic languages in the Neolithic, in conjunction with the dispersal of rice cultivation from the Yangtze River basin \[[@B39]\]. If this were the case, it would imply that the arrival of this linguistic phylum in India was not associated with female gene flow. Gene flow from West Eurasia --------------------------- Broadly, the average proportion of mtDNAs from West Eurasia among Indian caste populations is 17% (Table [2](#T2){ref-type="table"}). In the western States of India and in Pakistan their share is greater, reaching over 30% in Kashmir and Gujarat, nearly 40% in Indian Punjab, and peaking, expectedly, at approximately 50% in Pakistan (Table 11, see [Additional file 6](#S6){ref-type="supplementary-material"}, Figure [11](#F11){ref-type="fig"}, panel A). These frequencies demonstrate a general decline (SAA p \< 0.05 Figure [4](#F4){ref-type="fig"}) towards the south (23%, 11% and 15% in Maharashtra, Kerala and Sri Lanka, respectively) and even more so towards the east of India (13% in Uttar Pradesh and around 7% in West Bengal and Bangladesh). The low (\<3%) frequency of the western Eurasian mtDNAs in Rajasthan may be in part a statistical artifact due to the limited sample size of 35 Rajputs. ::: {#F11 .fig} Figure 11 ::: {.caption} ###### **The segregation of West Eurasian, East Eurasian and South Asian mtDNA pools.** Partial map of Eurasia illustrating the spatial frequency distribution of mtDNA haplogroups native to West Eurasia (panel A), South Asia (panel B) and East Eurasia (panel C). Data points represent states in India, geographic divisions in Iran and provinces in China and Thailand. Numbers adjacent to the data points indicate the applicable sample size. The distribution of West Eurasian-, South Asian- and East Eurasian-specific mtDNA haplogroups amongst the tribal populations of South and Southeast Asia are depicted as pie diagrams on panel D (colors as on panels A, B, and C, while the white color represents unclassified M\* and R\* mtDNAs). The data on tribal populations was not used for the isofrequency maps of panels A, B and C (see Materials and Methods for explanation). :::  ::: In comparison to an overall frequency of 17% (CR 15.1--19.3%) among the caste populations, only 7% (CR 6.2 -- 9.0%) of the mtDNAs from the tribal groups show affiliation to the West Eurasian haplogroups. The observed difference could be caused by differences in the extent of gene flow from the west to different social layers of the Indian society \[[@B19]\], and/or a more pronounced genetic drift among the tribal groups. This western Eurasian contribution into the Indian maternal gene pool can be broadly divided into two different components. Over two thirds of the West Eurasian-specific mtDNAs found in India are made up by haplogroups HV, TJ, N1 and West Eurasian-specific branches of haplogroup U. It is likely that these mtDNA haplogroups have been carried to western India both by relatively low-intensity long-lasting admixture at the border regions as well as a consequence of numerous but probably limited migrations during the last 10, 000 ybp \[[@B13]\]. The remaining one third of the West Eurasian-specific mtDNAs found in India is comprised of haplogroups U7, R2 and W showing much deeper time depths in India -- approximately forty thousand years before present (Table [4](#T4){ref-type="table"}). A large-scale immigration -- carrying these haplogroups -- could have introduced a substantial fraction of the diversity already present within the putative source areas. This would explain the deep coalescence times of these haplogroups in India, while their actual arrival could have occurred later. Alternatively, the coalescence estimates may indeed reflect a deeper autochthonous history of these mtDNA clades in India. It is worthwhile to stress that while in India the share of U7, R2 and W in the West Eurasian-specific mtDNAs mounts to nearly a third, in Iran it stays below 15%. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Coalescence estimates and diversity values for mtDNA haplogroups U7, W and R2 in India, Central Asia, Near and Middle East. ::: INDIA NEAR AND MIDDLE EAST CENTRAL ASIA ---- ----------------- ---------------------- ----------------- ------- ----------------- ------- U7 41,400 ± 15,800 0.941 41,200 ± 14,800 0.91 34,400 ± 15,500 0.941 W 37,900 ± 11,100 0.883 32,000 ± 8,700 0.934 27,400 ± 8,300 0.758 R2 40,400 ± 14,300 0.923 36,100 ± 11,100 0.955 25,200 ± 13,300 0.889 ::: Similarly to HV, TJ, N1, the spread of U7, W and R2 in India is geographically uneven -- the three haplogroups are much more frequent in the northwestern states (SAA p \< 0.05, Figure [4](#F4){ref-type="fig"}). Together, they constitute nearly 14% of the maternal gene pool in Indian Punjab, Gujarat and Rajasthan. Their frequency is also high in Iran (13%) and Pakistan (10%) but declines in Central Asia (5%). Although haplogroup W is not highly frequent in European populations, it is nevertheless quite common \[[@B9]\], reaching the highest frequencies among the central-northern Finns (9% \[[@B40]\]). Yet, it is virtually absent from the Finno-Ugric speaking populations of the Volga basin \[[@B41]\]. In Central Asia the frequency of haplogroup W stays below 2%. Many European populations are lacking haplogroup U7 \[[@B9]\], but its frequency climbs over 4% in the Near East and up to 5% in Pakistan, reaching nearly 10% level in Iranians (Table 9, see [Additional file 4](#S4){ref-type="supplementary-material"}). In India, haplogroup U7 frequency peaks at over 12% in Gujarat, in the westernmost state of India, while for the whole of India its frequency stays around 2%. Expansion times and haplotype diversities for the Indian and Near and Middle Eastern U7 mtDNAs are strikingly similar (Table [4](#T4){ref-type="table"}). The possible homeland of this haplogroup spans likely in Gujarat and Iran because from there its frequency declines steeply both to the east and to the west. If the origin were in Iran rather than in India, then its equally high frequency as well as diversity in Gujarat favors a scenario whereby U7 has been introduced to the coastal western India either very early, or by multiple founders. Notably, the overlap of the Indian and Iranian lineages is largely restricted to the ancestral nodes while the coalescence age estimates for the nearly exclusively Indian (16207-16309-16318T) and West Eurasian (16126-16309-16318T) founder HVS-I motifs both yield time depths of about 20,000 to 30,000 ybp. Haplogroup R2 appears at low frequencies in Near and Middle East and India and is virtually absent elsewhere. The spread of haplogroup R2 in Europe is restricted to a few populations in the Volga basin where it is represented by nodal haplotypes and by a region-specific subclade characterized by the HVS-I motif 16037--16172 \[[@B41]\]. The coalescence estimate of this sub-clade is 11,400 ± 9,000 ybp. However, its wide error range prevents us from drawing any firm conclusions. To summarize, the West and South Asian phylogeography of haplogroups W, U7 and R2 can be viewed as a genetic continuum that spans from the Near East into India, extending north into Central Asia. The coalescence times of these haplogroups suggest that this continuum took shape somewhere between 30,000 to 50,000 ybp (Table [4](#T4){ref-type="table"}), thus falling within the climatically favorable interglacial period. We notice that the extant U7 and W frequencies along the proposed continuum are not uniform. U7 is more predominant in Iran, Pakistan, northwestern India and the Arabian peninsula, while W is more frequent in the western Near-East, Anatolia and the Caucasus. The coalescence ages of the Indian- and Iranian-specific U7 clades suggest that the time-window of this continuum was closed by ca. 20,000 ybp. The inferred extreme aridity of eastern Iran and western India during the last glacial maximum, which is well documented in paleovegetation reconstructions \[[@B42]\] may explain the observed segregation. It has been suggested that the Jews settled in southwest India on the coast of the Arabian Sea sometime during the early Middle Ages. However, the mtDNA pool of the extant Cochin Jews is overwhelmingly Indian-specific (Table 10, see [Additional file 5](#S5){ref-type="supplementary-material"}). We found exact or close matches to the fourteen HVS-I haplotypes observed among the Cochin Jews in other Indian populations. It is not clear whether the Near Eastern mtDNA lineages have been lost or the initial Jewish settlers did not include women. Gene flow from East Eurasia --------------------------- The East Eurasian-specific mtDNA haplogroups are less common in India and more sharply geographically segregated than the haplogroups of western Eurasian ancestry (Table [2](#T2){ref-type="table"}; Figure [11](#F11){ref-type="fig"}, panel C). Indian caste populations harbor only about 4% of such mtDNAs, compared to 17% of the West Eurasian ones (Table [2](#T2){ref-type="table"}). Elevated frequencies of haplogroups common in eastern Eurasia are observed in Bangladesh (17%) and Indian Kashmir (21%) and may be explained by admixture with the adjacent populations of Tibet and Myanmar (and possibly further east: from China and perhaps Thailand). On the other hand, the high frequencies of East Eurasian-specific mtDNAs found in the southern Indian state of Tamil Nadu (21%) are unexpected when considering their relatively low frequencies (\~1%) in West Bengal and Andhra Pradesh. We notice, however, that the haplogroup assignments used here for the Tamil Nadu sample (A4, B4, F1a and M7) (Table 7, see [Additional file 2](#S2){ref-type="supplementary-material"}) are based on HVS-I sequences alone \[[@B29]\]. As shown and discussed elsewhere \[[@B7]\], such type of assignment is prone to mistakes. Two varieties of haplogroup M, D4c and G2a, were recently identified as largely specific to Central Asia \[[@B43]\]. In spite of geographical proximity we did not find these haplogroups in northern or northwestern India. Haplogroup G2a did, though in marginally low frequency, turn up in Iran (CR: 0.1 -- 1.6%) and in southern Indian states Andhra Pradesh (CR: 0.3 -- 2.9%) and Sri Lanka (CR: 0.2 -- 4.1%). Tibeto-Burman speaking tribal populations of eastern and northern India exhibit the highest frequencies of East Eurasian-specific mtDNA haplogroups. As inferred from the published HVS-I sequences \[[@B29]\], their share sums up to approximately two thirds of mtDNAs among the tribal groups in Assam, Nagaland, Arunachal Pradesh and Tripura, (Table 8, see [Additional file 3](#S3){ref-type="supplementary-material"}; Figure [11](#F11){ref-type="fig"}, panel D). MtDNA haplogroups native to East Eurasia are also highly frequent in the northern states of India, reaching a peak of nearly 50% among the Kanet of Himachal Pradesh. Papiha and colleagues have previously demonstrated through the typing of immunoglobin allotypes that the Tibetan admixture among the regional Kanet groups decreases as the distance from the Tibetan border increases \[[@B44]\]. Thus, mtDNA data are consistent with an ancestral origin of the Tibeto-Burman speaking tribal populations outside (east of) India in the neighboring Tibet and Myanmar \[[@B30],[@B45]\]. Haplotype sharing between populations ------------------------------------- The majority (70%) of the 1136 mtDNA haplotypes found among continental Indians (including Pakistan and Bangladesh) are singletons and 41% of those that occur more than once are restricted to a single population. Only a few haplotypes are shared among five or more populations. The number of shared haplotypes between pairs of social, linguistic and geographic groups of Indian populations is slightly (but in most cases insignificantly) lower than that between random groups of Indian populations taken for reference (see Materials and Methods). Where the decline of shared haplotypes is significant relative to the reference, it is most probably caused by large differences in the sample sizes of the groups under comparison (Table [5](#T5){ref-type="table"}). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Proportion of mtDNA haplotype sharing between population groups of South Asia ::: ----------------- ------------ ----------- -------------- ----------------- ------------ ----------- -------------- ------------------ Group 1 Group 2 pop. group n n^b^ 1/2^d^ pop. group n n^b^ 2/1^e^ n^c^ Random 1^g^ 1256 ± 215 617 ± 109 .399 -- .453 Random 2^g^ 1426 ± 215 643 ± 114 .396 -- .447 132 (112 -- 154) Tribals 1197 437 .411 -- .467 Castes 1485 806 .324 -- .372 107 (90 -- 128) Northern states 1204 647 .277 -- .329 Southern states 1478 585 .345 -- .394 96 (80 -- 116) Western states 1142 508 .453 -- .511 Eastern states 1540 735 .331 -- .379 107 (90 -- 128) Dravidians 974 380 .428 -- 490 Others 1708 846 .273 -- .317 90 (74 -- 109) Indo-Europeans 1322 686 .328 -- .379 Others 1360 556 .393 -- .446 106 (89 -- 127) AA tribals 90 35 .552 -- .746 Others 2592 1110 .014 -- .025 9 (5 -- 17) TB tribals 249 142 .174 -- .277 Others 2433 1018 .091 -- .115 24 (16 -- 35) Indo-Europeans 1322 686 .295 -- .346 Dravidians 974 380 .425 -- .487 87 (71 -- 106) Total 2682 1136 340 ^f^ ----------------- ------------ ----------- -------------- ----------------- ------------ ----------- -------------- ------------------ ^a^continental Indians including Pakistan and Bangladesh (excluding Andaman & Nicobar) ^b^number of haplotypes ^c^number of shared haplotypes between group 1 and group 2 (95% credible regions; CR) ^d^CR of the proportion of those haplotypes in group 1 that occur also in group 2 ^e^CR of the proportion of those haplotypes in group 2 that occur also in group 1 ^f^41% of the shared haplotypes are shared only between members of the same population ^g^± indicate SD ::: An alternative method that assesses the degree of haplotype sharing between populations is to investigate the combined frequency of the shared haplotypes in two population groups. Thus, amongst the northern and the southern population groups the combined frequency of the haplotypes present also in the other group is significantly lower than that which we observed in the case of random groups. This is not surprising because West Eurasian-specific mtDNA haplogroups are rather frequent in northwest India. Because the Indo-European and the Dravidic speakers of India are largely concentrated to the northern and southern parts of the subcontinent, respectively, the differences arising from geographic division of the Indian populations also correspond to these linguistic groupings (Table [5](#T5){ref-type="table"}). Conclusions =========== Three Indian-specific haplogroups, M2, U2i and R5, which encompass about 15% of the Indian mtDNA pool, exhibit equally deep coalescence ages of about 50,000 -- 70,000 years. Thus, their spread can be associated with the initial peopling of South Asia. Haplogroups U7, W and R2 harbor a number of similar traits. Their overlapping geographic distributions and coalescence times suggest some degree of genetic continuum in the area spanning from the Near and Middle East through northwest India and reaching north into Central Asia. The coalescence estimates for these haplogroups are equally deep (around 30,000 -- 50,000 years) in these different regions. That may be a result of either relatively more recent albeit large in scale migrations that brought along most of the diversity or may indeed reflect the region-specific expansions of these haplogroups. The former explanation could be ruled out since it is impossible to envisage a substantial movement of mtDNAs from South Asia that would not include haplogroup M. The same is true for the opposite -- the share of U7, W and R2 within the West Eurasian-specific mtDNA haplogroups is two-fold higher in India than it is in Iran. Moreover, the South- and West Asian-specific sub-branches of haplogroup U7 predate the last glacial maximum. Therefore, deep autochthonous history of these haplogroups in the region remains to be the most parsimonious explanation. Through the use of mtDNA coding region markers, we were able to classify altogether a quarter of the Indian M and R mtDNAs into a number of Indian-specific mtDNA haplogroups, four of which we newly identified. Several of these are characterized by clear patterns in their geographic distribution and/or different frequencies among different socio-cultural groups of India. Additional efforts should be undertaken to identify new coding region markers in order to further improve the classification of Indian mtDNAs. Indeed, much of the information is still hidden by the poor resolution of the Indian mtDNA tree and further piling of HVS-I datasets would add little to deepen our understanding of the demographic history of South and Southwest Asia. We found that haplogroup M frequency drops abruptly from about 60% in India to about 5% in Iran, marking the western border of the haplogroup M distribution. A similarly sharp border cuts the distribution of Indian-specific mtDNA haplogroups to the east and to the north of the subcontinent. We therefore propose that the initial mtDNA pool established upon the peopling of South Asia has not been replaced but has rather been reshaped *in situ*by major demographic episodes in the past and garnished by relatively minor events of gene flow both from the West and the East during more recent chapters of the demographic history in the region. Methods ======= Subjects -------- MtDNA sequence variation in a total of 796 Indian samples most of which are held in a collection at Newcastle University \[[@B46]\] was analyzed. The samples cover a wide geographical range that spans from Himachal Pradesh in the north, Sri Lanka in the south, West Bengal in the east and Gujarat in the west (Table [1](#T1){ref-type="table"}). Tribal populations constitute 15% of the total sample size. The Lodha (n = 56) live mostly in the western part of Midnapore district of West Bengal where they are also known as Kheria or Kharia. Their total population size was \~59000 according to the 1981 census. Their language belongs to the Mundari branch of the Austro-Asiatic language family \[[@B47]\]. The Kanet (n = 37) make up two thirds of the \~50000 inhabitants of the Kinnaur district in Himachal Pradesh \[[@B48]\]. Their language belongs to the Himalayan group of the Tibeto-Burman language family \[[@B47]\]. Five Bhoksa and twenty-six Tharu individuals were included in the present study in addition to those from the same populations that we have previously reported \[[@B13]\]. Most of the Tharu live in the Terai areas (a belt of marshy land at the foot of the Himalayas) of Nepal (n = 720000). Approximately 96000 reside in Uttar Pradesh and Uttaranchal, Indian states adjacent to Nepal. Around 32000 Bhoksas live in the lowland areas of Uttaranchal and the neighboring Bijnor district of Uttar Pradesh. Both these tribal groups speak languages belonging to the Indo-European phylum \[[@B47]\]. The non-tribal Indian samples analyzed contained 105 West Bengalis of different caste rank, 58 Konkanastha Brahmins from Bombay, 53 Gujaratis, 50 Moors and 82 Sinhalese from Sri Lanka, 109 Punjabis of different caste rank from the Punjab, 25 Brahmins from Uttar Pradesh, 35 Rajputs from Rajasthan, 55 Parsees from Maharashtra and 100 subjects from Cochin, Kerala (including 45 Jews who have moved to Israel) (Table [1](#T1){ref-type="table"}). The Iranian sample of 436 individuals was collected in different locations mainly from southwestern and northwestern Iran (Table [1](#T1){ref-type="table"}). The new Indian mtDNA sequence data was combined with that previously published on Indian populations \[[@B3],[@B13],[@B15],[@B17],[@B18],[@B29],[@B35],[@B49]-[@B51]\] to produce a pooled dataset (n = 2572) in which the tribal populations constitute slightly over 50% (Table 6, see [Additional file 1](#S1){ref-type="supplementary-material"}). By including also the data on Iranian and published data on Pakistani \[[@B13],[@B27],[@B29]\], Bangladeshi \[[@B29]\], Chinese \[[@B7],[@B22],[@B52]-[@B54]\] and Thai \[[@B53],[@B55],[@B56]\] populations (n = 145, 29, 919 and 552, respectively), a dataset of over 4600 samples spanning from West to East Asia (Table 6 and 7, see [Additional files 1](#S1){ref-type="supplementary-material"} and [2](#S2){ref-type="supplementary-material"}) was obtained. In many cases the published data was reanalyzed. Analogy with the newly obtained HVS-I motifs, that were classified into haplogroups using diagnostic markers of the mtDNA coding region, served as basis for haplogroup assignments of the published HVS-I sequences. An even wider range of data was used for the production of the isofrequency maps: the published data from Armenia (n = 192) \[[@B32]\] and Kyrgyz (n = 95) \[[@B57]\], and the unpublished data from Yemen (n = 118), Oman (n = 79), Saudi Arabia (n = 205), Ethiopia (n = 270), Uzbekistan (n = 114) and Tajikistan (n = 41). Further details about the mtDNA variation of these populations will be published elsewhere. MtDNA molecular analyses ------------------------ DNA was extracted using standard phenol-chloroform methods \[[@B58]\]. The hypervariable segment (HVS)-I (between nucleotide positions (np) 16024--16400) of the control region was sequenced in all the 796 Indian and 436 Iranian samples. Preparation of sequencing templates was carried out following Kaessmann et al. \[[@B59]\]. Purified products were sequenced with the DYEnamic™ ET terminator cycle sequencing kit (Amersham Pharmacia Biotech) and analyzed on ABI 377 DNA or Megabace2000 Sequencers. Sequences were aligned and analyzed with the Wisconsin Package (GCG). In addition, informative mtDNA coding region positions \[[@B3],[@B11],[@B27]\] were assayed (Table 7, see [Additional file 2](#S2){ref-type="supplementary-material"}) in selected individuals from different HVS-I haplotypes to determine haplogroup affiliations. Data analysis ------------- Median networks \[[@B60],[@B61]\] were constructed using Network 3.111 and Network 2.10B programs \[[@B62]\] with default settings (r = 2; ε = 0). MtDNA coding region markers were given five times the weight of the HVS-I positions. Coalescence of mtDNA haplogroups and sub-haplogroups was calculated using ρ (the averaged distance to a specified founder haplotype) and a mutation rate of one transition per 20,180 years between nps 16090--16365 \[[@B63]\]. Standard errors for coalescence estimates and efficiency of a sample for coalescence time calculation (ρ/nδ^2^) were calculated following \[[@B64]\]. Coalescence times were further calculated with the exclusion of tribal populations sequences, yet preserving the cluster topology (coalescence times marked with an asterisk). A more intense genetic drift (particularly founder effects) could introduce a bias into the coalescence time calculation (for example, see below the coalescence time of haplogroup M18 with and without the Lodha sample). The software kindly provided by Vincent Macaulay was adopted in order to calculate the 95% credible regions (CR) from the posterior distribution of the proportion of a haplogroup/sub-haplogroup in the population. Haplotype diversity was estimated as  where n is the number of sequences, k the number of distinct haplotypes, and n~i~number of sequences with one distinct haplotype. Haplogroup isofrequency maps were generated using the Kriging method in Surfer 7 program of Golden Software. Haplogroup frequencies were averaged over populations from the same state in India, provinces in China and Thailand and geographic divisions in Iran. The data points for Kriging are shown as black dots, while the sample size applicable to the data point is given adjacent to the dot. In relatively small and isolated groups (e.g. tribal groups) random genetic drift might seriously affect the haplogroup frequencies, which may become uninformative when a whole region (e.g. state) is considered (e.g. M18 among the Lodha, see below). Therefore, the tribal data were excluded from the haplogroup isofrequency maps calculation. When illustrating the spread of mtDNA haplogroups native to West Eurasia, East Eurasia and India (Figure [11](#F11){ref-type="fig"}, panel D) we present these data as pie diagrams. The respective sample size and origin are indicated adjacent to the diagrams. Spatial autocorrelation analysis was done using the PASSAGE software packet \[[@B65],[@B66]\]. The correlograms of Moran\'s I autocorrelation coefficient were calculated using binary weight matrix with five distance classes. Data on tribal populations was not used (see previous paragraph for explanation). Haplogroup frequencies based admixture proportions were calculated using the ADMIX 2.0 software \[[@B67]\]. Haplotypes were defined as the HVS-I motif combined with the haplogroup label for haplotype analysis. In this way it was possible to discriminate between mtDNAs with identical HVS-I motifs but otherwise known to belong to different haplogroups (e.g. sequences with the CRS motif in HVS-I belonging to haplogroups H or R). In order to assess the results of haplotype sharing analysis between population groups (e.g. tribal and caste populations) we divided the Indian populations ten times randomly into two sets and analyzed the level of haplotype sharing between these sets. Authors\' contributions ======================= SM and SSP collected most of the Indian samples (in 1970\'s) and have been keeping the collection in Newcastle University, United Kingdom. MM, TK, GH and KK carried out the RFLP analysis and mtDNA sequencing of the majority of the Indian samples. EM, JP, PS and MK carried out the RFLP analysis and mtDNA sequencing of the Iranian samples. DMB collected the DNA and analyzed mtDNA variation amongst the Indian samples from Cochin and the Cochin Jews collected (the latter) in Israel under supervision of KS, who also contributed to the editing of the manuscript. AT provided some critical new information and, in addition to KS, MTPG and PE, engaged into valuable discussions during manuscript preparation. MM did most of the phylogenetic analysis and was in charge of manuscript writing while TK and RV contributed significantly during both stages and were responsible for conceiving and designing the study. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 3 Table 8. Excel spreadsheet. MtDNA haplogroup frequencies among tribal populations. Frequencies of mtDNA haplogroups amongst the tribal populations of India, China and Thailand as averaged over states of India and provinces in China and Thailand. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 7 Table 12. Excel spreadsheet. Comparison of the mtDNA haplogroup frequencies amongst the Indian caste and tribal populations. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 Table 9. Excel spreadsheet. MtDNA haplogroup frequencies in India and Iran. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 6 Table 11. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst the Indian caste populations as averaged over states of India. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 8 Figure 12. Image file in PNG format. Network of HVS-I haplotypes belonging to haplogroup R5. Circle areas are proportional to haplotype frequencies. Variant bases of the HVS-I are numbered as in (Anderson et al. 1981) minus 16,000 and shown along links between haplotypes. The diagnostic R5 coding region marker 8594 is shown in bold and numbered as in (Anderson et al. 1981). Character changes are specified only for transversions. Underlined samples are those in which the marker 8594 was assayed by either RFLP analysis (-8592 MboI) or direct sequencing. Sample codes are as in Table 6 (see Additional file 1). Coalescence estimates marked with an asterisk are calculated excluding tribal populations (see Materials and Methods for explanation). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 Table 10. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst the Indian populations. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Table 7. Excel spreadsheet. MtDNA variation in the studied populations (raw data). MtDNA control and coding region variation in the populations that were used in the study. The database includes both the newly obtained datasets and the previously published datasets. The latter were in many cases reanalyzed both *in silico*and by typing for additional mtDNA coding region markers. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 1 Table 6. Excel spreadsheet. The list of studied populations. List and details of the populations whose mtDNA were used in the study. This includes both newly obtained datasets and previously published datasets ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 9 Table 13. Excel spreadsheet. Frequencies of mtDNA haplogroups amongst different linguistic groupings of Indian populations. ::: ::: {.caption} ###### Click here for file ::: Acknowledgments =============== We thank Lluis Quintana-Murci for providing data prior to publication, Vincent Macaulay for the algorithm for calculating credible regions for haplogroup frequencies, and Jaan Lind and Hille Hilpus for technical assistance. This work was supported by Estonian Science Foundation grants 514 (to RV), 5574 (to TK), 5807 (to EM), the Italian Ministry of the University: Progetti Ricerca Interesse Nazionale 2002 and 2003 (to AT), and European Commission grants ICA1CT20070006 and QLG2-CT-2002-90455 (to RV).

PubMed Central

2024-06-05T03:55:47.553371

2004-8-31

PMC516769

Background ========== Due to their isogenicity, inbred mouse strains demonstrate low biological variability within each strain\[[@B1],[@B2]\]. Genetic variation between inbred strains is considerable and has recently been characterized in detail using single nucleotide polymorphisms\[[@B3]\]. Differences in genetic background between strains affect the gene expression levels of a subset of genes, which probably explains phenotypic differences. Indeed, several reports have been published in which gene expression profiles have been used as QTLs in genetic mapping studies to identify complex traits \[[@B4]-[@B6]\]. From literature \[[@B7]-[@B9]\], it appears that the subset of genes for which expression is significantly affected by genetic background is small. However, this has never been related to the extent of gene expression changes observed due to disease-causing mutations. We are studying differential gene expression between affected and healthy muscle in a range of murine models for neuromuscular disorders with different genetic backgrounds (Turk *et al*., manuscript in preparation). We, therefore, determined gene expression levels in hindlimb muscles from five frequently used wildtype mouse inbred strains, and compared these to the differential gene expression levels in affected muscle tissue from a mouse model (*mdx*) for Duchenne muscular dystrophy with healthy muscle tissue. Both the number of differentially expressed genes between strains as well as the fold-change levels are lower when compared to the differences found in affected versus healthy muscle tissue. Results ======= Gene expression levels in hindlimb muscle tissue from five different inbred strains (CBA, BALB, BL6, DBA, and BL10) were determined. Total RNA from two individuals per strain was isolated, reversed transcribed, and subsequently labelled according to a recently developed protocol (adapted from Xiang *et al.*, 2002), which requires an input of only 1 μg total RNA. Labelled cDNA was hybridised to murine microarrays containing 7,776 65-mer oligonucleotides spotted in duplicate. Significance levels (p-values) between the five mouse inbred strains were calculated using analysis of variance\[[@B10]\]. Significance levels among two individual mice within each strain were determined using a hierarchical *t*-test providing higher statistical power than conservative methods for low (2--4) replicate numbers\[[@B11]\]. The higher power is yielded by borrowing information across genes to produce a better expression variance estimator. The gain in power is reported via an increase in the degrees of freedom associated with the t-test. Differentially expressed genes for both computations were selected by controlling the false discovery rate (FDR), as suggested by Benjamini and Hochberg (1995), rather than using pre-defined cut-offs for p-values or corrections for multiple testing. The FDR represents an expectation of the proportion of false positives among the selected differentially expressed genes, which increases dramatically during multiple testing, inherent in microarray experiments\[[@B12]\]. Using an FDR of 10% we selected 88 out of 6144 (1.4%) expressed genes that are differentially expressed between strains (Fig. [1](#F1){ref-type="fig"}). A lower number of differentially expressed genes was found in the analysis of variation within strains with identical FDRs of 10% (Table [1](#T1){ref-type="table"}). Results with other FDR levels are available online as additional file. Correlation between gene expression levels of the two samples from each strain was high (Pearson correlation coefficient ranging from 0.87 to 0.95), also indicating low internal variation (Table [1](#T1){ref-type="table"}). A considerable amount of differentially expressed genes (718 genes) were selected when pre-defined cut-off values (p \< 0.05) were used to determine the differential gene expression between strains. However, adjusted FDR levels indicated a proportion of false positives equal to 42%. On the other hand, adjusting for multiple testing using Bonferroni correction proved to be too stringent, leaving no or few differentially expressed genes. Controlling the FDR, therefore, appears to be an optimal method for both selecting differential gene expression and simultaneously determining the validity of the experimental outcome. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Differentially expressed genes between mouse inbred strains Relative expression levels of differentially expressed genes between mouse inbred strains are depicted in colour as relative intensity levels. Shown for each gene are GenBank accession number, description, functional annotation according to Gene Ontology, and UniGene cluster IDs. Relative expression levels are calculated by subtracting the average intensity value per gene from the strain-dependent intensity values. Differential expression was determined by selecting p-values from analysis of variance based on a false discovery rate of 10%. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Number of differentially expressed genes using several cut-off strategies ::: **Between strains** **Within strains** ------------------- --------------------- -------------------- ------ ------ ------ ------ Correlation 0.95 0.95 0.87 0.87 0.92 Naive (p \< 0.05) 718 737 610 963 1043 483 Bonferroni 0 2 4 1 0 3 FDR 10% 88 2 4 14 0 16 Correlation between two individuals per strain was calculated using Pearson\'s correlation coefficient. Significance levels (p-values) between strains were calculated with MA-ANOVA, and within strains using the hierarchical *t*-test. Differential gene expression was determined by selecting genes with p-values lower than a specified threshold. Thresholds were selected using three different strategies; naive, Bonferroni corrected, and False Discovery Rate (10%), and resulted in different numbers of significantly differentially expressed genes. ::: To put the influence of differential gene expression due to genetic background in perspective, we studied gene expression between affected and healthy tissue from hindlimb muscle derived from *mdx*mice, and from control mice with identical genetic backgrounds. Selection with an FDR of 10% resulted in 1298 differentially expressed genes. Differential gene expression between the two most divergent mouse inbred strains (BL6 and CBA, data not shown) was determined to allow a direct comparison with identical statistical methods. Selection with an FDR of 10% showed an approximately ten-fold decrease in the number of differentially expressed genes (126). Absolute fold changes were calculated and subsequently a comparison of the distribution was made (Fig. [2](#F2){ref-type="fig"}). Median gene expression levels are equal between affected/control and inbred/inbred. However, the number of large fold changes (\>3) between affected/healthy (221) is much higher than between inbred/inbred (7), consistent with low contribution of differential expression due to genetic background. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effect of different genetic background on differential gene expression The distribution of absolute fold changes of differentially expressed genes (n = 1298) between affected (*mdx*) and healthy (WT) muscle were compared to the distribution of absolute fold changes of differentially expressed genes (n = 126) between two mouse inbred strains (CBA and BL6). Selections were based on a FDR of 10%. :::  ::: Although overall expression levels are similar between strains, a relatively high number of differentially expressed genes was due to deviating gene expression levels in BL6. We performed quantitative real-time RT-PCR (qPCR) on five genes to verify our microarray data. Two genes myomesin 1 and tropomodulin 1, which were 2.2-fold and 1.8-fold lower expressed in BL6 compared to the other strains on our microarrays, were also found to be lower expressed (2.0-fold and 2.2-fold respectively) in our qPCR assay (Fig. [3](#F3){ref-type="fig"}). Three other genes (dysferlin, cystatin B, and thrombospondin 4) showed no differential expression between any strains. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Validation of BL6-dependent gene expression with qPCR Relative gene expression levels between mouse inbred strains of tropomodulin 1 (*Tmod1*) and myomesin 1 (*Myom1*) as determined by quantitative RT-PCR. Significantly lower expression (p \< 0.01, marked by \*) for both genes was shown in BL6 compared to other strains. :::  ::: Discussion ========== This study shows that variation in overall gene expression levels between mouse inbred strains is relatively low in hindlimb muscle tissue. This is particularly evident when the number of differentially expressed genes between two mouse inbred strains (C57 vs. Bl6, 126 genes with 7 genes having a fold-change \> 3) is compared to that between diseased and healthy muscle tissue (*mdx*vs. wild-type, 1298 genes with 221 genes having a fold-change \>3). Therefore, the use of mice with deviating genetic background may be justified in disease-related studies. Alternatively, strain-dependent gene expression differences may be evaluated in the initial study phase of gene targeting experiments, although the effect of hybrid backgrounds is difficult to assess. Gene expression studies in the brain revealed that approximately 1% of expressed genes differ between two mouse strains\[[@B8]\]. Application of alternative statistical methods, similar to those used in our study, on this dataset resulted in an increase in the number of differentially expressed genes (approx. 3%) between the two mouse strains\[[@B7]\], demonstrating that the number of differentially expressed genes is highly dependent on the statistical criteria used. A similar number of differentially expressed genes was found in a comparison of hippocampal gene expression between 8 different mouse strains\[[@B9]\]. The results of our study in muscle tissue demonstrated that approximately 1.4% of the expressed genes show differential expression between mouse strains. Based on these results, strain differences in gene expression seem to have a similar magnitude across different tissues. Genomic variability could be correlated with high levels of single nucleotide polymorphisms (SNPs) occurring in specific blocks between mouse inbred strains. The presence of cis-acting (single nucleotide) polymorphisms may be associated with regulatory variation affecting gene expression levels. It was estimated that probably a consistent amount (up to 6%) of the roughly estimated 35,000 mouse genes contain such functional regulatory variants\[[@B13]\]. We investigated if differentially expressed genes were localized in blocks with high genomic variability, but our number of differentially expressed genes was too low to obtain statistically significant answers (data not shown). This study suggests an additional method for phenotyping mouse inbred strains and provides a list of genes with significant differential expression based upon false discovery rate selection. Although overall gene expression profiles are highly similar, most significant differences are determined by low gene expression levels of BL6 compared to the other strains. A large proportion of these BL6-specific genes function as structural muscle proteins (i.e. nebulin, alpha-actinin 2, myomesin 1 and radixin). To date, however, no major differences in muscle physiology in BL6-mice have been described which can be attributed to these reduced gene expression levels. Perfectly isogenic backgrounds are sometimes difficult to obtain. This explorative study demonstrates that the effect of genetic background on muscle expression profiles is significant but rather limited compared to other effects, e.g. the dystrophic genetic defect (*mdx*) we study. As such, the genetic background will only marginally interfere with data analysis. Determination of gene expression profiles between mouse strains enables flagging a modest number of differentially expressed genes, and is an efficient and sensible approach to circumvent tedious backcrossings, necessary to obtain isogenic animals. Methods ======= Mouse breeding, tissue preparation and total RNA isolation ---------------------------------------------------------- We obtained CBA/CaOlaHsd (CBA), BALB/cOlaHsd (BALB), C57Bl/6JOlaHsd (BL6), DBA/2OlaHsd (DBA), and C57Bl/10ScSnOlaHsd (BL10) mice from Harland Laboratories, and C57Bl/10ScSn-Dmd^mdx/J^(*mdx*) mice from Jackson Laboratory at the age of 6 weeks. Mice were kept under standard conditions and were sacrificed by cervical dislocation when 8 weeks old. Hindlimb muscles (m. quadriceps femoris) were dissected and promptly snap-frozen in isopentane at -80°C. Total RNA was prepared by disrupting tissue using mortar and pestle and subsequent homogenisation by a rotor-stator homogenizor (Ultra-Turrax T25, Janke & Kunkel IKA-Labortechnik) in RNA-Bee (Campro Scientific) until uniformly homogenous (15--45 sec). Total RNA was isolated according to manufacturer\'s instructions followed by purification using RN-easy columns (Qiagen). Quality and yield was determined using Lab-on-a-chip (BioAnalyzer, Agilent). Target preparation and hybridisation ------------------------------------ Aminoallyl labelled cDNA (aa-cDNA) was prepared based on a previously described protocol\[[@B14]\]. Aliquots of 1 μg of total RNA in the presence of 2 μg amino-TN~6~primer (5\'-NH~2~-(CH~2~)~6~-TN~6~, Eurogentec) were adjusted to a volume of 21 μl with DEPC-treated H~2~O (diethyl pyrocarbonate, Sigma), heated for 10 minutes at 70°C and chilled on ice for 10 minutes. Reverse transcription mastermix (1.8 μl RevertAid RNaseH-M-MuLV reverse transcriptase (200 U/μl, MBI Fermentas), 6 μl 5x first-strand buffer (MBI Fermentas), and 1.2 μl 25x aa-dUTP / dNTP solution (2 μl 50 mM dATP, 2 μl 50 mM dCTP, 2 μl 50 mM dGTP, 1.2 μl 50 mM dTTP, 0.8 μl 50 mM aminoallyl-dUTP (Ambion)) was added per reaction and incubated at room temperature for 10 minutes followed by 2 hours at 42°C. RNA was hydrolysed by addition of 10 μl 0.5 M EDTA and 10 μl 1 M NaOH and incubation at 65°C for 30 minutes followed by neutralization by addition of 10 μl 1 M HCl. Aminoallyl labelled cDNA was then purified by combining 300 μl of PB-buffer (Qiagen) to 60 μl of the neutralized sample and centrifuged through a Qiaquick column (Qiagen) at 13000 rpm for 1 minute. Two washing steps were performed by spinning 500 μl of 75% EtOH at 13000 rpm for 1 minute while discarding the flow-through. To remove ethanol-traces the columns were centrifuged for an additional minute. cDNA was recovered by eluting three times using 30 μl basic H~2~O (3.3 mM NaHCO~3~buffer, pH 9.0) and concentrated to a volume of 6.66 μl using a speedvac. Aliquots of Cy3 and Cy5 reactive dyes (PA23001, PA25001, Amersham) were prepared by dissolving each vial of monoreactive dye in 40 μl fresh anhydrous DMSO (Sigma) and dividing into aliquots of 2 μl followed by vaccuumdrying until dry and subsequent storage at 4°C in the presence of silica. Fluorescent dyes were coupled by adding 3.33 μl of bicarbonate buffer (1 M NaHCO~3~buffer, pH 9.0) to the aa-cDNA sample and dissolving the dried aliquot of reactive dye, followed by incubation at room temperature for 1 hour in the dark. To the samples 4.5 μl 4 M hydroxylamine (Sigma) was added and incubated at room temperature in the dark for 15 minutes, followed by addition of 186 μl TE^-3^-buffer. Hybridisation mixtures were prepared by combining a Cy3-labeled cDNA sample with a Cy5-labeled cDNA sample and 10 μl Mouse-Hybloc (1 μg/μl, Applied Genetics Laboratories) followed by removing uncoupled dyes by spinning through a pre-wetted Microcon column (YM30, Amicon) for 8 minutes at 13000 rpm. Hybridisation mixture was washed by spinning 500 μl TE^-3^-buffer through the column and discarding the flow-through. This step was repeated two times as 2 μl yeast-tRNA (10 μg/μl, Sigma) and 2 μl polyA-RNA (10 μg/μl, Sigma) were added during the last step. Mixture was collected by inverting the column and spinning for 1 minute at 13000 rpm. Hybridisation mixture was finalized by adding TE^-3^-buffer to 84 μl together with 17 μl 20x SSC and 3 μl 10% SDS followed by denaturing at 100°C for 2 minutes, renaturing at room temperature for 15 minutes and spinning at 13000 rpm for 10 minutes. Labelled target was hybridised overnight on murine oligonucleotide microarrays (65-mer with 5\'-hexylaminolinker, Sigma-Genosys mouse 7.5 K oligonucleotide library, spotted in duplicate). Hybridisation occurred in a automatic hybridisation station (GeneTac, Perkin Elmer) and was followed by washing with 5x 2xSSC + 0.1% SDS at 30°C, 5x 1xSSC at 30°C, 3x 0.2xSSC at 30°C, 1x 0.2xSSC at 65°C, 2x 0.2xSSC at 30°C, and subsequently scanned as described previously\[[@B15]\]. Experimental design, data extraction and analysis ------------------------------------------------- Gene expression profiles from hindlimb muscle derived from 2 male animals of each strain were generated using dye-swap experiments. Subsequent duplicate spots on each array resulted in 8 replicate measurements per gene. Targets were assigned at random to the arrays, while avoiding co-hybridisation of samples from the same strain. GenePix Pro 3.0 (Axon) was used for feature extraction and quantification. Genes were considered as being expressed when the corresponding feature was not flagged by the algorithm provided by GenePix. Local background corrected spot intensities were normalized using Variance Stabilization and Normalization (VSN) in R \[[@B16]\]. Array data has been made available through the GEO data repository of the National Center for Biotechnology Information under series GSE662. Correlation between individuals was calculated using Pearson\'s correlation coefficient. Significantly differential expression levels were determined using MA-ANOVA (**MAANOVA2.0 The Jackson Laboratory**<http://www.jax.org/staff/churchill/labsite/software/anova/>), hierarchical *t*-test \[[@B11]\] and the False Discovery Rate \[[@B17]\] selection procedure. Quantitative Reverse Transcription Polymerase Chain Reaction ------------------------------------------------------------ qPCR was performed in duplicate for each individual resulting in four measurements per strain per gene. cDNA was prepared by reverse transcription using 1 μg total RNA as template. Random hexamers (40 ng) were used to prime the transcription after heating 10 minutes at 70°C followed by chilling on ice for 10 minutes. cDNA was synthesized by RevertAid RNaseH^-^MuLV reverse transcriptase and accompanying buffer (MBI-Fermentas) using 1 mM dNTPs. The mixture was incubated at room temperature for 10 minutes before a 2 hour incubation step at 42°C, followed by 10 minutes at 70°C. Quantitative PCR was performed using the Lightcycler (Roche). PCR mixture was prepared by combining cDNA dilution, 10 pmol forward and reverse primer, MgCl~2~(4 mM) with 4x home-made LC mastermix (0.9 mM dNTPs, BSA (1 μl/μl, Pharmacia Biotech), Taq polymerase (0.8 U/μl), 4x SYBR Green I (Molecular Probes), 4x AmpliTaq Reaction Buffer (Perkin Elmer)) to a total volume of 20 μl. Amplicons were generated during 45 cycles with annealing temperature set at 55°C. Optimal cDNA dilutions and relative concentrations were determined using a dilution series per gene. Replicate experiments (n = 4) were normalized to 1 and relative expression values were determined by calculating the ratio per gene over the average relative expression of genes, which show no differential expression on both microarray and qPCR (dysferlin, cystatin B, and thrombospondin 4). Significance levels were calculated with a one-sample *t*-test. PCR primer pairs were designed using the Primer3 search engine, available at: **Primer3 Software Distribution**<http://frodo.wi.mit.edu/primer3/primer3_code.html>. The screened genes and the oligonucleotide primer pairs used for each of the genes in this study correspond to the following nucleotides: myomesin1, 4761--4780 and 4865--4884 (NM\_010867); tropomodulin1, 670--689 and 878--897 (NM\_021883); dysferlin, 4218--4237 and 4353--4372 (AF188290); cystatinB, 3--22 and 151--170 (NM\_007793); thrombospondin4, 2167--2186 and 2289--2308 (NM\_011582). Authors\' contributions ======================= RT carried out the tissue preparation, total RNA isolation, target preparation, hybridisations, experimental design, data extraction, data analysis, rt-PCR, and the drafting of the manuscript. PH participated in the experimental design, analysis, rt-PCR, and study coordination. ES participated in the experimental design and analysis. RM provided statistical support. EM was responsible for mouse breeding and tissue preparation. JM participated in experimental design. GO and JD coordinated the study. All authors read the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Differentially expressed genes between mouse inbred strains are selected with a false discovery rate of 10, 15, and 20%. Selected genes are indicated with 1, genes not selected by the specified criteria are indicated with 0. The mean of the relative gene expression levels of each of the five mouse strains is shown. For each gene the GenBank accession number is shown as well as the UniGene ID, gene description and the gene ontology description. The additional file is formatted as Comma Separated Values (CSV) file, and is named Turketal2004\_Additional\_File.csv. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank Claire Wade and Mark Daly (Whitehead/MIT Center for Genome Research) for providing SNP density levels, and Stefan White (LUMC) for critical comments on the manuscript. This work was supported by the Nederlandse Stichting voor Wetenschappelijk Onderzoek (NWO), the Center for Medical Systems Biology (CMSB) established by the Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research (NGI/NWO), and the Muscular Dystrophy Campaign (UK). Technical support was provided by the Leiden Genome Technology Center.

PubMed Central

2024-06-05T03:55:47.561359

2004-8-18

PMC516770

Background ========== Today, many genetics laboratories have access to modern capillary DNA sequencing machines, such as the ABI PRISM 3100, 3700 or 3730. These machines generate vast amounts of raw sequence data with little user intervention. Consequently, the amount of data to be analyzed has expanded and the bottleneck now is the analysis capacity. Data analysis capacity can be increased by higher levels of automation. Investments in infrastructures to process the raw sequencing data in sophisticated but rigid pipelines might be justified for larger laboratories and larger projects but might be too costly for smaller laboratories. In addition, rigid pipelines are too impractical if different projects share run-time on the same machine while requiring (slightly) different analysis procedures (e.g. vector trimming is needed in plasmid sequencing, but needless when sequencing PCR products). Nucleotide sequence analysis can be performed with a variety of software tools. Although the number of console and web-based software tools has grown rapidly, the routine use of data input, output and storage may be inconvenient. Furthermore, for performing a series of analyses with different software tools, the sequence data need to be reformatted to the required data structure. Alternatively, sophisticated software suites that provide an integrated environment often are expensive. Several of the available software solutions are designed to facilitate automated DNA sequence analysis at low cost. Well-known solutions are the Staden package and Bioperl. The Staden Package contains *pregap4*and *gap4*, full-featured applications with an intuitive graphical user interface \[[@B1]\]. These programs handle a list of raw sequence reads method-by-method. The programs in the Staden Package typically require a degree of user intervention and thus hands-on time. Alternatively, Bioperl is a group of perl modules describing many genetics and genomics concepts \[[@B2]\]. For example, it includes the Bio::Seq::SeqWithQuality object that provides some of the basic properties of a raw sequence (i.e. its nucleotide sequence and quality values); the Bio::Tools::Primer3 object provides methods to work with primer3 input and output. However, to build custom DNA sequencing data pipelines, basic programming skills are needed to combine all these modules. Smaller laboratory sites, however, often need to implement versatile pipelines that can be adjusted for any research question that suits the project best; at the same time, they often also do not have dedicated programmers available. Although (semi-)automated procedures have been published by other groups \[[@B3],[@B4]\], these are mostly focused on one particular pipeline and environment. Here, we present POSA, a set of two new perl objects (Read.pm and Contig.pm) that describe a raw sequence and a *Phrap*contig in detail and are easily implemented in perl-based pipelines. Because these objects provide building blocks for sequencing data analysis pipelines and the actual pipelines are built using perl-scripts, the POSA objects can be used in very diverse settings. Implementation ============== The POSA source code is entirely coded in object-oriented Perl and consists of two objects: Read.pm and Contig.pm. In general, there are two important concepts associated with objects: methods (built-in procedures that can be performed on the object) and properties (describing some of the characteristics of the object). Most methods in the objects rely on the availability of other third-party programs (see Dependencies). Basically, POSA provides a wrapper around these programs and provides easy design and implementation of these programs in automated data analysis. The Read.pm object describes a DNA sequence trace and includes methods for data import from a variety of formats. It relies on *Phred*\[[@B5],[@B6]\] for import and interpretation of raw sequence data. The original trace data are stored in binary (*scf*) format within the object. Other methods of Read.pm use modules of the Staden Package \[[@B1]\], such as *qclip*and *vector\_clip*(if installed). Properties of Read.pm include e.g. the DNA sequence, quality scores, template and vector names and read direction. The Contig.pm object contains a method to assemble contigs of reads using the *Phrap*program \[[@B6]\]. The object typically is created based on a list of Read.pm objects and can be exported as alignments or screened for polymorphisms using *PolyPhred*\[[@B7]\]. Both the Read.pm and Contig.pm objects were designed with flexibility in mind. To allow a (virtually) unlimited amount of data to be processed, the perl scripts using these objects work sequence-by-sequence rather than method-by-method. Typically, these objects are called from straightforward perl scripts that outline the analysis steps to be performed. Example scripts using the objects can be accessed from the download website. An example of a script and output using the two objects to process a set of reads and annotate sequence polymorphisms from the assembled contig is given in Figure [1](#F1){ref-type="fig"} and Figure [2](#F2){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### A typical script that takes a list of ab1 files for analysis and assembly, reports the contig, and lists the putative SNP positions and SBE primers. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Typical output as generated by the script in Figure 1. :::  ::: POSA was developed with perl 5.6.1 and tested on a SuSE linux 8.1 system for *abi*-files from the ABI PRISM 377 DNA Sequencer and 3100 Genetic Analyzer (Applied Biosystems). *Phred*, *Phrap*and *PolyPhred*versions were 0.000925.c, 0.990329 and 4.05, respectively. Results and Discussion ====================== Functionality ------------- POSA provides an interface to design and implement automated sequencing data analysis. Sequencing data may be used in a variety of formats and originate from a variety of sources, e.g. data in *fasta, abi/ab1*or *scf*format retrieved from websites or from newly generated traces. In addition, new objects can be initiated from a text file or can be opened from previous stored objects. Subsequently, a variety of methods can be applied, including basecalling and assessment of quality codes (by *Phred*), quality clipping, vector clipping, screening for *E. coli*(or other) sequence, contig assembly (by *Phrap*) and analysis. The method *asPrimer3*can automatically generate input for the primer3 program \[[@B8]\] and is available in both objects. To facilitate automated SNP discovery or typing, the *SearchSnp*s method will generate output as shown in Figure [2](#F2){ref-type="fig"}. This method is based on the *PolyPhred*program and uses the \'rank\' argument to set the stringency. Finally, data can be stored in objects, or in files in either *exp*, *scf*or *fasta*format. In addition, the data can be saved in a *primer3*input file to allow automated PCR primer design, or data can be saved in MIPE format (i.e. an XML format to store information on PCR experiments; see <http://mipe.sourceforge.net>). Data on assembled contigs can be exported as a list of reads in a contig, as consensus sequence, as alignment, as putative SNPs, as SBE primers for SNP genotyping or as *gff*file for visualization in Gbrowse \[[@B9]\]. Combinations of the diversity of input, analysis and output options allow for a wide spectrum of possible implementations. Examples of possible analysis pipelines include (but are not limited to) BAC-end sequencing with automated PCR primer design for chromosome walking and resequencing of PCR products with SNP annotation either for SNP genotyping or for SNP discovery and SBE primer design. Examples of scripts are provided on the web site <http://posa.sourceforge.net>. Performance ----------- Although it represents only one of the numerous possible POSA-based pipelines, performance of POSA was validated by comparison of SNP discovery with the data after analysis using the Staden package. To do so, 5 PCR products were resequenced from a panel of 16 individuals to identify SNPs. Manual editing using the Staden Package revealed a total of 48 SNPs. Automated analysis using POSA also yielded a total of 48 SNPs with SNP ranking codes 1-3. Together, 41 SNPs were assigned with both manual editing and POSA. The remaining 7 SNPs assigned in manual editing corresponded to SNPs with ranks 4-6 in the POSA analysis. The 7 SNPs that were only assigned by POSA all originated from regions with lower quality sequence. While analysis time was reduced from several hours to a few minutes, POSA assigned SNPs in a way that was highly consistent with manual editing. This was expected because POSA provides options for an integrated analysis pipeline, but essentially is a wrapper around well-established sequence analysis tools like *Phred*, *Phrap*and *PolyPhred*. Intended use and benefits for users ----------------------------------- POSA is a tool that provides easy and highly automated DNA sequence and contig data analysis using popular analysis tools. Automated sequence analysis reduces analysis time from several hours to a few minutes. Pipelines can easily be expanded or adapted through perl scripts. Writing or altering the perl scripts is straightforward to do for people with only basic computer skills, although more linux/unix experience might be necessary to install the required software (e.g. *Phred*and *Phrap*). Overall, this guaranties easy implementation of highly automated quality pipelines in combination with high flexibility in setup and design. The perl objects are released under an open source license, allowing code improvements by the user community. Conclusions =========== POSA describes a DNA sequence read and a *Phrap*contig assembly in detail. These objects allow a flexible and easy setup of perl-based pipelines to handle DNA sequencing data, including generating primer3 input and automated SNP discovery, while requiring only little programming skills. Availability and requirements ============================= Project name: POSA Project home page: <http://posa.sourceforge.net> Operating system: platform independent Programming language: Perl 5.6.1 License: Artistic License (Open Source) Requirements ------------ • Perl modules: Carp; Statistics::Descriptive; Tie::File; IO::File; POSIX:: Storable. • Phred, Phrap, PolyPhred • Pregap4, gap4 (Staden Package (optional)) • Primer3 (optional) List of abbreviations ===================== POSA Perl objects for DNA sequencing data analysis SNP single nucleotide polymorphism abi/ab1ABI PRISM trace file format scf standard chromatogram format exp experiment file format, developed by Staden (see <http://staden.sourceforge.net>) MIPE minimum information on PCR experiments (see <http://mipe.sourceforge.net>) BAC bacterial artificial chromosome PCR polymerase chain reaction SBE single base extension Authors\' contributions ======================= JA programmed the Perl objects and participated in development of concept and architecture of the software; BJ participated in development of concept and architecture and wrote the manuscript; MG supervised the project. All authors read and approved the final manuscript. Acknowledgements ================ The authors wish to thank Tineke Veenendaal for testing.

PubMed Central

2024-06-05T03:55:47.563378

2004-8-27

PMC516771

Background ========== *Neisseria meningitidis*, the causative agent of meningococcal meningitis and septicaemia, is a common inhabitant of the human nasopharynx, being asymptomatically carried by approximately 10% of the population \[[@B1],[@B2]\]. There is evidence for extensive horizontal genetic exchange in populations of this antigenically and genetically variable Gram-negative bacterium \[[@B3]-[@B7]\] but, despite the diversity of carried meningococci, only a limited number of genotypes -- the hyper-invasive lineages -- are responsible for most reported disease \[[@B8]\]. These lineages have been identified by the techniques of multilocus enzyme electrophoresis \[[@B9]\] and multilocus sequence typing (MLST) \[[@B10]\] as clonal complexes. In recent years, the ST-1 complex (formerly subgroup I), ST-4 complex (subgroup IV), and ST-5 complex (subgroup III) have dominated meningococcal disease in Africa and Asia, while members of the ST-11 (ET-37) complex, ST-8 complex (cluster A4), ST-41/44 complex (lineage 3), and ST-32 (ET-5) complex have caused most disease in other parts of the world. Meningococci occasionally cause epidemic outbreaks of varying scale up to global pandemics, although at a given point in time disease in a given geographical locale is often dominated by a limited number of clonal complexes \[[@B8]\]. DNA adenine methyltransferase (Dam) is an enzyme involved in the mismatch repair system of bacteria. During DNA replication, only the parental strand is fully methylated by the enzyme, since methylation is not immediate, allowing a mismatch on the newly synthesised strand to be excised and replaced \[[@B11]\]. Disruption of Dam activity suggests that mismatch repair will be less effective, potentially allowing the creation of frameshift mutations in the homopolymeric or simple repeat motifs within the promoter or coding regions of surface antigen genes leading to the reversible switching of their expression state. This is an important mechanism found at bacterial loci that encode gene products that are advantageous under certain conditions but not others; such genes have been termed contingency loci \[[@B12]\]. There is extensive evidence for such loci in the meningococcal genome \[[@B13]\]. In particular, the ability to vary the expression state of surface-exposed cellular components is important for within host adaptation, for example by allowing evasion of the immune response in the nasopharynx. These mechanisms are also important in the occasional transition from harmless colonisation to invasive disease \[[@B14]-[@B16]\]. Examples of such loci described in the meningococcus to date include: the capsular polysaccharide \[[@B17]\]; the Opc outer membrane protein \[[@B18]-[@B20]\]; pili \[[@B21]\]; the PorA protein \[[@B22]\]; and opacity proteins \[[@B19],[@B23]\]. Dam methylation has been implicated in modifying the virulence of a number of bacterial pathogens \[[@B24]-[@B26]\], but its role in *N. meningitidis*has been contentious. Bucci *et al*., 1999 \[[@B27]\] suggested that the absence of Dam activity was responsible for high rates of phase variation in the *siaD*capsule gene, resulting in an increased virulence of some strains. In addition, the latter work suggested that all pathogenic isolates lacked Dam activity, the *dam*gene inactivated by the insertion of a putative restriction endonuclease, named \'*dam*replacing gene\' (*drg*), with the genotypes *dam*^+^/*drg*^-^and *dam*^-^/*drg*^+^being mutually exclusive. However, two subsequent studies \[[@B28],[@B29]\] have found no effect. The *drg*gene encodes a restriction enzyme, *Nme*BII that is similar to the *Streptococcus pneumoniae Dpn*I restriction endonuclease which cleaves at GmeATC, but not at GATC, sequences \[[@B30],[@B31]\]. In the present study the prevalence of *drg*and its association with disease-associated isolates of *N. meningitidis*was examined by a survey of its distribution in a collection of isolates chosen to represent the known diversity of pathogenic meningococci \[[@B10]\]. The results suggest that, while *drg*is present in some lineages but not others, this gene has spread in the meningococcal population by horizontal genetic exchange, possibly after the introduction of the *drg*gene from an exogenous source. Results ======= Distribution of *dam*and *drg*among invasive meningococci --------------------------------------------------------- In all cases, there was an exact correlation with the presence of *dam*and the absence of *drg*, and vice-versa. Of the 84 isolates tested, 23 were indicated to be *dam*^+^/*drg*^-^and 61 to be *dam*^-^/*drg*^+^by restriction analysis and PCR amplification (see [Additional file 1](#S1){ref-type="supplementary-material"}). The presence of *dam*or *drg*was associated with particular clonal complexes. Of the hyper-invasive lineages included in the analysis, all ST-8 complex and ST-11 complex isolates were *dam*^+^/*drg*^-^, while all of the representatives of the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes were *dam*^-^/*drg*^+^. Diversity of *drg*gene fragment sequences ----------------------------------------- The level of nucleotide diversity, as represented by number of alleles and polymorphic sites, along with the p-distances seen within *drg*gene fragments was similar to that seen with the housekeeping genes used for MLST (Table [1](#T1){ref-type="table"}), with a total of 19 different fragment sequences (*drg-1*-- *drg-19*) present in this collection. The ratio of non-synonymous to synonymous substitutions (*d*~*N*~/*d*~*S*~, 0.11) was also similar to that seen in the MLST gene fragments. Three *drg*gene fragments (*drg-13*, *drg-14*and *drg-15*) exhibited polymorphisms resulting in single base frameshifts, two resulting in stop codons (Figure [1](#F1){ref-type="fig"}) and one fragment (*drg-16*) had an insertion corresponding to a 24-base direct repeat. The G+C content of *drg*at 42.4% was lower than that of the housekeeping genes, which were in the range 51.1--57.4%. Split decomposition analysis of the *drg*fragment sequences generated a star phylogeny with some net-like phylogenetic structure involving the *drg-2*allele (Figure [2](#F2){ref-type="fig"}). The distribution of the different *drg*sequences amongst the clonal complexes is also shown in Figure [2](#F2){ref-type="fig"}. Discussion ========== The meningococcus is an example of an accidental pathogen, a commensal organism that rarely causes disease and which gains no evolutionary benefit from this process \[[@B32]\]. A complete understanding of the diseases caused by such organisms is dependent upon an appreciation of the mechanisms by which harmless carriage develops into invasive disease \[[@B33]\]. The observation that some meningococcal genotypes, the hyper-invasive lineages, are more likely to cause disease than others \[[@B8],[@B10]\] suggests that comparative studies of disease-associated and carried meningococci may identify genetic factors responsible for disease \[[@B34]\]. In this context, the suggestion that the possession of a single genetic change, the insertion of the *drg*gene at the *dam*locus, was essential for virulence in meningococci \[[@B27]\] was attractive as it provided a single characteristic associated with the disease phenotype. Further, this proposal provided a plausible mechanistic explanation, namely the promotion of rapid switching of expression of contingency genes, for example those encoding the capsule which represents the best defined meningococcal virulence determinant \[[@B35]\]. This is consistent with the evidence that inactivation of Dam has been shown to affect rates of recombination and phase change in other species \[[@B25],[@B36]\]. The data presented here, however, do not support the contention that the *drg*insertion is preferentially associated with disease-associated meningococcal isolates, even those expressing serogroup B capsular polysaccharide. There was no evidence of a difference between disease-associated and carried meningococcal isolates with 76% (45/59) of disease-associated isolates and 75% (11/15) of carrier isolates possessing the *drg*insertion. The isolate collection employed by the present investigation was chosen to be globally representative of meningococcal disease in the latter part of the 20^th^century, with multiple examples of each of the major hyper-invasive meningococci identified over this period. When analysed by clonal complex, which identifies hyper-invasive lineages, it was apparent that the *drg*insertion was absent from all of the isolates representing the ST-8 and ST-11 clonal complexes, both major hyper-invasive lineages but, conversely, *drg*was present in all representatives of the hyper invasive lineages represented by the ST-1, ST-4, ST-5, ST-41/44, and ST-32 complexes. This association was independent of the serogroup expressed by the isolates and provided a likely explanation of the earlier observations \[[@B27]\]. As meningococcal disease in a given locale at a given time tends to be dominated by a limited number of clonal complexes \[[@B8]\], it is likely from the results provided here that a given sample of disease-associated isolates collected from a given locale will be uniform at the *dam*locus. At a given point in time they may be dominated by *drg*containing meningococci, while at other times isolates without this insertion might dominate. For example, as both the ST-1 and ST 32 complexes contain the *drg*insertion, this insertion would be prevalent among the disease isolates recovered by the pandemic outbreaks caused by these clonal complexes \[[@B37],[@B38]\]; conversely, the spread of ST-11 meningococci \[[@B39],[@B40]\] would result in an increased prevalence of disease-associated meningococci without the *drg*insertion. This observation highlights the importance of assembling genetically defined isolate collections with a prescribed sampling frame if comparisons of meningococci with potential for virulence are to be undertaken. It has been suggested that the *drg*gene has been introduced into *Neisseria*from an exogenous source \[[@B30]\]. The difference in the G+C% content of the *drg*gene compared to the meningococcal housekeeping genes was consistent with this idea; however, the levels of diversity among the *drg*alleles, which was similar to that observed in the meningococcal housekeeping genes, along with the reported occurrence of *drg*in other *Neisseria*species \[[@B30]\], suggested that this was a relatively old event predating the emergence of present-day clonal complexes. The ratio of non-synonymous to synonymous substitutions (*d*~N~/*d*~S~), which was also similar to that observed in meningococcal housekeeping genes, suggested that the *drg*gene had been subject to stabilising selection for conservation of function since its putative introduction into the meningococcal population, although the possession of frame shift mutations in some of the *drg*alleles demonstrated that, as with *N. lactamica*, not all meningococci possessing the *drg*insertion expressed the endonuclease \[[@B30]\]. In other respects the distribution of the gene among clonal complexes and the patterns of nucleotide sequence variation observed were similar to those seen in meningococcal housekeeping genes, suggesting that it is subject to similar selection pressures \[[@B41]\]. The presence of multiple methylation systems is a characteristic of the *Neisseria*and may be related to the transformable nature of these organisms \[[@B42]\]. Whole genome comparisons of meningococci belonging to different clonal complexes frequently yield different restriction modification systems as the principal detectable genetic differences \[[@B43],[@B44]\]. It is attractive to speculate that the genetic isolation of clonal complexes may be promoted by such differences \[[@B45]\] and in this respect the insertion of *drg*in the *dam*locus, replacing a methylase with an endonuclease, may provide a potent barrier to genetic exchange from *dam*^+^to *drg*^+^meningococci; indeed, while there was evidence for the occasional horizontal genetic exchange of *drg*alleles among the genetically diverse meningococci that possess the insertion, there was no evidence for the transformational loss of *drg*from a given clonal complex among the isolates examined here. This concept of genetic isolation among lineages receives some support from the possession of *porB2*alleles by members of the *dam*^+^ST-8 and ST-11 complexes and *porB3*alleles by the clonal complexes with the *drg*insertion \[[@B9],[@B39]\], but the evidence for limitations in gene flow between these subgroups of meningococci is not at present conclusive. Conclusions =========== While it is possible that the possession of the *drg*insertion may influence meningococcal population structure, the data presented here do not support a direct association of this genotype with meningococcal virulence. Methods ======= Bacterial isolates ------------------ The 84 isolates used in this study (see [Additional file 1](#S1){ref-type="supplementary-material"}) were a subset of a collection of 107 diverse meningococci assembled to develop and validate the MLST scheme for *Neisseria meningitidis*\[[@B10]\]. The collection included representatives of the major hyper-invasive lineages and has been characterised at many genetic loci (full details of this isolate collection are available at <http://pubmlst.org/neisseria/>). Restriction digestion of chromosomal DNA ---------------------------------------- The methylation status of the chromosomal DNA of each isolate was determined by restriction digestion with the restriction endonucleases *Dpn*I, *Dpn*II, and *Sau*3AI, followed by separation of the digestion products by agarose gel electrophoresis. Each enzyme specifically recognises a cognate target site of GATC and cleaves this site depending on whether it is methylated or not: *Dpn*I cleaves this target sequence only when N^6^-methyladenine is present within the recognition sequence; *Dpn*II cleaves only unmethylated sites, and *Sau*3AI cleaves both methylated and unmethylated target sequences. PCR amplification and nucleotide sequence determination ------------------------------------------------------- The presence of the *dam*gene was determined by polymerase chain reaction (PCR) performed using *dam*specific primers, DamF1 (5\' -- TAAAATGGGCAGGCGGCA -- 3\') and DamB2 (5\' -- CGTAAGGGGGATCGCAAT -- 3\'). These amplify a 534 bp fragment from the 5\' end of the *dam*gene in *dam*+ strains but not from *dam*-strains. The presence of *drg*was determined by PCR using primers DrgF1 (5\' -- CATGAATTTATTTTTCGATA -- 3\') and DrgB2 (5\' -- AATTTGCAACTGTTGGCG -- 3\') that bind to *drg*internal sites and produce a 705 bp fragment in isolates containing the *drg*gene. PCR amplification was also performed using primer pairs Drg5F (5\' -- TGTCTAAAGAACTCAAAG -- 3\') / DrgB3 (5\' -- CGGTATCGAAAAATAAAT -- 3\') and Drg3F (5\' -- ATCCATCCAATTTCCCCA -- 3\') / DamB5 (5\' -- AAATGCCGTCTGAA -- 3\') based on *dam*and *drg*coding regions in order to confirm that dam inactivation was due to *drg*insertion. Amplicons corresponding to the *drg*gene were purified by precipitation with poylethylene glycol and sodium chloride as described previously \[[@B46]\] and their nucleotide sequences determined on both strands by cycle sequencing. BigDye™ terminators (ABI, Foster city, California) and the same primers as used for amplification were employed in the extension reactions and the labelled reaction products separated on an ABI 3700 automated DNA sequencer. Each fragment was sequenced at least once on each strand and the sequences assembled with the STADEN suite of computer programs. Each unique *drg*sequence was assigned an arbitrary allele number in order of discovery. Data analysis ------------- Percentage G+C was calculated using the program START \[[@B47]\]. Split decomposition analysis of the *drg*gene fragments was performed using SPLITSTREE, version 3.1 \[[@B48]\]. The proportion of non-synonymous to synonymous substitutions was calculated by the method of Nei and Gojobori \[[@B49]\] using the program MEGA2 \[[@B50]\]. Authors\' contributions ======================= KAJ carried out the nucleotide sequencing and analysis of the data. LS performed the PCR screening of the isolates. ERM and MCJM conceived of the study and participated in its design and coordination. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Bacterial isolates used in the study ::: ::: {.caption} ###### Click here for file ::: Acknowledegements ================= MCJM is a Wellcome Trust Senior Research Fellow. This work was supported by an award from the Wellcome Trust to MCJM (grant 055104). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Variable sites in *drg*gene fragments :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Splits graph of *drg*gene fragments.**The *drg*alleles found in the major *drg*^+^hyper-invasive lineages are annotated. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Genetic variation in MLST and *drg*loci ::: Locus No. of alleles (no./100 isolates) No. (%) of polymorphic sites dN/dS Average p-distance \%G+C -------- ----------------------------------- ------------------------------ ------- -------------------- ------- *abcZ* 15 (14) 75 (17.4) 0.05 0.052 51.1 *adk* 10 (9.4) 17 (3.7) 0.02 0.012 52.3 *aroE* 18 (16.8) 166 (34) 0.293 0.106 55.6 *fumC* 19 (17.8) 38 (8.2) 0.024 0.022 57.4 *gdh* 16 (15) 28 (5.6) 0.05 0.019 52.3 *pdhC* 24 (22.4) 80 (16.7) 0.07 0.054 55.9 *pgm* 21 (19.6) 77 (17) 0.121 0.050 54.2 *drg* 19 (31.1\*) 48 (7.4) † 0.11 0.021 42.4 \*only 61 isolates contained *drg* † excluding indels -- length of fragment: 650 :::

PubMed Central

2024-06-05T03:55:47.564547

2004-8-31

PMC516772

Background ========== DNA sequence periodicity with the period about 10--11 base pairs (bp) has been long known in eukaryotic DNA sequences. It was discovered recently in prokaryotic sequences as well \[[@B1]-[@B6]\]. The periodicity in Eubacteria sequences usually shows the period close to 11 bp \[[@B1]\]. This period is clearly different from the structural helical period of 10.5--10.6 bp/turn \[[@B7],[@B8]\]. The difference was interpreted \[[@B1],[@B2]\] as a possible reflection of the sequence dependent writhe of prokaryotic DNA. In the work \[[@B9]\] it was demonstrated that the periodicity in the bacterial genomes, in *E. coli*as well, is distributed in a non-uniform way, in scattered segments of the size 100--150 bases. It was also known for a long time that quite a few DNA promoter regions of *E. coli*possess the sequence periodicity of AA and TT dinucleotides \[[@B10]\]. The sequence periodicity of AA/TT dinucleotides is frequently associated with sequence-dependent DNA curvature, which is known to play an important role in the initiation of transcription of many genes (for reviews, see \[[@B11]-[@B15]\]). Using different models and approaches for prediction of intrinsic DNA curvature it was shown that many *E. coli*promoters have upstream curved sequences \[[@B16],[@B17]\]. Pedersen et al. \[[@B18]\] showed that promoter area frequently has an unusual sequence structure. This region possesses higher DNA curvature, more rigid and less stable. Moreover, in our study of prokaryotic terminators of transcription (Hosid and Bolshoy, submitted) we have found that in *E. coli*DNA curvature peaks are frequently located downstream of the CDS. Since the dinucleotide periodicity with the period close to the helical repeat is associated with DNA intrinsic curvature \[[@B19]-[@B23]\], the curvature distribution along DNA would suggest similar distribution of DNA sequence periodicity. In this work, the sequence dinucleotide periodicity in *E. coli*and its distribution along the genome are systematically analyzed. A strong preference of intergenic regions to express the sequence periodicity of AA, AC, GC, and TT dinucleotides is discovered. Results and Discussion ====================== Positional autocorrelation analysis of the nucleotide sequences is an appropriate tool to detect all major characteristic distances in the sequences, the periodicities in particular. The complete genome of *E. coli,*as well as its coding and noncoding regions, was subjected to this procedure. Resulting autocorrelation profiles for all 16 dinucleotides (data not shown) were further analyzed by Fourier transform. In Fig. [1](#F1){ref-type="fig"} the corresponding spectra are shown. The analysis demonstrates presence of the sequence periodicity of AA and TT dinucleotides with a period close to 11 bp mostly in intergenic regions, and weaker periodicity of AC and GC notably exclusively in intergenic regions. All 16 dinucleotides show periodicity of 3 bp, a well-known characteristics of the coding sequences, e.g. \[[@B24],[@B25]\]. Weak 2 bp periodicity of AT and TA is also observed in intergenic regions. It indicates, perhaps, presence of tandem ApT repeats. A weak 10 bp periodicity of GC in intergenic regions, probably, corresponds to terminator regions (work in progress). The amplitudes of the 11 bp periodicity of AA and TT are the highest, even comparable with 3 bp coding periodicity. We, thus, focused on AA and TT distributions. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Periodograms of the distance distributions of 16 dinucleotides in *E. coli*genome. The complete nucleotide sequence of *E. coli,*as well as subsets of its coding and noncoding regions, was subjected to the positional autocorrelation analysis for all 16 dinucleotides separately. Resulting autocorrelation profiles were after that analyzed by Fourier transform. The black lines correspond to the whole genome, the blue curves -- to the coding sequences, and the red curves -- to the noncoding sequences. :::  ::: To screen the genome of *E. coli*and find out where the periodical regions are located, we chose the period 11.2 bp \[[@B1],[@B2],[@B5]\] and this study (Fig. [1](#F1){ref-type="fig"}); and the window of 150 bp \[[@B9],[@B26]\]. We used periodical AA and TT probes with the above periodicity to correlate with the *E. coli*genome sequence and to detect the periodical sites. This calculation shows that the periodicity is not evenly distributed along the *E. coli*genome. In Fig. [2](#F2){ref-type="fig"}, the typical maps for several large segments of the *E. coli*genome are shown. The periodicity is distinctly located in certain regions. Many of the peaks observed are found to correspond to the intergenic regions (indicated by the black bars at the top). For example, two such peaks of periodicity in Fig. [2a](#F2){ref-type="fig"} correspond to the intergenic regions. Three such maxima are observed in Fig. [2b](#F2){ref-type="fig"}, three in Fig. [2c](#F2){ref-type="fig"}, and two in Fig. [2d](#F2){ref-type="fig"}. For the genome sections in Fig. [2](#F2){ref-type="fig"} about 2/3 of the intergenic regions are associated with the local periodicity. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Four examples of periodicity maps for fragments of *E. coli*genome. The maps were smoothed by running average with window 51 bp. The black bars on the top of the plot correspond to positions of intergenic regions. :::  ::: To verify the apparent strong correlation between the intergenic regions and AA/TT periodicity, we split intergenic regions in several families by size and analyzed the subsets separately by aligning (centering) the regions and summing up the respective local periodicity distributions. The combined maps for intergenic regions with a size from 50 to 150 bp, from 150 to 250 bp, from 250 to 350 bp, from 350 to 450 bp, and from 450 to 550 bp are shown in Fig. [3](#F3){ref-type="fig"}. This figure demonstrates, indeed, that intergenic regions are typically periodic, irrespective of the size. The average amplitudes of the observed periodicities -- 0.1--0.25 units -- are comparable with the amplitudes in Fig. [2](#F2){ref-type="fig"}, which indicates that, indeed a large proportion of the intergenic regions are periodical. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The averaged maps of periodicity are synchronized at the centers of intergenic regions and smoothed by a running average of 51 bp. Five families of the intergenic regions with different lengths are presented: a) 100 ± 50, bp 1073 sequences, b) 200 ± 50 bp, 602 sequences, c) 300 ± 50 bp, 319 sequences, d) 400 ± 50 bp, 160 sequences, and e) 500 ± 50 bp, 78 sequences. The black bars at the bottom of the each figure correspond to the average intergenic region. The gray bands around black dashed lines correspond to standard deviations around randomized background. :::  ::: To verify the choice of the period 11.2 bases, we calculated the periodicity maps for highly populated group of the regions of the size 200 ± 50 bp, by assuming different periods in the range 10.5--12.5 bases. The resonance 3D plot in Fig. [4](#F4){ref-type="fig"} indicates that the best-fit period is 11.3 ± 0.4 bp, which confirms earlier estimates of the *E. coli*DNA sequence periodicity. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### The 3D resonance plot for the intergenic regions of length 200 ± 50 bp. The maximum of resonance the plot corresponds to period 11.3 ± 0.4 bp. The contour around the maximum is also shown as a projection at the base line level. :::  ::: The spectral analysis (Fig. [1](#F1){ref-type="fig"}) and examples of the periodicity distribution maps (Fig. [2](#F2){ref-type="fig"}) show that apart from described correlation among the intergenic regions and AA/TT periodicity, there are numerous sites of periodicity located within coding sequences. Work is in progress to find out the functional relevance, if any, of these sites. Conclusions =========== The observed concentration of the sequence periodicity in the intergenic regions corroborates earlier results and suggests that the periodicity is a typical property of the intergenic regions. Methods ======= Genome data ----------- The sequence of the whole genome of *Escherichia coli*K-12 MG1655, locus U00096, 4639221 base pairs, was taken from the National Center of Biotechnology Information <ftp://ftp.ncbi.nih.gov/genbank/genomes>. Intergenic regions were identified in accordance with the annotation to this genome of *E. coli*and gathered in a separate dataset. Fourier transform of positional autocorrelation function -------------------------------------------------------- Autocorrelation profile *X*was calculated for each dinucleotide separately. For the calculation of ApA autocorrelation, for example, we calculated the number of occurrences of pairs ApA -- ApA in a distance *k*, and designated it by *X*~*k*~. Spectral analysis of autocorrelation profile *X*was obtained using the following formulae:  where *f*~*p*~is normalized wave-function amplitude of period *p*, *X*is an autocorrelation profile for one chosen dinucleotide, *X*~*i*~is its value in position *i*,  is its average value, and *W*is a maximal considered autocorrelation distance (in our case 100 bp). Sequence periodicity -------------------- As a probe of periodicity the sine waves with period *T*were taken to describe idealized periodical distribution of AA and TT dinucleotides within window *W*. The probes were correlated with *E. coli*sequences by moving the probes along the sequences and calculating the value *C*for every position.  where *i*is an index of a dinucleotide position in the window *W*and  The value *C*~max~is introduced for the normalization purposes. It is calculated as follows:  where *i*is a position in the window *W*and  Ideally periodical sequence segments would be, therefore, described by *C*= 1, while segments with no periodicity would correspond to *C*= 0. The results of these calculations are presented as maps of the sequence periodicity. The four sample maps are shown in Fig. [2a,2b,2c,2d](#F2){ref-type="fig"}. Synchronization of the maps --------------------------- The maps around intergenic regions were combined (summed) separately for the groups of similar sizes of the intergenic regions. Five such groups were analyzed: 100 ± 50 bp, 200 ± 50 bp, 300 ± 50 bp, 400 ± 50 bp, and 500 ± 50 bp. For each group the maps were synchronized at the respective intergenic centers and the sums of the maps were calculated and smoothed by a running average within 51 bp. The standard deviations for the combined plots were estimated by generating random sequences of the same size and dinucleotides composition for each group separately and averaging the respective periodicity maps. The resonance plot ------------------ The resonance 3D plot for the intergenic regions of length 200 ± 50 bp was built from calculations with different periods *T*in the interval 10--12.5 bp. One-third (202) of the most periodic maps of this group was taken for the calculation. The maps for different periods *T*were smoothed five times by a running average over 51 bp. The baselines were set to 0. The surface of 3D plot was smoothed 3 times by a running average over 9 point square elements, on the grid with separations 0.1 bp for *T*, and 20 bp for sequence position. Competing interests =================== None declared. Authors\' contributions ======================= SH carried out all graphics. ENT and AB participated in the design of the study and analysis of results. All authors drafted the manuscript. All authors read and approved the final manuscript. Acknowledgments =============== We thank V. Kirzhner and all members of the Genome Diversity Center for fruitful discussions and critical comments on the paper. A.B. is grateful to Professors T. Ratiu and J.H. Maddocks from the Bernoulli Institute at the Swiss Federal Institute of Technology for the kind invitation to visit \"Centre Bernoulli\" for two months. S.H. and A.B. are partially supported by the FIRST Foundation of the Israel Academy of Science and Humanities.

PubMed Central

2024-06-05T03:55:47.566232

2004-8-26

PMC516773

Background ========== Neurons in the mammalian pretectal nuclear complex (PNC) are involved in the control of various oculomotor reflexes, like the pupillary light reflex and the optokinetic reflex (OKR). Pupil constriction is controlled by neurons in the olivary pretectal nucleus that project bilaterally to the Edinger-Westphal nucleus \[[@B1]-[@B7]\]. Slow eye movements during OKR are generated by neurons in the nucleus of the optic tract (NOT) and in the adjacent dorsal terminal nucleus (DTN) of the accessory optic system (AOS) which project to the inferior olive (IO) and the nucleus prepositus hypoglossi \[[@B8]-[@B13]\]. In addition, PNC neurons carry signals related to saccadic eye movements to the dorsal lateral geniculate nucleus (LGNd) \[[@B14]-[@B16]\] and to the extrageniculate thalamus \[[@B17]\]. Other, reciprocal, projections connect the PNC to its contralateral counterpart, to the ipsilateral superior colliculus, and to the other AOS nuclei. The functions of those projections, however, are still under debate \[[@B18]-[@B23]\]. Each projection target receives input from an independent PNC neuronal population. Therefore, multiple retrograde tracing, e.g. from contralateral PNC and IO \[[@B24]\], or LGNd and Pulvinar \[[@B25]\], does not double label PNC cells. Furthermore, neurons with different projection targets show different response properties *in vivo*. Thus, neurons involved in the pupillary light reflex respond tonically to the overall retinal luminance \[[@B1],[@B3],[@B6],[@B26]\]. OKR-related neurons are directionally selective in response to slow movements of large visual stimuli \[[@B8],[@B27]-[@B33]\]. Finally, PNC neurons that project to thalamic visual centers only respond to fast moving visual stimuli without directional selectivity \[[@B15],[@B17],[@B25],[@B34]-[@B36]\]. Furthermore, activity patterns of visual responses also differ significantly between PNC cell populations. Thus, saccade-related PNC neurons show short high frequency activity bursts, while luminance neurons or OKR-related neurons exhibit tonic activity at moderate firing levels. Although such differences to some extent directly reflect the response properties of specific input systems, different intracellular properties might enforce activity patterns provided by different input systems. We therefore studied intrinsic properties of rat PNC cells *in vitro.*In particular*,*we describe a population of cells in the caudo-lateral PNC that is characterized by intrinsically generated spontaneous activity *in vitro*, which is an unusual property for neurons in a sensory relay structure. Results ======= In total, we obtained whole-cell recordings from 114 pretectal nuclear complex (PNC) neurons. Slices included the caudal part of the pretectum (Fig. [1](#F1){ref-type="fig"}), cells were recorded from the NOT, the posterior pretectal nucleus (PPN), and the olivary pretectal nucleus (OPN). Depolarizing current injections induced various spike patterns, like bursting (Fig. [2A,2B,2C](#F2){ref-type="fig"}), non-adapting regular spiking (Fig. [2D](#F2){ref-type="fig"}), or irregular spiking (Fig. [2E](#F2){ref-type="fig"}). Usually, increasing the current amplitude also increased the firing rate, however, in about 31% (n = 35) of the cells, the firing rate showed a clear maximum in response to intermediate depolarizing current injections and decreased upon further depolarization (Fig. [2C](#F2){ref-type="fig"}). Input resistances ranged from 201.0 to 776.3 MΩ (mean 410 ± 166.5 MΩ), resting potentials varied between -41.0 and -74.3 mV (mean -54.6 ± 8.5 mV). All cells tested, irrespective of the response type, responded to OT stimulation. Characteristics of spontaneous activity --------------------------------------- Of the cells recorded, 73 PNC neurons showed spontaneous firing at resting potential. Camera lucida reconstructions revealed that these cells were characterized by large fusiform cell bodies (diam. 15 μm and above) and multipolar dendritic trees that did not show any preference in their orientation. Whenever axons were also visible, they could be followed to leave the pretectal area in a ventro-lateral direction which indicates that these cells project to extrapretectal targets (arrowheads in Fig. [3A](#F3){ref-type="fig"}). Their dendritic morphologies, however, did not allow to distinguish spontaneously active cells from PNC neurons that were not spontaneously active. Physiologically, all spontaneously active PNC neurons were characterized by a regular firing pattern when recorded at resting potential without any injected current (Fig. [3C](#F3){ref-type="fig"}). The firing rate at resting potential of spontaneously active PNC cells varied between 0.9 and 9.4 imp/s (mean 3.0 ± 2.1 imp/s). Depolarizing current injections induced tonic firing patterns with only marginal adaptation (Fig. [3D](#F3){ref-type="fig"}). Responses to hyperpolarizing current injections showed no sign of inward rectification. Furthermore, following cessation of hyperpolarizing current steps we never observed rebound spikes. Spontaneously active PNC cells on average had significantly higher input resistances (mean 454.1 ± 164.7 MΩ, p \< 0.001), more positive resting potentials (mean -50.4 ± 7.0 mV, p \< 0.001) and lower spike thresholds (mean -55.0 ± 3.96 mV, p \< 0.001) than cells that did not show spontaneous activity (331.44 ± 137.1 MΩ, -58.4 ± 8.0 mV, and -40.66 ± 6.44 mV, respectively). In order to characterize the spike adaptation behavior of spontaneously active PNC cells, the holding potentials were increased in 5 mV steps by appropriate current injections in all recorded cells. In response to these depolarization steps, cells showed tonic increases of their firing rate without any sign of firing rate adaptation (Fig. [4](#F4){ref-type="fig"}). Also, no phasic firing rate increases were observed following the depolarizations. As could be already derived from current injections, the firing rate was directly correlated with the membrane potential. Increasing the membrane potential by positive current injections increased the firing rate until a maximum level was reached that could not be exceeded by further depolarization (Fig. [5A](#F5){ref-type="fig"}). Consequently, when the firing rate is plotted against the membrane potential, the course of the resulting function is sigmoidal (Fig. [5B](#F5){ref-type="fig"}). In order to get an impression about the regularity of the firing of spontaneously active PNC cells, interspike intervals (ISI) during maintained firing were analyzed in more detail (Fig. [6](#F6){ref-type="fig"}). Thus, maintained firing was recorded over a 10 s period at different membrane potentials and ISI histograms were generated from the recorded activity. ISIs obtained from these recordings followed a narrow unimodal Gaussian distribution with only little variation (Fig. [6B](#F6){ref-type="fig"}). According to the correlation between the firing rate and the membrane potential, depolarization of the cells resulted in shifts of the maximum of the Gaussian distribution towards lower ISI values. Depolarization, however, did not change the shape of the distribution. The regularity of the maintained firing of spontaneously active PNC cells is also supported by autocorrelograms of the recorded spike trains (Fig. [6C](#F6){ref-type="fig"}). The appearance of multiple equally spaced peaks in the autocorrelogram results from the regular timing of single spikes. Generation of spontaneous activity *in vitro* --------------------------------------------- In order to test whether the spontaneous activity of PNC neurons *in vitro*depends on excitatory input, we first suppressed glutamatergic synaptic transmission and pharmacologically blocked AMPA receptors in 13 spontaneously active PNC cells (Fig. [7](#F7){ref-type="fig"}). As a control for the effectiveness of AMPA receptor blockade, the influence of the AMPA receptor antagonist CNQX on postsynaptic responses was monitored. In all cells tested, bath application of 20 μM CNQX resulted in a complete loss of EPSCs after electrical stimulation of optic tract afferent fibers (Fig. [7A,7E](#F7){ref-type="fig"}). Although excitatory input was obviously blocked by CNQX application, the maintained firing remained unchanged (Fig. [7B,7F](#F7){ref-type="fig"}). In particular, no drop in the firing rate was observed that could have been induced by a possible loss of excitatory input. Furthermore, the comparison of both the ISI distribution (Fig. [7C,7G](#F7){ref-type="fig"}) and the autocorrelograms (Fig. [7D,7H](#F7){ref-type="fig"}) obtained from spike trains before and during CNQX application did not show any significant difference. Hence, both the generation of spontaneous activity and its patterning seem to be independent from excitatory input via AMPA receptors. Similar results were achieved when NMDA receptors were blocked by bath application of 50 μM APV or when 2 mM kynurenic acid was applied to simultaneously block AMPA and NMDA receptors (N = 19). After having excluded glutamatergic synaptic inputs as a trigger for maintained firing, we tried to remove all synaptic input by adding cobalt to the extracellular medium in 12 spontaneously active PNC cells. This blocks the influx of calcium into the presynaptic terminal and thus prevents vesicular neurotransmitter release. Adding 1.5 mM CoCl~2~to the bath completely suppressed all electrically evoked postsynaptic currents (Fig. [8A,8D](#F8){ref-type="fig"}) in all cells tested. In contrast to the complete loss of postsynaptic currents, however, the maintained firing always remained unchanged (Fig. [8B,8E](#F8){ref-type="fig"}). As during glutamate receptor blockade, no reduction of the firing rate was observed that could have indicated the removal of an excitatory input. In addition, no increase of the firing rate appeared that could have indicated a loss in tonic inhibitory input regulating maintained activity. Finally, examination of the ISI distribution in the spike trains demonstrated that the patterning of the maintained activity also did not show any significant difference in the presence of Cobalt (Fig. [8C,8F](#F8){ref-type="fig"}). This indicates that spontaneously active PNC cells generate their firing intrinsically without any external synaptic input. In current-clamp mode, each action potential was preceded by a depolarizing ramp (see, for example, Figs. [3C](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}). When cells were hyperpolarized to membrane potentials just below their resting potential single depolarizing ramps appeared that were not followed by an action potential. Concomitantly, in voltage-clamp mode, each unclamped action potential was preceded by an depolarizing inward current (Fig. [9A](#F9){ref-type="fig"}). Because they did not disappear after substitution of calcium by cobalt in the external solution these current ramps were calcium independent. However, when 1 μM tetrodotoxin (TTX), a selective blocker of sodium channels, was added to the bath solution current ramps were eliminated together with the action potentials (Fig. [9B,9C](#F9){ref-type="fig"}) in all seven cells tested. Discussion ========== We have examined neurons in the rat PNC that are characterized by maintained activity *in vitro*. These spontaneously active PNC cells do not differ in their dendritic morphology from PNC cells that are not spontaneously active, but they show higher input resistances, more positive resting potentials, and lower spike thresholds. Furthermore, our results indicate that, firstly, all PNC cells that display spontaneous activity share firing characteristics, such as very regularly patterned spike trains and pure tonic firing rate increases in response to intracellular depolarizations. Secondly, the generation of the maintained firing of these cells is independent from excitatory synaptic input which suggests that these cells exhibit specific intrinsic properties that underly the generation of spontaneous activity. Finally, the patterning of the maintained firing is also independent from synaptic input, both excitatory and inhibitory, which indicates that their intrinsic membrane properties determine the firing pattern. To our knowledge, this is the first demonstration of spontaneous activity generated *in vitro*by cells in a subcortical visual relay structure. Generation of spontaneous activity *in vitro* --------------------------------------------- Neurons that show spontaneous activity *in vitro*have been reported to exist in various mammalian CNS structures. Most extensively studied, spontaneously active neurons exist in the suprachiasmatic nucleus (SCN) which contains the biological clock that generates circadian rhythmicity. Thus, SCN cells not only generate spontaneous activity *in vitro*but they also maintain their circadian firing pattern \[[@B37]-[@B39]\]. Other populations of cells spontaneously active *in vitro*are the cholinergic interneurons in the neostriatum \[[@B40]\], dopaminergic cells in the substantia nigra \[[@B41],[@B42]\], neurons in the subthalamic nucleus \[[@B43],[@B44]\], neurons in the medial vestibular nucleus \[[@B45]-[@B47]\], cells from deep cerebellar nuclei \[[@B48],[@B49]\], and cerebellar Purkinje cells \[[@B48],[@B50],[@B51]\]. Within the visual system, particularly the subcortical portion, spontaneous activity *in vitro*has been described to occur in thalamocortical neurons \[[@B52]\] and in isolated dopanimergic cells from the retina \[[@B53]\]. However, in the mammalian PNC cells that show maintained activity *in vitro*have not yet been reported. These cells are characterized by very regular firing pattern and monotonically increasing firing rates in response to intracellular depolarization. They differed from PNC neurons not spontaneously active by higher input resistance and more positive resting membrane potentials. Because glutamate receptor blockade did not change the firing characteristics, neither the firing rate nor the patterning of the firing, excitatory synaptic input through glutamate receptors seems unnecessary for the generation of the spontaneous activity. Furthermore, maintaining spontaneous firing in these PNC neurons also seems to be independent from synaptic input through other neurotransmitter systems because blockade of synaptic transmission by bath application of Cobalt did not change the firing. Thus, we conclude that the firing pattern is neither shaped by tonic excitation nor by tonic inhibition. Similar to spontaneously active cells in the structures noted above, our PNC neurons must possess intrinsic membrane properties that allow the generation of maintained activity. As far as the ionic mechanisms underlying the generation of spontaneous activity are concerned our results suggest that it critically depends on a TTX-sensitive sodium conductance. This sodium conductance leads to a steady inward current following spike afterhyperpolarization which induces membrane depolarization to spike threshold. This is similar to the ionic mechanism that is responsible for spike generation in spontaneously active neurons in the suprachiasmatic nucleus \[[@B54]\]. Because spontaneous firing in PNC neurons was unchanged by calcium substitution with cobalt the spontaneous activity generation seems independent from calcium conductances. Possible functional implications -------------------------------- A characteristic response property of all spontaneously active PNC cells was that the firing rate increases to depolarizing voltage steps did not show any phasic components. This makes these cells perfectly suited to code maintained or tonic neuronal information. Of course, one has to keep in mind that neuronal response properties *in vivo*are shaped by numeous afferent input systems most of which are absent in the slice preparation. Thus, tonic inhibitory input could mask the maintained firing of spontaneously active PNC cells leading to a very different response pattern *in vivo*. Consequently, the maintained firing might become apparent only under very specific stimulus conditions upon withdrawal of the inhibitory input. However, from a functionl point of view, we regard it more reasonable to assume that PNC cells which are spontaneously active *in vitro*also exhibit tonic firing *in vivo.* Reviewing the known functions served by PNC neurons *in vivo*reveals only few reasonable suggestions for the possible functions which spontaneously active cells might accomplish. Thus, cells that typically show sustained activity *in vivo*are involved in the pupillary light reflex \[[@B2],[@B3],[@B6],[@B26],[@B55]\]. These cells are characterized by tonic increases of their firing rate to increases in the background luminance. However, these cells are predominantly found in the olivary pretectal nucleus (OPN) which is located in the rostro-medial PNC \[reviewed in \[[@B56]\]\]. Because our recordings were topographically restricted to the caudo-lateral PNC, particularly to the nucleus of the optic tract (NOT) and the posterior pretectal nucleus (PPN), it seems unlikely that we recorded from luminance neurons in the OPN. Neurons found in NOT and PPN include various functional cell populations. One of them has been associated with the generation of slow phase eye movements during OKR while others seem to transfer visual information linked to the execution of saccadic eye movements. Cells from these latter populations are all characterized by short duration, high frequency burst responses to fast image motions or rapid eye movements \[[@B15],[@B17],[@B25],[@B34]-[@B36],[@B57]\] and thus seem unlikely to correlate with cells that show maintained activity *in vitro*. Furthermore, because the timing of postsynaptic spikes with respect to their presynaptic input might be of considerable functional importance for saccade-related neurons such cells should exhibit lower input resistance than neurons for which spike time precision is less important. Low input resistances allow faster depolarization of the postsynaptic membrane and, hence, less temporal variance or \"jitter\" between presynaptic and postsynaptic spikes will occur. However, spontaneously active PNC cells on average showed higher input resistances in our sample and we therefore do not think that they represent saccade-related PNC neurons. On the other hand, cells that control compensatory eye movements during OKN are characterized by tonic firing *in vivo*when appropriately stimulated by low speed horizontal movements of whole field visual stimuli. In all mammals studied, neurons in the right PNC are excited by rightward stimulus motion and control eye movements to the right, while neurons in the left PNC are activated by leftward stimulus motion and control eye movements to the left \[[@B8],[@B27]-[@B33]\]. The response properties of OKN-related PNC neurons to a large extent reflect response characteristics of their retinal afferents which are also activated by slow stimulus movements and show strong directional selectivity \[[@B58]\]. However, it may be functionally important to assure a constant level of maintained activity in OKN-related PNC neurons by additional intrinsic mechanisms in the absence of appropriate visual stimuli. Unilateral inactivation of PNC neuronal activity by focal injections of muscimol or lidocaine leads to spontaneous eye movements in darkness \[[@B11],[@B59]\]. Because of the directional specificity in the PNC, inactivation of the right PNC elicits eye movements to the left, while inactivation of the left PNC elicits eye movements to the right. Thus, eye movements that appear after PNC inactivation seem to result from a distortion of a balanced activity between the two PNCs. Whenever the balance is distorted, premotor target structures postsynaptic to OKR-related PNC neurons receive stronger input from the PNC of one side and eye movements are elicited accordingly. It is therefore reasonable to assume that maintained activity spontaneously generated by OKR-related PNC assures this activity balance which is necessary for normal oculomotor function. In order to verify our hypothesis that OKR-related PNC neurons generate spontaneous activity that can be observed *in vitro*, it will be necessary to identify the postsynaptic targets of the spontaneously active PNC neurons. Conclusions =========== We have been able to demonstrate a specific population of neurons in the PNC that is capable of generating spontaneous activity *in vitro*. The spontaneous firing depends on a sodium conductance and is independent from afferent synaptic input. Although the postsynaptic target and, consequently, the functional role of the spontaneously active PNC cells remain to be determined it is reasonable to assume that these cells also show spontaneous activity *in vivo*. Therefore, one likely candidate to represent spontaneously active cells *in vivo*are PNC neurons that are involved in the generation of slow compensatory eye movements during optokinetic nystagmus. If this is true, spontaneous firing might help to maintain an activity balance between neurons in the right and in the left PNC and thus stabilize eye position in the absence of retinal image motion. Methods ======= Slice preparation ----------------- Acute brain slices were obtained from 3 to 6 week-old Long-Evans hooded rats of either sex that had been raised at the institute\'s own colony. All experimental procedures were in strict compliance with governmental regulations and in accordance with the *Guidelines for the Use of Animals in Neuroscience Research*of the Society for Neuroscience. Animals were deeply anesthetized with halothane and a subcutaneous injection of ketamine (100 mg/kg body weight) and thiazine hydrochloride (1 mg/kg), and transcardially perfused with ice-cold artificial cerebro-spinal fluid (ACSF) containing (in mM), NaCl 123, KCl 2.5, NaH~2~PO~4~1, NaHCO~3~26, MgSO~4~1.3, CaCl~2~1.8, glucose 11, that was continuously gassed with 5% CO~2~/ 95% O~2~. After the brain had been removed from the skull, 350 μ m-thick coronal slices were cut on a vibratome in ice cold ACSF. Three to four single slices that included the caudal PNC were obtained from each experimental animal. Slices were kept in ACSF at 36°C for at least one hour to allow recovery from the slicing procedure. For recording, they were transferred to a submerged type recording chamber where they were superfused at 3 ml/min with ACSF at 34°C during patch clamp experiments. Whole-cell patch clamp ---------------------- Whole-cell recordings from neurons in the caudo-lateral PNC were performed under visual guidance using infrared differential interference videomicroscopy \[[@B60]\]. For recording, borosilicate micropipettes (impedance 5--8 MΩ) were filled with internal solution composed of (in mM) potassium gluconate 130, sodium gluconate 5, HEPES 20, MgCl~2~4, Na~2~ATP 4, Na~3~GTP 0.4, EGTA 0.5, to which 0.5% biocytin was added for morphological single cell reconstruction. Measured membrane potentials were corrected for the junction potential of -10 mV. Postsynaptic responses were evoked with a concentric bipolar stimulation electrode (SNEX-100X, Rhodes Medical Instruments, Tujunga, CA) placed in the optic tract (OT) at the lateral PNC border. Electrical stimuli delivered were 0.5 to 2 mA in amplitude and had a duration of 100 to 500 μs. The neuronal signals were amplified and filtered using an EPC9 amplifier (Heka, Lambrecht, Germany), digitized at 20 kHz, and displayed, stored, and analyzed using PULSE/PULSEFIT software (Heka, Lambrecht, Germany). Unless otherwise stated, postsynaptic current responses evoked by OT stimuli were averaged over three consecutive stimulus applications. All drug effects are given as mean values ± standard deviation, they were statistically tested for significance using the Student\'s t-test. Drug delivery ------------- All drugs used were obtained from Sigma-Aldrich (Deisenhofen, Germany) and were bath applied. A10-minute application time proved sufficient to achieve stable responses. Application of 20 μM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) was used to block AMPA receptors. Either 50 μM APV or 2 mM kynurenic acid were used to block NMDA receptors. Na currents were suppressed by application of 1 μM tetrodotoxin (TTX). Histochemistry -------------- At the end of each recording session, slices were immersion fixed in 4% parafomaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C. After at least 24 h in fixative, slices were processed using standard histochemical techniques for visualization of biocytin with 3,3-diaminobenzidine (Sigma-Aldrich, Deisenhofen, Germany). Morphological reconstruction of stained cells was done with the aid of a camera lucida. Authors\' contributions ======================= NP participated in the design of the study and executed all aspects including data collection, analysis and drafting the manuscript. MS initially designed the study, assisted with data collection and analysis, and edited the manuscript. Both authors read and approved the final manuscript. Acknowledgements ================ We thank K. Engelhardt for participating in some of the experiments and M. Möllmann for excellent technical assistance. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 509, \"Neurovision\", TP A8). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic view of stimulation and recording sites. The box in the reconstructed section from the right midbrain in the left panel indicates the position of the PNC shown at higher magnification in the right panel (dorsal is up, medial is to the left). Neurons were recorded from the most dorsal and caudal nuclei of the PNC, the nucleus of the optic tract (NOT) and the posterior pretectal nucleus (PPN). No topographical segregation between spontaneously active cells (indicated by asterisks) and neurons without spontaneous activity (filled circles) was observed within the PNC. The stimulation electrode (black bar) was placed in the optic tract at the dorso-lateral border of the PNC medial to the lateral posterior thalamic nucleus (LP). Other abbreviations used, APN, anterior pretectal nucleus, OPN, olivary pretectal nucleus, LGN, lateral geniculate nucleus, MGB, medial geniculate body, SN, substantia nigra. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Different types of responses in PNC neurons evoked by depolarizing current injections. *A-C*, Whole-cell recordings from three individual PNC cells that showed burst firing in response to depolarizing currents. While the firing rate of cells in *A*and *B*monotonically increased with increasing currents, the cell in *C*only showed spikes in response to intermediate currents. *D*, Regular non-adapting spiking with monotonically increasing firing rates to increasing currents was observed in another population of cells. *E*, Responses from a PNC cell that showed irregular fast-adapting spiking. Although increasing currents in this cell decreased interspike intervals the total number of spikes elicited remained constant. Resting potentials of the cells were -61.6 mV, A, -62.5 mV, B, -61.2 mV, C, -58.4 mV, D, and -50.9 mV, E. Current amplitudes applied to each cell were 5, 10, 15, and 20 pA. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Morphological and physiological characteristics of PNC neurons with spontaneous activity *in vitro*. *A*, *B*, Reconstruction drawings of two biocytin-filled PNC neurons, insets indicate the cells\' position within the PNC. Horizontal lines mark the dorsal border of the PNC, arrowheads in *A*point to the cell\'s axon. *C*, in the absence of injected currents, spontaneous regular spiking was observed in this neuron. *D*, Responses to intracellular current injection identify this cell as regular non-adapting. *E*, continuous depolarization starting at a membrane potential of -90 mV revealed this cell\'s spike threshold at -66.4 mV. d, dorsal, m, medial, v, ventral, l, lateral. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Response to intracellular depolarization of spontaneously active PNC cells. Depolarizing current steps induce tonic firing increases in this spontaneously active PNC cell. No phasic component appears in the response to the depolarization step. This behavior was a characteristic property of all spontaneously active PNC cells. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Correlation between firing rate and membrane potential in spontaneously active PNC cells. *A*, Incremental intracellular depolarization leads to increasing firing rates without changing the regular firing pattern. *B*, When the firing rate is plotted as a function of membrane potential a sinusoidal curve appears that frequently saturates at membrane potentials above -20 mV. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Regularity of the firing pattern of spontaneously active PNC cells. *A*, Current clamp recording from a PNC cell at slightly depolarized membrane potential. *B*, The interspike interval histogram shows a narrow Gaussian distribution with only little variation of interspike intervals. *C*, The regularity of the firing can also be derived from the autocorrelogram which exhibits multiple equally spaced peaks due to the little variation in the interspike intervals. :::  ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Spontaneous activity of PNC is independent from tonic excitatory input. A single electric shock delivered to the optic tract lateral from the recorded neuron evokes a single peak EPSC (*A*) that completely disappears after AMPA receptor blockade by bath application of 20 μM CNQX (*E*). In contrast, the spontaneous firing rate recorded in control situation (*B*) does not change after AMPA receptor blockade (*F*). Removing excitatory input does also not change the regularity of the firing as derived from the distribution of interspike intervals (*C*vs. *G*) or from the autocorrelogram (*D*vs. *H*). :::  ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Tonic synaptic input does not contribute to the generation of spontaneous activity in PNC cells. Postsynaptic responses obtained by electrical optic tract stimulation (*A*) can also be blocked by bath application of 1.5 mM CoCl~2~(*D*). However, the spontaneous firing rate recorded in control situation (*B*) does not change after removal of synaptic input (*E*). Removing excitatory input does also not change the regularity of the firing as derived from the distribution of interspike intervals (*C*vs. *F*). :::  ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Spontaneous activity in PNC cells depends on a sodium conductance. In control solution (*A*) this cell showed regular firing pattern with depolarizing inward currents preceding each action potential. When 1 μM TTX was added to the bath the firing rate dropped at the beginning of TTX application (*B*). In the presence of TTX, both action potentials and depolarizing inward currents completely disappeared 5 min later (*C*). :::  :::

PubMed Central

2024-06-05T03:55:47.567749

2004-8-27

PMC516774

Background ========== ATP-sensitive K^+^(K~ATP~) channels are important regulators of cell function, coupling energy metabolism with electrical activity. K~ATP~channels are comprised of two proteins, derived from the sulphonylurea receptor (SUR) family and an inwardly rectifying K^+^channel (Kir6.x family), the exact composition of these being dependent upon tissue \[[@B1],[@B2]\]. For example, pancreatic β-cells and insulin-secreting clonal cell lines express K~ATP~channels consisting of Kir6.2 and SUR1 subunits \[[@B3]\]. K~ATP~channels are present in numerous tissues and are the target for drugs that inhibit or increase channel activity \[[@B4],[@B5]\]. The archetypal inhibitors of these channels are the sulphonylurea class of drugs, which bind to the SUR subunit of the channel. Modulation of K~ATP~channel activity in pancreatic β-cells has profound effects on insulin secretion and glucose homeostasis \[[@B6]\]. Sulphonylureas such as tolbutamide and glibenclamide inhibit channel activity, resulting in β-cell depolarization, increased electrical activity, enhanced calcium entry and consequently increased insulin secretion \[[@B7]\]. In contrast, pancreatic β-cell K~ATP~channel activation induces hyperglycaemia in animals and man \[[@B8]\]. This latter action is caused by membrane hyperpolarization, reduction in cell excitability and decreased intracellular calcium resulting in reduced secretion of insulin. Such effects have been reported following application of the benzothiadiazine, diazoxide, which has been used on occasion to treat persistent hyperinsulinemic hypoglycaemia of infancy \[[@B8]\]. It has been demonstrated that diazoxide interacts with the sulphonylurea receptor subunit, SUR1, encompassing transmembrane domains 6--11 and the first nucleotide binding fold \[[@B9]\]. A similar conclusion has also been reached using a novel diazoxide analogue \[[@B10]\]. The presence of K~ATP~channels in many other tissues, notably muscle and central neurons, has stimulated interest in the development of novel, selective K~ATP~channel openers for the treatment of various diseases \[[@B10],[@B11]\]. The *ob*gene product leptin has been demonstrated to activate K~ATP~channels in pancreatic β-cells \[[@B12]\] and insulin-secreting cell lines \[[@B13]\], consistent with a potential role in modifying insulin secretion \[[@B14]\]. One of the primary functions for this hormone is its role in the regulation of food intake and body weight \[[@B15]\]. Interestingly, leptin also activates K~ATP~channels of hypothalamic glucose-responsive neurones \[[@B16],[@B17]\] indicating a possible role for this channel in the control of energy homeostasis and body weight. In addition, Kir6.2 knock-out mice have deficits in central glucose sensing leading to loss of glucose mediated feeding response and a defective hypoglycaemic compensatory response \[[@B18]\]. These latter findings suggest that hypothalamic K~ATP~channels may also be an important target for drug manipulation with respect to centrally driven control of glucose and energy homeostasis. The aminoguanidine carboxylate, BVT.12777 (Figure [1](#F1){ref-type="fig"}), is one of a series of structurally related molecules based on the anti-diabetic/anti-obesity agent 3-guanidinopropionic acid \[[@B19]\], which, like leptin, have been demonstrated to reduce body weight in obese diabetic (*ob/ob*) mice \[[@B20]\]. Here we demonstrate that BVT.12777 opens K~ATP~channels in the CRI-G1 insulin secreting cell line, a useful model for pancreatic β-cells \[[@B21]\], and for analysing the mechanism by which leptin opens K~ATP~channels \[[@B13],[@B22],[@B23]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Structure of BVT.12777**(\[2-(hydrazinoiminomethyl)-hydrazino\] acetic acid) :::  ::: Results ======= BVT.12777 activates K~ATP~channels ---------------------------------- Under current clamp conditions with 5 mM ATP in the pipette solution to maintain K~ATP~channels in the closed state, the mean resting potential was -38.7 ± 1.7 mV (n = 10), similar to values reported in previous studies \[[@B13],[@B22]\] under these recording conditions. Application of BVT.12777 (100 μM) hyperpolarized CRI-G1 cells (Figure [2A](#F2){ref-type="fig"}) to -66.3 ± 2.7 mV (n = 10). Examination of the voltage-clamped macroscopic currents indicates that prior to the addition of BVT.12777 the slope conductance of the cells was 0.43 ± 0.03 nS (n = 10), and following exposure to BVT.12777 (100 μM), this increased to 3.45 ± 1.17 nS (n = 10). The reversal potential (obtained from the point of intersection of the current-voltage relationship) associated with the BVT.12777-induced conductance increase (Figure [2A](#F2){ref-type="fig"}) was -78.5 ± 0.8 mV (n = 10), close to the calculated value for E~k~of -84 mV in this system, indicating increased K^+^conductance. CRI-G1 cells responded to BVT.12777 in an all or none manner, with cells undergoing full hyperpolarization and increase in conductance, at all concentrations (100 -- 300 μM) examined. Such an effect has also been reported for leptin on CRI-G1 cells \[[@B13]\]. Removal of BVT.12777 from the bath solution did not fully recover the membrane potential and conductance to control values over the next 15--30 minutes (not shown). Application of the K~ATP~channel inhibitor, tolbutamide (100 μM) during BVT.12777 exposure (Figure [2A](#F2){ref-type="fig"}) completely reversed the BVT.12777-induced hyperpolarization and decreased conductance, to -41.0 ± 4.8 mV (n = 5) and 0.58 ± 0.07 nS (n = 5) respectively, values indistinguishable from control (P \> 0.05). These data indicate that BVT.12777 increases K~ATP~current in this cell line. This is demonstrated more clearly in cell-attached recordings from CRI-G1 cells, where bath application of BVT.12777 (100 μM) resulted in activation of single K~ATP~channel currents (Figure [2B](#F2){ref-type="fig"}; n = 7). The increase in channel activity was evident within 5 minutes of drug application, was sustained over the time course of exposure (\~30 minutes) and was not immediately reversed following removal of the drug. Figure [2C](#F2){ref-type="fig"} shows mean channel activity (N~f~.P~o~), normalised to the control for each recording, plotted against time of exposure to BVT.12777. BVT.12777 activation of K~ATP~channels was demonstrated to be reversibly inhibited by 100 μM tolbutamide (n = 4; Figure [2B,2C](#F2){ref-type="fig"}). Identical control experiments, in the absence of BVT.12777, resulted in no significant effect on K~ATP~channel activity, over a 30-minute test period (n = 8; P \> 0.05). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **BVT.12777 activates a tolbutamide-sensitive K+ current***A*, the upper trace shows a current clamp recording of a CRI-G1 cell following dialysis with a 5 mM ATP-containing solution. In this and subsequent current clamp figures the trace begins approximately 5 min after formation of the whole-cell configuration. Application of BVT.12777 (100 μM) for the time indicated hyperpolarized the cell from -50 mV to -76 mV, an action readily reversed by tolbutamide (100 μM), which returned membrane potential to -54 mV. Washout of all drugs from the bath resulted in a membrane potential of -70 mV, indicating the lack of reversibility of BVT.12777. The lower plot is the current-voltage relationship for the voltage clamped currents. Cells were voltage clamped at -50 mV and 10 mV steps of 100 ms duration were applied every 200 ms (range -120 to -30 mV). BVT.12777 increased the membrane conductance relative to control and tolbutamide reversed this BVT.12777-induced conductance increase with a reversal potential of -78 mV. *B*, cell-attached recording from a CRI-G1 cell, at 10 mV applied to the recording pipette. Single channel openings are shown as downward deflections. Addition of 100 μM BVT.12777 induced an increase in channel activity (N~f~.P~o~) from 0.17 in control to 0.31, and 1.25 at 10 and 20 minutes respectively, after BVT addition. Application of 100 μM tolbutamide induced a substantial inhibition of activity (to 0.02), which was reversed on washout of all drugs, with activity increasing to 0.74. The symbol C refers to the closed state of the channel in this and subsequent figures. *C*, diary plot of N~f~.P~o~against time from cell-attached experiments in the presence and absence of BVT.12777, where channel activity was calculated every 2 minutes. Each point is the mean of 4--7 separate determinations. :::  ::: The effect of BVT.12777 on K~ATP~channel activity in excised membrane patches was also examined. Recordings were made from inside-out patches in symmetrical (140 mM KCl in pipette and bath solutions) K^+^at a membrane potential of -40 mV. K~ATP~channels were identified by inhibition of channel activity following application of 100 μM MgATP to the inner membrane aspect of the patch, which reduced normalised N~f~P~o~from 1.0 to 0.23 ± 0.05 (n = 4; P \< 0.05). Subsequent application of 100 μM BVT.12777, in the continued presence of MgATP, induced a gradual increase in K~ATP~channel activity (Figure [3](#F3){ref-type="fig"}), to levels similar to that of control (in the absence of MgATP). For example 15 minutes after 100 μM BVT.12777 application normalised mean channel activity had recovered to 1.18 ± 0.46 (n = 4). In experiments where no drug was added, K~ATP~channel currents, in the presence of 100 μM MgATP, did not activate spontaneously (n = 4). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **BVT.12777 activates K~ATP~channels in inside-out patches**Continuous single channel currents recorded from an inside-out patch at a holding potential of -40 mV. Application of 100 μM MgATP reversibly inhibited channel activity by \>90%, demonstrating K~ATP~identity. Addition of 100 μM BVT.12777, in the presence of 100 μM MgATP to the cytoplasmic aspect of the patch resulted in K~ATP~channel activation. N~f~.P~o~values were 2.96 (control, after first MgATP challenge), and 0.25 in the presence of MgATP, which increased to 0.72, 1.06 and 2.74 at 5, 10 and 20 minutes respectively, after BVT.12777 addition. :::  ::: BVT.12777 activates K~ATP~channels independently of PI 3-kinase activity ------------------------------------------------------------------------ Leptin and diazoxide hyperpolarized CRI-G1 cells, in a manner similar to that of BVT.12777 (data not shown). Leptin (10 nM) induced a hyperpolarization from a mean membrane potential of -47.6 ± 1.6 mV to -68.5 ± 1.9 mV (n = 8; P \< 0.05), and application of tolbutamide (100 μM) reversed this action, returning the membrane potential to -47.5 ± 1.9 mV (n = 4). Diazoxide (200 μM) rapidly hyperpolarized CRI-G1 cells from a mean membrane potential of -49.9 ± 1.7 mV to -74.0 ± 1.5 mV (n = 6; P \< 0.05), with tolbutamide (100 μM) also reversing this action, returning membrane potential to -46.9 ± 3.8 mV (n = 6). Leptin, but not diazoxide activation of CRI-G1 K~ATP~channels is PI3K dependent \[[@B22],[@B23]\]. Thus, we investigated whether BVT.12777 activates K~ATP~channels in CRI-G1 cells by direct (like diazoxide) or indirect (like leptin) mechanisms. Pre-incubation of CRI-G1 cells (20 min) with inhibitors of PI 3-kinase, wortmannin (10 nM) or LY294002 (10 μM) had no significant effect on the mean resting membrane potential or slope conductance of CRI-G1 cells and did not prevent BVT.12777 from causing hyperpolarization and increased cell conductance (Figure [4A](#F4){ref-type="fig"}). In the presence of 10 nM wortmannin, values for mean membrane potential and slope conductance were -44.3 ± 1.2 mV (n = 6) and 0.86 ± 0.10 nS (n = 5), and addition of 200 μM BVT.12777 hyperpolarized cells to -68.9 ± 0.8 mV (n = 6) with an increase in slope conductance to 3.10 ± 0.38 nS (n = 5). Identical results were obtained in the presence of 10 μM LY294002 (data not shown), with corresponding control values of -40.8 ± 2.8 mV (n = 6) and 0.79 ± 0.11 nS (n = 4), and in the presence of 200 μM BVT.12777, -67.9 ± 0.6 mV (n = 6) and 2.69 ± 0.35 nS (n = 4) for membrane potential and slope conductance respectively. In all experiments (i.e with either PI3K inhibitor) addition of tolbutamide (100 μM) recovered the membrane potential (-41.8 ± 1.5 and -34.0 ± 1.7 mV; n = 6) and slope conductance (0.89 ± 0.11 (n = 5) and 0.58 ± 0.06 (n = 4) nS) for wortmannin and LY294002 respectively, to values indistinguishable from controls (P \> 0.1). Cell-attached recordings from CRI-G1 cells also show that wortmannin (10 -- 100 nM) did not occlude BVT.12777 activation of K~ATP~channels (Figure [4B](#F4){ref-type="fig"}). Mean channel activity in the presence of wortmannin (10 nM) was 0.02 ± 0.00 which increased to 0.16 ± 0.02, 20 minutes after exposure to 100 μM BVT.12777 (n = 3; P \< 0.05). Control experiments where no BVT.12777 was added show no change in channel activity over a 30-minute period (N~f~.P~o~= 0.01 ± 0.00 and 0.04 ± 0.00 after 5 and 30 minutes respectively; n = 4). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Wortmannin does not inhibit BVT.12777 activation of K~ATP~***A*, current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP, following exposure of cells to 10 nM wortmannin for 15--20 minutes. Application of BVT.12777 (200 μM), in the continued presence of wortmannin hyperpolarized the cell from -46 to -77 mV. Tolbutamide (100 μM), applied after the BVT-induced hyperpolarization, recovered the membrane potential (to -40 mV). *B*, cell-attached recordings from CRI-G1 cells, following exposure of cells to 10 nM wortmannin for 15--20 minutes. Upper trace; in the continued presence of wortmannin, N~f~.P~o~was 0.01 and 0.03 after 5 and 30 minutes respectively. Lower trace, application of BVT.12777 (100 μM) to cell-attached recording in the presence of 10 nM wortmannin resulted in K~ATP~activation, with N~f~.P~o~values of 0.01, 0.12 and 0.27 prior to, and 10 and 30 minutes after, BVT.12777, respectively. Addition of 100 nM wortmannin did not inhibit channel activity. :::  ::: Heterologously expressed K~ATP~currents are not activated by BVT.12777 ---------------------------------------------------------------------- Oocytes injected with Kir6.2 and SUR1 cRNAs were challenged with sodium azide (3 mM) to elicit a reversible increase in current, which was completely blocked by 1 μM glibenclamide or 0.5 mM tolbutamide, indicating that the current was due to K~ATP~activation, as described previously \[[@B24],[@B25]\]. In oocytes, previously exposed to sodium azide in order to verify Kir6.2-SUR1 expression, application of BVT.12777 (10 μM -- 1 mM) did not produce any consistent increase in K~ATP~current (n = 16; data not shown). Consequently, we utilized an alternative expression system, the HEK 293 cell line \[[@B25]\]. Application of BVT.12777 (100 μM) to the bathing solution using the cell-attached recording configuration resulted in no significant increase in mean channel activity above control levels over a 30-minute period, although subsequent addition of sodium azide (3 mM) did cause a rapid increase in channel activity, which was reversed by the addition of 100 μM tolbutamide (n = 4, data not shown). Similarly, application of BVT.12777 in the presence of 0.1 mM MgATP to inside-out patches from HEK 293 cells transiently expressing Kir6.2-SUR1, did not cause activation of channel activity following 30 minutes exposure (n = 4; data not shown). Thus BVT.12777 does not appear to be capable of activating heterologously expressed Kir6.2-SUR1 currents. MAPK does not mediate BVT.12777-activation of K~ATP~ ---------------------------------------------------- Exposure of CRI-G1 cells to BVT.12777 (100 μM) for up to 30 minutes had no consistent effect on the phosphorylation of enzymes downstream of PI3K (PKB and its downstream target, GSK3), but did increase the phosphorylation of STAT3 (n = 4) and MAPK (n = 4; data not shown). These data are in agreement with the lack of BVT.12777 sensitivity to PI3K inhibitors on activation of K~ATP~channels. However, activation of MAPK has been implicated as a significant intermediate for both insulin and leptin signalling pathways in various cell types \[[@B26]-[@B29]\]. Thus, we examined the effect of UO126, a potent and specific inhibitor of the activation of the classical MAPK cascade \[[@B30]\], on BVT.12777 opening of K~ATP~channels. Application of UO126 (25 μM) inhibited approximately 90 % of K~ATP~channel activity in cell-attached or inside-out recordings, whereas 1--10 μM UO126, concentrations that suppresses activation of MAPKK \[[@B30]\], had no significant effect on channel activity (data not shown). Control cell attached recordings had a mean channel activity of 0.07 ± 0.02, which increased to 1.29 ± 0.82 (n = 3) in the presence of BVT.12777 (100 μM). Subsequent application of UO126 (1 μM) in the continued presence of BVT.12777 did not alter channel activity (data not shown), over a 15-minute period (N~f~.P~o~was 1.86 ± 1.45 (n = 3) and 2.44 ± 2.00 (n = 3), at 5 and 15 minutes respectively; P \< 0.05). In addition, increasing UO126 to 10 μM had no effect on BVT.12777 induced K~ATP~channel activation. BVT.12777 activation of K~ATP~channels is dependent on actin cytoskeleton dynamics ---------------------------------------------------------------------------------- Leptin activation of K~ATP~channels in the CRI-G1 cell line is dependent upon reorganisation of the cytoskeleton, a process downstream from PI3K activation \[[@B31]\]. Therefore, we examined whether BVT.12777 opening of CRI-G1 K~ATP~channels occurs through alteration of actin filament dynamics. For this series of experiments the heptapeptide mushroom toxin phalloidin \[[@B32]\] was used to stabilise the polymerised form of actin (F-actin). As phalloidin is membrane-impermeant, it was directly applied to the internal aspect of the cell membrane. In whole-cell experiments, 10 μM phalloidin was added to the electrode solution and allowed to dialyse into the cell. The mean resting potential and slope conductance were -38.0 ± 0.6 mV and 0.66 ± 0.04 nS (n = 4) respectively, and following addition of 200 μM BVT.12777 no significant change in these parameters was observed (Figure [5A](#F5){ref-type="fig"}), with a mean membrane potential of -41.7 ± 1.1 mV and slope conductance of 0.60 ± 0.08 nS (n = 4; P \> 0.05). The presence of phalloidin (10 μM) in the bath solution also prevented K~ATP~channel activation by BVT.12777 in the inside-out isolated patch configuration (Figure [5B](#F5){ref-type="fig"}). Application of 0.1 mM MgATP to the cytoplasmic aspect of inside-out patches caused 97.5 ± 2.1% inhibition of K~ATP~channel activity (n = 3; P \< 0.05) and subsequent addition of 10 μM phalloidin had no further effect, as reported previously \[[@B29]\]. Subsequent addition of BVT.12777 (100 μM) failed to increase K~ATP~channel activity, with mean Nf.Po values of 0.06 ± 0.05 and 0.03 ± 0.01 in the absence and presence of BVT.12777 respectively (n = 3; P \> 0.05). In contrast, the direct K~ATP~channel opener, diazoxide activates K~ATP~channels in the presence of phalloidin. In whole-cell experiments (Figure [5C](#F5){ref-type="fig"}), diazoxide (200 μM) hyperpolarized CRI-G1 cells from a mean membrane potential of -42.6 ± 0.1 mV to -70.1 ± 0.8 mV (n = 4; P \< 0.05), and increased slope conductance from 0.87 ± 0.23 to 7.39 ± 0.72, actions reversed by tolbutamide (100 μM). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Phalloidin prevents BVT.12777 activation of K~ATP~***A*, current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP and 10 μM phalloidin. Application of BVT.12777 (200 μM) had no effect on the membrane potential of the cell (-41 mV) in the presence of phalloidin. *B*, continuous single channel currents recorded from an inside-out patch at a holding potential of -40 mV. Application of 100 μM MgATP reversibly inhibited N~f~.P~o~from 1.25 to 0.02. Addition of 10 μM phalloidin and subsequently 100 μM BVT.12777, in the presence of 100 μM MgATP, to the cytoplasmic aspect of the patch resulted in no effect on K~ATP~, with N~f~.P~o~values of 0.01 and 0.03 respectively. *C*, current clamp record of a CRI-G1 cell dialysed with 5 mM MgATP and 10 μM phalloidin. Application of diazoxide (200 μM) induced rapid cell membrane hyperpolarization, from -55 to -72 mV, an action reversed (to -45 mV) by tolbutamide (100 μM). :::  ::: F-actin is disrupted by BVT.12777 --------------------------------- The prevention of BVT.12777-induced K~ATP~activation by phalloidin mirrors the effect of this toxin on leptin activation of K~ATP~\[[@B31]\]. Thus, we visualised F-actin by staining with rhodamine-conjugated phalloidin. In untreated CRI-G1 cells there was pronounced phalloidin-positive labelling of the cell membrane, with more diffuse, granular staining within the cytoplasm (Figure [6A](#F6){ref-type="fig"}). In contrast, cells treated with BVT.12777 (100 μM) or leptin (10 nM) for 40 min showed a marked reduction in phalloidin fluorescence intensity, with disjointed labelling at the cell membrane (Figure [6A](#F6){ref-type="fig"}). The actin filament disrupter cytochalasin B \[[@B33]\] also reduced the intensity of phalloidin labelling but in a more punctate manner on visualisation of treated cells compared with controls (data not shown). Analysis of the mean fluorescence intensity at the cell membrane following the actions of BVT.12777 and leptin demonstrated that both treatments caused a significant reduction of the intensity of rhodamine-phalloidin labelling, by 43.0 ± 4.2% (n = 6; P \< 0.05) and 62.2 ± 6.0% (n = 6; P \< 0.05), respectively, compared to untreated cells (Figure [6B](#F6){ref-type="fig"}). However, the directly acting K~ATP~channel opener, diazoxide did not cause disruption of the actin cytoskeleton (Figure [6A,6B](#F6){ref-type="fig"}), with a relative intensity of rhodamine-phalloidin staining of 0.98 ± 0.16 (P \> 0.05). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **BVT.12777 disrupts the actin cytoskeleton***A*, images of rhodamine-conjugated phalloidin fluorescence in CRI-G1 cells in control conditions and following incubation with leptin (10 nM), BVT.12777 (100 μM) or diazoxide (200 μM) for 30 minutes. All panels show representative X-Y images. Note the marked reduction in phalloidin staining in cells pre-treated with leptin or BVT.12777, and not diazoxide. Scale bars are 50 μm. *B*, histogram comparing the normalised fluorescence intensity relative to control in the membrane periphery of randomly selected CRI-G1 cells for each condition; (control (n = 13; cells = 195), 10 nM leptin (n = 6; cells = 90), BVT.12777 (n = 6; cells = 90) and 200 μM diazoxide (n = 4, cells = 60). Error bars indicate s.e.m. and \* significance of P \< 0.001. :::  ::: Discussion ========== BVT.12777 induced hyperpolarization of CRI-G1 cells, with an associated increase in K^+^conductance, an action likely caused by the activation of K~ATP~channels, as the sulphonylurea tolbutamide completely reversed its effects. Cell-attached and inside-out single channel current recordings demonstrate directly that BVT.12777 activates K~ATP~channels. The increased K~ATP~current generated in isolated membrane patches resembles the effects of K~ATP~activators such as diazoxide \[[@B34]\] and sodium azide \[[@B35]\], which have also been shown to activate insulinoma or pancreatic β-cell K~ATP~channels in isolated patches in the presence of Mg-ATP. Thus, although not tested here, BVT.12777 as an activator of K~ATP~would be expected, as observed for diazoxide, to inhibit insulin release from CRI-G1 cells stimulated by metabolizable substrates or tolbutamide \[[@B36]\], although this would clearly be dependent on its action on other β-cell conductances, notably calcium channels. BVT.12777 activation of K~ATP~channels was only slowly reversed on withdrawal of the drug, unlike the actions of diazoxide or sodium azide, which are rapidly reversed on washout \[[@B35],[@B36]\]. Indeed, following removal of BVT.12777 in the absence or presence of tolbutamide, enhanced K~ATP~channel activity was apparent for a considerable time. The slow reversibility on washout of BVT.12777 resembles the effects of the hormone leptin on CRI-G1 cell membrane potential and K~ATP~channel activation \[[@B13]\]. Leptin, via activation of the main signalling form of the leptin receptor (ObRb), has been shown to increase the phosphorylation of STAT3, MAPK and to stimulate PI3K pathways in various peripheral tissues, cell lines \[[@B37]\], and in hypothalamic neurones \[[@B38]\]. BVT.12777 although stimulating phosphorylation of STAT3 and MAPK did not stimulate PI3K dependent pathways as demonstrated by the lack of effect on the phosphorylation status of the PI3K output indicators, PKB and GSK3. It is unclear at present how this molecule induces STAT3 and MAPK phosphorylation. As K~ATP~activation by BVT.12777 is rapid and occurs in isolated membrane patches it is unlikely that any JAK-STAT pathway (which drives changes in transcription) contributes to this action. Leptin activation of K~ATP~channel currents in CRI-G1 cells has previously been shown to be independent of MAPK, but prevented by the inhibitors of PI3K \[[@B22]\]. However, BVT.12777 activation was not only insensitive to inhibition by the MAPKK inhibitor, UO126, it was also insensitive to the presence of the PI3-kinase inhibitors, wortmannin and LY294002, at concentrations sufficient to prevent leptin activation of K~ATP~in this cell line. These data led us to suspect that BVT.12777, irrespective of its ability to initiate various signalling cascades in this cell line, increased K~ATP~channel activity by a more direct effect on the channel subunits in a manner analogous to diazoxide, which is purported to interact directly with the SUR1 subunit \[[@B9],[@B10]\]. This possibility was tested by heterologous expression of the β-cell subunits of K~ATP~channels, Kir6.2 and SUR1, in Xenopus oocytes, a commonly utilised expression system for electrophysiological studies of these recombinant channels \[[@B24],[@B25]\]. However, BVT.12777 did not activate Kir6.2-SUR1 currents in oocytes, demonstrated to express functional K~ATP~channel currents. Thus we explored this question further by utilising a second heterologous expression system for Kir6.2-SUR1, HEK293 cells. Recordings from inside-out patches demonstrated that BVT.12777 did not activate Kir6.2-SUR1 currents in the presence of Mg-ATP, in contrast to diazoxide \[[@B39]\] or sodium azide \[[@B35]\]. Overall these data strongly suggest that expression of the K~ATP~channel subunits, Kir6.2 and SUR1 are insufficient *per se*to bring about sensitivity to BVT.12777, and indicate that this opener may activate this channel type by an indirect mechanism (which is not available in oocytes or HEK cells). Although the activation of K~ATP~channels by leptin in CRI-G1 cells is PI3-kinase dependent the lipid products of this enzyme system, such as PtdIns(3,4,5)P~3~also do not interact directly with K~ATP~channels \[[@B22]\]. Recent studies demonstrate that both leptin and PtdIns(3,4,5)P~3~increase K~ATP~channel activity indirectly, through changes in cytoskeletal dynamics \[[@B31]\]. It is well established that many ion channels and transporters are anchored in the membrane by either direct or indirect association with the cytoskeleton. In addition, there is growing evidence that altering the integrity of cytoskeletal elements, in particular actin filaments, can modulate the activity of a variety of ion channels \[[@B40]\] and receptors \[[@B41]\]. For example, disruption of actin filaments with cytochalasin is shown to increase K~ATP~channel activity in cardiac myocytes \[[@B42]\] and CRI-G1 cells \[[@B31]\]. Indeed, a number of lipid kinases, including PI 3-kinase, are also localised to the cytoskeleton and their activities are modulated by a variety of cytoskeletal proteins, especially those associated with actin \[[@B40]\]. Actin filament structure is controlled by reversible polymerisation of G-actin, which forms F-actin, and this process is under the dynamic control of various actin-binding proteins \[[@B43]\]. The heptapeptide mushroom toxin phalloidin \[[@B32]\] binds to filamentous F-actin with high affinity and stabilises the actin in this form. The addition of phalloidin to the intracellular aspect of CRI-G1 cells prevented BVT.12777, but not diazoxide, from activation of K~ATP~channel currents in whole cell and inside out recording configurations indicating that this molecule likely causes the opening of K~ATP~channels by a membrane delimited alteration of cytoskeletal dynamics. This mechanism of action is identical to that proposed for leptin and PtdIns(3,4,5)P~3~activation of K~ATP~in this cell line \[[@B31]\]. Fluorescence staining of CRI-G1 cells with rhodamine-conjugated phalloidin revealed disassembly of actin filaments by both BVT.12777 and leptin, but not diazoxide. These data provide direct support for an important role for cytoskeletal dynamics in the control of K~ATP~channel activity by both leptin and BVT.12777. The lack of effect of diazoxide on the actin filament structure is also supportive of this opener acting directly on the K~ATP~channel subunits. Conclusions =========== BVT.12777 activation of K~ATP~channels in CRI-G1 cells was evident regardless of whether it was applied to the external or internal surface of the cell. BVT.12777 signalling to K~ATP~channels is not mediated by PI 3-kinase or MAPK, but does appear to depend on actin filament re-modelling. As leptin hyperpolarizes a sub-population of hypothalamic neurones by opening K~ATP~channels \[[@B16]\], it is feasible that at least part of the anti-obesity action of BVT.12777 may be through the activation of this potassium channel. Furthermore, as BVT.12777 acts downstream of PI3K, such an agent may act to overcome the putative central leptin resistance associated with the obese state \[[@B37]\]. Thus, although BVT.12777 and its close structural analogues are unlikely *per se*to be useful anti-obesity agents as they display hepatotoxicity \[[@B44]\], understanding the general principles underlying their mechanism of action may reveal clues for future anti-obesity drug development. Methods ======= Cell culture and transfection ----------------------------- Cells from the insulin secreting cell line, CRI-G1, and the human embryonic kidney cell line, HEK 293, were grown as described previously \[[@B25],[@B35]\]. The preparation of mouse Kir6.2 (provided by Professor F. Ashcroft, University of Oxford), rat SUR 1 (provided by Dr G. Bell, University of Chicago) and CD4 cDNAs and transfection procedures were as described by \[[@B25]\]. Transfected cells were selected by visible binding of anti-CD4 coated beads (Dynal, Oslo) following incubation with the beads for 20 min. Oocyte collection and preparation --------------------------------- Ovarian lobes were removed from mature female *Xenopus laevis*frogs (Blades Biological, UK) following killing of the animal by destruction of the brain. The use of animals was in accordance with the Home Office Animals (Scientific Procedures) Act (1986) and approved by the local ethics committee. Separation and selection of oocytes and the preparation and injection of cRNAs were performed as described by \[[@B25]\]. Western blotting ---------------- CRI-G1 cells, in normal saline (containing in mM; NaCl 135, KCl 5, MgCl~2~1, CaCl~2~, 1, HEPES 10 with glucose 10 (pH 7.4) were treated with BVT.12777 (100 μM) for 0, 1, 5, 15 or 30 minutes and whole-cell extracts were prepared as described \[[@B23]\]. Proteins (10 μg) were suspended in loading buffer (Invitrogen) and after denaturation, loaded on to NuPage 4--12% Bis-Tris mini-gels (Invitrogen) and run at 200 V for 1 hr. Subsequently, proteins were transferred to Hybond-C Extra nitrocellulose membranes (Amersham) at 25 V for 80 minutes at room temperature. Membranes were incubated in blocking buffer (5% non-fat milk in TBST (20 mM Tris HCl, 150 mM NaCl, 0.5% Tween, pH 7.4)) for 1 hr at room temperature after which antibodies to phospho-MAPK, phospho-STAT3, phospho PKB, phospho-GSK3 and PKB (all at 1:1000) were applied at 4°C with gentle shaking, overnight. The membranes were washed with TBST (4 × 30 minutes) and incubated for 1 hr at room temperature with HRP conjugated ImmunoPure goat anti-rabbit IgG (1:5000). After washing with TBST (5 × 15 minutes), immunoreactive bands were visualised by the enhanced chemiluminescence (ECL) detection reagent (Amersham). Cytoskeletal fluorescence imaging and analysis ---------------------------------------------- CRI-G1 cells were gently washed in normal saline (containing in mM): NaCl 135, KCl 5, MgCl~2~1, CaCl~2~1, HEPES 10, pH 7.4, and incubated for 40 min with either 100 μM BVT.12777, 10 nM leptin, 200 μM diazoxide or 3 mM sodium azide for 30 min with the cytoskeletal disrupter, cytochalasin B (10 μM). Cells were then fixed, permeabilised, stained with rhodamine-conjugated phalloidin (2.66 U ml^-1^) and visualised using a BioRad Microradiance, confocal imaging system as described by \[[@B31]\]. The intensity of rhodamine-conjugated phalloidin staining in the plasma membrane was determined using BioRad Lasersharp processing software (Bio-Rad, CA, USA). Analysis lines were drawn along randomly selected regions of the plasma membrane and the fluorescence intensity determined. A histogram giving the mean fluorescence intensity was constructed for a minimum of 5 cells on each stimulated or control dish on at least 3 separate occasions. Within a given experimental series all conditions for capturing images were constant. In order to allow for quantification of experimental data obtained on separate days, the results were normalised relative to the mean plasma membrane fluorescence measured in the control cells for each day and presented as mean ± S.E.M. Statistical analyses were performed using Student\'s unpaired *t*test. p \< 0.05 was considered significant. Electrophysiological recording and analysis ------------------------------------------- Whole cell currents from Xenopus oocytes were measured using a two-electrode voltage clamp technique as described by \[[@B25]\]. Recordings were made in a high-potassium bath solution, KD96 containing (mM): KCl 96, NaCl 2, CaCl~2~1.8, HEPES 5 (pH 7.4 with KOH). Working concentrations of drugs were prepared in KD96 and superfused into the bath. Whole-cell current-clamp recordings with excursions to voltage clamp mode were used to monitor membrane potential and macroscopic currents from CRI-G1 cells. Cell-attached and excised inside-out recordings were made from CRI-G1 cells and HEK cells expressing Kir6.2 and SUR1 to examine single channel responses as described previously \[[@B25],[@B35]\]. Single channel data were analysed for current amplitude and channel activity (N~f~.P~o~; where N~f~is the number of functional channels in the patch and P~o~is the open probability) as described previously \[[@B45]\]. All data were normalised to control and are expressed as mean ± S.E.M. Statistical analyses were performed using Student\'s unpaired *t*test. P \< 0.05 was considered significant. Recording electrodes were pulled from borosilicate glass and had resistances of 2--5 MΩ for whole cell recordings and 7--10 MΩ for cell-attached and inside-out experiments when filled with electrolyte solution. The pipette solution for whole-cell recordings comprised (in mM): KCl 140, MgCl~2~0.6, CaCl~2~2.73, Mg-ATP 5.0, EGTA 10, HEPES 10, pH 7.2 (free \[Ca^2+^\] of 100 nM), whereas for single channel recordings the pipette solution contained (in mM): KCl 140, CaCl~2~1, MgCl~2~1, HEPES 10, pH 7.2. The bath solution for whole-cell and cell-attached recordings was normal saline whereas for inside-out patches the bath solution contained (in mM): KCl 140, MgCl~2~1, CaCl~2~2, EGTA 10, HEPES 10, pH 7.2 (free \[Ca^2+^\] of 30 nM). All solution changes were achieved by superfusing the bath with a gravity feed system at a rate of 10 ml min^-1^, which allowed complete exchange within 2 min. All experiments were performed at room temperature (22--25°C). Antibodies & drugs ------------------ Anti-PKB, which recognises all three isoforms of PKB, and the phospho-specific PKB (Thr308), GSK3α/β (Ser21/9), STAT3 (Tyr705) and p44/42 MAPK (Thr202/Tyr204) antibodies were obtained from Cell Signalling Technology Inc. Recombinant human leptin, wortmannin and LY 294002 were obtained from Novachem-Calbiochem and BVT.12777 (\[2-(hydrazinoiminomethyl) hydrazino\] acetic acid) was a gift from Biovitrum (Stockholm, Sweden). Tolbutamide, Mg-ATP, diazoxide, sodium azide, phalloidin and cytochalasin B were obtained from Sigma. Rhodamine-conjugated phalloidin was obtained from Molecular Probes and UO126 from Promega. BVT.12777 was prepared as a 100 mM stock solution in normal saline and stored at -70°C prior to use. Leptin was prepared as a 10 μM stock solution in normal saline containing 0.2 % bovine serum albumin as carrier. Rhodamine-conjugated phalloidin (200 U ml^-1^) and LY 294002 (10 mM) were stored as stock solutions in 1% methanol at -20°C. Cytochalasin B was stored as a 10 mM stock solution, and diazoxide and tolbutamide as 100 mM solutions, all in DMSO at 2--4°C. Mg-ATP was stored at -20°C as a 100 mM solution in 10 mM HEPES (pH 7.2). Wortmannin and UO126 were stored as 10 mM stock solutions in Me~2~SO at -20°C. List of abbreviations used ========================== CRI-G1, Cambridge Rat Insulinoma-G1; GSK3, glycogen synthase kinase-3; HEK293, human embryonic kidney 293; JAK, janus kinase; K~ATP~, ATP-sensitive potassium; Kir6.2, potassium channel inward rectifier-6.2; MAPK, p42, p44 mitogen-activated protein kinase; MAPKK, MAPK kinase; ObRb, Obese (leptin) receptor-b; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; PtdIns(3,4,5)P~3~, phosphatidylinositol 3,4,5 tris-phosphate; STAT3, signal transducer and activator of transcription-3; SUR, sulphonylurea receptor Authors\' contributions ======================= JK carried out the majority of the electrophysiology and cytoskeletal fluorescence studies. HL carried out the western blot experiments. TT and JH participated in the electrophysiological experiments. CS participated in the design and implementation of the western blot experiments. MA conceived of the study, participated in its design and co-ordination and drafted the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ This work was supported in part by grants from The Wellcome Trust (042726 & 065287), Biovitrum AB and Tenovus, Scotland. CS is a Diabetes UK Senior Fellow.

PubMed Central

2024-06-05T03:55:47.570558

2004-8-24

PMC516775

Background ========== Infection of plants by necrotizing pathogens can lead to the induction of a complex set of defense responses resulting in a restriction of pathogen growth and spread. The infected leaves develop a hypersensitive response (HR), i.e., rapid, localized cell death occurring at the sites of pathogen entry \[[@B1]\]. Concomitant with the HR is the accumulation of salicylic acid and several classes of pathogenesis-related (PR) proteins, many of which exhibiting antimicrobial activity \[[@B2]\]. Subsequently, an enhancement of the plant defensive capacity against a broad spectrum of pathogens is observed. This resistance is expressed locally as well as in distal, uninfected tissues and can last for several weeks to months. It is known as Systemic Acquired Resistance or SAR \[[@B3]\]. Salicylic acid was identified as an important signal in the SAR transduction pathway. It was shown to accumulate during the onset of SAR in cucumber \[[@B4]\], tobacco \[[@B5]\], and *Arabidopsis thaliana*\[[@B6]\]. Exogenously supplied salicylic acid induces the same set of genes and resistance against the same spectrum of pathogens, as with pathogen-induced SAR \[[@B3]\]. Plants in which salicylic acid accumulation is prevented by over expression of a bacterial salicylate hydroxylase gene failed to develop SAR and/or exhibited increased susceptibility to pathogen infection \[[@B7],[@B8]\]. Analysis of *A. thaliana*mutants has revealed that the NPR1 protein (also known as NIM1) is required for SAR induction and acts downstream of salicylic acid in the SAR pathway. Plants carrying mutations in this gene fail to express several *PR*genes and display enhanced susceptibility to infection \[[@B9],[@B10]\]. The NPR1/NIM1 protein interacts with members of the TGA/OBF family of basic leucine zipper transcription factors. Some of these factors have been shown to bind to salicylic acid-responsive promoter elements of the *PR-1*gene \[[@B11]-[@B13]\]. The salicylic acid induced interaction of NPR1 with TGA factors is localized in the nucleus \[[@B14]\], while cytoplasmic NPR1 appears to modulate crosstalk between salicylic acid- and jasmonate-dependent defense pathways \[[@B15]\]. Other components of the salicylic acid signaling pathway have been identified using genetic approaches in *Arabidopsis thaliana*\[[@B16]\]. They include PAD4 and EDS3 which activate EDS4, SID2 and EDS5 leading to salicylic acid accumulation \[[@B17]\]. Besides pathogens, SAR is also induced by exogenously applied chemicals. In addition to salicylic acid, well-known chemical inducers of *PR*genes include 2,6-dichloroisonicotinic acid (INA), benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) and L-α-amino butyric acid (ABA) \[[@B18]-[@B23]\]. SAR induction appears to be governed by a complex signal transduction process \[[@B24]\] that involves a signal originating at the site of infection or treatment, and moving throughout the plant. Chemical inducers provide tools for dissection of the signal transduction pathway that regulates the defense responses (they enter the pathway at different points). On another hand, the cucumber plant provides a good model system for studying induced disease resistance \[[@B25]\]. Local and systemic increases in chitinase and/or peroxidase activity have been observed in response to inoculation with necrotizing pathogens or to treatment with SA, INA and BTH \[[@B26]-[@B28]\]. The aim of the present work was to identify and clone mRNAs accumulating after induction of SAR in cucumber plants. These sequences could correspond to proteins potentially involved in the signal transduction pathway leading to SAR or to proteins responsible for the state of resistance. In a first set of experiments, potential SAR inducing chemicals were tested in order to set up a reproducible system of activation of this resistance in cucumber. The mRNA RT-PCR differential display method \[[@B29]\] was then used to identify genes activated during SAR induction. Several differentially expressed cDNA were identified and some of them were cloned and sequenced. Their expression was analyzed by Northern blotting. Results ======= Chemically induced resistance in cucumber ----------------------------------------- The SAR inducing activity of different chemicals has already been documented in one or several plant species \[[@B18]-[@B23]\]. To evaluate their activity on cucumber, cotyledons of two-week-old plants were infiltrated with 0.5 mM salicylic acid, 50 mM L-α-amino butyric acid, 0.78 mM 2-thiouracil, 1 mM thiamine or 20 mM BaCl~2~. Control plants were infiltrated with water. In a parallel experiment, four-week-old cucumber plants (with two true leaves) were sprayed with 5 mM salicylic acid, 50 mM L-α-amino butyric acid, 0.78 mM 2-thiouracil, 1 mM thiamine, 20 mM BaCl~2~or 1 mM BTH. Control plants were sprayed with water or left untreated. Total RNAs were extracted from treated leaves 6 h, 24 h or 48 h after treatment. Induction of *PR-8*gene was assayed by Northern blotting followed by hybridization with a cucumber PR-8 cDNA probe. *PR-8*cucumber gene encodes an acidic chitinase which accumulates after tobacco necrosis virus (TNV) infection or salicylic acid induction and is considered as a SAR molecular marker \[[@B30]\]. In infiltration experiments (results not shown), control plants showed positive hybridization signals. To check whether this activation of the *PR-8*gene could be due to wounding during infiltration, the effect of wounding alone was evaluated by making a cut from the midvein to the outer edge of the cotyledons. A strong signal was obtained after wounding which could be explained by induction of the acidic chitinase itself or of an isoform. Therefore infiltration did not prove to be an appropriate method for applying SAR inducers in cucumber, because it probably induced a wound response. When applied by spraying, BTH, L-α-amino butyric acid and 2-thiouracil strongly induced PR-8 mRNA accumulation, while SA treatment led to a lower induction level (Fig. [1](#F1){ref-type="fig"}). Thiamine and BaCl~2~induced slightly *PR-8*expression (results not shown). RNA from control plants did not show any hybridization signal indicating that spraying itself did not induce *PR-8*expression. BTH, which is an analogue of salicylic acid, showed the strongest *PR-8*induction. But cucumber plants treated with 1 mM BTH showed a reduced growth (smaller leaves area and shorter internodes). As BTH was shown to be a very efficient activator of disease resistance in different plant species \[[@B20]-[@B23]\], we tried to induce SAR in cucumber plants with lower BTH concentrations (10 μM, 30 μM and 100 μM). PR-8 transcript accumulation was quite similar for the three concentrations (see [Additional file 1](#S1){ref-type="supplementary-material"}) and no adverse effect on plant growth was observed. In cucumber, SAR has been shown to be effective against anthracnose caused by *Colletotrichum lagenarium*and salicylic acid treatment was shown to induce resistance against this pathogen \[[@B31]\]. To confirm that BTH acts as a chemical inducer of SAR in cucumber, four-week-old plants were sprayed with water, 10 μM BTH, 50 μM BTH, or left untreated, 5 days prior to inoculation of the second true leaf with *C. lagenarium*. Two weeks after inoculation, the plants were scored for spreading lesions, i.e. lesions showing a larger size than the initial inoculated area. As shown in figure [2](#F2){ref-type="fig"} and table [1](#T1){ref-type="table"}, BTH treatment resulted in decreased symptoms. Control leaves inoculated with *C. lagenarium*showed large spreading lesions and thus extensive fungal growth. Treatment of plants with BTH prior to *C. lagenarium*inoculation resulted in a markedly decreased lesion formation (Fig. [2](#F2){ref-type="fig"}). In untreated plants and water treated plants, 67 and 64 % of inoculation sites showed spreading lesions respectively (Table [1](#T1){ref-type="table"}). In BTH treated plants, only 23 % (10 μM BTH) or 9 % (50 μM BTH) of inoculated sites developed spreading lesions (Table [1](#T1){ref-type="table"}). To check whether BTH is able to act systemically or is transported through cucumber plants, the third leaf of four-week-old plants was wrapped with a plastic film and the plants were sprayed with H~2~O, 10 μM BTH, 50 μM BTH or left untreated. This leaf was inoculated with *C. lagenarium*7 days later. The percentage of spreading lesions on third leaves from pathogen-challenged untreated or H~2~O treated cucumber plants (infected controls) were respectively 86% and 83.5%. In BTH treated plants, 38 % (10 μM BTH) or 12 % (50 μM BTH) of inoculated sites developed spreading lesions (Table [2](#T2){ref-type="table"}). These results suggest that BTH can activate resistance in untreated leaves. Differential expression of genes in BTH induced cucumber plants --------------------------------------------------------------- Plants were sprayed with 10 μM BTH or water. Sampling was performed 24 h after treatment in order to point out early-induced differences in gene expression. RT-PCR products were separated on polyacrylamide gels. Bands that appeared on the display of RNA only from BTH treated plants, or only from water treated plants were likely to correspond to differentially expressed mRNAs. Twelve differentially expressed bands were identified (Table [3](#T3){ref-type="table"}). Their sizes ranged between 250 and 450 bp. They corresponded to genes expressed in the BTH treated plants and not in the control ones or presenting a higher expression in the BTH treated plants compared to the control ones (Fig. [3](#F3){ref-type="fig"}). These bands were eluted from the gel, PCR amplified and cloned in the pGEM-T vector. In order to confirm the differential expression of these genes, reverse slot blot hybridizations were performed: DNA from four to ten clones obtained from each differentially displayed band was fixed on a membrane and hybridized with the corresponding reverse transcribed products from either BTH or H~2~O treated plants (Fig. [4](#F4){ref-type="fig"}). Six from the twelve fragments were confirmed to be differentially expressed: dd3, dd4, dd5, dd6, dd7 and dd11. Five bands (dd1, dd8, dd9, dd10 and dd12) were either expressed evenly between BTH and H~2~O treated plants (differential display false positives or signal masked by the expression of homologous genes). In the remaining case (dd2), no signal could be observed on the slot blots. This absence of hybridization signal could be due to a lack of sensitivity of the slot blot hybridization procedure. The dd3, dd4, dd5, dd6, dd7 and dd11 cDNA fragments were sequenced. Blast analysis did not lead to identification of any homology with known sequences. This is not surprising as mRNA differential display generates fragments corresponding to 3\'untranslated regions for which homologies are difficult to find. Two of the six fragments were proved to be part of the same gene (dd5 and dd7). A cDNA library was constructed from cucumber plants sprayed with 10 μM BTH (leaves were sampled 24 h after treatment). The library was screened with the five differentially expressed fragments. Hits were obtained for four of these probes (corresponding to dd3, dd4, dd6 and dd11). Full length cDNAs corresponding to dd4, dd6 and dd11 were obtained. Homology between cloned cDNA and sequences in databases is presented in table [4](#T4){ref-type="table"}. The cDNA dd3 matched to a putative RAS-related GTP-binding protein from *A. thaliana*. It will be referred to as CRG (cucumber RAS-related GTP-binding protein). The cDNA dd4 matched to a translation releasing factor 2. It will be referred to as CTR (cucumber translation releasing factor 2). The cDNA dd6 matched to a putative β-1,4-*N*-Acetylglucosaminyltranferase III from *A. thaliana*. This protein was named CGT (cucumber acetylglucosaminyltransferase III). The cDNA dd11 matched to a putative pathogenesis related protein from *A. thaliana*and, with slightly less homology, to a pathogenesis related protein from barley induced by fungal infection \[[@B32]\]. It will be referred to as CPR (cucumber pathogenesis-related protein). *PR-8*, *CPR*, *CRG and CGT*expression analysis ----------------------------------------------- To investigate the expression of *PR-8*, *CPR*, *CRG*and *CGT*genes in cucumber, the two first leaves of four-week old plants were sprayed with 10 μM BTH. In another batch of plants, the first leaf was inoculated with *C. lagenarium*. Total RNA was extracted from treated leaves or upper untreated leaves at various times after treatment. Accumulation of the PR-8, CPR, CRG and CGT mRNAs was assayed by Northern blotting followed by hybridization with the corresponding cDNA probes (fig. [5](#F5){ref-type="fig"}). Expression of *PR-8*gene was induced at a high level by both 10 μM BTH spraying and *C. lagenarium*infection in the leaves submitted to the treatment. PR-8 mRNA could be detected as soon as 5 h after BTH application and a maximum of expression could be observed after 72 h. *PR-8*expression was observed as from 72 h after inoculation with *C. lagenarium*. No significant PR-8 mRNA accumulation could be detected in the untreated leaves from BTH sprayed plants, while in non infected leaves *PR-8*expression was observed 72 h after infection by *C. lagenarium*; the level of expression was still increasing 2 weeks after *C. lagenarium*inoculation. *CPR*gene expression was strongly induced in both 10 μM BTH treated and *C. lagenarium*inoculated leaves. CPR mRNA could be detected as soon as 5 h after BTH application and a maximum of expression could be observed after 24 h. *CPR*expression was observed 72 h after infection by *C. lagenarium*. No expression of *CPR*gene could be observed in untreated leaves from BTH sprayed plants, while CPR mRNA accumulation could be detected in uninfected leaves from *C. lagenarium*infected plants 72 h after inoculation. *CGT*gene showed a significant basal level of expression as it could be observed in control plants (fig. [5](#F5){ref-type="fig"}). Nevertheless the expression of *CGT*was increased both by spraying with 10 μM BTH (with a maximum at 24 h) and by inoculation with *C. lagenarium*(with a maximum at 72 h) in the leaves submitted to the treatment. A systemic induction could also be observed in untreated leaves from *C. lagenarium*inoculated plants (as from 72 h after infection). The expression level of the *CRG*gene was too low to allow us to show an induction of the expression (data not shown). Expression of the same genes was analyzed in wounded cucumber plants. A cut was made from the midvein to the outer edge of the first leaf. Total RNA was extracted from the cut leaf or the upper leaf, 24 and 48 h after wounding. An induction of the expression of *PR-8*and *CPR*genes was only observed in the cut leaf 24 h after wounding. No expression was observed later nor in the uncut upper leaf. The *CGT*and *CRG*genes were not induced by wounding (results not shown). Expression of these genes was also analyzed in cucumber plants showing rhizobacteria-induced systemic resistance. Plants were grown in compost drenched with a *Pseudomonas putida*BTP1 suspension, a plant growth-promoting rhizobacteria known to promote induced systemic resistance in cucumber \[[@B33]\]. Total RNA was extracted from cotyledons collected three weeks after sowing. *PR-8*, *CPR*, *CGT*and *CRG*did not show any induction of expression in these conditions (results not shown). Discussion ========== We tested here two different techniques and several compounds to induce SAR in cucumber. The expression of *PR-8*gene, which encodes an acidic chitinase from cucumber, was used as an indicator of the activation of the defense response. The accumulation of this acidic chitinase after tobacco necrosis virus infection or salicylic acid induction correlates with induced resistance \[[@B30]\]. Among the different compounds tested, BTH applied by spraying proved to be the stronger inducer of PR-8 mRNA accumulation. BTH sprayed at a concentration of 10 μM was still able to induce *PR-8*gene expression. The protective effects of BTH on cucumber were confirmed in challenge experiments with *C. lagenarium*. Spraying cucumber plants with BTH (10 μM or 50 μM) allowed effective local and systemic protection against *C. lagenarium*. BTH was recently identified as a safe, reliable and non phytotoxic plant protection agent by scientists at Novartis. Exogenous application of BTH to tobacco, *A. thaliana*, wheat and cucumber has been shown to activate a number of SAR associated genes leading to enhanced plant protection against various pathogens \[[@B20]-[@B22],[@B28]\]. Identifying SAR induced genes could provide clues to elucidate the signal transduction pathway leading to plant defense responses. We used RT-PCR differential display in BTH-treated cucumber plants to detect SAR associated mRNAs. We have identified four BTH-inducible genes: a putative ras-related GTP binding protein (CRG), a putative translation releasing factor 2 (CTR), a putative β-1,4-*N*-acetylglucosaminyltranferase III (CGT) and a putative pathogenesis related protein (CPR). Expression of *PR-8*, *CPR*and *CGT*genes was shown to be induced by 10 μM BTH treatment as well as *C. lagenarium*infection. The response to BTH was observed earlier (5 h after treatment) than the one developed after pathogen inoculation (72 h after infection). However, systemic expression of these genes was only observed in *C. lagenarium*infected plants. Small GTP-binding proteins form a large family of nucleotide triphosphatases whose activity is related to the binding, hydrolysis and release of guanosine triphosphate \[[@B34]\]. MAP kinase cascade represents an important downstream effector pathway for RAS in most cells \[[@B35]\] and recent studies have shown that MAP kinases are activated in plants in response to pathogen attack and wounding \[[@B36],[@B37]\]. Small GTP-binding proteins have been shown to be involved in important cell mechanisms like cell division, transduction of external signals across the plasma membrane, endocytosis and exocytosis, cell death in plants and establishment of plant defense reaction \[[@B34],[@B38]-[@B42]\]. The β-1,4-*N*-Acetylglucosaminyltranferase III catalyses the addition of *N*-acetylglucosamine to the β-mannoside of the tri-mannose core in *N*-glycans, resulting in the suppression of further processing and elongation. In mammals, *N*-acetylglucosaminyltransferase III expression is associated with differentiation, cell adhesion and tumor progression \[[@B43],[@B44]\]. It was shown recently to interfere with epidermal growth factor signaling and H~2~O~2~-induced activation of protein kinase C \[[@B45],[@B46]\]. *N*-linked glycans were shown to be widely distributed in plants and highly expressed at the cell surface, which might suggest a putative function in cell/cell communication \[[@B47]\]. To our knowledge β-1,4-*N*-Acetylglucosaminyltranferase has never been described in the context of a plant-pathogen interaction. Several cDNAs or gene encoding enzymes involved in the biosynthesis of complex *N*-linked glycans have been cloned from plants. They include *N*-acetylglucosaminyltranferase I cDNAs from *A. thaliana*, potato and tobacco \[[@B48]\], *N*-acetylglucosaminyltransferase II cDNA from *A. thaliana*\[[@B49]\] and defense-related glucosyl transferase gene from tomato \[[@B50]\]. GTP-binding proteins and *N*-acetylglucosaminyltranferases are involved in important mechanisms, notably signaling pathways and therefore seem of high interest. The putative PR protein, which has been first identified in barley after fungal infection \[[@B32]\] has no known function. Only *PR-8*and *CPR*genes were induced by wounding but not systemically. *PR-8*and *CPR*genes were not induced by *P. putida*BTP1 treatment, which triggers induced systemic resistance (ISR) in cucumber. ISR is phenotypically similar to SAR but the mechanisms of this resistance were shown to be different: although this was not tested with *P. putida*BTP1, in other experimental systems ISR was shown to be dependent on NPR/NIM1 function, but does not involve salicylic acid, nor PR protein accumulation \[[@B51]\]. Our results correlate with those of Pieterse et al. who showed that induced systemic resistance in *A. thaliana*was independent of *PR*genes expression \[[@B51]\]. In the same way, ISR also seems to be independent on *CGT*and *CRG*expression, as these genes were not induced by *P. putida*BTP1. Conclusions =========== Among different SAR inducers tested on cucumber, BTH proved to be very efficient. Applied by spraying, BTH induced PR-8 mRNA accumulation and allowed effective local and systemic protection against *C. lagenarium*. Using RT-PCR differential display, we identified four BTH-inducible genes, including a putative ras-related GTP binding protein (CRG), a translation releasing factor 2 (CTR), a putative β-1,4-*N*-acetylglucosaminyltranferase III (CGT) and a putative pathogenesis related protein (CPR). Genes expression studies confirmed the local induction of *CGT*and *CPR*after BTH treatment or *C. lagenarium*infection and their systemic induction in response to *C. lagenarium*infection. Moreover *CPR*was locally induced by wounding. The significant level of expression of *CGT*and *CPR*genes during defense responses suggests a potential role for the gene products in the SAR pathway or in the state of resistance in cucumber. Methods ======= Organisms and growth conditions ------------------------------- Cucumber plants (*Cucumis sativus*cv. *Marketer*) were grown in compost at 22°C with a 16 h light photoperiod. Cultures of *Colletotrichum lagenarium*were maintained on malt agar at 25 °C. Spores suspensions in water supplemented with 0.01 % tween 80 were prepared from four- to six-week-old cultures grown in petri plates. Suspensions were filtered through cheesecloth and the spore concentration determined with a hemacytometer. Chemical treatments ------------------- All chemicals were obtained from Sigma except for BTH (provided by Novartis). Chemicals were dissolved in water and the pH adjusted to 6.6 with NaOH except for thiamine, BaCl~2~and BTH. Infiltration experiments: cotyledons of two-week-old plants were infiltrated through a vein until the whole leaf was impregnated with the solution. Spraying experiments: the two first true leaves of four-week-old cucumber plants were sprayed with +/- 3 ml solution/plant. The upper leaves were protected with a plastic sheet when systemic resistance was to be tested. ISR induction ------------- Prior to planting, disinfected cucumber seeds were soaked for 10 min in a *Pseudomonas putida*BTP1 suspension (4.10^8^CFU/ml in 0.85% NaCl). Control seeds were soaked in 0.85% NaCl. Cucumber seeds were sown in 10 cm-pots containing sterilized potting soil previously mixed with a *P. putida*BTP1 inoculum to a final concentration of 3.10^7^CFU/g or with an equal volume of sterile water for control plants. Cucumber were germinated at 25°C with a 16 h light photoperiod. Six and 12 days after sowing, 20 ml of a bacterial suspension at 10^8^CFU/ml were added as a drench to the roots. Control plants were watered with 20 ml of 0.85% NaCl. *C. lagenarium*infection ------------------------ Plants were placed in a humidity chamber 48 hours before infection experiments. The first true leaf was inoculated with 10 drops (10 μl) of a conidial suspension of *C. lagenarium*(10^6^spores/ml). In challenge experiments, inoculation was performed 5 (or 7) days after chemical treatment on the second or third leaf. Plant RNA extraction and analysis --------------------------------- The extraction of total RNA from cucumber leaves (5 plants per treatment) was performed using a phenol/SDS method \[[@B52]\]. Northern hybridization of RNA fractionated by agarose-formaldehyde gel electrophoresis \[[@B52]\] was performed with a α-^32^P PR-8, CPR, CGT or CRG cDNA probe (Random Primed DNA labeling kit, Roche). mRNA Differential Display ------------------------- The mRNA differential display was performed using the differential display kit from Eurogentec (Belgium) and following the instructions of the supplier. PCR products were labeled with α-^35^S dATP, separated by electrophoresis on denaturing 6% polyacrylamide gels and visualized by autoradiography. PCR reactions showing differentially displayed bands were repeated twice on two different RNA samples in order to reduce the number of false-positives. Cloning and sequencing ---------------------- Differentially displayed bands were excised and eluted during 30 min in 100 μl of H~2~O followed by boiling during 10 min, then precipitated and suspended in 10 μl of H~2~O. A second PCR amplification was performed on 4 μl of DNA following the same protocol than for the differential display itself. Products were cloned in pGEM-T vector (Promega) and manually sequenced by the dideoxy sequencing method. Available public databases (i.e. EMBL) were searched for homology with our sequences using the GCG software package (Blastall, Fasta). Slot-Blot hybridisation ----------------------- PCR amplified DNA from four to ten clones of each differentially displayed band was blotted on a positively charged nylon membrane using a filtration manifold and following the instructions of the supplier (Hoefer). Blots were hybridized with the ^32^P labeled RT-PCR corresponding products from either BTH or H~2~O treated plants. cDNA library construction and screening --------------------------------------- mRNA from cucumber plants sprayed with 10 μM BTH (leaves were sampled 24 h after treatment) were purified with the PolyATractmRNA Isolation System III from Promega. The cDNA library was performed with the SMART cDNA Library Construction Kit from Clontech following the instructions of the supplier for cDNA synthesis by primer extension. The library was screened with ^32^P labeled cDNA probes. cDNA sequencing was performed at Genome Express, France. Available public databases (i.e. EMBL) were searched for homology with our sequences using the GCG software package (Blastall, Fasta). Authors\' contributions ======================= CB carried out the plants treatments, the molecular biology studies and the database research and drafted the manuscript. MO supplied the pathogen and participated in the challenge and ISR induction experiments. JD and PT participated in the design and coordination of the study. All authors read and approved the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 **PR-8 mRNA accumulation in cucumber leaves in response to different concentrations of BTH**. Total RNA was extracted from cucumber leaves 24 h and 48 h after spraying with water, 10 μM BTH, 30 μM BTH, 100 μM BTH or from untreated leaves (UT). RNA was fractionated by electrophoresis on agarose gel. Northern blot was probed with α-^32^P labeled PR-8 cDNA. Loading of equal amounts of RNA was confirmed by ethidium bromide (Et Br) staining. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work was supported by a grant from the National Funds for Scientific Research (FNRS, Belgium) (Program FRFC n° 2.4.570.00). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Time course of PR-8 mRNA accumulation in cucumber leaves in response to chemical treatments.**Total RNA was extracted from cucumber leaves at various time after spraying with water, 5 mM salicylic acid, 1 mM BTH, 50 mM L-α-amino butyric acid (ABA), 0.78 mM 2-thiouracil (2T-U) or from untreated leaves (UT). RNA (10 μg) was fractionated by electrophoresis on agarose gel. Northern blot was probed with α-^32^P labeled PR-8 cDNA (loading of equal amounts of RNA was confirmed by ethidium bromide staining). :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Local acquired resistance induced by 10 μM or 50 μM BTH.**Four-week-old cucumber plants (with two true fully expanded leaves) were sprayed with H~2~O, 10 μM BTH, 50 μM BTH or left untreated. The second true leaf was infected with *C. lagenarium*5 days later. Plants were photographed 2 weeks postinoculation. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Zoom in on an auto-radiogram of differentially displayed RT-PCR products.**The RT-PCR were performed with primers 5\'T~11~GA and 5\'GATCTAACCG (lane 1 to 4), primers 5\'T~11~GA and 5\'GATCAATCGC (lane 5 to 8), primers 5\'T~11~GA and 5\'TACAACGAGG (lanes 9 to 12). Uneven lanes: H~2~O treated plants. Even lanes: 10 μM BTH treated plants. The arrows show bands corresponding to transcripts accumulating only or presenting a higher expression in BTH treated plants. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Slot blotting of PCR amplified fragments identified in differential display experiments.**PCR amplified DNA from four dd11 clones, nine dd6 clones and four dd5 clones respectively were hybridized with the ^32^P labeled RT-PCR corresponding products from either H~2~O or BTH treated plants. The arrows show DNA from clones containing differentially displayed fragments. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Time course of mRNA accumulation in cucumber leaves after BTH treatment (A) or *C. lagenarium*inoculation (B).**A: Total RNA was extracted from cucumber leaves at various time after application of 10 μM BTH or water (mock treatment). RNA (15 μg) from leaves 1 and 2 (treated) and leaves 3 and 4 (untreated) was fractionated by electrophoresis on agarose gels. Identical Northern blots were probed with ^32^P-labeled PR-8, CPR and CGT cDNAs. B: Total RNA was extracted from cucumber leaves at various time after *C. lagenarium*inoculation. RNA (15 μg) from untreated leaves (UT), leaves 1 (infected) and leaves 2, 3 and 4 (non infected) was fractionated by electrophoresis on agarose gels. Identical Northern blots were probed with ^32^P-labeled PR-8, CPR and CGT cDNAs. Equal loading of RNA was confirmed by ethidium bromide (Et Br) staining. A representative gel is shown. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Local acquired resistance induced by 10 μM or 50 μM BTH. ::: untreated H~2~O 10 μM BTH 50 μM BTH ------------ ----------- ----------- ----------- ----------- Exp.1 66 ± 24 % 57 ± 25 % 12 ± 9 % 13 ± 11 % Exp.2 68 ± 28 % 71 ± 23 % 34 ± 30 % 5 ± 6 % Mean value 67 % 64 % 23 % 9 % Four-week-old cucumber plants (with two true fully expanded leaves) were sprayed with H~2~O, 10 μM BTH, 50 μM BTH or left untreated. The second true leaf was infected with *C. lagenarium*5 days later (ten infection sites per leaf). The percentages of spreading lesions (i.e. lesions showing a larger size than the initial inoculated area) observed 2 weeks after inoculation with *C. lagenarium*were calculated on the basis of 14 plants/treatment (experiment 1) and 16 plants/treatment (experiment 2) (means +/- standard deviations). ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Systemic acquired resistance induced by 10 μM or 50 μM BTH. ::: untreated H~2~O 10 μM BTH 50 μM BTH ------------ ----------- ----------- ----------- ----------- Exp.1 76 ± 20 % 69 ± 17 % 37 ± 19 % 18 ± 16 % Exp.2 96 ± 6 % 98 ± 3 % 39 ± 15 % 7 ± 9 % Mean value 86 % 83.5 % 38 % 12,5 % Four-week-old cucumber plants (with two true fully expanded leaves) were sprayed with H~2~O, 10 μM BTH, 50 μM BTH or left untreated. The third true leaf was infected with *C. lagenarium*7 days later (ten infection sites per leaf). The percentages of spreading lesions (i.e. lesions showing a larger size than the initial inoculated area) observed 2 weeks after inoculation with *C. lagenarium*were calculated on the basis of 18 plants/treatment (experiment 1) and 17 plants/treatment (experiment 2) (means +/- standard deviations). ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Oligonucleotide primers used in RT-PCR that allowed to amplify the differentially displayed cDNA fragments. ::: Clone name Downstream primer used Upstream primer used ------------ ------------------------ ---------------------- dd1 5\' T~11~AA 5\' CTGCTTGATG dd2 5\' T~11~CG 5\' GATCAATCGC dd3 5\' T~11~CG 5\' GATCAATCGC dd4 5\' T~11~CG 5\' TCGGTCATAG dd5 5\' T~11~GA 5\' AAACTCCGTC dd6 5\' T~11~GA 5\' TTTTGGCTCC dd7 5\' T~11~GA 5\' GATCTAACCG dd8 5\' T~11~GA 5\' GATCAATCGC dd9 5\' T~11~GA 5\' GATCAATCGC dd10 5\' T~11~GA 5\' TCGATACAGG dd11 5\' T~11~GA 5\' TACAACGAGG dd12 5\' T~11~GG 5\' TACAACGAGG ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Characteristics of isolated cDNA clones. ::: Clone name Clone length (bp) Accession N° Matching sequence from data base Origin of matching sequence and accession number Amino acid sequence % identity (\*) ------------ ------------------- -------------- ---------------------------------------- -------------------------------------------------- ------------------------------------- dd3 774 AJ634908 Ras-related GTP-binding protein *A. thaliana*NP 177505 65 % (1e^-73^) dd4 1533 AJ635428 Translation releasing factor 2 *A. thaliana*NP 851097 70 % (7e^-170^) dd6 2192 AJ629867 β-1,4-*N*-acetylglucosaminyltranferase *A. thaliana*NP 172759 77 % (0.0) dd11 1588 AJ629866 Pathogenesis-related protein *A. thaliana* NP 565189 51 % (1e^-51^) \* expected value of best match :::

PubMed Central

2024-06-05T03:55:47.573416

2004-8-26

PMC516776

Background ========== Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) share common routes of transmission, mainly sexual, parenteral and vertical. Therefore, the prevalence of HBV serological markers is higher among HIV infected patients than in non-HIV infected individuals \[[@B1],[@B2]\]. Considerable variations in the prevalence of HBV markers have been observed in HIV patients according to the geographical region and risk of exposure \[[@B3]\]. Recently, a prevalence of 68% of antibodies against hepatitis B core antigen (anti-HBc) was observed in HIV infected patients living in Rio de Janeiro, Brazil \[[@B4]\]. Co-infection with HIV interferes with the natural history of HBV infection and is associated with higher HBV DNA levels \[[@B5],[@B6]\]. A more common progression to cirrhosis, despite a milder histological necro-inflammatory activity, has also been observed in cases of HIV-HBV co-infection \[[@B5]\]. Lamivudine is a nucleoside analogue that inhibits the reverse transcriptase activity of both HIV and HBV \[[@B7]\] and is commonly used in the treatment of both viral infections \[[@B8],[@B9]\]. The HBV DNA loss due to lamivudine treatment is usually accompanied by significant histological and biochemical improvement \[[@B10]\]. The major limitation in the use of lamivudine is the selection of resistant mutations that may arise and accumulate during therapy. Most of these mutations usually affect the YMDD motif of the HBV DNA polymerase, by replacement of a methionine residue at position 550 with either valine (M550V) or isoleucine (M550I). Such mutations have notably been reported in HIV-HBV co-infected patients \[[@B11],[@B12]\] who develop lamivudine resistance at an annual rate of 20%, with a projected rate of 90% after four years of therapy \[[@B13]\]. The consequences of drug resistance mutations for the evolution of HBV induced liver disease are currently under study. Hepatitis B acute exacerbation has been described after withdrawal of lamivudine therapy \[[@B14]\]. Such an exacerbation has also been associated with the appearance of YMDD mutants \[[@B14],[@B15]\] and with a rapid increase of viral load \[[@B16]\]. The present study was performed to characterize HBV variants and genetic patterns of lamivudine resistant HBV strains in a patient co-infected with HIV-1 at different periods of an antiretroviral treatment with and without lamivudine. Methods ======= Patient characteristics and serological markers ----------------------------------------------- A 30-year-old male patient initiated a hemodialysis treatment in August 1995. At that time, he was negative for hepatitis B surface antigen (HBsAg) as well as for anti-HBc and anti-HIV antibodies. The patient received three blood transfusions during the first trimester of 1996 and developed a posttransfusional hepatitis. He became HBsAg and anti-HIV positive and remained positive for both markers in all routine tests performed between 1996 and 2002. Data of other tests, such as serum transaminases, CD4 levels, and HIV load, performed in external laboratories between 1995 and 2002, were available. Resistance to different HIV therapies was evaluated by the medical staff based on HIV load and CD4 levels. HIV treatment started in April 1996, with a combination of zidovudine (AZT) and didanosine (ddI). In November 1996, this treatment was modified to a combination of AZT and lamivudine which was maintained until March 1997, when treatment was once again changed to triple therapy with AZT, lamivudine and invirase. In May 1999, high HIV load (3.1 × 10^5^copies/ml) was observed, indicating HIV resistance. At that time the patient started an anti-tuberculosis treatment with rifampicin. However, due to interaction of antiretroviral drugs with rifampicin, the administration of antiretroviral drugs was discontinued. This resulted in a decrease of CD4 counts to 156 cells/ml, while elevated levels of HIV persisted at the end of 1999. Antiretroviral treatment resumed in January 2000, composed of four drugs, namely ddI, stavudine, nevirapine (NVP) and nelfinavir (NFV). As a consequence, CD4 cell counts increased noticeably, and HIV RNA became undetectable. Unfortunately, resistance to therapy was observed during the second semester of 2001, when HIV load reached 1.2 × 10^5^copies/ml and CD4 counts decreased to 120 cells/ml. The treatment was then replaced by another including lamivudine associated to AZT, NVP and NFV. No HIV resistance was observed until the end of the follow-up (November 2002). The patient died of renal failure at the end of 2003. DNA extraction and PCR assays for DNA sequencing ------------------------------------------------ Three serum samples collected in March 1999, August 2000 and November 2002, were available for HBV DNA analysis. Sera were submitted to DNA extraction by the phenol-chloroform method after treatment with proteinase K, as described previously \[[@B17]\]. HBV pre-S/S genomic region was amplified by PCR using sense primer PS1 (5\'-CCATATTCTTGGGAACAAGA-3\', nt 2826-2845) and a mix of antisense primers S2 (5\'-GGGTTTAAATGTATACCCAAAGA-3\', nt 841-819) and S22 (5\'-GTATTTAAATGGATACCCACAGA-3\', nt 841-819), able to amplify all HBV genotypes. PCR assays were performed under the following conditions: 94°C, 30 s; 52°C, 1 min; 72°C, 2 min; 35 cycles, followed by a final elongation of 7 min at 72°C. Amplification products (10 μL) were loaded on a 2% agarose gel, electrophoresed, stained with ethidium bromide, and visualized under UV light. Quantification of HBV DNA ------------------------- Quantification of HBV DNA was performed by endpoint dilution. DNA samples were diluted to a tenfold series up to 10^-8^dilution. Each dilution was submitted to PCR with oligonucleotide pairs PS1--PS2, designed in the pre-S region and C1--C2 (core region), as described previously \[[@B4]\]. Serial dilutions and PCR assays for HBV DNA quantification were performed in triplicate. The sensitivity of the method has been estimated to about 100 copies per PCR reaction \[[@B18]\], equivalent to 10^4^HBV genome copies per milliliter of serum. Quantitative results were estimated by dividing 10^4^copies/ml by the last positive dilution. Molecular cloning and nucleotide sequencing ------------------------------------------- Pre-S/S PCR products were cloned into pCRII plasmid vector using TA cloning kit (Invitrogen, San Diego, CA). For nucleotide sequencing, recombinant plasmid DNAs were purified by a commercially available kit (Plasmid midi kit, Qiagen, Hilden, Germany). Nucleotide sequences were determined using the Cy5 auto read sequencing kit (Amersham Biosciences, Little Chalfont, UK) with M13 universal and reverse primers, as well as internal, HBV specific primers. For direct sequencing (S region), PCR products were extracted from low melting agarose gels (Qiaquick gel extraction kit, Qiagen). Sequencing reactions with HBV specific primers were done using the thermo sequenase Cy5 dye terminator sequencing kit and analyzed on an ALFexpress automated sequencer (Amersham Biosciences). Independent plus and minus strand sequencing was completed. Phylogenetic analysis --------------------- Nucleotide sequences were aligned using PILEUP (Wisconsin Sequences Analysis Package GCG, Madison, WI). A phylogenetic tree was generated by neighbour-joining analysis of genetic distances, using the TREECON software package for Windows \[[@B19]\]. HBV sequences available from the GenBank database (accession numbers AY090458, M57663, U55220-U55222, J02201 and X51970) were used for the construction of a phylogenetic tree. Results ======= Markers of HBV infection ------------------------ The patient under study developed an acute hepatitis and became anti-HIV positive in 1996, soon after receiving blood transfusions during the initial period of hemodialysis treatment. He became an HBV chronic carrier, with all blood samples collected between 1996 and 2002 being positive for HBsAg and HBeAg. During this period, the patient remained asymptomatic without clinical signs for HBV infection. Aspartate aminotransferase and alkaline phosphatase levels remained normal all during the follow-up. However, an increase of alanine aminotransferase (ALT) levels to 180 IU/L was observed in March 1999 (Figure [1](#F1){ref-type="fig"}). HIV treatment started in April 1996. Lamivudine was introduced as part of antiretroviral treatment in November 1996. HBV DNA was detected at high levels (10^8^copies/ml) in the blood sample collected in March 1999, during this first period of lamivudine treatment (Figure [1](#F1){ref-type="fig"}). In May 1999, lamivudine was discontinued, and the sample analyzed during the period of lamivudine interruption (August 2001) showed the highest (10^9^copies/ml) HBV DNA titre mesured in this study. HBV DNA was again detected at high levels (10^8^copies/ml) after reintroduction of lamivudine (November 2002, Figure [1](#F1){ref-type="fig"}). HBV variants ------------ Nucleotide sequences of HBVs (pre-S/S region) derived from HBsAg/HBeAg positive samples collected in March 1999, August 2001 and November 2002 were determined (three clones each). Phylogenetic analysis showed that all nine clones belonged to genotype A. This genotype has been subdivided into two subgenomic groups, designated A-A\' (genotype A excluding A\') and A\' \[[@B20]\]. Recently, subgroups A-A\' and A\' were designated respectively as A1 and A2 \[[@B21]\] or Ae and Aa \[[@B22]\]. Isolates belonging to subgroup A\' have been first identified in South Africa and circulate in a high proportion among HBV Brazilian isolates \[[@B23]\]. As can be observed on the phylogenetic tree represented in Figure [2](#F2){ref-type="fig"}, all clones belonged to subgroup A\' and were closely related to each other. The six clones obtained during the two periods of lamivudine treatment (1999 and 2002) clustered separately (with a bootstrap value of 86%) from those obtained in 2001, during the period of lamivudine interruption. Table [1](#T1){ref-type="table"} shows the main amino acid changes observed in the S region of the genome. Two substitutions, H359Y in the polymerase and Y100C in the surface antigen (small S), were observed in all HBV sequences. These two changes were natural variation, characteristics of the HBV strain infecting the patient under study. All three clones derived from the sample collected in 1999, during the first lamivudine treatment, showed two lamivudine resistant mutations (L526M and M550V), also detected in the major viral population by direct sequencing. One clone (1-B57) showed an additional mutation related to drug resistance (V519L) that was not detected by direct sequencing. None of these three mutations associated with lamivudine resistance was observed in sequences derived from the second sample (2001). In contrast, all three clones derived from the third sample (2002) showed the three lamivudine resistant mutations. However, by direct sequencing of PCR products of this last sample, the electropherogram could detect two nucleotides (A and G) at the same sequence position indicating a mixture of V519 and L519 residues. Curiously, all HBV sequences obtained during the period without lamivudine treatment (2001) displayed a unique G473E substitution in the polymerase gene. Stop mutations were also observed in the small S protein, which was truncated at position 182 in clone 2-B14 and at position 216 in clone 2-B62 (Table [1](#T1){ref-type="table"}). Due to overlapping of polymerase and S genes on the HBV genome, mutations at positions 473, 519 and 550 of the polymerase were accompanied by mutations at positions 119, 164 and 195 of the small S protein (Table [1](#T1){ref-type="table"}). Discussion ========== Among lamivudine resistant mutations, those in the YMDD motif of the HBV DNA polymerase are the most common. The resistance occurs by replacement of a methionine residue at position 550 by either valine (M550V) or isoleucine (M550I). More rarely, HBV variant presenting M550S replacement may be selected during lamivudine treatment \[[@B24]\] The M550V variant may be accompanied by a mutation (leucine to methionine) at position 526 \[[@B25]\]. In the absence of HIV infection, the mutation at position 550 alone has been found in up to two-thirds of patients with lamivudine-resistant chronic hepatitis B. However, more than 90% of HIV-HBV co-infected patients display the double lamivudine resistant mutation at positions 526 and 550 \[[@B12]\]. Furthermore, the presence of L526M mutation in addition to mutation at position 550 seems to be associated with prolonged lamivudine treatment \[[@B26]\]. The double mutant has been shown to exhibit a 15-fold decrease of the *in vitro*susceptibility to lamivudine \[[@B27]\], since each mutation contributes to the loss of lamivudine sensitivity \[[@B28]\]. It is believed that the L526M mutation, when not accompanied with a mutation in the YMDD motif, does not confer lamivudine resistance \[[@B29]\]. Even so, the L526M mutation has been found alone in patients under lamivudine therapy \[[@B30]\]. Here, both L526M and M550V mutations were detected in all HBV sequences derived from the two blood samples collected during lamivudine treatment (1999 and 2002). Besides these common mutations, a third lamivudine resistant, rare mutation \[[@B11],[@B31]\], namely V519L, was detected in one clone derived from the sample collected in 1999 as well as in all HBV sequences derived from the sample collected in 2002. This was in agreement with recent observations, showing that long lamivudine treatment may result in the predominance of this rare mutant \[[@B32]\]. The triple mutation V519L, L526M, M550V causes the concomitant amino acid substitutions E164D and I195M in the small S protein. It has been shown that this triple mutant has a reduced *in vitro*affinity to anti-HBs antibodies, similar to the hepatitis B vaccine escape mutant G145R \[[@B11],[@B31]\]. The accumulation of mutations in the HBV genome should be monitored in order to guide patient management adequately. There is a general consensus that the lamivudine resistant single mutants in the YMDD motif (M550V/I) replicate substantially more slowly than the wild type. The addition of the mutation at position 526 may act as a compensatory change that partially restores the replication fitness of the virus \[[@B33]\]. Here, in agreement with this observation, HBV DNA was detected at high levels (about 10^8^copies/ml) in the blood sample collected in 1999, in which the double mutant represents the major viral population. Similar high HBV loads were observed in the sample containing the triple mutant (2002). This result is in agreement with a recent study, showing that V519L mutation also enhances viral replication \[[@B34]\]. The HBV population that emerged during the interruption of lamivudine treatment did not show mutations at polymerase positions 519, 526 and 550. However, another substitution, namely G473E, was observed that was associated to G119R substitution in the small S protein. Such a virus might be present as a minor population but not detected during the first lamivudine treatment. Although two out three clones of this population possessed stop codon mutations in S gene, HBV load (10^9^copies/ml) was moderately higher during the lamivudine interruption period than that found during lamivudine periods. HBV chronically infected patients submitted to lamivudine treatment may have acute exacerbation after withdrawal of drug therapy or when lamivudine resistance emerges \[[@B8],[@B14]-[@B16],[@B26],[@B35]-[@B37]\]. In HIV-HBV co-infected patients, withdrawal of lamivudine may result in severe \[[@B8],[@B35],[@B37]\] or fulminant \[[@B37]\] hepatitis. Factors that trigger severe hepatitis in these cases are not well known. Both viral load and genome variations have been implicated in the pathogenesis of acute exacerbation. Here, the patient under study did not present clinical signs of HBV infection. The absence of acute exacerbation after the emergence of lamivudine resistant variants may be correlated to the absence of significant variations of the viral load. Indeed, HBV DNA was detected at high levels (10^8^--10^9^copies/ml) at different periods of antiretroviral treatment with and without lamivudine. Conclusions =========== A rare HBV triple mutant, belonging to genotype A, subgroup A\', appeared predominantly in a patient submitted to lamivudine as part of HIV treatment. This type of mutant, previously found in isolates belonging to genotypes A, D and G \[[@B15],[@B36]\], may behave as a vaccine escape mutant. Understanding the circumstances leading to the appearance of such HBV strains may help to guide future therapies in HIV-HBV co-infected patients. A correlation may exist between acute exacerbation of hepatitis B and HBV load in lamivudine treated patients. Competing interests =================== None declared. Authors\' contributions ======================= EAS and MVS carried out cloning and sequencing of HBV DNA. JA was involved in clinical evaluation of the patient and supervised the antiretroviral treatment. SAG conceived the study, participated in its design and coordination, and wrote the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/29/prepub> Acknowledgements ================ The authors acknowledge Drs Christian Niel and Regina MB Martins for the critical reading of the manuscript. This work was supported by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### HBV load (vertical bars) and ALT levels (curve) during antiretroviral therapies. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Phylogenetic tree of HBV isolates constructed with the neighbor-joining method, and based on the nucleotide sequences of the entire pre-S/S region (nt positions 2854 to 833). Isolates whose names begin by 1,2,3 were molecular clones from this study derived from samples collected in 1999, 2001, and 2002, respectively. The other isolates are designated by their GenBank accession numbers. Genotype A isolates clustered in two subgroups, designated A-A\' (genotype A excluding A\') and A\' \[27\]. The sequence AY090458 belongs to genotype F and was used as an outgroup. Numbers at internal nodes indicate percentage of 100 bootstrap replicates that support the branch. Only values \> 85% are indicated. The horizontal bar provides a genetic distance. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Amino acid replacements in polymerase and surface antigen (small S) ::: Year of collect Lamivudine HBV sequences Polymerase residues S residues --------------------------- ------------ --------------- --------------------- ------------ --------- ------- ------- --- --- --------- ---- ------- ---- 1999 Yes Direct Y G V **M** **V** C G E W **M** L 1-B40 Y G V **M** **V** C G E W **M** L 1-B52 Y G V **M** **V** C G E W **M** L 1-B57 Y G **L** **M** **V** C G **D** W **M** L 2001 No Direct Y E V L M C R E W I L 2-B14 Y E V L M C R E \* I L 2-B62 Y E V L M C R E W I \* 2-B63 Y E V L M C R E W I L 2002 Yes Direct Y G **L/V** **M** **V** C G **D/E** W **M** L 3-B02 Y G **L** **M** **V** C G **D** W **M** L 3-B06 Y G **L** **M** **V** C G **D** W **M** L 3-B71 Y G **L** **M** **V** C G **D** W **M** L *Consensus of genotype A* H G V L M Y G E W I L Lamivudine resistant mutations in the viral DNA polymerase and their counterparts in the small surface antigen are marked in bold. Asterisks represent stop mutations. :::

PubMed Central

2024-06-05T03:55:47.576136

2004-8-31

PMC516777

Background ========== Prospective trials have the disadvantages of requiring a long time to complete, and using highly selected patient subgroups in tertiary centers. While one waits for the results to mature, this delays additional research to improve treatment. If there were a method that allowed earlier prediction of the results of prospective trials, advances in cancer treatment could be attained within a shorter time period. There is a parametric lognormal model, proposed by Boag \[[@B1]-[@B3]\] that had been retrospectively validated in the literature, and could be used prospectively for clinical trials to predict long-term survival rates several years earlier than would otherwise be possible using the standard life-table/actuarial Kaplan-Meier method of calculation \[[@B4]\]. The prognosis for metastatic breast cancer is generally poor and therefore it is believed that statistical prediction models for long-term survival rates are not needed. Nevertheless, specific subgroups of metastatic breast cancer patients exist, for which depending on the treatment given, the prognosis is improved so that some patients can survive for some time, particularly for those with limited organs involvement such as involvement with bone and/or skin only. In this situation, for which the present study was relevant, a prediction model, even for metastatic breast cancer, can be useful. Breast cancer, among other cancers, has the highest incidence in women, and many studies are currently in progress to assess treatment regimens. If, even for a subgroup of patients, the 10- and 15-year survival rates can be predicted from follow-up data available only 3 years after a 5-year diagnosis period, this would be a useful means of obtaining study results earlier than would otherwise have been possible. For example, a 15-year survival rate calculated by the Kaplan-Meier method requires at least some patients to have been followed for 15 years. In addition prediction model such as the lognormal model can also be used to review the progress of treatment results for a specific period from a treatment center, and to compare that with another specific period of the same treatment center to evaluate the potential impact for any possible change in treatment policy or guideline. Boag\'s lognormal model for long-term cancer survival rates has been available for use for some 50 years. When the lognormal model was first proposed in the 1940s, it was difficult to implement because of a lack of computing power, and lack of good quality long-term follow-up data from cancer registries. Since 1970s the model has been used by some authors in breast cancer, cervix uteri cancer, head and neck cancer, intraocular melanoma, choroidal-ciliary body melanoma, and small cell lung cancer \[[@B5]-[@B10]\]. Currently, although the computing power is sufficient, good quality follow-up data on a sufficient number of patients are seldom available, and it can be a limitation for its application. Large data registry such as the Surveillance, Epidemiology, and End Results (SEER) data \[[@B11]\] with good long-term follow-up data available can overcome this potential limitation. Methods ======= Between 1981 and 1985, 782 and 580 female patients of metastatic breast cancer were extracted respectively from the Connecticut and San Francisco-Oakland registries from the SEER database using SEER\*Stat 5.0 software. The two registries were chosen because they are two of the earliest registries, with a large population. The data used were survival time, vital status, cause of death, age at diagnosis, and race. The cause-specific survival was defined as the interval from the date of diagnosis to the date of death from breast cancer or the last follow-up date for censoring purposes, if the patient was alive and still being followed at the time of analysis. The survival time of the uncured group of patients who died of breast cancer had been verified to follow a lognormal distribution previously \[[@B12]\]. Next, between 1991 and 1995, 752 and 632 female patients of metastatic breast cancer were extracted respectively from the two registries. The data were used to estimate the 15-year cause-specific survival rates before the year 2005. To be comparable, for both the 1981--1985 and 1991--1995 cohorts, the staging system used was the SEER historical system (classified as localized, regional, or distant, based on combined pathologic and clinical data). The choice of 1981--1985 and 1991--1995 has the advantage that the two time periods are not too far apart otherwise there would be too much changes of medical practice. These time periods have a minimum of 5 years follow-up. The overall survival rates (OSR) of the two time periods were calculated using the Kaplan-Meier method. The actual relative survival rates (RSR) were calculated using SEER\*Stat 5.0 software. The modified version of period analysis \[[@B13]\] was applied using the Hakulinen method \[[@B14]\] to obtain more up-to-date absolute survival rates (ASR) and relative survival rates (RSR) for comparison purpose with a computer program run by Microsoft Excel software. Validation of the lognormal model --------------------------------- The validation of the lognormal model has two phases. Phase 1 tests the goodness of fit to a lognormal distribution of the survival time of those cancer patients who died with their disease present, termed an uncured group with a fraction of 1-C, where C is the cured proportion of patients. The lognormal distribution is similar to the normal distribution in that if the variable in the normal is time t, the variable in the lognormal is the logarithm of t. In other words, the investigators attempt to show that the logarithm of the survival time follows a normal distribution. Phase 2 attempts to show that the lognormal model, using short-term follow-up data, can predict long-term survival rates comparable to those calculated by the Kaplan-Meier life-table method with long-term available. This model can be used to estimate long-term cause-specific survival rates (CSSR) by a maximum likelihood method (e.g., 10-year and 15-year survival rates) from only short-term follow-up data. The maximum likelihood method is used to estimate the CSSR at time τ, and is calculated as \[C+(1-C)·Q\]·100%, where Q is the integral of the lognormal distribution between the limits time τ and infinity. The lognormal statistical model had been validated in stages III and IV breast cancer in a previous publication that survival rates could be estimated several years earlier than is possible using the standard life-table actuarial method \[[@B12]\]. The survival time of unsuccessfully treated cases could be represented by a lognormal distribution, the long-term survival rates were predicted by Boag\'s method using a computer program run by Microsoft Excel. In this parametric lognormal model, the standard deviation S was fixed, and only the two remaining parameters, mean M and proportion cured C, were kept floating when using the maximum likelihood method. Multiple iterations converged to a stable solution for C. A 5-year period of diagnosis could be selected and patients followed as a cohort for an additional 3 years. The current study was for metastatic breast cancer patients treated between 1981 and 1985, with follow-up to the end of year 2000, making the series ideal for validating purposes. For example, for cases diagnosed during the 5-year period, prediction of the 15-year survival rate was made using data at the follow-up cutoff date of December 31, 1988 (i.e., 3 years after 1985). The 15-year survival rate prediction was then validated by Kaplan-Meier life-table calculations using the follow-up data available in 2000. For metastatic breast cancer patients treated between 1991 and 1995, and follow-up to the end of year 2000, prediction of the 15-year survival rate was made using data at the follow-up cutoff date of December 31, 1998 (i.e., 3 years after 1995) before the year 2005. Results ======= From the cohort of 1981--1985 inclusively, 782 patients from the Connecticut registry were followed to the end of 1988. The lognormal model predicted the 15-year CSSR to be 7.1% (95% CI, 1.8--12.4). The 15-year CSSR was 8.3% (95% CI, 5.8--10.8) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. From the cohort of 1981--1985 inclusively, 580 patients from the San Francisco-Oakland registry were followed to the end of 1988. The lognormal model predicted the 15-year CSSR to be 9.2% (95% CI, 3.9--14.5). The 15-year CSSR was 7.0% (95% CI, 4.3--9.7) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. Using the same method, the cohort of 1991--1995 inclusively, 752 patients from the Connecticut registry were followed to the end of 1998. The lognormal model predicted the 10-year CSSR to be 12.6% (95% CI, 7.3--17.9). The 10-year CSSR was 11.3% (95% CI, 7.8--14.8) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. The lognormal model predicted the 15-year CSSR to be 9.1% (95% CI, 3.8--14.4), which cannot be validated before 2005. For the cohort of 1991--1995 inclusively, 632 patients from the San Francisco-Oakland registry were followed to the end of 1998. The lognormal model predicted the 10-year CSSR to be 17.0% (95% CI, 12.1--21.9). The 10-year CSSR was 15.9% (95% CI, 11.4--20.4) validated by the Kaplan-Meier calculation using actuarial follow-up data up to the end of year 2000. The lognormal model predicted the 15-year CSSR to be 14.7% (95% CI, 9.8--19.6), which cannot be validated before 2005. For the period 1991--1995, there was not much change of only about 2% absolute percentage point in the predicted 15-year CSSR for the Connecticut registry, but there was an improvement of about 6% absolute percentage points for the San Francisco-Oakland registry when compared with the period 1981--1985 15-year CSSR, which was validated by the Kaplan-Meier calculation. (Table [1](#T1){ref-type="table"}) For comparison purpose, the actual OSR and RSR were compared with the ASR and RSR obtained by the period analysis. (Tables [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}) It was found that there were more patient survival improvements shown in the actual OSR and RSR for the San Francisco-Oakland registry, but not much for the Connecticut registry. However the period analysis results did not show such improvements. Discussion ========== Lognormal model --------------- Rutqvist studied the fit of Boag\'s lognormal model to the survival times of 8170 breast cancer cases reported to the Swedish Cancer Registry during 1961--1963. The model fitted the 1961--1963 data well for the entire case material and for patients aged less than 70 years. In this registry, the lognormal model did not fit the data for patients aged greater than 70 years, who were more likely to be censored because of coincidental causes of death. Another disadvantage stated by the author was that large number of patients was required to obtain estimates with reasonably small standard errors for breast cancer. With another series of the Norwegian Cancer Registry of 14,000 breast cancer cases, Rutqvist *et al*. \[[@B15]\] deduced that lognormal is the best model because other models did not fit the observed survival in all stages, ages, and time periods (two-parameter models, such as exponential or extrapolated actuarial, or three-parameter models, such as sum of two exponential, exponential with shoulder, Weibull). Both the exponential and extrapolated actuarial models assume that the conditional relative survival is lowest immediately after treatment. With the lognormal model, the survival curve has a low initial mortality that rapidly increases to a maximum, with a slow decrease in the mortality after the maximum has occurred. Requirements for using the lognormal model ------------------------------------------ The lognormal model can only predict cause-specific survival, because other coincidental causes of death are too unpredictable (e.g., the rate of stroke). Therefore, overall survival cannot be predicted. The maximum likelihood method is the most accurate method for fitting the lognormal model with the smallest mean squared error. However, there are some requirements for its use. The maximum likelihood method fails to converge to a stable solution using the initial estimates if there is extensive censoring within the data. This occurs if patients are lost to follow-up or die from coincidental non-cancerous causes. The frequency of failure to yield a successful fit for lognormality was greater when one-fourth of cases were designated as lost to follow-up. Gamel *et al*. established a stable linear algorithm for fitting the lognormal model to survival data. To achieve convergence, some authors have fixed one or two parameters of the lognormal model to pre-selected values to simplify the iterative procedure required for convergence \[[@B6]\]. Some prognostic factors follow lognormal distribution ----------------------------------------------------- Prognostic factors in patients with distant metastases at the time of diagnosis were investigated by Rudan *et al*. \[[@B16]\], and Chapman *et al*. \[[@B17]\], primary tumor size was a significant prognostic factor. Engel *et al*. \[[@B18]\] found that the number of metastatic cases and the time to metastasis depended on the tumor diameter at diagnosis. Cell growth is essential for the development of tumors. Tumor size is therefore the most important factor in describing tumor biology. As the tumor size increases, the probability of node-positivity increases. Another study group also found this correlation up to 5 cm \[[@B19]\]. Tubiana and Koscielny \[[@B20]\] have found a highly significant correlation between tumor size and the probability of distant metastasis. The distribution of tumor sizes at metastatic spread was *lognormal*with a median diameter equal to 3.5 cm. The patients were subdivided into 3 groups according to the histological grade. In each subgroup there was a significant correlation between tumor size and the probability of distant spread. The distributions were lognormal and the median size was markedly larger for grade 1 tumors. A number of quantitative postmortem observations regarding the size distribution of metastases have been published \[[@B21]-[@B23]\]. These studies revealed a skewed distribution with a high proportion of smaller metastases, and a significant tail extending to the larger metastases, consistent with a lognormal distribution. The more detailed measurements from human liver metastases provided by Yamanami *et al*. were found to approximate the lognormal distribution reasonably well. A hypothesis was proposed by Kendal \[[@B24]\] that the time available for the growth of metastases is normally distributed, presumably as a consequence of the summation of multiple independently distributed time intervals from each of the steps and of the Central Limit Theorem. For exponentially growing metastases, the corresponding size distribution would be lognormal; Gompertzian growth would imply a modified (Gompertz-normal) distribution, where larger metastases would occur less frequently as a consequence of a decreased growth rate. These two size distributions were evaluated against 18 human autopsy cases where precise size measurements had been collected from over 3900 macroscopic hematogenous organ metastases. The lognormal distribution provided an approximate agreement. Its main deficiency was a tendency to over-represent metastases greater than 10 mm diameter. These observations supported the hypothesis of normally distributed growth times, and qualified the utility of the lognormal and Gompertz-normal distributions for the size distribution of metastases. Why is the lognormal model applicable to so many organ sites \[[@B3],[@B6]-[@B10],[@B12],[@B25]-[@B36]\] (Table [4](#T4){ref-type="table"})? Boag\'s explanation for the lognormal survival time distribution was that if the patient was not cured by treatment, the length of the remaining survival time would be dependent principally on the growth rate of the tumor remnants. Pearlman \[[@B37]\] estimated the growth rates of breast cancer that recurred in the scar, assuming that the recurrence started from a single cell. He found that the growth rates were approximately lognormally distributed. Likewise, von Fournier *et al*. \[[@B38]\] found that the growth rates of breast cancers followed by serial mammography were lognormally distributed. Variation of survival rates over time ------------------------------------- In order to determine whether current programs for the management of metastatic breast cancer have led to improved patient survival, Debonis *et al*. \[[@B39]\] determined the median survival times for five-year intervals of 849 patients admitted to the City of Hope National Medical Center with metastatic breast cancer from 1955 to 1980. Survival times in each of the clinical subsets remained unchanged during the period of observation, regardless of the therapeutic modalities included in the treatment regimens. The study indicates that changes in palliative therapy for metastatic breast cancer during the 25 years of observation have not influenced overall survival. On the contrary, Dickman *et al*. \[[@B40]\] studied the survival of cancer patients in Finland during the years 1955--1994. The 5-year RSR for distant metastases breast cancer had increased from 10% for the period 1955--1964 to 22% for 1985--1994. The tumor registry at Yale-New Haven Hospital, which began recording data in 1920, was utilized by Todd *et al*. \[[@B41]\] to examine the ultimate outcome of all breast cancer patients who were initially diagnosed at Yale with metastatic breast cancer. The median survival of these patients increased steadily from 21 months in 1920 to 41 months in the decade from 1970 to 1980. The percentage of women actually surviving 5 years increased from 5% in the 1920s to approximately 25% in the 1960s. Despite the use of combination drug programs in the 1970s, the percentage of these patients remaining alive at 5 years remained near 25%. Firm conclusions cannot be made from a retrospective study spanning 60 years, although the trends depicted the lack of continued improvement indicate that the current therapeutic approach to metastatic breast cancer in that period may not result in dramatic improvement in overall survival. Geographical variation of survival rates ---------------------------------------- Farrow *et al*. \[[@B42]\] documented substantial geographical variation in patterns of treatment of cancer and other diseases. Because cancer treatment is not uniform nationwide in the States, survival following the diagnosis of cancer might also be expected to vary geographically. Survival data from the nine population-based registries in the SEER Program were analyzed for cancers of the stomach, colon, rectum, lung, breast, uterus, ovary, prostate, and bladder. The patients included all non-Hispanic white patients diagnosed with cancer of one of the selected sites during 1983--1991. Regional variation in crude five-year survival rates across the nine SEER areas was most marked for cancers of the uterus and prostate. For uterine cancer, for example, five-year survival ranged from 73.2% in Connecticut to 84.0% in Hawaii. Less marked variation was observed for cancers of the colon, rectum, and breast. For cancers of the bladder, ovary, stomach, and lung, survival rates five years after diagnosis were relatively invariant across the SEER areas. Maggard *et al*. \[[@B43]\] also found that variations in the breast cancer mortality rates exist between states. A nearly 50% increase is observed between the states with the highest and lowest mortality rates. Adjusted analyses demonstrated that stage at presentation is a more important predictor of mortality variation than treatment differences. Goodwin *et al*. \[[@B44]\] examined breast cancer incidence, survival, and mortality in the 66 health service areas covered by the SEER program for women aged 65 and older at diagnosis. They found that there was considerable geographic variation in survival from breast cancer among older women, and this contributed to variation in breast cancer mortality. The elevated mortality in the Northeast is apparent only in older women \[[@B45]\]. For women aged 65 years and older, breast cancer mortality is 26% higher in New England than in the South, while incidence is only 3% higher. Breast cancer mortality for older women by state correlates poorly with incidence (r = 0.28). The above-mentioned results are consistent with that from the present study: the Connecticut registry has lower CSSR than the San Francisco-Oakland registry for the period 1991--1995. The Connecticut cohort has median age at diagnosis of 66 (range 25--103), while the San Francisco-Oakland cohort has lower median age of 63 (range 26--96). It could be argued that new treatments evolved in the recent decade have improved the survival of the breast cancer patients, and younger patients benefit more than the older patients. Apart from treatment offered, changes of survival rates over time or geographical areas can be due to co-morbidities or other characteristics such as race, age, and differences in staging procedures. Conclusions =========== For the period 1981--1985, the 15-year cause-specific survival for the Connecticut and the San Francisco-Oakland registries were comparable. For the period 1991--1995, there was not much change in survival for the Connecticut registry patients, but there was an improvement in survival for the San Francisco-Oakland registry patients. List of abbreviations ===================== CSSR: Cause-specific survival. SEER: Surveillance, Epidemiology, and End Results. OSR: Overall survival rate. ASR: Absolute survival rate. RSR: Relative survival rate. Competing interests =================== None declared. Authors contributions ===================== PT: Data analysis and writing of manuscript. EY, VVH, GC, GV: Critical appraisal of manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/4/60/prepub> Acknowledgments =============== PT: Saskatchewan Cancer Agency Research Grant Award 2792. GC: János Bolyai Research Fellowship from the Hungarian Academy of Sciences. Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### 10- and 15-year cause-specific survival rates for Connecticut and San Francisco-Oakland registries for 1981--1985 and 1991--1995 cohorts calculated by lognormal model (LN) and Kaplan-Meier (KM) method with 95% confidence intervals in brackets ::: Connecticut San Francisco-Oakland ---- ------------------ ----------------------- ------------------ ------------------ ------------------ ---------------- ------------------- ------------------- 10 11.5 (6.2--16.8) 11.0 (8.5--13.5) 12.6 (7.3--17.9) 11.3 (7.8--14.8) 12.3 (7.0--17.6) 9.7(6.8--12.6) 17.0 (12.1--21.9) 15.9 (11.4--20.4) 15 7.1 (1.8--12.4) 8.3 (5.8--10.8) 9.1 (3.8--14.4) N.A. 9.2 (3.9--14.5) 7.0 (4.3--9.7) 14.7 (9.8--19.6) N.A. N.A. = not available ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### 10- and 15-year OSR^a^, RSR^b^, ASR^c^, RSR^d^for Connecticut registry for 1981--1985 and 1991--1995 cohorts with 95% confidence intervals in brackets ::: Connecticut ------------- ---------------- ----------------- ------------------- ------------------- ----------------- ----------------- ------------------- ------------------- Year 1981--1985 1991--1995 OSR^a^ RSR^b^ ASR^c^ RSR^d^ OSR^a^ RSR^b^ ASR^c^ RSR^d^ 10 6.4 (4.6--8.2) 9.1 (6.7--11.5) 13.0 (12.2--13.8) 16.7 (15.7--17.7) 7.2 (3.7--10.7) 9.7 (6.4--13.0) 15.6 (14.6--16.6) 20.7 (19.5--21.9) 15 3.2 (2.0--4.4) 5.7 (3.5--7.9) 4.4 (4.0--4.8) 6.4 (6.0--6.8) N.A. N.A. 5.8 (5.4--6.2) 8.9 (6.2--9.5) OSR^a^= overall survival rate calculate by Kaplan-Meier method RSR^b^= relative survival rate calculated using SEER\*Stat 5.0 ASR^c^= absolute survival rate calculated by period analysis RSR^d^= relative survival rate calculated by period analysis N.A. = not available ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### 10- and 15-year OSR^a^, RSR^b^, ASR^c^, RSR^d^for San Francisco-Oakland registry for 1981--1985 and 1991--1995 cohorts with 95% confidence intervals in brackets ::: San Francisco-Oakland ----------------------- ---------------- ---------------- ------------------- ------------------- ----------------- ------------------ ------------------- ------------------- Year 1981--1985 1991--1995 OSR^a^ RSR^b^ ASR^c^ RSR^d^ OSR^a^ RSR^b^ ASR^c^ RSR^d^ 10 4.6 (2.8--6.4) 6.6 (4.2--9.0) 13.3 (12.3--14.3) 16.9 (15.7--18.1) 8.8 (5.7--11.9) 11.4 (7.9--14.9) 14.7 (13.7--15.7) 18.4 (17.2--19.6) 15 2.4 (1.2--3.6) 4.1 (1.9--6.3) 4.6 (4.2--5.0) 6.6 (6.0--7.2) N.A. N.A. 5.3 (4.9--5.7) 7.4 (6.8--8.0) OSR^a^= overall survival rate calculate by Kaplan-Meier method RSR^b^= relative survival rate calculated using SEER\*Stat 5.0 ASR^c^= absolute survival rate calculated by period analysis RSR^d^= relative survival rate calculated by period analysis N.A. = not available ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Cancer sites with survival times demonstrated to follow the lognormal distribution in the literature as at year 2004 ::: Cancer sites Author ---------------------------- ------------------------------------------------------------------------------------------------------------ Head and neck cancer Berg \[25\], Mould\[7\] Nasal sinus cancer Berg \[25\] Mouth and throat cancer Boag \[3\] Mouth Berg \[25\] Thyroid Tai\*\[26\] Larynx, tongue Mould & Tai\*\[27\] Non small cell lung cancer Berg \[25\] Small cell lung cancer Tai\*\[10\] Intraocular melanoma Gamel \[8\] Cutaneous melanoma Gamel \[9\] Breast cancer Boag \[3\], Berg \[25\], Rutqvist \[5,15\], Gamel \[28,29\], Haybittle \[30\], Royston \[31\], Tai\*\[12\] Bone sarcomas Berg \[25\] Cancer of uterine cerivx Mould & Boag\*\[6\], Berg \[25\] Ovarian cancer Berg \[25\], Tai\*\[32\], Royston \[31\] Hypernephroma Berg \[25\] Bladder cancer Berg \[25\] Prostate Cancer Mould & Tai\*\[33\] Gastric cancer Berg \[25\], Maetani \[34\] Lymphoma Berg \[25\] Chronic leukemia Tivey \[35\] Brain tumors Berg \[25\] 42 SEER cancer sites Tai \[36\] SEER, Surveillance, Epidemiology, and End Results; \*, phase 2 validation also performed, i.e. concordance between Kaplan-Meier method and lognormal model. :::

PubMed Central

2024-06-05T03:55:47.577882

2004-9-2

PMC516778

Background ========== Posttraumatic stress disorder (PTSD) is a difficult-to-treat condition that over a lifetime affects approximately 10% of the general population \[[@B1]\]. The condition develops after traumatic events such as combat, terror activities, disaster, or rape, and has 3 main features: (1) reexperiencing the trauma through recollection, dreams, and reliving, (2) avoidance of thoughts, activities, and emotions associated with the trauma, and (3) hyperarousal \[[@B2]\]. PTSD is usually a chronic disorder, with one third of patients displaying symptoms for ≥ 10 years after experiencing the traumatic event \[[@B3],[@B4]\]. Generally the response to pharmacotherapy has been poor, with many patients completely unresponsive and others only marginally responsive \[[@B5]\]. Some tricyclic antidepressants, monoamine oxidase inhibitors, and selective serotonin reuptake inhibitors have demonstrated efficacy in double-blind trials \[[@B3]\]. The complex neurobiology of PTSD involves a number of systems, including dopaminergic, serotonergic, sympathetic, hypothalamic-pituitary-adrenal, and various anatomic regions of the amygdala and other parts of the limbic system \[[@B3]\]. It has been suggested that after traumatic events limbic nuclei may become kindled or abnormally sensitized \[[@B6]\], leading to increased susceptibility for psychic and physical arousal and psychiatric disturbance \[[@B2]\]. Because of the suggested involvement of the kindling phenomenon, several anticonvulsants have been assessed in the treatment of PTSD, including carbamazepine, valproate, lamotrigine, gabapentin, tiagabine, and topiramate \[[@B3]\]. Topiramate has a broad spectrum of pharmacologic properties, including Na^+^channel blockade \[[@B7]-[@B11]\], inhibition of some high voltage-activated Ca^2+^channels \[[@B12]\], enhanced γ-aminobutyric acid (GABA) neuroinhibition at novel GABA~A~receptors \[[@B13],[@B14]\], glutamate inhibition at kainate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors \[[@B15],[@B16]\], and promotion of protein phosphorylation of neuronal conductance channels \[[@B15]\]. These properties, together with inhibitory activity in animal kindling models \[[@B17],[@B18]\], suggest that topiramate may have therapeutic potential in PTSD. Treatment with topiramate has been reported to improve reexperiencing symptoms associated with civilian PTSD \[[@B6]\]. Results from that study, which included a number of patients who were classified as treatment resistant, suggested that topiramate fully or partially suppressed both intrusions (distressing recollections or nonhallucinatory flashbacks) and nightmares, if present, in 89% of patients with nonhallucinatory PTSD. Although the DSM-IV definition of PTSD lists hallucinations among reexperiencing symptoms \[[@B19]\], because a few patients displayed varying degrees of impaired reality testing of hallucinations, it was difficult to determine if hallucinations were due to PTSD, to an independent psychotic disorder, or, in some individuals, to both. Although psychotic variants of PTSD have been reported in as many as 40% of veterans with chronic combat-associated PTSD without evidence of a primary psychotic disorder \[[@B20],[@B21]\], it is also possible for patients with psychotic disorders to have comorbid PTSD, leaving questions about whether specific hallucinations should be attributable to the PTSD reexperiencing cluster or to a psychotic disorder. Because the prior study found a more robust effect in the group of PTSD patients without hallucinations \[[@B6]\], it was decided to focus further on the core group of PTSD patients without ambiguous symptoms confounded by the possible presence of independent psychotic disorders. Based on these earlier results of topiramate in PTSD, a fully prospective open-label study was begun to see whether the previously observed signal of a therapeutic benefit of topiramate for chronic civilian PTSD could be confirmed. None of the patients reported in the earlier study were included in the present study. In order to address methodological limitations of the prior study, the current study is modified in 4 major ways: (1) it excludes patients with hallucinations, (2) it limits the study to 12 weeks, (3) it prospectively employs the PTSD Checklist-Civilian Version (PCL-C) as a validated clinical assessment instrument in all patients, and (4) it determines a clinical response rate as of week 4, using a conventional, predetermined definition for positive response as a ≥ 30% improvement in a standardized clinical rating scale. Methods ======= This sample included all consecutive adults (n = 33) meeting the DSM-IV criteria for chronic civilian PTSD in an outpatient private practice who started open-label topiramate between January 2000 and November 2002 in the course of clinical care. The study excluded patients with concurrent auditory or visual hallucinations associated with either PTSD or a possible psychotic disorder. Topiramate, administered either as monotherapy (n = 5) or added to the patients\' existing therapeutic regimen (n = 28) (Table [1](#T1){ref-type="table"}), was initiated at a starting dosage of 12.5 to 25 mg/day and increased in 25- to 50-mg/day increments every 3 to 4 days as tolerated until a clinical response was achieved. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Patient characteristics ::: All subjects N = 33 ------------------------------------------- --------------------- Mean age, years ± SD 46 ± 6.5  Range 29--55 Sex, %  Women 85  Men 15 Mean age at onset of PTSD, years ± SD 29 ± 15  Range 2--53 Mean duration of PTSD history, years ± SD 18 ± 15  Range 0--46 Patients with comorbid disorders, N (%)  Bipolar disorder 10 (30)  Major depressive disorder 21 (64)  Substance abuse   Current 3 (9)   In past 5 (15) SD =standard deviation; PTSD = posttraumatic stress disorder. ::: The study was performed according to the Declaration of Helsinki. Each participant provided verbal informed consent based on disclosure of off-label usage, the availability of alternative medications for PTSD such as sertraline and paroxetine, and expected adverse events and risks. Verbal consent was deemed appropriate and sufficient because of the nonexperimental nature of the treatment. Because this was a descriptive study using aggregate data gathered in the course of treating patients in clinical practice for their own benefit and not an experimental design, there was no protocol that required Institutional Review Board approval. Patients were asked to measure their symptoms using standardized clinical instruments already routinely used as a part of individual care. The study included no features of clinical studies, such as randomization, concealment of treatment, or surrender of patient decision-making rights, as an integral part of conducting the study. The primary outcome was a change in PTSD symptoms calculated from change in PCL-C score from baseline through 4 weeks of treatment \[[@B22]\]. The PCL-C, a validated self-report instrument, strongly correlates (*k*\~0.90) with the Clinician-Administered PTSD Scale (CAPS), a structured clinical interview widely used in PTSD studies \[[@B23]\]. The PCL-C is scored on a Likert scale of 1 to 5 (1 = not at all; 5 = extremely) for each of 17 items that cover all 3 symptom clusters (B, reexperiencing; C, avoidance/numbing; D, hyperarousal), yielding a score range of 17 to 85. A total score of ≥ 50 meets the threshold for active PTSD. In accordance with conventional usage, a 30% reduction in clinical symptoms of PTSD was defined as a positive clinical response. The secondary measures included were Cluster B symptoms of reexperiencing of trauma, including trauma-related intrusions and nightmares. Secondary measures were limited to Cluster B symptoms because of the practical difficulty patients in clinical practice can have linking a symptom (such as avoidance, numbing, hyperarousal, insomnia, or startling) specifically to a trauma as opposed to some other source, as required by the DSM-IV definitions. Also, Cluster B symptoms appeared to be among the most distressing and salient for many patients, thereby making tracking these distinctive symptoms comparatively easy. Data on patient demographics, concomitant medication, comorbidities, primary trauma, and duration of PTSD were collected at baseline. Assessments were made through clinical interviews over a 12-week period. Cessation of reexperiencing symptoms was considered partial if subjects reported a definite reduction in intensity and frequency of nightmares or intrusive recollections or flashbacks. Full cessation was defined as complete elimination of nightmares and intrusions for a sustained period. Adverse effects were recorded if they resulted in treatment discontinuation. Statistical analyses were performed using Jandel SigmaStat^®^Version 2.0 (Jandel Corporation, San Rafael, CA, USA). A single paired *t*-test of before and after total PCL-C scores was used to analyze the data. Subscale scores for PCL-C Clusters B, C, and D were also evaluated. Results ======= Patient characteristics ----------------------- Baseline patient characteristics and concomitantly administered medications are listed in Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}, respectively. All monotherapy patients were in the nonbipolar subgroup. Primary traumas for which symptoms were reported are detailed in Table [3](#T3){ref-type="table"}. Of these, 9 were of childhood onset, with 4 involving childhood physical assault, 3 unwanted sex or sexual assault, and 2 sudden unexpected or violent deaths. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Concomitant medications ::: **Medication type** **Number of patients (N = 33)** ----------------------- --------------------------------- SSRIs 12 Benzodiazepines 10 Stimulants 6 Atypical neuroleptics 3 Gabapentin 3 Lamotrigine 5 Mirtazapine 3 Venlafaxine 3 Verapamil 1 Other 8 Monotherapy 5 SSRIs = selective serotonin reuptake inhibitors. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Primary traumas ::: **Trauma type** **Number of patients, N = 33** -------------------------------------- -------------------------------- Unwanted sex 11 Physical assault 6 Sudden violent death 4 Sudden unexpected death of loved one 3 Sexual assault 2 Fire/explosion 1 Weapon assault 1 Combat 1 Life-threatening illness 1 Other 3 ::: Primary measures ---------------- ### Reduction in PCL-C symptoms Ninety-one percent of patients (30/33) with baseline PCL-C measurements completed a PCL-C at week 4. In those patients, the total score was 62.6 at baseline and 40.3 at week 4. In addition, the scores for reexperiencing, avoidance, and hyperarousal clusters were 18.2, 25.1, and 19.2 at baseline and 11.2, 17.2, and 12.3 at week 4, respectively. To calculate percentage reduction in PTSD symptoms, to correct for an absence of symptoms being scored as \"1\" for each item, the change in score between baseline and week 4 was divided by the baseline score minus the minimum score for no symptoms. Therefore, the change in total symptoms was calculated as (baseline -- week 4)/(baseline -- 17). Total symptoms declined significantly (49%) by week 4 (paired *t*-test, *P*\< 0.001). In addition, subscale symptoms for reexperiencing (Cluster B), avoidance/numbing (Cluster C), and hyperarousal (Cluster D) decreased by 53%, 43%, and 48%, respectively, from baseline (Figure [1](#F1){ref-type="fig"}). By the end of week 4, 70% of all entrants in the study (n = 33) and 77% of those with both baseline and week 4 PCL-C scores (n = 30) were positive responders, defined as a ≥ 30% improvement in symptoms. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Mean percentage symptom reduction at week 4.**Symptom reduction in those 30 subjects who completed a PCL-C at baseline and after 4 weeks. \*Paired *t*-test, *P*\< 0.001 versus baseline. PTSD = posttraumatic stress disorder; PCL-C = PTSD Checklist-Civilian Version. :::  ::: ### Predictors of reduction in PCL-C symptom improvement Comparisons for bipolar versus nonbipolar patients (50% versus 49%, analysis of variance \[ANOVA\]), duration of symptoms (r = 0.37), age at onset of PTSD (r = -0.36), severity of baseline PCL-C score (r = 0.02), age (r = -0.02), female sex (49% versus 50%, ANOVA), and monotherapy versus add-on medication administration (53% versus 48%, ANOVA) found none significantly associated with reduction in PCL-C symptoms. Secondary measures ------------------ ### Results for combined suppression of nightmares and intrusions are shown in Table [4](#T4){ref-type="table"} ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Secondary efficacy measures ::: **Combined suppression of nightmares and intrusions** ------------------------------------------------------- ----------------- Responder status, n (%)  Full 26/33 (79%)  Partial 3/33 (9%)  None 4/33 (12%) Mean time to response, days ± SD, (range)  Full response (n = 25) 15 ± 18 (1--83)  Partial response (n = 17) 11 ± 13 (2--46) Median time to full response, days 9 Median time to partial response, days 5 Mean dosage at time of response, mg/day  Full response 60 ± 47  Partial response 32 ± 15 Median dosage at time of response, mg/day  Full response 50  Partial response 25 Modal daily dosage for full response, mg/day 25  % of patients in the 12.5--50-mg/day range 65 Modal dosage at time of partial response, mg/day 25  % of patients in the 12.5--50-mg/day range 100 **Improvements in nightmares** Full cessation of nightmares 17/18 (94%) **Improvements in intrusions** Full cessation of intrusions 26/33 (79%) Partial improvement 3/33 (9%) No improvement 4/33 (12%) SD = standard deviation. ::: Of the 33 patients who entered the study, 79% fully ceased having reexperiencing symptoms with a mean time to full cessation of 15 days. The onset of full cessation for all patients occurred at ≤ 200 mg/day and the onset of partial cessation for all patients at ≤ 50 mg/day. Ninety-four percent of patients with nightmares and 79% of patients with intrusions at baseline reported full cessation at the end of week 4. Discontinuations ---------------- ### Serious adverse events There were no serious adverse events during this study. Of the 33 patients who entered the study, 7 (21%) discontinued due to adverse events and 5 (15%) for other reasons. The single most common adverse event resulting in discontinuation was intolerable nervous system overstimulation (panic/nervousness/overstimulation/ shakiness), reported by 3 patients. Other adverse events resulting in discontinuation included clumsiness (n = 2), cognitive impairment (n = 1), and severe headache (n = 1). Additional reasons for discontinuation included personal choice (n = 1) and lack of relapse of PTSD symptoms after interrupting medication (n = 4). Of the 12 patients that discontinued, 54% (n = 7) fully ceased having reexperiencing symptoms of nightmares/intrusions and 15% (n = 2) partially ceased having reexperiencing symptoms of nightmares/intrusions. The median time to termination was 25.5 days (range, 4--77). ### Predictors of discontinuation There were no statistically significant predictors of panic-like adverse events resulting in discontinuation from the study (age, sex, symptom duration, age at onset, bipolarity, substance abuse, baseline severity of illness), although the coadministration of a benzodiazepine nearly reached significance (Fisher exact test, *P*= 0.05). No monotherapy patients discontinued because of overstimulation/panic-like responses, so the possibility of drug interactions or conditions such as comorbid panic disorder contributing to this reaction should be considered in future investigations. Discussion ========== Results of this prospective open-label study suggest that the use of topiramate for the treatment of chronic PTSD in civilians is indeed promising and warrants controlled clinical trials. Topiramate was able to improve symptoms of reexperiencing, avoidance, and hyperarousal by 49% overall, compared with baseline, and decreased intrusions in 94% of patients and nightmares in 79% of those with this symptom. Although there are numerous other reports describing the use of antiepileptic agents for the treatment of PTSD, most are case reports and case series, and some lack the use of standardized PTSD scales. The first published trials using anticonvulsants date back almost 20 years, when a 5-week trial of carbamazepine in 10 combat veterans produced a 36% reduction in a nonvalidated interviewer-scored instrument \[[@B24]\]. Studies on adjunctive therapy with valproate reported improved global scores in avoidance and hyperarousal, but not reexperiencing symptoms in veterans over a 14-month period \[[@B25]\], whereas a subsequent 8-week open-label trial reported a small, 17% reduction in CAPS score in 13 subjects with significant reductions in all 3 cluster criteria \[[@B26]\]. Two studies in which valproate was used as monotherapy reported varying results, with one showing a 30% reduction in total CAPS scores in 28 veterans \[[@B27]\], whereas the other in 10 civilian patients with non-combat-related PTSD showed no improvement \[[@B28]\].The only double-blind placebo-controlled trial evaluating an anticonvulsant in PTSD was conducted with lamotrigine \[[@B29]\]. Overall, after 10 weeks of treatment, 5 of 10 subjects were characterized as responders, compared with 1 of 4 subjects receiving placebo. However, a last observation carried forward analysis revealed only a 23% decrease in mean pretreatment scores with lamotrigine, compared with 20% in the placebo group. A retrospective clinical series of adjunctive gabapentin therapy in 30 consecutive patients with PTSD assessed improvement of target symptoms such as nightmares, insomnia, and irritability. It was reported that the majority of patients (77%) showed moderate or greater improvement in duration of sleep, and most notably a decrease in the frequency of nightmares \[[@B30]\]. These studies highlight the need for double-blind placebo-controlled trials using standardized and validated questionnaires. It is encouraging that the results of this current study with topiramate were consistent with the earlier, preliminary data obtained in patients with PTSD. Percentage responders (77% versus 75%), median time to full response (9 d versus 8 d), suppression of intrusions and nightmares (88% versus 89%), and discontinuation rate (39% versus 32%) were all similar. Overall, the open-label experience with topiramate, based on patient statements and self-rating reports, suggests that the drug may have a rapid rate of response with limited risk of clinically significant adverse events and no evidence of tolerance developing over time \[[@B6],[@B31]\]. Moreover, from topiramate studies in epilepsy \[Topamax prescribing information\] it is known that the drug is not associated with the cardiac, pancreatic, and hematologic toxicity found with valproate or carbamazepine \[[@B3]\]. In addition, topiramate is not associated with weight gain, a side effect reported with valproate that can predispose patients to diabetes mellitus \[[@B32]\]. Topiramate also offers potential clinical advantages over serotonin reuptake inhibitors (SSRIs) because it does not appear to destabilize mood in patients with comorbid bipolar disorder and is relatively devoid of common adverse effects of SSRIs, such as sexual dysfunction, weight gain with chronic use, or sedation when used as monotherapy. Limitations of the present study include: (1) the absence of structured assessment data following week 4 to test the maintenance of response over time, (2) use of self-report instruments that, although validated and correlated with results of structured clinical interviews, may be less accurate than structured interviews, and (3) the absence of clinical assessment scores at the time patients discontinued the trial. From a broader perspective, the greatest limitation of this study is characteristic of all open trials: the lack of standard features of clinical trials such as placebo controls, randomization, and blinding of raters. Conclusions =========== Fundamentally, the findings of current and prior topiramate studies are convergent and consistently signal a potentially beneficial therapeutic effect for all 3 criteria clusters of chronic PTSD in civilian adults. In both studies, there is a positive response in a large proportion of patients, at dosages considerably below the usual anticonvulsant dosage levels of 200 to 600 mg/day, and a rapid onset. The effect appears independent of comorbidity with bipolar disorder, age, sex, duration of symptoms, baseline severity of illness, or administration alone or with other psychotropic medications. The purpose of this study was to prospectively test the results of an earlier open-label trial in a new sample; however, the limitations of those open studies will have to be addressed in double-blind, placebo-controlled studies, which are currently in progress. Competing interests =================== The author is a consultant to and a participant in the Speaker\'s Bureau for Ortho-McNeil Pharmaceutical, Inc. He also holds a licensing agreement with Ortho-McNeil based on issuance of a United States Government patent for topiramate therapy of PTSD. Author\'s contributions ======================= JLB conceived of the study and its design, coordination, statistical analysis, and drafted the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-244X/4/24/prepub>

PubMed Central

2024-06-05T03:55:47.580499

2004-8-18

PMC516779

Background ========== Autoimmune thyroid disease may be linked to depression \[[@B1]\] and anxiety \[[@B2]\]. Autoimmune disease and depression are not uncommon: the prevalence of autoimmune thyroid disease in the community ranged from 4 to 25% \[[@B3]\] and lifetime prevalence of Major Depressive Disorder ranged from 6 to 17% \[[@B4]\]. Thus the association may have a great relevance in terms of public health and prevention. The purpose of this investigation was to evaluate the relationship between mood and anxiety disorders and thyroid autoimmunity in a community survey. This research was carried out on the data base of two epidemiological studies aimed at defining the prevalence of psychiatric \[[@B5]\] and thyroid diseases \[[@B6]\] in Sardinia. On planning these surveys, researchers agreed to evaluate a representative sub-sample of a defined geographical area common to both endocrinological and psychiatric epidemiological surveys. This paper present the results of the cross psychiatric and endocrinological evaluation from the common areas of the two surveys. Methods ======= The sample was extracted by randomization (1/10) subsequent to stratification according to age and sex, from the records of 2 Sardinian villages. Probands were interviewed face to face in their homes by specifically trained physicians. Two standardized forms were used to acquire information concerning: demographic data, state of health and use of social and health services. Psychiatric diagnosis was made using the Italian Simplified version of the Composite International Diagnostic Interview (CIDIS) \[[@B7]\]. The computer elaboration of data obtained enabled prevalence of psychiatric disorders according to DSM-IV \[[@B8]\] diagnostic criteria to be calculated. Anti-thyroid peroxidase autoantibodies (anti-TPO), considered as the most sensitive and specific marker of thyroid autoimmunity \[[@B9]\] was determined by RIA (Sorin Biomedica Diagnostics, Saluggia, Italy) with a cut-off value of 20 IU/ml. All subjects underwent a complete thyroid evaluation including physical examination, thyroid echography and measure of serum free T4 (FT4), free T3 (FT3), thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase autoantibodies (anti-TPO). FT4 and FT3 were measured by means of a chromatographic method based on separation of free T4 on Lisophase columns (Technogenetics, Milan, Italy; normal values: FT4 6.6--16 pg/ml; FT3 2.8--5.6 pg/ml). TSH was measured by a chemiluminescent method (Ortho-Clinical Diagnostics Amersham, U.K.) with normal values ranging from 0.3--3.0 μU/ml. Thyroid echography was performed using a \"real time\" echograph (ALOKA Mod SSD 500 with a small parts 7.5). The association of anti- TPO+ with the main diagnoses deriving from CIDIS interview was calculated using Odds Ratio. Statistical significance was calculated using the X^2^ test in 2 × 2 tables. Odds Ratio confidence intervals were calculated through application of the method of Miettinen \[[@B10]\]. Multivariate Logistic Regression was performed in order to evaluate the possible influences of gender and age on the association between anti-TPO+ and mood or anxiety disorders. The analysis was carried out considering mood (or anxiety) disorders as dependent variable, and anti-TPO+ (presence vs absence), gender (female vs male) and age (≤ 44 vs \> 44) and their second order interactions as independent variables, by means of backward stepwise procedure; interactions lacking evidence of association (p \> 0.20) were eliminated from the models. Results ======= From a total of 261 subjects identified (age \>18 years), 222 (85.1%), 127 females (57.2%), and 95 males (42.7%); over 44 years 127 (57.2%), 79 females (62.2%,) 48 males (37.7%), agreed to take part in the study whilst 20 (8,7%) refused to participate and 19 (7.3%) could not be traced. The final sample did not differ respect to the population of origin with reference to the variables applied in stratification. The lifetime prevalence of anxiety disorders in the sample was: Generalized Anxiety Disorder (GAD) 11.3%, Panic Disorder (PD) 2.7%, Anxiety Disorder Not Otherwise Specified (ADNOS) 5.4%, Social Phobia (SP) 5.4%; 18.5% had been diagnosed with at least one of these anxiety disorders. With regard to mood disorders, Major Depressive Episode (MDE) was present in 14.4%, Dysthymic Disorder (DD) in 2.7%, Depressive Disorder Not Otherwise Specified (DDNOS) in 4.0%; 18.9% had at least one of the above mentioned depressive disorders. 1.1% of the overall sample was affected by hypothyroidism, 16.6% had an anti-TPO value above the normal cut-off (anti-TPO+). Table [1](#T1){ref-type="table"} shows the association between anti-TPO+ and lifetime psychiatric diagnosis. Subjects with at least one lifetime diagnosis of anxiety disorders or one lifetime diagnosis of mood disorders presented anti-TPO+ more frequently than subjects without mood or anxiety disorders. An association with anti-TPO+ was found in ADNOS, in MDE and DDNOS. The attributable risk for ADNOS was 0.54, 0.37 for MDE and 0.34 for DDNOS, other conditions as PD and DD presented very high attributable risk. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Association between positivity anti-TPO (anti-TPO+), mood and anxiety diagnosis. ::: Diagnosis Prev.nce N (%) Anti-TPO+ N (%) OR IC 95% χ2 P Att.able Risk --------------------------------------- ---------------- ----------------- ----- ---------- ------ ------- --------------- One Anxiety Diagnosis GAD+PD+SP+ADNOS 41 (18.5) 15 (36.6) 4.2 1.9/38.8 12.6 0.001 0.54 One Mood Diagnosis MDE+DD+DDNOS 42 (18.9) 13 (30.9) 2.9 1.4/6.6 6.4 0.011 0.37 GAD 25 (11.3) 8 (32) 2.7 0.97/7.5 3.6 0.058 0.35 PD 6 (2.7) 3 (50) 5.4 0.7/37.3 2.8 0.096 0.68 SP 12 (5.4) 4 (40) 3.6 0.7/7.6 2.5 0.111 0.52 ADNOS 12 (5.4) 5 (41.7) 4.0 1.1/15.5 3.9 0.045 0.55 MDE 32 (14.4) 10 (31.2) 2.7 1.1/6.7 4.6 0.033 0.34 DD 6 (2.7) 2 (50) 5.2 0.3/16.8 1.3 0.250 0.67 DDNOS 9 (4.0) 4 (44.4) 4.4 1/19.3 3.8 0.049 0.60 GAD: Generalized Anxiety Disorder; PD: Panic Disorder; SP: Social Phobia; ADNOS: Anxiety Disorder Not Otherwise Specified; MDE: Major Depressive Episode; DD: Dysthymic Disorder; DDNOS: Depressive Disorder Not Otherwise Specified. ::: The Multivariate Logistic Regression showed that gender and age do not interact with anti-TPO either in mood (interaction anti-TPO-gender p = 0.97, OR 1.03, CI 95%, 0.19--5.48; interaction anti-TPO-age p = 0.62, OR = 1.52, CI 95%, 0.30--7.77), or in anxiety disorders (interaction anti-TPO-gender p = 0.93, OR = 0.93, CI 95%, 0.18--4.80; interaction anti-TPO-age p = 0.67, OR = 0.70, CI 95%, 0.14--3.64). Interactions were therefore eliminated from the models. The final Logistic Regression models clearly indicated that gender and age do not influence the risk of one mood or anxiety diagnosis either as independent variables or as confounders (Table [2](#T2){ref-type="table"}, Table [3](#T3){ref-type="table"} and Table [4](#T4){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Frequency of anti-TPO+ in to the sample according sex and age and one mood diagnosis (OMD) and one anxiety diagnosis (OAD) diagnosis for multivariate logistic regression. ::: Age Sex N anti-TPO+ (%) OMD (%) OMD (%) \#anti-TPO+ OAD (%) OAD (%) \#anti-TPO+ ------ -------- ---- --------------- ----------- --------------------- ----------- --------------------- \<45 Female 79 17 (21.5) 17 (21.5) 6 (35.2) 13 (16.4) 6 (35.2) \<45 Male 48 8 (16.6) 7 (14.5) 2 (24) 7 (14.5) 3 (37.5) \>44 Female 48 7 (14.5) 10 (20.8) 3 (42.8) 14 (29.1) 4 (57.1) \>44 Male 47 5 (10.6) 8 (17.2) 2 (40) 7 (15.1) 2 (40) \#TPO+ in the sub-group is the total ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Multivariate logistic regression: effect of anti-TPO+ on risk of one mood diagnosis considering gender (female vs male) and age (≤ 44 vs \> 44) effects. ::: p OR CI 95% ------------------------ ------ ------ ------------ anti-TPO+ vs anti-TPO- 0.01 2.89 1.31--6.38 Gender (F vs M) 0.37 1.38 0.68--2.82 Age (≤ 44 vs \> 44) 0.71 1.14 0.57--2.30 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Multivariate Logistic Regression: effect of anti-TPO+ on risk of one anxiety diagnosis considering gender (Female vs Male) and age (≤ 44 vs \> 44) effects. ::: p OR CI 95% ------------------------ ------- ------ --------------- anti-TPO+ vs anti-TPO- 0.001 4.50 2.02 -- 10.04 Gender (F vs M) 0.23 1.58 0.75 -- 3.31 Age (≤ 44 vs \> 44) 0.08 1.91 0.92 -- 3.96 ::: Discussion ========== The present study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+ in a general population sample which had not been selected from medical or psychiatric health facilities. This association is independent by gender and age. Regarding specific diagnosis, MDE, DDNOS and ADNOS were associated with anti-TPO+. This finding is consistent with several previous clinical studies providing evidence for a significant association of mood disorders or post-partum depression and symptomless autoimmune thyroiditis with or without sub-clinical hypothyroidism \[[@B11]\]. Association between hypothyroidism and mood disorders is however controversial as other authors maintain that bipolar disorders rather than unipolar depression are characterized by an increased risk for the presence of anti-thyroid antibodies \[[@B11]\]. However, a study performed by Fountoulakis and collaborators \[[@B1]\] recently found a link between autoimmune thyroid disease and Unipolar Depression. In this study, compared to control patients all depressive subtypes had significantly higher thyroid binding inhibitory immunoglobulins, and atypical patients had significantly higher thyroid microsomal antibodies. Thyroid function markers Free Triiodothyronine (FT3), Free Thyroxine (FT4), and Thyroid Stimulating Hormone (TSH) were normal in all subjects suggesting that Unipolar Depression might be characterized by a \"low-thyroid function syndrome\". A sub-clinical dysfunction of axis Thyrotropin Releasing Hormone (TRH) -- Thyroid Stimulating Hormone (TSH) with consequent alteration of circadian rhythms of TSH has been hypothesized in some depressive disorders. Indeed, this hypothesis may explain why some forms of mood disorders were associated with anti-TPO+ or thyroid autoimmunity without hypothyroidism, as defined by routine blood tests. A slight reduction in thyroid hormone secretion such as that found in sub-clinical hypothyroidism may affect cognition and mood \[[@B12]\]. At variance with other tissues which mainly rely on peripherally generated Triiodothyronine, the brain utilizes preferentially circulating thyroxine directly secreted by the thyroid gland and may become hypothyroid before other organs \[[@B13]\]. Moreover, a study carried out in a large community sample found no association between thyroid dysfunction, including hypothyroidism defined by thyroid blood test, and the presence of depression or anxiety symptoms \[[@B14]\]. This survey is rather limited due to the fact that the presence of depression or anxiety symptoms was defined using a self-rating scale, whilst in the present study depression and anxiety disorders were diagnosed by means of a structured psychiatric interview according to an international classifications. However, the link between thyroid autoimmunity and depression may involve other mechanisms related to the autoimmune pathogenesis of thyroid disease rather than hypothyroidism. Since several neuroendocrine secretory systems are involved in the control of immune reaction, a common neuroendocrine dysregulation involving cytokines might concur towards the pathogenesis of both affective disorders and autoimmune disease. Recent evidence suggests that thyroid autoimmunity may be affected by the Hypothalamic-Pituitary-Adrenal axis (HPA) through the balance of proinflammatory and antiinflammatory cytokines \[[@B15]\]. In line with this view, the increased frequency of post-partum depression, associated to the fact that pregnancy would seem to be a \"protected\" period, could explain at least in part the consequences on thyroid autoimmunity elicited by HPA-related modifications to the immunitary axis. Indeed, similar phenomena are observed in rheumatoid arthritis and multiple sclerosis \[[@B16],[@B17]\]. With regard to the public heath aspects of this research, should these findings be confirmed, they would constitute a most important public health issue, due to the high attributable risk found. The attributable risk is a useful measure to document the burden of risk to a community. Attributable risk depends both on the magnitude of relative risk and on the prevalence of the risk factor in the population \[[@B4]\]. The high attributable risk of autoimmune thyroid for Major Depressive Disorder and anxiety and depressive sub-threshold syndromes may have implication for the development of preventive interventions. Particularly, further longitudinal studies will be required to confirm whether anxiety and depressive disorders are a consequence and not a cause of thyroid autoimmunity. Limitations =========== The potential of the study is reduced by the small sample size, particularly regard to psychiatric diagnoses less frequently observed in the general population, such as Panic Disorder; the extension of the findings is therefore rather limited. Conclusions =========== This study indicates an association between the presence of a lifetime diagnosis of mood or anxiety disorder and anti-TPO+. The psychiatric disorders and the autoimmune reaction seem to be rooted in a same (and not easy correctable) aberrancy in the immuno-endocrine system. If the findings are confirmed, they may prove to be of interest for future projects of case finding: a systematic screening for mood disorders in anti-TPO+ subjects and a systematic evaluation for thyroid diseases and thyroid autoimmunity in subjects with mood disorders may be advisable. Competing interests =================== None declared. Authors\' contributions ======================= MGC conceived the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. AL, SM and CS participated in the statistical analysis and drafted the manuscript. MCH, SM, MC, BC, LDO participated in its design and coordination. All authors have read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-244X/4/25/prepub>

PubMed Central

2024-06-05T03:55:47.582790

2004-8-18

PMC516780

Background ========== Infection due to Salmonella is a major public health problem in England and Wales with reports of over 14,400 infections due to Salmonella in the year 2003 \[[@B1]\]. The most common serotypes responsible for human infection are *S*Enteritidis, *S*Typhimurium and *S*Virchow \[[@B2]\]. Although *Salmonella*enterica serovar Enteritidis phage types 4, 21 and 6 have been reported in previous outbreaks \[[@B3]\], phage type 34a is rare and reports of outbreaks due to this serotype are scarce in the literature. Apart from one report from Wales \[[@B4]\], we are not aware of any other outbreaks due to *S*Enteritidis PT 34a infection reported in the literature from United Kingdom. In this report, we present the results of an epidemiological investigation of an outbreak due to this rare phage type associated with a Chinese restaurant in Suffolk, United Kingdom. Methods ======= On 30^th^July 2002, the Suffolk Communicable Disease Control team (SCDC) was informed by the consultant microbiologist that *S*. Enteritidis had been isolated from stool samples of five patients. All had recently eaten a meal in a local Chinese restaurant. Further enquires revealed that there were more patients with similar food history and gastrointestinal symptoms. An Outbreak Control Team was convened on 31^st^July and it was decided that a full investigation should be carried out to identify the extent of the outbreak, the probable vehicle of infection and to advise on the appropriate control measures. Epidemiological --------------- The environmental health department (EHD) staff initially gathered information from people who had become ill on a standard data collection form. In the initial stages of the investigation, it became apparent that all those who became ill had eaten or had bought a take away at the restaurant on 27^th^July 2002. The information collected included name, address, sex, their symptoms and date of onset. The restaurant provided the list of food items that were served/sold on the day in question. This menu extended to 40 food items. This list was shown to the restaurant patrons and they were asked to state the food items they had eaten. A variety of ways was used to identify further cases including the technique of snowball sampling. This involved asking the patrons whether they were aware of any others who had similar symptoms and had eaten in the restaurant. General Practitioners providing primary care in the area were contacted and were requested to check for patients with gastrointestinal symptoms. The presenting symptoms of the patrons were diarrhoea, headache, abdominal pain and fever. The next step involved interviewing all those who had eaten/bought food on 27^th^July whether they became ill or not. The restaurant provided table-booking details. The following case definition was adopted for the outbreak. \"Symptoms of acute gastroenteritis including one of the following: diarrhoea, vomiting or abdominal pain up to 96 hrs after having had a meal from the said restaurant including takeaway between 22 and 30 July 2002 and/or individuals who have positive stool sample for *S*. Enteritidis up to 96 hrs after having a meal from the restaurant including a takeaway between 22 and 30 July 2002\". An analytical investigation was carried out using a retrospective cohort design. Efforts were made to identify anyone who ate or bought food at the restaurant on 27^th^July. Eligibility for membership of the cohort was defined as a person having the opportunity to eat any of the food items available on the day. Statistical methods ------------------- Data was entered in to Statistical Package of Social Sciences version 10 \[[@B5]\]. Food specific attack rates and the corresponding two tailed p vales were derived by Fisher\'s exact test \[[@B6]\]. Univariate relative risk (RR) and 95% Confidence Intervals (CI) were calculated using standard cohort analysis \[[@B7]\]. Microbiological --------------- Stool samples were requested from all who had eaten food from the restaurant on 27^th^of July. Environmental sampling was not carried out as this was considered to be of limited value. There was no food left over from 27^th^July, but three food samples were taken on 30^th^July and sent for analysis to the food laboratory at Chelmsford Public Health Laboratory. Stool specimens were sent to Ipswich Hospital microbiology laboratory and were cultured for the presence of *Salmonella*sp. Isolates of Salmonella were forwarded to the Laboratory of Enteric Pathogens at Central Public Health Laboratory, Colindale for phage typing. Standard procedures were adopted for phage typing at the laboratory \[[@B8]\]. Environmental ------------- The EHD staff inspected the premises including verifying the procedures for hazard analysis and critical control point (HACCP). Egg storage and preparation of egg items were also investigated during the visit. Efforts were made to trace the egg trail back to the supplier. Results ======= Epidemiological --------------- Data were gathered from 52 subjects who had eaten food from the restaurant on 27^th^July of whom 38 developed symptoms and 14 were free of symptoms. Of the 38 who became ill 16 were male and 22 were female. The mean age was 27 years. The mean incubation period was 30 hours with a range of 6 to 90 hours suggesting a point source outbreak. Two patients received hospital care and there were no deaths. No gastrointestinal illness was reported among the kitchen staff of the restaurant in the weeks before or during the outbreak. On investigation of food preparation practices at the restaurant, it appeared dishes containing egg were the most likely vehicle for this outbreak. However, this information was not discussed when gathering data from the subjects. Data was gathered in a standardised format from all subjects to avoid any interviewer or recall bias. We had a strong \"a priori\" hypothesis that people who had eaten egg or food that had come in to contact with egg were at an increased risk even before looking at the data and these were analysed first. During analysis it became apparent that illness was significantly associated with the three food items that contained egg or the egg rice mixture as shown by the food specific attack rates (Table [1](#T1){ref-type="table"}) and the increased relative risks (Table [2](#T2){ref-type="table"}). When many other food items eaten on the day including pork-fried rice and a variety of fish dishes were analysed none of which showed an increased attack rate or was significant in the cohort analysis. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Specific attack rates of suspected foods ::: Food item Eaten Not eaten p\* value -------------------- ------- ----------- ----------- ---- ---- ------ ------- Egg fried rice 31 5 86.1 7 9 43.8 0.002 Special fried rice 13 0 100.0 25 14 64.0 0.009 Chicken fried rice 9 0 100.0 29 14 67.4 0.04 \* Fisher exact test ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Relative risk and 95% Confidence Intervals of suspected foods ::: Food Item Relative Risk 95% Confidence Interval -------------------- --------------- ------------------------- Egg fried rice 1.97 1.11 -- 3.48 Special fried rice 1.56 1.23 -- 1.97 Chicken fried rice 1.48 1.20 -- 1.83 ::: Microbiological --------------- A total of 31 stool specimens were submitted to the laboratory from which *S*. Enteritidis was isolated in 29. Twenty-eight of these 29 isolates were confirmed to be PT 34a and one as PT4. No pathogens were isolated from food samples taken from the restaurant on 30^th^July. Environmental ------------- During the visit to the restaurant, the EHD staff reported that there was no evidence of hazard analysis and noted many cleaning and maintenance issues. Hand wash facilities were inadequate. The chef explained that after preparing the egg rice mixture, it was left out at room temperature for the rest of the evening, and reheated when ordered. This mixture was used for some of the other fried rice items. It was estimated that on the evening of the 27^th^of July the egg rice mixture was left at room temperature for seven hours. The restaurant received eggs from a supplier in London every week and they were not refrigerated. Attempts to trace the egg trail were not successful. Control measures ---------------- The restaurant closed voluntarily on 31^st^July and the EHD staff reassessed the situation on the evening of 1^st^of August. As they were satisfied with the arrangements, the restaurant was allowed to reopen. The owner and restaurant staff were provided with information on proper cooking methods and the importance of undertaking HACCP. Discussion ========== The epidemiological investigation showed that eggs used in the preparation of egg-fried rice, which in turn was used in the preparation of some of the other rice items was the vehicle of infection. Isolation of the unusual phage type 34a strengthened the conclusion that eating from the restaurant was linked to a point source outbreak. In general, by the time investigations are initiated often no food material is available for laboratory analysis and the investigator has to rely on epidemiological evidence. The first step in identifying the source of an outbreak is the calculation of attack rates and the responsible food should have a significantly higher attack rate \[[@B9]\]. In this study, three types of food were found to have higher attack rates and were considered responsible for the outbreak (Table [1](#T1){ref-type="table"}). All these items contained egg or egg rice mixture, which was left at room temperature for a long time. Cohort analysis also showed elevated RRs which were significant (Table [2](#T2){ref-type="table"}). Seven sick patrons did not eat egg-fried rice. Descriptive analysis showed that all except one gave a history of eating chicken and or special fried rice. Statistical methods have better power while there is an \"a priori\" hypothesis as shown in the study of summer excess of leukaemia \[[@B10]\]. We had an \"a priori\" hypothesis that food items containing eggs increased the risk of illness. To our knowledge, this is the third report of an outbreak due to phage type 34a and the first of its kind in England published in the literature. In the UK, this phage type has been associated with travel abroad especially to southern Spain \[[@B11]\] and indigenous infections are rare. The restaurant received eggs from two sources and one of which was a packaging firm. Hence, it was not possible to determine the origin of the eggs. An outbreak due to closely related phage type 34, associated with an egg-containing dish in a Mexican restaurant in the United States has also been described \[[@B12]\]. There have been earlier reports of *S*. Enteritidis outbreaks associated with Chinese food businesses in England \[[@B13]\], Scotland \[[@B14]\] and the United States \[[@B15]\] although it is not clear whether any shortfalls in specific food handling techniques are responsible. In one instance \[[@B15]\], egg roll batter was made from pooled shelled eggs which were left at room temperature throughout the day. The proportion of eggs infected with *S*. Enteritidis has been reported to be low \[[@B16]\] and hence the risk of acquiring infection from consuming a single raw egg is much lower. However, the practice of pooling shelled eggs together with storage at room temperature as happened in our outbreak promotes bacterial multiplication and a single contaminated egg can contaminate different types of food. The role of *S*. Enteritidis in causing food borne outbreaks is well known as it has the ability to contaminate eggs without causing discernible illness in the birds affected \[[@B17]\]. Eggs have been implicated as the source of Salmonella infection in many previous outbreaks \[[@B18]-[@B22]\]. Hayes *et al*\[[@B23]\] in their case control study in Wales found that undercooked hens eggs are an important risk factor for sporadic Salmonella infections. We could not find any veterinary data on phage type 34 in British flocks. We searched the literature to determine whether there is any molecular relationship between phage type 34a and phage type 4. Hudson et al \[[@B24]\] based on the results of pulsed-field gel electrophoresis concluded that different *S*. Enteritidis phage types appear to be genetically related or clonal. Discussion on stability of phage types of S. Enteritidis can also be found in the literature. Conversion of phage type 4 to 24, phage type 23 to 8 and 4 to 7 have been reported. We could not find reports linking phage type 34a and phage type 4. In a recent public health investigation \[[@B25]\] of *S*. Enteritidis in raw eggshells, various serotypes of Salmonella were isolated from 23 out of 449 (5.1%) pooled samples labelled as originating from Spain. These sero/phage types included S. Enteritidis PT6a, PT5c, 13a, 14b, 58, PT6d, PT1, PT1c and PT12. A few limitations of this study are to be noted. The origin of the suspected contaminated eggs could not be traced. Although trace back exercises are key in epidemic investigations, often they are not successful due to logistic and practical reasons. In our outbreak one of the suppliers to the restaurant turned out to be a packaging firm. During our investigation, we found that there were problems with the distribution system, which prevented us from pin pointing the origin of the contaminated eggs. A possibility always exists that we missed a few subjects from this investigation and not all could be persuaded to provide a stool sample. There was no single list of all the patrons who ate/purchased food on the evening. However, all efforts were made to contact the patrons and the outbreak caused considerable publicity in the local media. Hence, we are confident that we have included most patrons. Although the precise number of patrons who were not included will never be known we are confident that their number is small and might be in the region of 10 to 15. Palmer \[[@B26]\] has pointed out the need to undertake outbreak investigations rapidly but at the same time with sound methodology. We tried to adopt the standard approach to investigating an outbreak including a retrospective cohort study. However, we did not attempt multivariate analysis due to the small number of subjects involved in the investigation. *S*. Enteritidis PT4 was isolated from one of the subjects. Further investigation revealed that this subject had recently returned from holiday in continental Europe and had suffered mild symptoms before the meal. In response to this and other outbreaks associated with eggs, a Public Health Investigation was launched in October 2002 in the UK to determine the rate of Salmonella contamination in eggs. Tests of nearly 4000 eggs showed that Salmonella was recovered from 5.3% of pooled eggs \[[@B25]\]. The Food Standards Agency has also produced a leaflet titled \"Eggs -- what caterers need to know\" \[[@B27]\] which emphasises the importance of thoroughly cooking the eggs, buying eggs from reputable suppliers and use of pasteurised eggs when serving a vulnerable individuals. Conclusions =========== Investigation of this outbreak was greatly facilitated by the close cooperation between local EHD, Communicable Disease Control Team, microbiological laboratory and local health care providers. Although food samples from this point source outbreak were not available for microbiological culture, epidemiological evidence pointed to eggs containing dishes as the most likely source of the outbreak. This outbreak highlights the continuing hazards of raw eggs. It is likely that the use of pasteurised eggs and the adoption of safe food preparation practices would have prevented this outbreak. Competing interests =================== The first author (PB) served as the expert witness for the prosecution during the court proceedings. Authors\' contributions ======================= PB and TS conceived and designed the study and drafted the manuscript. PB analysed the data RK oversaw the microbiological investigation. PB, TS, RK and HM all interpreted the results of the analysis and critically reviewed the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/4/40/prepub> Acknowledgements ================ The authors would like to thank Philip Gore, Jonathan Hayes and Wai Lowe of the local environmental health department and Martin Lodge and Dalma Marossy from the food laboratory, Chelmsford Public Health Laboratory for their in put in to the investigation. We are grateful to staff of the Laboratory of Enteric Pathogens at the Central Public Health Laboratory, Colindale for carrying out the phage typing and for helpful discussions. The authors gratefully acknowledge the constructive comments and useful suggestions from both the reviewers. PB would like to thank Pat Murray, Southend Primary Care Trust for her comments on the earlier version of the document.

PubMed Central

2024-06-05T03:55:47.584688

2004-9-1

PMC516781

Background ========== Acute respiratory distress syndrome (ARDS) is characterized by respiratory failure of acute onset as a result of acute lung injury (ALI) either directly or indirectly via the blood. The main characteristics of the syndrome are diffuse inflammation and increased microvascular permeability that cause diffuses interstitial and alveolar oedema and persistent refractory hypoxemia \[[@B1]\]. Although a variety of insults may lead to ARDS, a common pathway may probably result in the lung damage \[[@B2]-[@B4]\]. A complex series of inflammatory events have been recognized during the development of ARDS but the exact sequence of the events remains unclear. Leukocyte activation and free radical release, proteases, arachidonic acid metabolites, inflammatory and anti-inflammatory cytokines results in the increased alveolar-capillary membrane permeability \[[@B5]-[@B7]\]. Cytokines are produced in the lung by local resident cells such as alveolar macrophages, lung epithelial cells, and fibroblasts or by cells such as neutrophils, lymphocytes, monocytes and platelets as a response to local or systemic injury \[[@B8]-[@B12]\]. Cytokines involved in the early phase of inflammatory response, such as IL-1, IL-2, IL-6, IL-8, \[[@B8],[@B13],[@B14]\] are secreted in response to injurious agents. Inflammatory cytokines are of critical importance in the pathophysiology of septic shock, a condition frequently leading to ARDS \[[@B15]\]. It has been hypothesized that the inability of lung to repair after ALI is due to a persisted inflammatory stimulus \[[@B16]\]. Predictive levels of inflammatory cytokines (IL-1, IL-2, IL-6, IL-8) for ARDS development in at risk patients have been reported with controversial results \[[@B5],[@B7],[@B11],[@B15],[@B16]\]. Cut-off values above which ARDS development occurs in at risk patients have been also reported for IL-4 and IL-10 \[[@B16]\]. Schutte et al \[[@B17]\] compared ARDS to pneumonia and cardiogenic pulmonary oedema patients and found higher IL-6 and IL-8 values in ARDS compared to remaining populations. A systematic study of the role of all main inflammatory cytokines at the same time in the pathogenesis and development of the ARDS has not been undertaken. The purpose of this study is to evaluate the role of inflammatory cytokines IL-4, IL-6, IL-6r, IL-8, and IL-10 in serum and bronchoalveolar lavage (BALF) as possible prognostic indicators for the development, severity, and outcome of patients with ARDS or at risk for ARDS. Methods ======= Patients -------- We studied prospectively 59 consecutive patients who were admitted in our Intensive Care Units (ICU) (Table [1](#T1){ref-type="table"}). The first group (group A) included 20 patients fulfilling the criteria of ARDS \[[@B1]\]. All these patients were supported mechanically for their respiratory failure. The second group (group B) included 12 patients on mechanical respiratory support who had at least one condition from those suggested by Fowler et al \[[@B2]\] as risk factors for ARDS development. All patients in this group developed ARDS within 48 hours. The third group (group C) included 27 patients on high risk for ARDS development who never developed ARDS (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Clinical features of the studied population on admission. ::: Group N Sex Age (yr) Diagnosis PaO2/FiO2 APACHE II ------- ---- -------- ---------- ---------------- ----------- -------------- A 20 M = 14 53 ± 19 Trauma 9 121 ± 10 19.8 ± 1.4 F = 6 Pneumonia 3 Sepsis 2 Transfusion 2 Pancreatitis 2 Intoxication 1 Burns 1 B 12 M = 8 56 ± 20 Sepsis 3 239 ± 30 20.5 ± 1.3 F = 4 Pneumonia 4 Trauma 4 Pancreatitis 1 C 27 M = 21 49 ± 18 Sepsis 12 276 ± 16 16.0 ± 1.1\* F = 6 Trauma 5 Pneumonia 2 Transfusion 2 Intoxication 2 Arrest 2 Pancreatitis 2 D 33 M = 20 36 ± 16 F = 13 M = male, F = female, burns = \>40% of the body surface. \* p \< 0.05 group A vs groups B and C. ::: For patients\' classification, the following criteria were employed: 1. The ARDS criteria of the American-European Consensus Conference on ARDS (1): a. acute onset, b. bilateral chest radiographic infiltrates, c. pulmonary artery occlusion pressure of ≤18 mm Hg, or no evidence of left atrial hypertension, and d. impaired oxygenation regardless of the PEEP concentration, with a PaO~2~/FiO~2~ratio of ≤ 300 torr for ALI and ≤ 200 torr for ARDS. 2. The high-risk criteria for ARDS development according to Fowler et al \[[@B2]\]). 3. The criteria for pneumonia according to EPIC study \[[@B18]\], and 4. The criteria for septic syndrome according to Bone et al \[[@B19]\]. Acute Physiology and Chronic Health Evaluation-II (APACHE II) scoring system was used for grading the disease severity \[[@B20]\]). The main clinical features of the patients are shown in Table [1](#T1){ref-type="table"}. The protocol was approved by the Ethics Committee of our institutions. After admission to the ICU blood samples were obtained from a central venous line within 2 hours. APACHE II score and PaO~2~/FiO~2~values were obtained at the time of sample collection. The blood was collected in a heparinized vacutainer tube and kept immediately at 4°C. After centrifugation at 1500 g at 4°C, the plasma was kept at -80°C until the measurement. Immediately after blood collection BALF was obtained by fiberoptic bronchoscopy. The fluid was filtered through nylon net to remove the mucous secretions, and centrifuged at 500 g for 10 min to remove cells. The supernatant was kept in cryotubes at -80°C in aliquots of 0.5 ml. The method of micro-lavage was used as described previously \[[@B21]\]. The following criteria were used for an acceptable sample: a. The procedure should be shorter than 1 min, while the time of saline staying into the lungs should be less than 20 sec, b. recovery of more than 50% of the saline used for the lavage, c. absence of obvious blood contamination in the BALF, and d. the level of urea in the BALF should be more than 0.4 mmol. The urea level was used as an index of BALF dilution \[[@B21]\]. To check the accuracy of the method, two subsequent lavages were taken in 8 patients and all the studied parameters in the two samples did not differ significantly. Measurement of the plasma cytokines ----------------------------------- The assay method for cytokine measurement was the same for blood and BALF samples. Determination of plasma cytokines was done with solid phase enzyme-linked immunosorbent assay (ELISA) methodology based on the quantitative immunometric sandwich enzyme immunoassay technique \[[@B22]\]. Reagents for the studied cytokines were obtained from several sources (kits of R&D systems, Inc. Minneapolis, MN, USA, for IL-6R, kits of Genzyme Diagnostics, Cambridge, MA, USA for IL-8, IL-10, and RIA kits of Amersham, Buckinghamshire, UK, for IL-4, IL-6) were used according to manufacturer\'s instructions. Intra-assay and inter-assay reproducibility was checked and found more than 90%. To calculate the dilution factor of the BALF, urea values in the plasma and BALF were used because this low molecular weight substance is found to be in the body fluids at the same concentration as in the blood. Statistical analysis -------------------- Data analysis was carried out using SPSS 8.0 statistical software (SPSS Inc., Chicago, IL). Results are expressed as mean ± 1SD, or median (range), unless otherwise indicated. The Mann-Whitney non-parametric test was used to compare the mean values of the cytokines in the blood and BALF in the various groups. Receiver-operating characteristic (ROC) correlation was used to find the optimal cut-off values of the studied cytokines for ARDS development in patient at risk and survival of the patient population \[[@B23]\]. For tests of association, we calculated Spearman\'s correlation coefficient. A p value \<0.05 was considered to be statistically significant. Results ======= There was no significant difference in the mean age of the patients among the three studied groups. Using APACHE-II score to determine the severity of the disease, significant difference between group A and group C (p = 0.04) and group B and C (p = 0.045) was found, and not between groups A and B (p = 0.06). The mean time of staying in the ICU did not differ among the three groups. Predictive capabilities of BALF mediators for onset of ARDS ----------------------------------------------------------- The mean values (+/- SD) of the measured cytokines in BALF and serum in the three studied patient groups are shown in Table [2](#T2){ref-type="table"}. A significant difference was found for BALF IL-6r, which was higher in group A than in groups B and C (p \< 0.0001). Similarly, BALF IL-6 was higher in groups A and B compared to C (p \< 0.01). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Mean (+/-SD) BALF and serum levels of studied cytokines in the three groups of patients. ::: Group A (n = 20) Group B (n = 12) Group C (n = 27) ------- ------------------ ------------------ ------------------ ---------------- ------------ -------------- IL-4 260 ± 181 158 ± 68 284 ± 119 95 ± 35^1^ 242 ± 147 83 ± 68^1^ IL-6 538 ± 432^2^ 388 ± 324^3^ 1135 ± 1382^2^ 505 ± 217 318 ± 446 313 ± 373^3^ IL-6r 180 ± 52 30 ± 25 80 ± 37^4^ 34 ± 26 73 ± 22^4^ 39 ± 45 IL-8 480 ± 222 3525 ± 1523^5^ 492 ± 165 3543 ± 2265^5^ 467 ± 179 2553 ± 2824 IL-10 62 ± 24 117 ± 60 111 ± 98 177 ± 117 73 ± 50 118 ± 84 ^1^p \< 0.0001 versus group A, ^2^p \< 0.01 versus group C. ^3^p \< 0.05 versus group B. ^4^p \< 0.0001 versus group A. ^5^p \< 0.0001 versus group C. ::: Predictive capabilities of serum mediators for onset of ARDS ------------------------------------------------------------ Serum levels of IL-4 were higher in group A compared to groups B and C (p \< 0.0001). Serum IL-6 was higher in group B compared to group A and C (p \< 0.05). Serum levels of IL-8 was higher in group A and group B compared to group C (p \< 0.0001) (Table [2](#T2){ref-type="table"}). Predictive values for ARDS development in at risk patients (groups B and C) for BALF and serum IL-6 are shown in Table [3](#T3){ref-type="table"}. IL-6 negative predictive values for ARDS development were 100% and 95% for BALF cut off value of 195 pg/ml and serum cut off value of 255 pg/ml, respectively. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### BALF and serum IL-6 predictive values for ARDS development in patients at risk (n = 39, groups B+C). ::: Criterion PPV NPV Sensitivity Specificity Prevalence 95% CI ----------------- ----------- ----- ----- ------------- ------------- ------------ ------------ BALF IL-6, *(pg/mL)* \>195 44 100 100 62 24 0.62--0.91 Serum IL-6, *(pg/mL)* \>255 44 95 88 65 24 0.60--0.90 PPV: positive predictive value, NPV: negative predictive value. CI: confidence interval ::: Predictive capabilities of BALF mediators for survival of ARDS -------------------------------------------------------------- Mean (SEM) values in BALF and serum of the studied mediators in the survivors and non-survivors (groups A+B+C) are shown in Table [4](#T4){ref-type="table"}. BALF levels of IL-6, IL-6r and IL-8 were significantly higher in those who did not survive (p \< 0.05, p \< 0.05 and p \< 0.0001, respectively). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Mean (SEM) BALF and serum levels of the measured cytokines in all patients (Groups A+B+C) according to survival. ::: BALF Serum ------------------- -------------------- ------------------------ -------------------- ------------------------ Cytokines (pg/ml) survivors (n = 30) non-survivors (n = 29) survivors (n = 30) non-survivors (n = 29) IL-4 262 ± 188 247 ± 108 72 ± 51 154 ± 68\*\*\* IL-6 313 ± 427 743 ± 877\* 218 ± 191 530 ± 389\*\*\* IL-6r 94 ± 52 129 ± 69\* 18 ± 19 53 ± 42\*\* IL-8 340 ± 109 621 ± 144\*\*\* 1269 ± 830 4957 ± 1965\*\*\* IL-10 69 ± 37 82 ± 70 70 ± 16 188 ± 84\*\*\* \* p \< 0.05, \*\* p \< 0.001, \*\*\* p \< 0.0001 versus survivors group. ::: Patients with ARDS (group A) who did not survive had significantly higher BALF levels of IL-8 (p \< 0.0001) and significantly lower BALF levels of IL-10 (p \< 0.001) (Table [5](#T5){ref-type="table"}). Patients at risk (groups B and C) who did not survive had significantly higher BALF levels of IL-8 (p \< 0.0001) (Table [6](#T6){ref-type="table"}). IL-6, IL6-r and IL-8 BALF concentration cut off predictive values for surviving patients are shown in Table [7](#T7){ref-type="table"}. BALF IL-8 was also elevated in patients of group C who died (p \< 0.0001) (Table [8](#T8){ref-type="table"}). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Mean (SEM) BALF and serum levels of cytokines in ARDS patients according to survival (group A). ::: BALF Serum ------- ----------- ----------------- ------------ --------------- IL-4 352 ± 291 224 ± 115 155 ± 63 160 ± 73 IL-6 361 ± 238 606 ± 476 213 ± 140 455 ± 353 IL-6r 180 ± 38 180 ± 59 24 ± 24 31 ± 25 IL-8 218 ± 79 581 ± 167\*\*\* 2028 ± 700 4100 ± 1353\* IL-10 80 ± 12 55 ± 24\*\* 63 ± 11 138 ± 58\* \*p \< 0.05, \*\* p \< 0.001, \*\*\* p \< 0.0001 versus survivors group. ::: ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Mean (SEM) BALF and serum levels of cytokines in at risk patients who developed or not ARDS according to survival (Groups B and C). ::: BALF Serum ------- ----------- ----------------- ------------ ------------------- IL-4 242 ± 160 271 ± 100 53 ± 21 147 ± 65\*\*\* IL-6 302 ± 463 890 ± 1178 219 ± 203 612 ± 423\*\* IL-6r 74 ± 29 74 ± 18 17 ± 18 77 ± 44\*\*\* IL-8 368 ± 96 664 ± 105\*\*\* 1097 ± 770 5885 ± 2151\*\*\* IL-10 67 ± 40 111 ± 91 72 ± 17 243 ± 74\*\*\* \* p \< 0.01, \*\* p \< 0.001, \*\*\* p \< 0.0001 versus survivors group. ::: ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Predictive BALF and serum levels (pg/ml) for surviving patients of all groups (n = 59) ::: ------- ----------- ----- ----- ------------- ------------- ------------ Criterion PPV NPV Sensitivity Specificity 95 % CI BALF IL-6 299 68 70 68 70 0.57--0.83 IL-6r 101 65 63 52 74 0.52--0.79 IL-8 481 96 90 88 96 0.85--0.99 Serum IL-4 84 81 100 100 78 0.77--0.96 IL-6 160 69 94 96 59 0.69--0.92 IL-6r 18 76 78 76 78 0.66--0.89 IL-8 2340 92 96 96 93 0.90--0.99 IL-10 98 96 93 92 96 0.84--0.99 ------- ----------- ----- ----- ------------- ------------- ------------ PPV: positive predictive value, NPV: negative predictive value, CI: confidence interval. ::: ::: {#T8 .table-wrap} Table 8 ::: {.caption} ###### Mean (SEM) BALF and serum levels of cytokines in at risk patients who did not develop ARDS (group C) ::: BALF Serum ------- ----------- ----------------- ------------ ------------------- IL-4 242 ± 169 243 ± 69 51 ± 22 169 ± 78\*\* IL-6 297 ± 497 374 ± 285 199 ± 197 620 ± 559\* IL-6r 70 ± 25 80 ± 7 17 ± 19 98 ± 43\*\* IL-8 377 ± 95 711 ± 102\*\*\* 1102 ± 810 6496 ± 2543\*\*\* IL-10 69 ± 43 85 ± 69 73 ± 17 240 ± 70\*\*\* \**p \< 0.05,*\*\**p \< 0.001,*\*\*\**p \< 0.0001 versus survivors group.* ::: Predictive capabilities of serum mediators for survival of ARDS --------------------------------------------------------------- Cytokine concentration cut off predictive values for surviving patients are shown in Table [7](#T7){ref-type="table"}. All studied mediators were found at higher levels in the serum of non-survivors (p \< 0.001 to p \< 0.0001). In patients at risk (groups B and C) who did not survive all serum mediators were significantly elevated (p \< 0.001 to p \< 0.0001) (Table [6](#T6){ref-type="table"}). Serum levels of all the studied molecules were increased in all patients that did not survive (p \< 0.05 to p \< 0.0001) (Table [8](#T8){ref-type="table"}). In survivors BALF/serum ratios were significantly higher for IL-4, IL-8, IL-10 (p \< 0.0001, p \< 0.001 and p \< 0.0001, respectively), due to lower serum levels and not to higher BALF levels. Correlations of the studied cytokines ------------------------------------- Furthermore, the serum levels of all studied mediators were significantly correlated to APACHE II score. Serum IL-8 exhibited the strongest correlation with APACHE II score (Figure [1](#F1){ref-type="fig"}). The level of IL-8 in the BALF were found to be significantly correlated to APACHE II score (r = 0.60, p \< 0.0001). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Positive strong correlation of serum levels of IL-8 to APACHE II score (Spearman\'s rank order correlation coefficient). :::  ::: PaO~2~/FiO~2~ratio was significantly correlated to the BALF levels of IL-6 and IL-6r (r = -0.27, p \< 0.05; r = -0.55, p \< 0.0001; respectively) (Figure [2](#F2){ref-type="fig"}) and to serum levels of IL-4 (r = -0.36, p \< 0.05). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Negative correlation of BALF levels of IL-6, and IL6 receptor to PaO~2~/FiO~2~ratio (Spearman\'s rank order correlation coefficient). :::  ::: Discussion ========== We designed this study in order to explore factors that could have prognostic value for the development, the severity, and the outcome of patients with ARDS and at risk for ARDS. Prediction of ARDS development ------------------------------ We observed that BALF levels of IL-6r were significantly higher in group A than in groups B and C (p \< 0.0001), while no difference was observed in serum levels among the three groups of patients. Interestingly, the BALF and serum levels of cytokine IL-6 were significantly higher in patients at risk who developed ARDS (group B) compared to the other two groups. This observation differs from previous studies \[[@B24],[@B25]\], probably reflecting the different patient population from our study. However, our results are in agreement with previous reports regarding the luck of its prediction capacity for ARDS onset \[[@B25],[@B26]\], since both BALF and serum IL-6 levels, showed a low positive predictive value according to the ROC analysis. Patients of group A and group B had higher serum levels of the inflammatory cytokine IL-8 compared to the patients of group C, but neither serum nor BALF IL-8 levels were predictive for ARDS development. In two studies, Miller et al, \[[@B27]\] found that IL-8 in BAL at the beginning of ARDS was highest in patients who died, and Donnelly et al \[[@B28]\] found that IL-8 was highest in patients at risk for ARDS who later developed ARDS. Unfortunately, subsequent studies have found that IL-8 does not predict outcome either at the outset or during the course of ARDS \[[@B5]\]). Meduri et al \[[@B16]\] found that all cytokines measured remained high during the course of ARDS in patients who died. The importance of considering anti-inflammatory constituents of BALF is shown by Donnelly et al \[[@B28]\] who found that patients with ARDS who died had significantly lower initial concentrations of IL-10 in BAL than patients who lived. Parson et al \[[@B29]\]) studied serial levels of IL-1ra and IL-10 in patients who were identified as being at risk for the development of ARDS. Initial IL-1ra levels were significantly higher (p \< 0.0001) in the patients than in normal control subjects. Similarly, IL-10 levels were increased in patients compared with normal control subjects but did not predict the development of ARDS. Like IL-1ra levels, initial IL-10 levels were significantly higher (p = 0.005) in patients who died compared with survivors. However, in other studies increased levels of IL-4, and IL-10 in serum and/or BALF were found to have beneficial effect in pre-ARDS patients \[[@B13],[@B14]\]. Thus, the heterogeneity of patients in the various studies may be a reason for the contradictive results reported earlier. Prediction of outcome --------------------- Patients who died had significantly higher levels of IL-6, IL-6r and IL-8 in BALF than those who finally survived, while all mediators studied were significantly higher in the serum of non-survivors. The rationale for analysis of cytokine concentrations in BAL fluid is that inflammatory cytokines, like IL-6 and IL-8 are known to be produced by airway epithelial cells and activated pulmonary macrophages in response to a variety of infectious agents and other triggers of airway inflammation \[[@B30]\]. During ARDS, the alveolar epithelial-endothelial barrier is disrupted, and cytokines produced in the lung are released into the systemic circulation. This is believed to be a potential mechanism for the development of systemic inflammatory response syndrome \[[@B31],[@B32]\]. The relationship between circulatory and pulmonary cytokines levels and outcome provides support to the hypothesis that poor outcome in ARDS is related to a persistent inflammatory process \[[@B30]-[@B33]\]. In addition, in agreement with our findings, bronchoalveolar concentrations of the above cytokines have been reported to be increased in patients with or at risk for ARDS \[[@B33]\]. As demonstrated by Meduri and co-workers, BAL fluid concentrations of IL-8 and IL-6 were significantly higher in nonsurvivors than in survivors \[[@B31]\]. Increased BAL levels most likely indicate intrapulmonary overproduction and not increased permeability \[[@B33]\]. Therefore, determination of these selected inflammatory cytokines in BAL fluid in ARDS could be of prognostic relevance \[[@B33],[@B34]\]. Regarding serum levels, patients at risk (groups B and C) who died had all molecules significantly increased (Table [6](#T6){ref-type="table"}), suggesting that systemic inflammatory over-response in critically ill patients may be destructive leading to multiple organ dysfunction and poor outcome. Serum levels of all the studied molecules were increased taking all patients together (groups A+B+C, Table [4](#T4){ref-type="table"}) or separate (Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, and [8](#T8){ref-type="table"}) that did not survive, suggesting that cytokinemia might reflect the severity and extension of inflammation but is not the only factor related to ARDS development. Interestingly, only IL-8 and IL-10 both in BALF and serum were higher in ARDS patients who died. These results are consistent with those of Donnelly and coworkers, who found elevated concentrations of IL-10 in BALF of 28 patients with ARDS \[[@B35]\]. However, our results differ from those of Armstrong and Millar, who found significantly lower concentrations of IL-10 in a small number group of patients at risk for ARDS \[[@B36]\]. In addition, low concentrations of IL-10 in BALF from patients with ARDS were found to be associated with increased mortality \[[@B35],[@B37]\]. In contrast, all cytokines were elevated in those who died taking together all the patients at risk (groups B and C). Regarding the survival prediction, IL-8 and IL-10 showed the higher serum positive predictive value (92 and 96%, respectively), while IL-4 had the higher serum negative predictive value and sensitivity, taking together all patients. Relation to severity of lung injury ----------------------------------- Regarding the relation of the studied molecules and the severity of lung injury, a negative correlation was found between BALF IL-6, and IL-6r and PaO2/FiO2. The same was true for serum IL-4 and PaO2/FiO2. All the studied molecules in the serum were positively correlated with the APACHE II score, as was BALF IL-8. It is probable that this cytokine is closely related to the extension of tissue damage and organ failure. Conclusions =========== In conclusion, our data show that the predictive role of most of the studied molecules both in serum and BALF for ARDS development is valuable. In addition, almost all of them are good predictors of outcome in these patients. Further studies with greater number of patients with various subgroups of ARDS as well as stricter grouping criteria should be designed to investigate the complex network of these molecules and their receptors in ARDS and their value as predictive factors in these patients. Competing interest ================== None declared. Authors\' contributions ======================= DB conceived of the study, and participated in its design and coordination and drafted the manuscript MGA participated in the design of the study, carried out immunoassays and drafted the manuscript KMA carried out immunoassays and drafted the manuscript PA patients data and samples collection IP patients data and samples collection SA patients data and samples collection GP Performed statistical analysis AP patients data and samples collection NK carried out RIA measurements DP KMA carried out immunoassays and drafted the manuscript All authors read and approved the final manuscript Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2466/4/6/prepub> Acknowledgments =============== We thank Konstantinos Perisynakis for assisting in statistical analysis.

PubMed Central

2024-06-05T03:55:47.586654

2004-8-17

PMC516782

Background ========== Traditional non-steroidal anti-inflammatory drugs (NSAIDs) are a widely prescribed class of therapy used to relieve pain and inflammation. The drugs have been shown to be effective for a variety of common disorders (hence their widespread use), most notably chronic osteoarthritis and rheumatoid arthritis. They are relatively inexpensive due to the available generic versions, but unfortunately have clinically important drawbacks related to their gastrointestinal (GI) toxicity \[[@B1]\]. Each year, about 1% to 1.5% of patients taking traditional NSAIDs experience serious GI side effects such as perforations, ulcers, and bleeding \[[@B2]-[@B5]\]. When multiplied by the total number of NSAID users this translates into significant patient morbidity and mortality \[[@B1],[@B6]\] and is associated with considerable health care costs related to hospitalizations or to the prescribing of expensive gastro-protective agents (GPAs) \[[@B7]-[@B12]\]. The cause of this GI toxicity is the \"non-selective nature\" of traditional NSAIDs that block both cyclo-oxygenase-2 (Cox-2), an enzyme involved in the production of inflammation and pain, and Cox-1, a related molecule that functions in GI tract mucosal protection and platelet function \[[@B13]\]. In the last five years, a new class of non-steroidal anti-inflammatory agents has become available to physicians, fashioned specifically to be safer than the traditional NSAIDs but priced at least two to three times higher than the generic versions of traditional NSAIDs \[[@B14]\]. The new drugs preferentially inhibit Cox-2 enzymes as compared to COX-1 molecules, and therefore have been christened \"Cox-2 selective inhibitors\" or \"coxibs\" for short. Large clinical trials comparing the use of coxibs to traditional NSAIDs have lent support to the concept that the new agents offer an improved GI safety profile while maintaining comparable analgesic efficacy in patients with chronic arthritis \[[@B3],[@B4],[@B15]\]. The trials, however, have also hinted that the improved GI safety may be compromised by concomitant use of low-dose aspirin \[[@B4]\] and may come at the expense of some cardiovascular safety, although these data remain controversial \[[@B16],[@B17]\]. Regardless, the introduction of these new anti-inflammatory agents has prompted the question: when is it appropriate and cost-effective to prescribe coxibs versus traditional NSAID monotherapy or traditional NSAIDs in combination with GPAs? To assist physicians in selecting from the different classes of anti-inflammatory agents, various scientific bodies have published clinical guidelines and third party payers have issued restricted reimbursement criteria \[[@B18]-[@B24]\]. These guidelines and reimbursement criteria in general propose that, for the treatment of chronic osteoarthritis and rheumatoid arthritis, coxibs should be used in lieu of traditional NSAID monotherapy when patients have an elevated risk for serious GI events. Traditional NSAID therapy in combination with certain GPAs (misoprostol or proton pump inhibitors) is also recommended as an alternative for most high-risk patients. High-risk patients are identified as individuals who have one or more of the following clinical characteristics: a history of peptic ulcer disease; advanced age (over 65 years); concomitant use of corticosteroids or anticoagulants; multiple comorbid conditions; or use of high doses or multiple NSAIDs \[[@B1],[@B5],[@B6],[@B25],[@B26]\]. These high-risk patients are considered to benefit from the improved GI safety of coxibs or from the GI protection afforded by GPAs. Furthermore, coxibs and GPA co-therapy have been shown to be cost-effective in such high-risk patients, as the increased cost of the drugs is partially offset by significant reductions in morbidity and mortality and related expenses \[[@B14],[@B27]-[@B29]\]. With these guidelines and policies on prescribing in place, it is now important for physicians to adjust their prescribing practices accordingly. This is true both for rheumatology specialists and for physicians in the primary care setting where osteoarthritis patients are frequently managed. Unfortunately, experience and educational research show that simply making guidelines available does not elicit behavioural change from physicians \[[@B30]-[@B33]\]. In the case of anti-inflammatory drug prescribing, there is already evidence showing that despite the availability of guidelines and reimbursement policies, physicians\' choices of drugs remain suboptimal both in terms of the inappropriate use of traditional NSAIDs and the non-cost-effective use of coxibs \[[@B34]-[@B38]\]. The current use of anti-inflammatory drugs suggests a need for strategies that will prompt physicians to change their prescribing practice in accordance with the expert recommendations. Preferably, strategies should be investigated that have proved successful at altering physician behaviour in other settings. In this paper, we describe a randomized controlled study, which we are presently conducting, to evaluate the impact of an educational intervention on primary care physicians\' prescribing of non-steroidal anti-inflammatory therapies. The study, being conducted in the province of Manitoba, Canada, tests the hypothesis that this intervention will significantly improve physician appropriate prescribing of anti-inflammatory drugs in compliance with international clinical practice guidelines and Manitoba\'s restricted drug reimbursement policy \[[@B18]-[@B22]\]. The intervention being tested is modelled on proven approaches to changing physician behaviour; it consists of an audit and feedback mechanism with optional participation in a Continuing Medical Education (CME) interactive workshop. This manuscript summarizes Phases II to V of a larger initiative entitled the Manitoba Appropriate Anti-Inflammatory Utilization Initiative (MAAUI). The study began in November 2000 and is currently in the data analysis stage. In this paper, we relate in detail the protocol of this randomized controlled study. Methods ======= Study population ---------------- MAAUI is a province-wide population-based study. All primary care physicians (non-specialists) registered with the College of Physicians and Surgeons of Manitoba and practicing in Manitoba since July 1995 were eligible to enter MAAUI. The study excluded residents, new physician graduates, physicians registered as specialists, and physicians who started practicing in the province of Manitoba after July 1995. The eligible study population thus totalled approximately 884 physicians. All eligible physicians were automatically entered into the MAAUI protocol. Physicians who were randomized to intervention group of the research were given the option to withdraw from the study. Six family physicians who agreed to act as facilitators for the MAAUI CME workshop were not included in the study population. Study design ------------ The MAAUI study design is depicted in Figure [1](#F1){ref-type="fig"}. Primary care physicians were allocated to the control and intervention arms of the study using a stratified randomization process. Specifically, study participants were divided into groups according to the geographical area of their practice within Manitoba. These groups were stratified by physicians\' \"urban\" (within Winnipeg -- the provincial capital, population size 676,700) or \"rural\" (outside of Winnipeg, population size 468,300) practice location. Randomization within each stratum was then carried out at the level of the group. The MAAUI research team originally identified 12 urban and 11 rural areas in Manitoba based on existing community boundaries within Winnipeg and the presence of distinct regional health districts outside of Winnipeg. Because one urban and rural area each contained very few primary care physicians (less than five), these areas were joined to neighbouring regions resulting in 11 urban and 10 rural groups. Urban and rural groups were randomized in such a way as to achieve a 1:2 ratio of control to intervention groups. Four of the urban groups and three of the rural groups were randomized to the control arm, and the remaining seven urban and seven rural groups were randomized to the intervention arm. This study design was chosen for several reasons. First, it offered the scientific rigour that is associated with randomization and the use of a control group. It also ensured that both rural and urban Manitoban physician groups had an equal opportunity to take part in and learn from the study intervention. The study design was also particularly amenable to testing an intervention that required physicians to congregate in common locations (i.e. for the CME workshop). The 1:2 ratio of study groups helped to ensure that there were a sufficient number of study participants who would consider attending the CME workshops. The educational intervention ---------------------------- Physicians allocated to the intervention arm of MAAUI were mailed a package that included an introduction to the study, audit and feedback material, and an invitation to participate in a CME workshop. Physicians in the control arm received no package. ### Audit and feedback material The audit and feedback material consisted of a \"Personalised Prescribing Profile\" (see Figure [2](#F2){ref-type="fig"}). This profile illustrated for each physician his/her recent prescribing pattern of non-steroidal anti-inflammatory therapies and the appropriateness of this prescribing pattern in light of expert recommendations. Specifically, the profile contained a bar chart showing the number of patients to whom the physician had prescribed long-term treatment with coxibs, traditional NSAID monotherapy, or traditional NSAIDs in combination with GPAs between August 1999 and September 2000. The proportion of these patients with whom the prescription was appropriate was also included. Long-term treatment was defined as patients who received therapy for a minimum of 100 days during this period. The chart, in addition to showing the physician\'s own prescribing profile, also showed the average profile of primary care physicians in the same geographical area and the profile of Manitoban primary care physicians overall. The reverse side of the chart listed the criteria used to define appropriate prescribing. These criteria were based on international clinical practice guidelines and Manitoba\'s restricted drug reimbursement policy for coxibs, and were verified by a rheumatologist on the MAAUI research team \[[@B18]-[@B22]\]. The criteria identified coxibs or traditional NSAID/GPA combination therapy as the appropriate choice for patients at risk for serious GI complications. The criteria also identified coxibs as the only appropriate therapy for patients with bleeding disorders or patients taking concomitant anticoagulants (this latter recommendation was based in part on Manitoba\'s reimbursement policy for coxibs and in part on the Canadian Consensus guidelines, and reflected concerns over the anti-platelet effect of traditional NSAIDs \[[@B18],[@B22]\]). The format of the Personalised Prescribing Profiles was developed by the MAAUI research team and was validated prior to the study intervention using two focus groups of family physicians. The Personalized Prescribing Profiles were generated by Manitoba Health and the Manitoba Centre for Health Policy (MCHP) at the University of Manitoba, using administrative data from the Population Health Research Data Repository. This repository contains anonymized encounter-based records of individuals\' interactions with the provincial health care system and is derived from information received by the Department of Health, Province of Manitoba, as part of the routine provision of health care in the province. The repository includes Physician Registry files, Medical Claims and Hospital discharge files, as well as Drug Programs Information Network (DPIN) files. The DPIN contains records of all drugs dispensed by Manitoba pharmacies, regardless of who is responsible for payment. Because of the sensitive nature of the Personalised Prescribing Profiles, the individualized profiles were not seen by the MAAUI research team. Instead the packages containing the profiles were assembled and mailed out by Manitoba Health with the assistance of an independent researcher, hired by the MAAUI research team, who signed a confidentiality agreement with Manitoba Health. ### CME workshop The CME workshop for MAAUI was entitled \"The Utilization and Prescribing of Anti-inflammatory Drugs in Osteoarthritis\". The workshop focused specifically on the prescribing for osteoarthritis, as it was felt that people with this disease account for a large proportion of the chronic non-steroidal anti-inflammatory use by primary care practices \[[@B39]\]. Eleven CME workshops were held throughout Manitoba (four within Winnipeg and seven outside of Winnipeg) and physicians in the intervention group were invited to voluntarily attend one of the 11. The workshops were free of charge and were approved for 3.0 hours of MAINPRO-M1 credits (continuing education credits awarded by the College of Family Physicians of Canada for group learning activities). Each workshop was facilitated by a trained family physician. The workshops included three components: an introductory video, a decision tree, and a case study portion. The introductory video provided the history and rationale of MAAUI, explained aspects of the Personalised Prescribing Profile, and introduced the decision tree. The decision tree depicted a stepwise approach to the diagnosis and treatment of osteoarthritis. This aid was designed by a rheumatologist on the MAAUI research team specifically for use in the study and was based on international clinical practice guidelines and Manitoba\'s drug reimbursement policy \[[@B18]-[@B22]\]. A take-home copy of the decision tree was provided to each workshop participant (see Additional file [1](#S1){ref-type="supplementary-material"}). Finally, the case study portion of the workshop consisted of studies exploring both the diagnosis and appropriate treatment of osteoarthritis. These case studies were supplied from an existing CME curriculum entitled \"Clinical Scenarios in Osteoarthritis\" \[[@B40]\]. The overall MAAUI educational intervention, including both the audit and feedback material and the optional CME interactive workshop, was chosen by the MAAUI research team based on the success of similar interventions in the past. Audit and feedback mechanisms have shown moderate success in changing physician behaviour in past studies \[[@B41]\]. Equally, the addition of a continuing education workshop to an existing intervention has been shown to increase the impact on physician behaviour \[[@B42]\]. Workshops with interactive elements, such as case studies, have also been found to be more successful at influencing physician behaviour than purely didactic lectures \[[@B43]\]. Affordability, practicality, and reproducibility were also considered when designing the intervention. Outcomes -------- ### Principal outcomes The primary goal of MAAUI is to evaluate the impact of the educational intervention on physician appropriate prescribing of non-steroidal anti-inflammatory therapies for patients requiring long-term treatment. To this end, four principal outcome measures have been developed: • the change in appropriate prescribing of all non-steroidal anti-inflammatory therapy (including coxibs, traditional NSAIDs, and traditional NSAID/GPA combination therapy) from pre-to post-intervention; • the change in appropriate prescribing of coxibs from pre-to post-intervention; • the change in appropriate prescribing of traditional NSAID/GPA combination therapy from pre-to post-intervention; and • the change in appropriate prescribing of traditional NSAID monotherapy from pre-to post-intervention. These primary outcomes will be evaluated at the level of the individual physician and will be calculated using administrative data similar to that used to create the Personalised Prescribing Profiles. The change in appropriate prescribing (AP) for a given physician will be calculated by the physician\'s rate of appropriate prescribing during a 6-month period post-intervention (October 1^st^2001 to March 31^st^2002) minus this rate during a 10-month period immediately pre-intervention (October 1^st^, 2000 to July 31^st^, 2001). The rate of appropriate prescribing will be defined as the number of patients prescribed long-term treatment in whom the treatment was appropriate, divided by the total number of patients prescribed long-term treatment, multiplied by 100%. Otherwise expressed: Physician\'s ΔAP = Post-intervention AP - Pre-intervention AP  Appropriateness of prescribing will be calculated by a researcher who is blinded to participants\' study-arm allocation, and will be based on the same criteria as listed in the Personalised Prescribing Profiles (see Figure [2a](#F2){ref-type="fig"}). In the event that a physician has fewer than five patients on long-term treatment in either the pre- or post-intervention period, that physician\'s data will be withheld from analysis to ensure confidentiality of the patients. ### Secondary outcomes In addition to the four principal outcomes, the MAAUI study has a number of prospectively-defined secondary outcomes: • The study will re-evaluate the primary outcomes in the following two physician subgroups: physicians who received the audit and feedback material and attended the CME workshop; physicians who received the audit and feedback material and chose not to attend the CME workshop. This analysis will help to determine the impact that the different components of the educational intervention had on physician behaviour. Specifically in the subgroup of physicians who attended the CME workshop: • The study will measure the change in physician knowledge of osteoarthritis from pre-CME workshop to immediately post-CME workshop and the change in knowledge from pre-CME workshop to five months post-CME workshop. These two outcomes should provide insight into physicians\' retention of information following a CME workshop and will allow us to examine any relationship between change in physician knowledge and change in physician prescribing behaviour. • The study will also survey physicians\' perceived change in prescribing behaviour at five months post-intervention. This outcome, compared with the principal study outcomes, will provide insight into the accuracy with which physicians\' discern their own prescribing practices. • The study will collect process-related measures in order to mark areas for improvement in the study interventions. Specifically, physicians\' impressions of the Personalised Prescribing Profile and CME workshop will be surveyed using the questionnaire distributed immediately following the CME workshop and using focus-group discussions at the end of the study follow-up. All workshop participants will be invited to attend a follow-up focus group (one for urban participants and one for rural participants). A separate focus group will be held for the workshop facilitators. Baseline data collection ------------------------ The following data were collected at baseline (October 1^st^, 2000 to July 31^st^, 2001) as control measurements: i) physician demographics including sex, urban or rural practice location, and the number of years in practice; ii) physician volume of non-steroidal anti-inflammatory therapies prescribed annually for patients requiring chronic therapy; and iii) physician rate of appropriate prescribing of NSAIDs. These latter two measures were assessed for each of coxibs, traditional NSAID/GPA combination therapy, and traditional NSAID monotherapy. Sample size and statistics -------------------------- Sample size was calculated based on the need to detect at least a 10% improvement in physician practice patterns associated with the intervention, for clinical significance. Alpha error was set at 1% because of the multiple primary outcomes and power was set at 80%. It was also assumed in the sample size calculation that physician baseline demographic measures and baseline practice patterns would contribute an additional 10% variation to the outcomes. Based on these figures and using sample size techniques for multivariate regression analysis \[[@B44]\], we estimated the need for a minimum of 116 physicians in the control group and intervention group respectively. Multivariate regression analysis will be used to determine if the study intervention has a significant impact on the primary outcomes. Baseline data will be included as control measures and interaction effects between these baseline data and the intervention will also be evaluated. Ethics ------ The MAAUI protocol has been approved by the Health Research Ethics Board, Faculty of Medicine, University of Manitoba, and by the Health Information Privacy Committee of Manitoba Health which is responsible for approving research projects that use personal health information held by a government department. Competing interests =================== The MAAUI study is funded by Merck Frosst Canada Ltd., a pharmaceutical company that produces a range of non-steroidal anti-inflammatory therapies including a coxib and various traditional NSAIDs. Merck Frosst\'s involvement in the study is limited to representation (by L-JM) on the steering committee, which is the governing body responsible for approving all aspects of study development and implementation. Merck Frosst will not participate in data analysis and will not have access to confidential individual patient or physician data. AK, CM, and GT have received speaking fees from Merck Frosst Canada. KR is the scientific writer for this manuscript, employed by the PHCRU. All of these competing interests were not specifically disclosed to study participants. Merck Frosst\'s sponsorship of the study, however, was disclosed in the consent form signed by participants at the MAAUI CME workshops. Authors\' contributions ======================= MD, AK, BK, and CM conceived of the study. MD, AK, L-JM, CM, and GT contributed to the development of the protocol. GT created the decision tree and list of criteria for appropriate prescribing. GT also chaired the MAAUI project steering committee. LM is project managing the study. KR performed the literature review for this manuscript and wrote the first draft and coordinated all subsequent revisions. All authors approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/4/21/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional file 1 **Decision Tree: take-home copy**The decision tree was a component of the MAAUI CME workshop. Physicians were introduced to the decision tree during the workshop and were provided with a two-sided take-home copy. The decision tree depicts a stepwise approach to the diagnosis and treatment of osteoarthritis. a/ Side 1. b/ Side 2. Of note, on side 2 of the decision tree the term \"NSAID\" denotes traditional NSAIDs. ::: ::: {.caption} ###### click here for file ::: Acknowledgements ================ We are indebted to Health Information Management, Manitoba Health for providing data and to the Manitoba Centre for Health Policy for their maintenance and development of the database. We would also like to acknowledge Manitoba Health for their role on the MAAUI steering committee. The results and conclusions in this paper, however, are those of the authors and no official endorsement by Manitoba Health was intended or should be implied. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### MAAUI study design :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Generic example of a Personalised Prescribing Profile.**The Personalised Prescribing Profile constituted the audit and feedback mechanism in MAAUI. Every physician in the intervention arm of the study was sent a Personalised Prescribing Profile by mail. a/ Side 1 of the profile provides a brief introduction to the purpose of the profile and lists the criteria for appropriate prescribing of coxibs, traditional NSAIDs (referred to as \"NSAIDs\" throughout the Profile), and traditional NSAID/GPA combination therapy. b/ On side 2, a bar chart illustrates for the physician his/her recent prescribing pattern of chronic non-steroidal anti-inflammatory therapies. The chart includes the number of patients to whom the physician has prescribed long-term treatment with each of the anti-inflammatory therapies between August 1999 and September 2000 and the proportion of these patients for whom the prescription was appropriate (\"Recommended Treatment\") or inappropriate (\"Not a recommended treatment\"). The chart also provides averaged statistics on appropriate and inappropriate prescribing for the physicians in the same geographical area and for primary care physicians in Manitoba overall. :::  :::

PubMed Central

2024-06-05T03:55:47.589380

2004-8-24

PMC516783

Background ========== The QRS complexes and ventricular beats in an electrocardiogram represent the depolarization phenomenon of the ventricles and yield useful information about their behavior. Beat detection is a procedure preceding any kind of ECG processing and analysis. For morphological analysis this is the reference for detection of other ECG waves and parameter measurements. Rhythm analysis requires classification of QRS and other ventricular beat complexes as normal and abnormal. Real-time ventricular beat detection is essential for monitoring of patients in critical heart condition. Correct beats recognition is impeded by power-line interference, electromyogram noise and baseline wander often present in the ECG signal. In long-term monitoring electrode impedance can increase considerably, resulting in very low signal-to-noise ratio, which can make detection practically impossible in a single lead. Therefore, usually two or three leads are used for monitoring \[[@B1]\]. Friesen *et al.*\[[@B2]\] have presented a comparison of nine QRS detection algorithms, based on: i) amplitude and first derivative, ii) first derivative only, iii) first and second derivative, and iv) digital filtering. Daskalov *et al*. \[[@B3]\] applied these algorithms to selected signals containing records with pronounced baseline drift. The results were unsatisfactory, which was probably due to the use of fixed detection thresholds, whereas adaptive ones would be more appropriate. Poli *et al*. \[[@B4]\] used a generic algorithm for QRS detection. The complexes were emphasized with respect to the rest of the signal by polynomial filters and compared to an adaptive threshold. The authors reported 99.60 % sensitivity (Se) and 99.51 % specificity (Sp) with the MIT-BIH Arrhythmia Database. The method is inapplicable in real-time. Afonso *et al.*\[[@B5]\] proposed hardware filter banks for ECG signal decomposition, where several parameters were independently computed and combined in a decision rule. The authors reported Se = 99.59 % and Sp = 99.56 % for their real-time, single-channel beat detection algorithm tested with the MIT-BIH Arrhythmia Database. Dotsinsky and Stoyanov \[[@B6]\] developed a heuristic, pseudo-real-time algorithm for ventricular beat detection for single-channel ECG, based on steep edges and sharp peaks evaluation criteria. They reported Se = 99.04% and Sp = 99.62%, obtained with two channel recordings from AHA and MIT-BIH Arrhythmia Database Moraes *et al*. \[[@B1]\] combined logically two different algorithms working in parallel -- the first has been taken from the work of Englese and Zeelenberg \[[@B7]\] and the other was based on Pan and Tompkins \[[@B8]\], and Ligtenberg and Kunt \[[@B9]\]. Moraes *et al*. \[[@B1]\] reported Se = 99.22 % and Sp = 99.73 % after having excluded records of patients with pacemaker. After excluding a few more recordings 108, 200, 201 and 203, containing high amplitude noise (according to the authors), the statistical indices rises to Se = 99.56 % and Sp = 99.82 %. Li *et al*. \[[@B10]\] have used wavelet transforms for detection. They reported 0.15 % false detections out of 46 files from the MIT-BIH Arrhythmia Database, but with exclusion of files 214 and 215. In addition, we found some errors in their Table II. After correction, the reported accuracy slightly decreased. The large variety of QRS detection algorithms, and the continuous efforts for their enhancement, proves that universally acceptable solution has not been found yet. Difficulties arise mainly from the huge diversity of the QRS complex waveforms and the noise and artifacts accompanying the ECG signals. ECG databases ============= All 48 ECG recordings of MIT-BIH Arrhythmia database were used, without exception. Each one has a duration of 30 min and includes two leads -- the modified limb lead II and one of the modified leads V1, V2, V4 or V5 \[[@B11]\]. The sampling frequency is 360 Hz with resolution 5 μV/bit. Two cardiologists have annotated all beats. Approximately 70 % of the beats are annotated as *Normal*. Four of the records are from patients with pacemakers. The American Heart Association (AHA) database was also considered, during the evaluation of the method, mostly due to the fact that it contains patients with premature ventricular beats of contraction of R-over-T type, some of them very difficult to detect because of their closeness to the previous complex. Statistical indices for this database are not derived, because they can be compared with limited number of articles working with AHA. The database consists of 80 recordings: 2-leads, 250 Hz sampling rate and 5 μV/bit resolution. Method ====== The differentiated and summed signals from *L*leads are compared to the absolute value of a threshold *MFR*= *M*+ *F*+ *R*-- a combination of three independent adaptive thresholds, where: • *M*-- Steep-slope threshold; • *F*-- Integrating threshold for high-frequency signal components; • *R*-- Beat expectation threshold. Two algorithms were developed: *Algorithm 1*detects at the current beat. *Algorithm 2*Pseudo-real-time detection with additional triggering of potentially missed heart beat in the last interval by RR interval analyses. The algorithms are self-adjusting to the thresholds and weighting constants, regardless of resolution and sampling frequency used. They operate with any number *L*of ECG leads, self-synchronize to QRS or beat slopes and adapt to beat-to-beat intervals. Preprocessing ------------- • Moving averaging filter for power-line interference suppression: averages samples in one period of the power-line interference frequency with a first zero at this frequency. • Moving averaging of samples in 28 ms interval for electromyogram noise suppression -- a filter with first zero at about 35 Hz. • Moving averaging of a complex lead (the sintesis is explained in the next section) in 40 ms intervals -- a filter with first zero at about 25 Hz. It is suppressing the noise magnified by the differentiation procedure used in the process of the complex lead sintesis. Complex lead ------------ The algorithm operates with a complex lead *Y*of several primary leads *L*. In cases of 12-standard leads, synthesis of the three quasi-orthogonal Frank leads is recommended first \[[@B3],[@B12]\], thus determining the complex lead as a spatial vector. The complex lead is obtained as:  where *Xj(i)*is the amplitude value of the sample *i*in lead *j*, and *Y(i)*is the current complex lead. The above formula (except the normalizing coefficient 1/L and the absolute value) was initially adopted from the work of Bakardjian \[[@B13]\]. Operating with unsigned (absolute) values proved convenient when dealing with QRSs and extrasystoles having different, for example positive (in one lead) and negative (in the other lead) deflections. Adaptive steep-slope threshold -- M ----------------------------------- • Initially *M*= 0.6\**max(Y)*is set for the first 5 s of the signal, where at least 2 QRS complexes should occur. A buffer with 5 steep-slope threshold values is preset: *MM*= \[*M*~1~*M*~2~*M*~3~*M*~4~*M*~5~\], where *M*~1~÷ *M*~5~are equal to *M*. • QRS or beat complex is detected if *Yi*≥ *MFR*, • No detection is allowed 200 ms after the current one. In the interval QRS ÷ QRS~+200*ms*~a new value of *M*~5~is calculated: *newM*~5~= 0.6\**max(Yi)* The estimated *newM*~5~value can become quite high, if steep slope premature ventricular contraction or artifact appeared, and for that reason it is limited to *newM*~5~= 1.1\* *M*~5~if *newM*~5~\> 1.5\* *M*~5~. The *MM*buffer is refreshed excluding the oldest component, and including *M*~5~= *newM*~5~. *M*is calculated as an average value of *MM*. • *M*is decreased in an interval 200 to 1200 ms following the last QRS detection at a low slope, reaching 60 % of its refreshed value at 1200 ms. • After 1200 ms *M*remains unchanged. The thresholds definitions are presented in more detail with the help of several examples. Two ECG leads are shown in Fig. [1a](#F1){ref-type="fig"}. Detected QRSs are marked with \'red O\' on Lead 1. The summary lead and the steep-slope threshold are represented in Fig. [1b](#F1){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Adaptive steep-slope threshold :::  ::: Adaptive integrating threshold -- *F* ------------------------------------- The integrating threshold *F*is intended to raise the combined threshold if electromyogram noise is accompanying the ECG, thus protecting the algorithm against \'erroneous beat detection\'. Initially *F*is the mean value of the pseudo-spatial velocity *Y*for 350 ms. With every signal sample, *F*is updated adding the maximum of *Y*in the latest 50 ms of the 350 ms interval and subtracting *maxY*in the earliest 50 ms of the interval. F = F + (max(Y~in\ latest\ 50\ ms\ in\ the\ 350\ ms\ interval~) - max(Y~in\ earliest\ 50\ ms\ in\ the\ 350\ ms\ interval~))/150 The way *F*is updated means that not every sample in the interval is integrated, but just the envelope of the pseudo-spatial velocity *Y*. The weight coefficient 1/150 is empirically derived. Two ECG leads are shown in Fig. [2a](#F2){ref-type="fig"}. The pseudo-spatial velocity *Y*and the integrated threshold are presented in Fig. [2b](#F2){ref-type="fig"}. The correct detection is due to the rise of *F*(hence of *MFR*) with about 0.2 mV. The beat complex is included in the integration process (note the high rise of *F*after any of the complexes), thus making almost impossible a close detection to the previous complex. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Adaptive integrating threshold :::  ::: Adaptive beat expectation threshold -- *R* ------------------------------------------ The beat expectation threshold *R*is intended to deal with heartbeats of normal amplitude followed by a beat with very small amplitude (and respectively with very small slew rate). This can be observed for example in cases of electrode artifacts. Conversely to the integrating threshold protecting against erroneous QRS detection, *R*is protecting against \'QRS misdetection\'. A buffer with the 5 last RR intervals is updated at any new QRS detection. *Rm*is the mean value of the buffer. • *R*= 0 *V*in the interval from the last detected QRS to 2/3 of the expected *Rm*. • In the interval QRS + Rm \* 2/3 to QRS + Rm, *R*decreases 1.4 times slower then the decrease of the previously discussed steep slope threshold (*M*in the 200--1200 ms interval). • After QRS + Rm the decrease of *R*is stopped. The time-course of the beat expectation threshold *R*is shown in Fig. [3](#F3){ref-type="fig"}. The decrease of *R*(respectively *MFR*) with about 0.2 mV at the fourth QRS allows its detection, despite the lack of complex in Lead 2, which leads to a two-fold decrease of the summary lead amplitude *Y*(Fig. [3b](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Adaptive beat expectation threshold :::  ::: Combined adaptive threshold -- *MFR* ------------------------------------ The combined adaptive threshold is a sum of the adaptive steep-slope threshold, adaptive integrating threshold and adaptive beat expectation thresholds. (Fig. [4](#F4){ref-type="fig"}) ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Combined adaptive threshold :::  ::: *MFR*= *M*+ *F*+ *R* Algorithm 2: pseudo-real-time detection with additional triggering of eventually missed heart beat in the last detected RR interval ----------------------------------------------------------------------------------------------------------------------------------- All previous considerations relate to Algorithm 1, which detects a beat at its occurrence. Additional checking for an eventually missed heartbeat is performed by Algorithm 2. Its function is explained by the signal in Fig. [5](#F5){ref-type="fig"}. The fourth complex at the 15.2 s in Fig. [5b](#F5){ref-type="fig"} should be missed due to the fact that, *MFR*is greater then the summary lead *Y*. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Pseudo-real-time detection with additional triggering of eventually missed heart beat in the last RR interval. :::  ::: Let\'s mark the previous RR interval with *t1*and the last -- with *t2*(Fig. [5a](#F5){ref-type="fig"}). If *t1*is not shortened, which is tested by logic OR of the 2 conditions t1\>Rm OR Rm-t1\<0.12\*Rm AND in the same time *t2*is quite long to fulfill the condition abs(t2-2\*Rm)\<0.5\*Rm, the interval is subjected to check for a missed complex. A test is performed on each of the primary leads where a sharp peak is searched (defined as a product \> 4 μV of two signal differences having one central and two lateral points 8 ms apart). If the test is passed, a second one is carried out for the amplitude of the summary lead at that point, which should be bigger then 1/3 of the mean value of the buffer *MM*, in order to define this point as a missed QRS complex. Results and discussion ====================== Normally the statistical indices Se and Sp are derived from the following parameters: correctly detected beats TP (true positive), falsely detected beats FP (false positive) and undetected beats FN (false negative). In addition, we used two parameters, adopted from Dotsinsky and Stoyanov \[[@B6]\], as described below. SP -- shifted positive error was introduced in order to explain cases like the one shown in Fig. [6](#F6){ref-type="fig"}. Here the algorithm made a false positive error before the 3rd QRS and missed the following QRS. Formally, this is a false positive error, immediately followed by a false negative. However, if the time interval between these two detections is reasonably short, for example ≤ 200 ms, we accepted this as one error only, labeled as *Shifted False Positive Error*(SP). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Shifted positive error at the P wave :::  ::: Another example of SP error as a result of artifacts just before the normal complexes is shown in Fig. [7](#F7){ref-type="fig"}. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Shifted positive errors, false positive + false negative twins :::  ::: SN -- shifted negative error was assumed by the same principle as SP, but in the opposite sense. It also included twin FN+FP error occurring within 200 ms. The first incoming FP or FN error of the shifted is defining it as SP or SN. The logic of using shifted errors (instead of FP and FN or FN and FP in cases when they appear within 200 ms of each other) is that thus the total number of beats in a record retains its value. Otherwise it would change depending on the type and number of errors and thus impede correct computation of Se and Sp. The sensitivity Se is calculated by summing FN  SN, while the specificity Sp -- by summing FP+SP.  The method was developed in Matlab. All 48 recordings from the MIT-BIH Arrhythmia database, without any exception, were used for testing the two algorithms. The processed files containing detection marks were automatically compared with the original MIT-BIH annotated beats by specially designed software. It shows all cases where the annotation and detection marks differ by more than 60 ms. These cases were examined by an independent expert, thus excluding possible author\'s influence. The results for the two algorithms are presented in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Statistical results for the two algorithms ::: File Annotated beats Algorithm 1 Algorithm 2 ------ ----------------- ------------- ------------- ----- ---- ----- -------- ----- ----- ---- ----- 100 2273 2273 0 0 0 0 2273 0 0 0 0 101 1863 1862 1 4 0 0 1862 1 4 0 0 102 2187 2187 0 0 0 0 2187 0 0 0 0 103 2084 2062 2 54 11 9 2065 0 58 12 7 104 2212 2211 1 0 0 0 2211 1 0 0 0 105 2567 2543 2 35 8 14 2544 2 36 8 13 106 2027 2017 1 1 0 9 2018 0 1 0 9 107 2137 2135 2 0 0 0 2137 0 0 0 0 108 1763 1664 2 40 3 94 1674 1 42 3 85 109 2532 2521 11 1 0 0 2527 5 0 0 0 111 2124 2124 0 0 0 0 2124 0 0 0 0 112 2539 2539 0 0 0 0 2539 0 0 0 0 113 1797 1797 0 0 0 0 1797 0 0 0 0 114 1879 1879 0 0 0 0 1879 0 0 0 0 115 1953 1951 0 4 1 1 1952 0 4 0 1 116 2412 2389 22 2 0 1 2392 19 2 0 1 117 1535 1535 0 0 0 0 1535 0 0 0 0 118 2275 2275 0 0 0 0 2275 0 0 0 0 119 1987 1987 0 0 0 0 1987 0 0 0 0 121 1863 1863 0 0 0 0 1863 0 0 0 0 122 2476 2476 0 0 0 0 2476 0 0 0 0 123 1518 1516 2 0 0 0 1516 2 0 0 0 124 1619 1617 2 0 0 0 1619 0 0 0 0 200 2601 2549 9 39 18 25 2552 6 41 20 23 201 1963 1902 60 0 0 1 1902 60 0 0 1 202 2136 2130 6 0 0 0 2130 6 0 0 0 203 2978 2901 71 13 3 3 2911 62 27 3 2 205 2656 2652 4 0 0 0 2652 4 0 0 0 207 1862 1860 2 0 0 0 1862 0 1 0 0 208 2954 2937 14 7 2 1 2939 11 7 2 2 209 3004 3004 0 1 0 0 3004 0 1 0 0 210 2647 2591 56 1 0 0 2603 44 1 0 0 212 2748 2748 0 0 0 0 2748 0 0 0 0 213 3551 3548 3 0 0 0 3550 1 0 0 0 214 2260 2258 1 1 1 0 2256 4 1 0 0 215 3362 3362 0 0 0 0 3362 0 0 0 0 217 2208 2204 3 0 0 1 2205 2 0 0 1 219 2154 2153 1 0 0 0 2153 1 0 0 0 220 2048 2048 0 0 0 0 2048 0 0 0 0 221 2427 2426 1 0 0 0 2426 1 0 0 0 222 2483 2480 2 0 0 1 2482 0 0 0 1 223 2595 2585 10 0 0 0 2590 5 0 0 0 228 2053 2053 0 0 0 0 2053 0 1 0 0 230 2256 2256 0 0 0 0 2256 0 0 0 0 231 1886 1886 0 0 0 0 1886 0 0 0 0 232 1767 1766 0 12 0 1 1766 0 12 0 1 233 3076 3073 3 0 0 0 3074 2 0 0 0 234 2753 2753 0 0 0 0 2753 0 0 0 0 Sum 110050 109548 294 215 47 161 109615 240 239 48 147 ::: Of all 110050 annotated beats (\'unknown\' or \'questionable\' were excluded from the study), true detected are 109548 for Algorithm 1 and 109616 for Algorithm 2. The statistical indices are: **Algorithm 1**: Se = 99.69 %, Sp = 99.66 %; **Algorithm 2**: Se = 99.74 %, Sp = 99.65 %. The standard way of Se and Sp calculation,  considering the joint SP and SN errors as two separate errors gives the following results: **Algorithm 1**: Se = 99.54 %, Sp = 99.61 %; **Algorithm 2**: Se = 99.60 %, Sp = 99.60 %. Algorithm 2 improved the sensitivity by 0.05 % (0.06 % for the standard evaluation) as a result of decreased number of undetected beats. This result can be observed for example in recordings 109,203, 210 and 223, where the additionally detected beats are respectively 6, 9, 12 and 5. The performance of both algorithms was especially tested with the file A5001 from the AHA containing R-over-T premature ventricular complexes, very close to the previous normal QRS complex (Fig. [5a](#F5){ref-type="fig"}). An improvement of 74 undetected by Algorithm 1 R-on-T complexes was observed. The detection of such premature ventricular complexes occurring at the time of ventricular repolarization was considered important, having in mind possible risk of ventricular fibrillation triggering by R-on-T events. Conclusions =========== The proposed algorithms for real-time and pseudo-real-time implementation are adaptive, independent of thresholds and constants values. They are self-synchronized to the QRS steep slope and the heart rhythm, regardless of the resolution and sampling frequency used. Due to the integration threshold, the algorithms are practically insensitive to electromyogram and similar high-frequency noise. The algorithms can operate with one, two or more leads, using a combined lead signal derived from the sum of absolute values of the differentiated lead signals. The statistical indices are higher than, or comparable to those, cited in the scientific literature. Acknowledgements ================ The author gratefully acknowledges the contribution of Prof. Ivan Dotsinsky, Centre of Biomedical Engineering, Bulgarian Academy of Sciences, for examining and testing the results of the proposed algorithms.

PubMed Central

2024-06-05T03:55:47.591690

2004-8-27

PMC516784

Background ========== As with the analysis of any set of data, it is always good practice to begin by producing and inspecting graphs. A feel for the data can then be obtained and any outstanding features identified. In spatial epidemiology this is called disease mapping. Bithel \[[@B7]\], Diggle \[[@B14]\] and Lawson \[[@B25]\] provide recent reviews of disease mapping. Spatial epidemiology comprises at least three types of study focus \[[@B17],[@B25]\]. These are (i) disease mapping, (ii) disease clustering and (iii) geographical correlation analysis but these distinctions are not strict. For example a disease map is also used for reporting the results of a geographical correlation study or to highlight areas of high or low disease incidence, i.e. cluster locations in a cluster study \[[@B4],[@B14]\]. But in the following, disease mapping is considered as exploratory analysis used to get an impression of the geographical or spatial distribution of disease or the corresponding risk. For this the disease map should be based on smoothed estimates, clean of noise and adjusted for variation in \'at-risk\' population \[\[[@B24]\], p. 163\]. The resulting disease map should provide insight into possible causes, effects and trends in the vast amount of data. This will provide an invaluable starting point for epidemiologic enquiry. There are three basic types of disease maps corresponding to certain types of data. These are dot maps for point (or case-event) data, choropleth maps for regional data (also called lattice- or census-tract data), and lastly, isopleth maps for geostatistical data (also called point measurements) representing spatially continuous phenomena at a limited number of sampling locations. Spatial epidemiology is mainly concerned with the analysis of two types of data: case-event data and regional summary data, generally leading to dot maps and choropleth maps, respectively. However, epidemiology is also concerned with the identification of unknown risk factors, which may be part of the environment such as air pollution, radiation or magnetic fields. And such factors vary spatially continuously. Thus it is important to produce an isopleth map of disease occurrence or risk. Furthermore, as has been often pointed out in the literature \[\[[@B24]\], p. 131\], choropleth maps must be interpreted with caution, as the grey scale grouping is arbitrary and such a choice can affect interpretation, although choropleth maps imply a constant risk (incidence or prevalence) over regions with discontinuities at the border lines. Another point of criticism concerns the areal bias \[[@B12]\], as the varying shape and size of geographical regions make the patchy maps difficult to interpret. The visual impact of larger areas is higher and may dominate the map, leading to biased visual perception, whereas in human epidemiology it is the smaller, urban areas, and not the rural surroundings, that are of primarily interest due to population sizes. These objections may be circumvented by the use of continuous surface mappings, i.e. isopleth maps. Methods ======= Review of interpolation methods ------------------------------- For spatial point case-control data Bithell \[[@B6]\] introduced a spatial interpolation method based on kernel density estimation. The resulting map is called the \"spatial relative risk function\". Lawson and Williams \[[@B28]\] and Kellsall and Diggle \[[@B20]\] proposed modifications of this procedure. Regional data arise from summarising individual information for administrative regions such as census tracts. The basic model for the individual data, i.e. spatial point data, is the spatial Poisson process. In case of a rare disease, aggregation of the data over distinct regions results again in Poisson distributed data. For a more common disease the regional counts may be binomially distributed. When spatial interpolation of regional data is the objective of the disease mapping study a grid of surface interpolant co-ordinates must be provided. Then a number of techniques can be used to automatically interpolate the data by use of deterministic methods or to predict the values statistically at the grid co-ordinates. Among the possible choices are kernel smoothing, splines, loess and running medians; see \[[@B19]\] for a discussion of these methods. Kernel smoothing has the advantage of preserving the positivity condition implied in rate data. Brillinger \[[@B8]\] used kernel-type smoothing in the context of birth rate data, and Müller, Stadtmüller and Tabnak \[[@B33]\] applied locally weighted least squares adapted for spatial aggregation to AIDS incidence maps for the San Francisco Area. Another approach for the interpolation of regional data onto a continuous surface is the geostatistical prediction method of kriging. Some implementations of kriging have been proposed to obtain a risk surface \[[@B10],[@B23],[@B31],[@B34],[@B35],[@B38]\]. Kelsall and Wakefield \[[@B21]\] have proposed a more complex geostatistical approach based on generalised linear modelling (GLIM) of regional data, which is similar to the work by Diggle, Tawn and Moyeed \[[@B15]\]. The relative merits of different interpolation approaches haves not so far been systematically investigated \[\[[@B24]\], p. 131\]. Kriging the spatial risk function --------------------------------- The problems with the kriging method for generating isopleth maps of disease occurrence, i.e. the spatial risk function, are: (1) heterogeneous variances in the regional estimates and (2) the potential of negative interpolations. The first problem can be ameliorated by the use of empirical Bayes estimation to smooth the data prior to kriging. Appropriate geostatistical modelling can solve the second problem. Let the study area be divided into *N*disjunctive sub regions indexed by *i*(*i =*1*,\..., N*). Define *n*~*i*~as the size of the \'at-risk\' population of the *i*-th region, and denote the number of cases by *m*~*i*~. The proportion *p*~*i*~= *m*~*i*~*/ n*~*i*~is the crude estimate for the parameter of interest *θ*~*i*~. ### Smoothing by empirical Bayes estimation Mapping raw estimates of disease occurrence can lead to spurious spatial features. To overcome this problem Cressie and Read \[[@B13]\] have explored the use of several variance stabilising transformations, but the results on the transformed scale are difficult to interpret. Furthermore, empirical Bayesian methods have been developed based on the idea of pooling information across regions. The resulting smoothed regional estimates have a variance stabilising side effect by borrowing strength from (local or global) neighbourhood information. The outline of the empirical Bayes approach for smoothing regional rates of rare diseases is based on the Poisson model. For the case of more common diseases Martuzzi and Elliott \[[@B30]\] adapted the approach to the Binomial model. ### Poisson model for rare diseases Clayton and Kaldor \[[@B11]\] proposed empirical Bayes or shrinkage estimation for smoothing regional data along with maximum likelihood estimation (MLE) of the unknown prior parameters. Marshall \[[@B29]\] modified this approach using method of moments estimators (MME) instead of MLE. The resulting estimates provide starting values for the iterative maximum likelihood procedure; or they could be used for exploratory mapping purposes, which is the main interest of this work. Assume the cases in every region *i*are independently Poisson distributed with the unknown parameter *θ*~*i*~which has an unknown prior distribution associated with expectation E(*θ*~*i*~) = *π*and variance Var(*θ*~*i*~) = *φ*^2^. Then the totals of cases from the *i*-th region, e.g. *m*~*i*~(*i =*1*,\..., N*), are distributed as follows *m*~*i*~\| *θ*~*i*~, *n*~*i*~\~ Po(*n*~*i*~*θ*~*i*~) *θ*~*i*~\~ (*π*, *φ*^2^) The MME of the unknown hyperparameters are  for the prior mean and  for the prior variance, where  denotes the mean regional \'at-risk\' population and the summation is over the range of *i*. The empirical Bayesian estimates then becomes  with shrinkage weights . ### Tendency of variance homogeneity As stated above, the smoothing of the crude regional estimates *p*~*i*~has the side-effect of stabilising the variances for the regional empirical Bayes estimates, i.e. . Heuristically this is clear, because the empirical Bayes estimates are based on the whole sample information and not just on the individual regional sample, and thus are more stable. But this is difficult to prove, because this requires an analytical expression for the corresponding variance or an approximate variance estimate. Morris \[[@B32]\] proposed an approximate variance estimate for the empirical Bayes estimate in the Gaussian/Gaussian setting. However, an extension of this idea to the non-Gaussian setting is awkward \[\[[@B9]\], p. 80\]. Therefore, two facts are used to claim the conjecture that the empirical Bayes estimates of the regional estimates show up the tendency of variance homogeneity. First, it should be noted that Bayes estimates, i.e. , generally have a smaller associated variance than the corresponding frequentist estimates, i.e. *p*~*i*~\[[@B32]\]. This means the Bayesian smoothing generally results in reduced variances, thus reducing the absolute differences between regional variances, i.e. the variance heterogeneity. Secondly, by virtue of the shrinkage property of the empirical Bayes estimate, it follows that the variance will be shrunk back to the global variance in the case of small regional samples, which is responsible for a large part of the unstable estimates. Both points together make up what is called \"borrowing strength from the ensemble\" \[[@B32]\]. ### Geostatistical modelling The geostatistical method of kriging is widely accepted for the purpose of spatial prediction, i.e. interpolation and (moderate) extrapolation. It is proposed here to predict the smoothed regional data onto a fine meshed regular grid of points for isopleth mapping purposes. ### Spatial linear model Kriging of spatial data, say *Z*= (*Z*~1~,\..., *Z*~*N*~)\' at sample sites *s*~*i*~, i = 1,..,*N*, takes place within the framework of the spatial linear model \[[@B1],[@B12]\] *Z*= *μ*+ *δ*,     E(*Z*) = *μ*,     *δ*\~ Gau(0, Σ) An integral part of this model is spatial correlation, which must be taken into account to draw valid scientific inferences. Here Σ is a variance-covariance matrix, spatially structured according to the position and direction between sampling sites. The basic assumption for spatial data is that near things are more related than distant things. This was neglected for smoothing of the regional epidemiological measures (prevalence or incidence) via empirical Bayes estimators. ### Semivariogram For geostatistical modelling, the structure of spatial variation will be estimated through the semivariogram , where *h*denotes the translation between any two arbitrary sites *s*~*i*~and *s*~*j*~within the study region. See \[[@B12],[@B37]\] for diverse parameterised semivariogram models and estimation methods. ### Kriging In geostatistics kriging is used synonymously with optimal spatial prediction. However the optimality depends on the appropriateness of the spatial model. Cressie \[[@B12]\] gives a review on diverse kriging approaches. There are different models with respect to the knowledge and estimation of the spatial mean function, i.e. *μ*(*s*). Ordinary kriging is concerned with an unknown but constant mean function, i.e. *μ*(*s*) = *μ*. Furthermore, universal kriging is based on a polynomial trend surface model which is to be removed prior to estimation of the semivariogram from the residuals, i.e. *δ*(*s*). This technique may be the most widely-used in practice. An outlier-resistant alternative is median polish kriging. This method starts by the robust and non-parametric estimation of the non-constant mean surface via median polishing followed by robust semivariogram estimation. Berke \[[@B2]\] proposed a modification of median polish kriging for larger spatial data sets. Generally, kriging surfaces are the sum of an estimate for the trend surface *μ*(*s*) plus the kriging prediction for the residual process *δ*(*s*), formally . ### Kriging and smoothing Kriging is sometimes termed a smoothing method. This is due to the fact that the predicted residuals  are in absolute not larger than the model residuals *δ*(*s*) = *Z*(*s*) - *μ*(*s*), i.e. the variability of the predictions  around the estimated mean surface  is smaller than the variability of the observations *Z*(*s*). When the semivariogram is modelled without nugget effect, i.e. without small-scale variability at spatial scales smaller than the observational scale, then kriging leads to direct interpolation at the sampling sites. In this case the prediction equals the observation at the sample sites and thus the predicted residuals are equal to the model residuals, i.e. . Predictions at any other sites have the tendency to shrink towards the value of the estimated trend surface at that place. On the other hand, when a semivariogram with nugget effect is appropriate, than the prediction tends to be closer to the mean surface, which gives smaller residuals , i.e. a smoother prediction surface . Thus the prediction of invalid values for risk or for other epidemiologic measures, as has been criticised in the past, is a consequence of inappropriate spatial modelling. This could only happen when universal kriging is based on an estimated trend surface model , which exceeds the range of valid values. Results ======= The mapping technique proposed to generate isopleth risk maps from regional count data is now applied to two example data sets. Example 1 is based on the SIDS mortality rates in North Carolina that show no spatial trend. Example 2 is based on spatial trend contaminated data of tapeworm infections among red foxes in Lower Saxony. Example 1: sudden infant death syndrome (SIDS) in North Carolina ---------------------------------------------------------------- This by now classical spatial data set on SIDS mortality rates in North Carolina from 1974 to 1984 has been analysed by many researchers. Cressie \[[@B12]\] gives an introduction to the problem, earlier references and results on data modelling, mapping and cluster detection. More recently Kulldorff \[[@B22]\] applied the spatial scan test to detect clusters of disease and Lawson and Clark \[[@B27]\] applied a spatial mixture modelling approach to map the standardized mortality ratio (SMR) for the period 1974 -- 1978. For the years 1974 to 1984 the number of live births ranges from 567 to 52345 over North Carolina\'s 100 counties. The total number of reported SIDS cases is 1503 out of 753354 live births, which results in an annual mortality rate of approximately 2 per 1000 live births. The mean of the counties boundary files coordinates in longitude and latitude were used here as the geographic coordinates for the regions centres. Figure [1](#F1){ref-type="fig"} shows the shrinkage effect by using parallel box plots for the raw rates and the empirically Bayesian smoothed rates under the Poisson model for rare phenomena. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Comparison of raw and Bayesian smoothed SIDS mortality rates.**Parallel box plots for the raw annual SIDS mortality rate per 1000 live births (*r*) and the corresponding shrinkage estimates or empirical Bayesian estimated rates (*eber*) from 100 counties of North Carolina, 1974--1984. :::  ::: The smoothed rates are more appropriate for disease mapping than the raw rates and hence used here for choropleth mapping in Figure [2](#F2){ref-type="fig"}. Cut points of the grey scale shading are the 5%, 50% and 95% quantiles of the empirical distribution. These are used to highlight the upper and lower five percent of the distribution of the Bayesian smoothed mortality rates and to distinguish between higher and lower values. The smoothed rates vary from about 1.2 to 3.5 cases per 1000 live births. Visual map perception reveals some potential high and low risk areas in the north and southwest but no striking spatial trend pattern. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Choropleth map of Bayesian smoothed SIDS mortality rates of North Carolina.**Choropleth map of the empirical Bayesian smoothed mortalities per 1000 live births from 100 counties of North Carolina, 1974--1984. Cut points of the grey scale shading are the 5%, 50% and 95% quantiles of the empirical distribution. :::  ::: Geostatistical prediction, i.e. kriging of the empirical Bayesian smoothed rates, is based on appropriate modelling of the data. Here, the constant mean assumption for the spatial mean surface is chosen and justified by visual inspection of the empirical semivariogram which levels out and reaches a sill. The spatial dependence structure is modelled by an isotropic exponential semivariogram without nugget effect, which is fitted by weighted least squares estimation to the robustly estimated empirical semivariogram; see \[[@B12]\] for technical details. The result of this procedure is summarised in Figure [3](#F3){ref-type="fig"}. The close fitting of the empirical semivariogram to the model indicates appropriate model choice for the dependence structure as well as for the constant mean surface. See \[[@B1]\] for diagnostic methods and its applications in geostatistical modelling. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Empirical semivariogram from smoothed SIDS mortality rates and fitted exponential model.**Exponential model (sill = 0.22, range = 0.37) fitted by weighted least squares (WLSE) to the robust empirical semivariogram from empirical Bayesian smoothed annual SIDS mortality rates per 1000 live births from 100 counties of North Carolina, 1974--1984. :::  ::: Due to the constant mean assumption ordinary kriging is the appropriate spatial prediction method and without nugget effect this leads to direct interpolation of the data at the counties centres and to smoothed values shrunken towards the global mean for the rest of the study area. The resulting isopleth map or risk surface map is given in Figure [4](#F4){ref-type="fig"}. Now the grey scale shading is almost continuous, i.e. the patchy nature of Figure [2](#F2){ref-type="fig"} is replaced by a surface. Additional isolines are drawn for the same cut points as for the grey scale shadings in Figure [2](#F2){ref-type="fig"} (i.e. at the 5%, 50% and 95% level of the empirical distribution of the empirical Bayesian estimates) and will be useful to support map interpretation and comparison. The risk surface map may be more useful to identify potential environmental risk factors than the choropleth map. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Isopleth map from kriging the smoothed SIDS mortality rates of North Carolina.**Isopleth map based on kriging predictions of the empirical Bayesian smoothed annual SIDS mortality rates per 1000 live births from 100 counties of North Carolina, 1974--1984. :::  ::: Cross-validation can be used to explore the predictive performance of the kriging model. The cross-validation residuals (weighted with respect to the spatial dependence structure given by the semivariogram) should be approximately Gaussian distributed. Normal probability plots can be used to disclose grossly model inadequacies \[\[[@B12]\], p. 498\] as well as for outlier identification. Figure [5](#F5){ref-type="fig"} shows the normal probability plot of the cross-validation residuals based on the refitted model with the outliers removed. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Normal probability plot of cross-validation residuals from kriging smoothed SIDS mortality rates.**Normal probability plot of the cross-validation residuals from kriging the empirical Bayesian smoothed regional SIDS mortality rates. Observations 4, 42, 83, 8 and 24 as well as 77 and 59 may be outliers with respect to the spatial model. :::  ::: Aside from some positive and negative extreme values the general appearance of the residual process is Gaussian. The potential outliers are regions with steep gradients in risk and hence part of disease clusters (or their surroundings) which were previously identified \[[@B22]\] and are located in the north and south-west of North Carolina. Of course, disease clusters are of interest and the proposed modelling approach reveals their existence and points to the respective high risk areas. This is in line with the gross aims of exploratory disease mapping. Example 2: tapeworm infections in red foxes in Lower Saxony ----------------------------------------------------------- *Echinococcus multilocularis*(E.m.) is a tapeworm occurring in the northern hemisphere, including endemic regions in central Europe, most of northern and central Eurasia and parts of North America. In central Europe the red fox is the main definitive host with rodents such as mice or muskrats serving as intermediate hosts \[[@B16]\]. The parasite E.m. causes the zoonosis alveolar echinococcosis (A.E.), which has a potentially high fatality rate. Recent studies reflect an alarmingly wide geographic range of the parasite in foxes, with average prevalences varying up to 60% for central Europe. However, the spatial distribution of E.m. in foxes is complex and insufficiently known. There are indications of emerging risk factors for human A.E. such as increasing parasite prevalences in red foxes, growing fox population and progressive spread of foxes to cities. The federal state of Lower Saxony is part of northern Germany and contains the federal city-state of Bremen as an enclave. During the period from 1991 to 1997, 5365 red foxes were sampled in Lower Saxony and examined for infections with E.m.. The data are given in Table [1](#T1){ref-type="table"}\[[@B3]\]. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Regions and statistics for the fox tape worm example. The 43 investigated regions in Lower Saxony with their co-ordinates (x, y), the number of red foxes tested (n) and found positive for *E. multilocularis*(m), the raw period prevalence in % (PP) and the empirical Bayes smoothed period prevalence in % (BPP). The regions no. 5, 8, 13, 14 and 15 form a previously identified disease cluster \[4\]. ::: **Regions** **Statistics** --------------- --------------------------- -------- -------- ------ ----- ----- ----- 1 Braunschweig 7,57 -4,30 25 1 4 6 2 Salzgitter & Wolfenbüttel 7,47 -6,07 115 9 8 8 3 Wolfsburg 9,33 -2,83 22 6 27 23 4 Gifhorn 8,15 -1,37 158 13 8 8 5 Göttingen 3,44 -12,92 157 84 54 51 6 Goslar 6,68 -8,81 152 20 13 13 7 Helmstedt 10,11 -4,30 66 7 11 10 8 Northeim 2,86 -10,28 186 96 52 49 9 Osterode/Harz 6,68 -11,07 94 23 24 23 10 Peine 5,80 -4,11 115 10 9 9 11 Hannover 2,17 -2,64 327 41 13 12 12 Diepholz -4,39 0,88 143 10 7 7 13 Hameln-Pyrmont -0,17 -6,36 99 41 41 39 14 Hildesheim 3,74 -6,36 202 60 30 29 15 Holzminden 1,19 -8,62 60 16 27 24 16 Nienburg/Weser -1,74 -0,58 325 20 6 6 17 Schaumburg -1,25 -4,11 90 12 13 13 18 Celle 4,81 0,59 255 8 3 3 19 Cuxhaven -3,51 10,69 73 14 19 18 20 Harburg 3,64 7,06 285 17 6 6 21 Lüchow-Dannenberg 11,68 3,92 225 20 9 9 22 Lüneburg 7,37 6,37 278 22 8 8 23 Qsterholz-Scharmbeck -3,80 6,57 32 8 25 22 24 Rothenburg/Wümme -0,17 6,47 114 6 5 5 25 Soltau-Fallingbostel 2,46 2,84 137 8 6 6 26 Stade 0,41 10,00 84 5 6 6 27 Uelzen 7,76 3,53 214 14 7 6 28 Verden -1,45 3,44 107 11 10 10 29 Delmenhorst -4,88 4,31 8 1 13 12 30 Emden -14,74 8,19 4 0 0 9 31 Oldenburg -6,84 4,12 103 15 15 14 32 Osnabrück -9,19 -3,12 290 21 7 7 33 Wilhelmshaven -8,55 10,30 10 0 0 6 34 Ammerland -8,99 6,28 76 4 5 6 35 Aurich -13,11 9,42 111 15 14 13 36 Cloppenburg -9,78 2,84 110 8 7 7 37 Emsland -13,60 1,07 242 21 9 8 38 Friesland -9,10 9,32 15 1 7 9 39 Bentheim -15,95 -1,46 36 1 3 4 40 Leer -12,42 6,67 39 2 5 6 41 Vechta -8,12 0,20 83 2 2 3 42 Wesermarsch -6,54 7,55 45 9 20 18 43 Wittmund -10,76 10,10 53 4 8 8 Sum 5365 706 Median (in %) 8,2 9,0 ::: Figure [6](#F6){ref-type="fig"} is a choropleth map of the empirical Bayes smoothed period prevalences from 43 regions in the study area. The cut points of the grey scale shading are again the 5%, 50% and 95% quantiles of the empirical distribution or the smoothed data. The period prevalences range from 3% to 51% with the median at 9%. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Choropleth map of Bayesian smoothed prevalences in Lower Saxony.**Choropleth map of the empirical Bayesian smoothed period prevalences from 43 regions in Lower Saxony. Cut points of the grey scale shading are the 5%, 50% and 95% quantiles of the empirical distribution. :::  ::: The map indicates high period prevalences in the south and north of Lower Saxony. Extraordinarily high prevalences were observed in the southern regions, which indicate the presence of a positive disease cluster that is identified by use of the spatial scan statistic \[[@B4]\]. Figure [7](#F7){ref-type="fig"} shows the corresponding isopleth map resulting from universal kriging, with overlaid isolines. The isopleth map gives an impression of gradual changes instead of jumps at the regional borders. Furthermore, the missing region of Bremen in the central north of Lower Saxony has also been supplied with predicted values. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Isopleth map from kriging the smoothed prevalences in Lower Saxony.**Isopleth map with overlaid isolines of the kriging interpolated choropleth map in Figure 6. Isolines are at the 5%, 50% and 95% quantiles of the empirical distribution of the empirical Bayesian smoothed regional period prevalences. :::  ::: Figure [6](#F6){ref-type="fig"} points out the presence of heterogeneity in the spatial mean. Here, universal kriging is based on the assumption that the spatial mean function of the data could be represented by an incomplete quadratic polynomial in the spatial co-ordinates. Other potential explanatory variables are not at hand. Let *s*= (*x*, *y*)\' denote an arbitrary location in the study region, where *x*and *y*are the co-ordinates to the east and north. By inspection of the variogram cloud \[[@B12]\] based on trend residuals, two observations (Sites 3 and 13) were identified as outliers and removed from the structure analysis. In the second step of an iterated modelling approach then the trend polynomial fitted by ordinary least squares (OLSE) is given by *μ*(*s*) = *β*~0~+ *β*~1~*y*+ *β*~2~*y*^2^, with , , . Instead of OLSE one could use iterated WLSE, but iteration may lead to biased estimates. The residuals of the trend surface fit to the empirical Bayes smoothed regional period prevalences were then used to model a spherical semivariogram by weighted least squares estimation of the robustly estimated empirical semivariogram. Figure [8](#F8){ref-type="fig"} shows the empirical semivariogram as well as the fitted model for both, the detrended and trend contaminated data (sill = 0.0039, range = 5.29 and sill = 0.0047, range = 7.83 respectively), to indicate the benefit from the trend model as measured by the 20% decrease of the sill value. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Empirical semivariograms and fitted exponential models from detrended and trend-contaminated smoothed prevalences.**Robust empirical semivariograms of the detrended (black dot) and trend-contaminated data (circle) along with the WLSE fitted spherical models for the detrended (solid line) and the trend-contaminated (dashed line) data with observations 3 and 13 removed. :::  ::: The normal probability plot of the kriging cross-validation residuals in Figure [9](#F9){ref-type="fig"} draws attention to the presence of three or four outliers (Observations 3, 8 and 13 and possibly 15). Observation 3 and 15 were previously identified as extremes and excluded from the analysis. Regions 8 and 13 are the nearest neighbours of Region 15. All three are part of a positive spatial cluster \[[@B4]\], a spatial structure that could not be captured by the spatial linear model used for universal kriging. However, the general appearance of the data is Gaussian. This in turn justifies the appropriateness of the modelling and prediction approach based on the empirical Bayesian smoothed regional data. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Normal probability plot of cross-validation residuals from kriging smoothed prevalences.**Normal probability plot of the cross-validation residuals from kriging the empirical Bayesian smoothed regional period prevalences. Observations 3, 8 and 13 may be spatial outliers with respect to the model based on the data set with observations 3 and 15 removed. Regions 8 and 13 are nearest neighbours of region 15, all three of which are part of a positive cluster. :::  ::: Geostatistical modelling of the empirical Bayesian smoothed regional prevalences allows the calculation of an error map representing the kriging standard errors. The darker the error map in Figure [10](#F10){ref-type="fig"} the larger are the kriging standard errors, which range up from 0 to 0.06. ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **Error map.**Error map based on the universal kriging standard errors. :::  ::: Summary and discussion ====================== In this paper two sophisticated statistical methods were combined to solve an open problem in disease mapping. Mapping regional data using choropleth maps holds on to several problems that are overcome by isopleth mapping. To interpolate the regional, i.e. spatially discrete information, the geostatistical method of kriging is used here. Kriging requires variance homogeneous data. However, regional risk estimates are generally based on varying sample sizes and consequently turn up spatially varying standard errors. Therefore the spatial risk estimates are smoothed using linear empirical Bayes estimation. By borrowing-strength-from-the-ensemble, the impact of outliers is reduced and standard errors are stabilized over space. This cannot achieve variance homogeneity entirely nor could the tendency be shown analytically. Bootstrapping methods for spatially dependent data \[[@B18]\] promise an empirical justification and will be a source for future research. The purpose of exploratory disease mapping is to provide insight, as opposed to precise estimates of location, spread or trends \[[@B19]\]. Emphasise shall be on easy and intuitive statistical mapping methods. The method proposed here for exploratory disease mapping is on one hand based on empirical Bayesian estimation for smoothing which is related to internal standardization in epidemiology \[\[[@B9]\], p. 260\] and could thus be viewed as a natural choice for adjusting the data for spatially varying variances. On the other hand, the kriging predictions are weighted moving averages, where weights are chosen with respect to the spatial autocorrelation structure exhibited by the sample data. This concept is easily communicable to map users. Only monochrome colours or shades of grey should be used along with isopleth mapping \[[@B36]\], as has been done here in order to avoid the natural preference of the human eye for bright colours and in deference to colour-blind map users. Kriging is known to be a smoothing method and it may be argued that the proposed mapping method results in double or over-smoothing. But here kriging is based on a semivariogram model without nugget effect, which is known to be a direct interpolation method \[[@B12]\]. Therefore the risk map shows the Bayesian smoothed regional risks in the respective regional centres. The proposed disease mapping approach is a two-step procedure. The corresponding error map (Figure [10](#F10){ref-type="fig"}), however, neglects the error from the first step, i.e. the smoothing step. This is in line with error maps used along with similar sandwich predictors obtained via median polish kriging \[[@B3],[@B12]\]. Thus the error map is useful to investigate the predictive performance of the kriging step but less useful for analytical inferences. The empirical Bayes method as described here does not take into account spatial autocorrelation, but a modification to overcome this lack has been proposed \[[@B29]\]. The method described thus far is called global smoothing, because the Bayes estimates are shrunk towards the global mean of all regions. The modification consists of using the local mean based on neighbouring regions instead of shrinking the estimates, which is called local smoothing. In any case, a simulation-based evaluation of a wide range of estimation methods \[[@B26]\] shows that the global smoother performs better overall than the local smoother. (This result may be related to the fact, that also regions with higher or lower population density cluster into urbanized regions and rural surroundings.) Only the full Bayesian approach to hierarchical modelling of regional disease data \[[@B5]\] outperforms the global smoother. But this modelling approach is certainly not suited for exploratory work. Conclusions =========== Interpolation of the regional disease risk estimates overcomes the areal bias and related problems of choropleth disease mapping. Consequently, isopleth maps are easier to read and interpret than choropleth maps. The geostatistical method of kriging is appropriate for this task when based on linear empirical Bayesian smoothed data. Unlike non-parametric and mathematical interpolation methods, the spatial model underlying the exploratory spatial risk map offers ways of interpretation. Finally, the proposed concept for exploratory spatial risk mapping is easily communicable to map users. The Bayesian smoothing estimator is related to internal standardization in epidemiology. Also kriging can be viewed as weighted moving averaging.

PubMed Central

2024-06-05T03:55:47.594669

2004-8-26

PMC516785

Background ========== In its response to tissue damage and inflammation induced by a variety of xenobiotics, endotoxins and disease states (such as viral hepatitis), post-ischemic and regenerative injury, the liver produces a large quantity of nitric oxide (NO). Nearly all cell types in liver tissue, including hepatocytes, Kupffer cells, stellate cells and endothelial cells, have the capacity for generating NO. It has been documented that NO is capable of promoting apoptosis in a number of different cell types, generally classified as cGMP-dependent or cGMP-independent \[[@B1]-[@B4]\]. The potential of chemical NO donor sodium nitroprusside (SNP) to induce apoptosis directly from NO liberation has been established *in vitro*\[[@B5]\]. The fact that NO is capable of triggering apoptosis is consistent with its ability to induce DNA damage, the inhibition of DNA synthesis and cell cycle arrest \[[@B6],[@B7]\]. The reaction product formed between NO^•^and superoxide \[i.e., peroxynitrite (ONOO^-^)\] plays a critical role in the induction of inflammatory reaction and apoptosis, but is also associated with tumor promotion and/or progression. Potentially toxic levels of peroxynitrite can be achieved whenever NO^•^and O~2~^.-^production is stimulated, due to the fact that a 100-fold increase in the rate of peroxynitrite formation occurs for every 10-fold increase in NO^•^and O~2~^.-^concentration \[[@B8]\]. Apoptosis, frequently termed \"programmed cell death\", is the form of cell death that occurs in normal liver in the course of its development and organogenesis, and in adult liver during the renewal of hepatocytes. In addition, apoptosis can be triggered by several hepatotropic viruses and toxic drugs, as well as in various liver diseases and experimental liver conditions such as hepatic allograft rejection. Degradation of the nuclear DNA, a common phenomenon observed in many organisms throughout the evolutionary scale, is one of the best-characterized biochemical features of apoptotic cell death. It has been established that the cell undergoes epigenetic reprogramming in the D~1~phase of programmed cell death, the result of which is the activation of double-stranded DNA fragmentation in the F phase during which the nuclear morphology dramatically changes \[[@B9]\]. The cleavage of DNA may have a protective function in that it reduces the likelihood for genes in a potentially active site to be transferred from dying cells to the nuclei of viable neighboring cells. It is possible that various endonucleases exert a DNA degrading activity, as well as that many proteins can receive DNA degrading properties upon change of pH conditions \[[@B11]\]. The most important aspect of apoptosis is the universal property of some proteins to exert a dual function: the protection against proteolysis and the maintenance of the structure and function of normal cells. Being free from the inhibitory complex, however, these proteins may also contribute to protein or chromatin cleavage during apoptosis \[[@B12],[@B13]\]. Changes in DNA degradation may lead to the pathogenesis in various disorders, such as liver cancer \[[@B14],[@B15]\]. On the basis of the pharmacological data supporting the critical role of NO and peroxynitrite in apoptosis, current research studies have evaluated the activity of alkaline and acid DNase during the administration of SNP or of peroxynitrite, as well as the changes in numerous susceptible parameters of nitrosative stress, including SH group oxidation, carbonyl group formation, lipid peroxidation and DNA modification. An assay of enzyme activity was performed using liver tissue after *in vivo*administration and *in vitro*treatment of isolated rat hepatocytes or purified commercial enzymes either with SNP or authentic peroxynitrite. Results ======= There are few data concerning the *in vivo*susceptibility of liver tissue to NO donor SNP and authentic peroxynitrite. A considerable attention has been paid to the establishment of *in vivo*tolerability and to the markers of apoptotic effects. Both NO and peroxynitrite can directly react with aromatic and sulfhydryl nucleophiles and nitrate aromatic residues. Sulfhydryl groups oxidation was documented in the plasma and, almost equally, in liver tissue. Peroxynitrite administration led to a more pronounced decrease in the concentration of other plasma free radical scavengers such as uric acid, and was followed by an increase in plasma nitrate concentration (Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). According to the data suggesting that peroxynitrite decomposes rapidly to OH^•^and NO~2~^•^-like species at physiological pH, it was assumed that the carbonyl groups and the lipid peroxidation product \[i.e., malondyaldehyde (MDA)\] may play a significant role in liver cell toxicity. Neither plasma nor liver protein carbonyls showed any significant increase. This may be a likely consequence of the significant increase in aromatic amino acids nitration, presumably tyrosine the spectral contribution of which was substracted from the samples treated with 2,4-dinitrophenylhidrazine. Plasma and liver MDA concentrations were not significantly changed, either. The obtained results do not support the data suggesting that oxygen radicals, probably generated during cellular SNP metabolism, may mediate cell toxicity and apoptosis, but do confirm previous *in vitro*observations \[[@B16]\]. Plasma alanine aminotranferase (ALT) activity, used as the standard liver functional test, decreased in the peroxynitrite-treated group (Table [1](#T1){ref-type="table"}). The activity of liver alkaline DNase increased and that of acid DNase decreased after *in vivo*treatment with either SNP or peroxynitrite (Table [2](#T2){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Plasma levels of investigated parameters of Sprague-Dawley rats after *in vivo*treatment with nitric oxide donor (SNP) and peroxynitrite. Data expressed as Mean (SD); n = 8 per group. ::: **Parameters** **Control** **SNP** **Peroxynitrite** ----------------------------------- ---------------- ---------------- ------------------- Lipid peroxidation (MDA) (μmol/l) 7.10 (0.86) 6.78 (2.11) 6.64 (2.85) SH groups (μmol/l) 180.47 (36.08) 150.84 (4.78) 110.40 (11.96)\* NO~2~+NO~3~(μmol/l) 45.47 (18.80) 66.82 (4.12) 81.94 (10.18) Uric acid (μmol/l) 64.82 (4.18) 22.34 (8.32) 17.50 (3.21)\* Carbonyl groups (μmol/g protein) 179.59 (28.50) 144.90 (67.40) 169.06 (14.88) Nitrotyrosine (nmol/g protein) 96.02 (26.53) 136.17 (27.47) 135.11 (4.64) ALT (U/l) 31.9 (4.15) 34.44 (4.96) 28.5 (5.15) Male Sprague-Dawley rats three months old were allocated into three different groups. Either peroxynitrite (0.5 ml/kg BW of 30 mmol solution) or SNP (10 mg/kg BW) in a volume of 100 μl were administrated in bolus in systemic circulation. The control group received physiological saline solution at the same volume. All procedures were carried out as described in Methods. Asterisk: significantly different from the control (p \< 0.05). ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Liver levels of investigated parameters of Sprague-Dawley rats after *in vivo*treatment with nitric oxide donor (SNP) and peroxynitrite. Data expressed as Mean (SD); n = 8 per group. ::: **Parameters** **Control** **SNP** **Peroxynitrite** ------------------------------------------- ---------------- ---------------- ------------------- Lipid peroxidation (MDA) (μmol/g protein) 54.42 (8.68) 52.5 (4.34) 58.2 (6.11) SH groups (μmol/g protein) 636.04 (74.58) 538.67 (82.87) 505.52 (41.43) NO~2~+NO~3~(μmol/g protein) 6.15 (2.91) 6.04 (2.61) 5.74 (1.03) Carbonyl groups (μmol/g protein) 4.14 (0.13) 4.25 (0.12) 4.20 (0.26) Nitrotyrosine (nmol/g protein) 10.26 (2.62) 11.40 (1.24) 16.62 (3.58) DNA (mg/g tissue) 10.71 (1.75) 12.65 (1.01) 12.15 (1.25) DNA fragmentation (%) 11.99 (0.33) 13.21 (1.43) 13.0 (0.89) Alkaline-DNase (U/g protein) 11.95 (0.57) 15.30 (0.72) 16.45 (1.04) Acid-DNase (U/g protein) 15.28 (3.42) 12.34 (0.35) 9.34 (0.76) Male Sprague-Dawley rats three months old were allocated into three different groups. Either peroxynitrite (0.5 ml/kg BW of 30 mmol solution) or SNP (10 mg/kg BW) in a volume of 100 μl were administrated *in bolus*in systemic circulation. The control group received physiological saline solution at the same volume. All procedures were carried out as described in Methods. ::: The ultraviolet spectra of DNA were obtained by spectrophotometric scanning between 230--500 nm on a scan detecting system. According to the data by Yermilov et al. \[[@B17]\], the appearance of a peak between 375 and 405 nm (depending on pH) corresponds to 8-nitroguanine. In the scan analysis (Figure [1](#F1){ref-type="fig"}), the peak was between 390 and 410 nm with a maximum absorbance at 405 nm in alkaline conditions (DNA extract was adjusted to pH 10). This peak may correspond to the formation of nitro-derivatives, most probably of 8-nitroguanine. The nitroguanine peak at 405 nm was particularly apparent in peroxynitrite-treated samples. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **The peak appearance of isolated liver DNA**. The extraction oftissue DNA was performed according to the method of Wannemacher et al. \[50\], modified by Setaro & Morley \[51\], with the protein and nucleic acid precipitation by using ice-cold trichloroacetic acid after lipid extraction. DNA was separated from proteins by hydrolisis of resulting pellet at 96 ± 1°C for 45 min. Samples were analyzed for DNA concentration by ultraviolet absorption difference at 260 and 290 nm. Purified DNA was employed for spectral changes, monitored by using Beckman spectrophotometer. On the basis of the data obtained by Yermilov et al. \[13\], the appearance of a peak between 375 and 405 nm (depending on pH) corresponds to 8-nitroguanine. The peak appearance was between 390 and 410 nm with the maximum absorbance at 405 nm, obtained in alkaline conditions (DNA extract was adjusted to pH 10). :::  ::: *In vivo*administration of SNP or peroxynitrite tended to increase the rate of DNA fragmentation, but it was not statistically significant. The rate was estimated according to the percentage of DNA resisting centrifugation at 27 000 g (Table [2](#T2){ref-type="table"}). After *in vitro*exposure of isolated hepatocytes to SNP or peroxynitrite, the activity of both alkaline and acid DNase decreased in a dose-dependent fashion (Figure [2](#F2){ref-type="fig"}). During *in vitro*incubation of purified enzymes DNase I and DNase II with SNP or peroxynitrite, a dose-dependent decrease of enzyme activity was also documented (Figure [3](#F3){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **The activity of alkaline and acid DNase after *in vitro*treatment of isolated hepatocytes with NO donor (SNP) or peroxynitrite**. The isolation of hepatocytes was done according to the method already published \[42\], by using a 1% collagenase dissolved in RPMI 1640 medium. Hepatocytes, isolated from 8 Male Sprague-Dawley rats, were dissolved in a physiological saline solution in a concentration of approximately 10^8^cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol) for a period of 1 hour at 37°C. Given *in vitro*concentrations were calculated according to the literature data \[38\]. The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. \[43\] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The enzyme activity was expressed as U/g protein. Data (n = 8) in graph is putted as: Mean + SD. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **The activity of commercial DNase I and DNase II after in vitro treatment with NO donor (SNP) or peroxynitrite.**Purified enzymes DNase I (E.C. 3.1.21.1) and DNase II (E.C. 3.1.22.1) were dissolved in physiological saline solution. Hepatocytes, isolated from 8 Male Sprague-Dawley rats, were dissolved in a physiological saline solution in a concentration of approximately 10^8^cells/ml. They were divided into seven groups (each comprising 8 samples) exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol). The reducing agent (cysteine 1 mmol) was added to SNP to induce *in vitro*NO release \[39\]. The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. \[43\] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The defined units for purified DNase I and DNase II (increase in absorbance of 0.001/min in a sample containing 0.132 mg DNA, pH 7.4 or pH 5 and 3 ml of reaction mixture) were obtained from the Sigma catalogue label. Data (n = 8) in graph is putted as: Mean + SD. :::  ::: Discussion ========== NO^•^, a free radical gaseous molecule is one of the simplest compounds found to be continuously produced in humans and animals. It can be derived from L-arginine through the enzyme nitric oxide synthase (NOS) and by different NO donors, including SNP. NO has been shown to play an unprecedented range of roles in biological systems, acting as a universal intracellular and transcellular signaling molecule and the regulator of vascular tone, cell proliferation and apoptosis \[[@B18]-[@B20]\]. Peroxynitrite is a strong, relatively long-lived oxidant with a half-life of approximately 0.5--1 s under physiological conditions. Our study confirmed that in both plasma and liver tissue peroxynitrite causes a rapid oxidation of sulfhydryl groups and thioethers, as well as the nitration and hydroxylation of aromatic compounds (Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). A chronic exposure of hepatocytes to reactive nitrogen species exhibits a cytotoxic and cytostatic activity leading to functional and morphological alterations \[[@B8],[@B21]\]. Cell death after exposure to different NO-donors such as SNP has been to date established through the expression of tumor suppressor gene p53 and pro-apoptotic genes such as bax, cyclin-dependent kinase inhibitor p21, the inhibited expression of anti-apoptotic protein bcl-2, the inhibited NF-κB binding activity, ERK and p-38-dependent cytochrome c release, and caspase-3 activation \[[@B22]-[@B24]\]. In contrast, the anti-apoptotic effects of NO may be mediated through the mechanisms such as blockade of the recruitment of pro-caspase-9 to the Apaf-1 apoptosome, stimulation of c-GMP-dependent protein kinase, control of mitochondrial permeability transition, induction of the heat shock protein HSP 70, and interaction with the ceramide pathway \[[@B25],[@B26]\]. The prolonged damage of p53 gene by peroxynitrite has been associated with tumor formation. Recent results by Vincent and Maiese \[[@B3]\] indicate that NO donor SNP (at 300 μmol concentration) is capable of inducing strong apoptotic effects via DNA fragmentation and induction of Mg^2+^-dependent endonuclease activity in the culture of neuronal cells. In our *in vivo*study (Table [2](#T2){ref-type="table"}), the activity of alkaline DNase increased within 24 h after exposure to SNP (achieving approximately a similar blood concentration of about 250 μmol) or to authentic peroxynitrite. Several molecules involved in nuclear DNA fragmentation have been detected and characterized based on their ionic sensitivity. Besides the presence of constitutive Ca^2+^/Mg^2+^-dependent endonucleases, a great deal of endonuclease activity within a 7.2--8.0 pH range most probably represents the inducible form of DNase. The molecular weights of the constitutive (NO-independent) and inducible (NO-dependent) endonuclease are similar, as well as their optimum pH range (7.5--8.0). A likely conclusion is that Mg^2+^-dependent endonuclease seems to be a result of *de novo*synthesized or the pre-existing Ca^2+^/Mg^2+^-dependent endonuclease activation. Up to now, several Mg^2+^- or Ca^2+^/Mg^2+^-dependent alkaline DNases (DNase I) with an optimum activity within the range of 7.5--9.5 have been purified. Some of them, including specific caspase3-activated DNase (CAD), are active upon release of the specific inhibitor ICAD \[[@B27],[@B28]\]. DNase gamma has been documented as a critical component of apoptotic machinery, in that it cleaves the chromosomal DNA into nucleosomal units, thus leading to DNA ladder formation \[[@B29]\]. The alkaline DNase, active only during apoptosis, has been documented to be inherent to cyclophilins (A, B and C) as well, irrespective of their protein folding (peptidylprolyl cis-trans-isomerase) activity. All of them have the ability to degrade the supercoiled, single stranded and double stranded DNA \[[@B30],[@B31]\]. Besides alkaline DNases, the cation-independent endonuclease with an optimum activity at pH 5, known as acid or DNase II, was identified. One leucocyte elastase inhibitor (LEI) can also exert an acid DNase activity after post-translational modification through the proteolytic cleavage \[[@B32]\]. The specific involvement of DNase II in physiological nuclear degradation during apoptosis could not be excluded upon decrease of intracellular pH values below 7 with a proton ionophore. Three potential *N*-nitrosylation sites are important for DNase II regulation \[[@B32],[@B33]\]. Since our experimental data indicated a decrease in acid DNase activity 24 h after exposure to SNP or peroxynitrite (Table [2](#T2){ref-type="table"}), the inhibition of DNase II may be explained by the nitrosylation of its susceptible sites. Indeed, when isolated hepatocytes were exposed to SNP or peroxynitrite for 1 h, a dose-dependent inhibition of DNase II was also documented (Fig. [2](#F2){ref-type="fig"}). The same result was obtained after exposure of purified enzyme to SNP (in the presence of the reducing agent cysteine 1 mmol) or peroxynitrite (Fig. [3](#F3){ref-type="fig"}). The formation of 8-nitroguanine, 8-oxo-deoxyguanine and oxazolone and the oxidative modification of 2\'-deoxyribose into TBA-responsive compounds are the most prominent nucleotide modifications after reactive nitrogen species attack \[[@B34],[@B35]\]. A highly potential mutagenic product 8-nitroguanine can be depurinated yielding apurinic sites capable of inducing GC→TA transversions, GC→CG transversions and deletions \[[@B17],[@B36]\]. The appearance of the nitroguanine peak during the scan analysis of purified DNA at 405 nm was documented in our study (Fig. [1](#F1){ref-type="fig"}). The rate of DNA fragmentation tended to be increased, but the difference was not significant (Table [2](#T2){ref-type="table"}). Conclusions =========== *In vivo*administrated SNP and peroxynitrite increase the activity of alkaline DNase. They also induced DNA modifications, such as nitroguanine formation. The obtained DNase activation seems to be associated with the programmed destruction of the genome and cell death. Given the above results and observations, the elimination of damaged hepatic cells appears to be a likely factor in prevention against mutation and carcinogenesis. Methods ======= Chemicals --------- SNP, DNA, DNase I (E.C. 3.1.21.1.) and DNase II (E.C. 3.1.22.1) were obtained from Sigma-Aldrich Company. RPMI-1640, fetal calf serum (FCS) and collagenase were purchased from ICN (Costa Mesa, CA). Authentic peroxynitrite was freshly synthesized by the quench-flow technique \[[@B37]\] and its concentration was monitored in alkaline solution before use in each experiment by measuring the extinction coefficient at 302 nm \[[@B38]\]. All other chemicals were of the highest purity range. *In vivo*study -------------- Twenty-four male Sprague-Dawley rats, three months old, were divided into three different groups, each comprising 8 animals. Either SNP (10 mg/kg BW) or peroxynitrite (0.5 ml/kg BW of 30 mmol solution) in a volume of 100 μl were administrated *in bolus*in systemic circulation by intraventricular injection under penthobarbital sodium anesthesia. The concentrations were calculated according to the literature data concerning their *in vivo*tolerability and in vitro ability to induce apoptotic effects \[[@B39],[@B40]\]. The calculation of peroxynitrite intra-arterial concentration (6 nmol) was done according to its biological half-life of about 0.6 s, cardiac output of 40 ml/min/100 g and circulating volume of 20 ml and 250 g of rat BW \[[@B41]\]. The corresponding control group received physiological saline solution in the same volume. The rats were killed 24 h afterwards, under the same anesthesia. Blood was collected from the abdominal aorta and livers were quickly removed, frozen and homogenised on ice. Isolation of hepatocytes ------------------------ The isolation of hepatocytes was done according to a method already published \[[@B42]\], by using a 1% collagenase dissolved in RPMI 1640 medium. Collagenase was inhibited by using 10% FCS and cells were washed twice in physiological saline solution. Hepatocytes were isolated from 8 Male Sprague-Dawley rats. They were dissolved in a physiological saline solution in a concentration approximately 10^8^cells/ml. They were divided into seven groups (each comprising 8 samples), exposed to either SNP (0.1, 1 and 10 mmol) or peroxynitrite (0.03, 0.3 and 3 mmol) for a period of 1 hour at 37°C. Given *in vitro*concentrations were calculated according to the literature data \[[@B38]\]. Purified enzymes DNase I and DNase II were dissolved in physiological saline solution, exposed to the same concentrations of SNP and peroxynitrite, except that the reducing agent (cysteine 1 mmol) was added to SNP to induce *in vitro*NO release \[[@B39]\]. Methods for alkaline and acid-DNase ----------------------------------- The activity of alkaline and acid-DNase was measured by the methods of Bartholeyns et al. \[[@B43]\] and acid soluble nucleotides were determined spectrophotometrically at 260 nm. The enzyme activity was expressed as U/g protein, for tissue and cell samples. The defined units for purified DNase I and DNase II (increase in absorbance of 0.001 / min in a sample containing 0.132 mg DNA, pH 7.4 or pH 5 and 3 ml of reaction mixture) were obtained from the Sigma catalogue label. Extraction of DNA and proteins ------------------------------ The extraction of tissue DNA and proteins was performed according to the method of Wannemacher et al. \[[@B44]\] modified by Setaro & Morley \[[@B45]\] by protein and nucleic acid precipitation using ice-cold trichloroacetic acid (TCA), 0.6 N, after lipid extraction. RNA and DNA were isolated by using cold 60% perchloric acid (PCA). DNA was separated from proteins by hydrolysis of resulting pellet at 96 ± 1°C for 45 min after adding 0.5 N PCA. Tissue protein content was measured according to the Lowry et al. procedure \[[@B46]\]. Samples were analysed for DNA concentration by an ultraviolet absorption difference at 260 and 290 nm. Purified DNA was employed for spectral changes monitored by using Beckman DU 530 spectrophotometer. Protein carbonyls and protein nitrotyrosine were measured in plasma proteins and the remaining protein pellet according to the method of Oliver et al. \[[@B47]\] modified by Tien et al. \[[@B48]\]. DNA fragmentation assay was performed according to the method of Jones et al. \[[@B49]\] based on the percentage of DNA resisting centrifugation at 27 000 g for 20 min. The proportion is expressed as percentage of the total DNA in the uncentrifugated sample. Protein carbonyls were quantified by spectrophotometric measurement of their 2,4 dinitrophenylhydrazone derivatives (ε 370 nm = 22000 M-1 cm^-1^). The difference between the spectrum of the DNPH-treated sample and that of the HCl control was determined and expressed as μmol DNPH/g protein. As nitrotyrosine also absorbs at 370 nm, it was measured according to its spectral contribution at 370 nm. Plasma and tissue SH groups were measured by using DTMB according to the Elman method \[[@B50]\]. Plasma and tissue lipid peroxidation product MDA was measured according to the method of Ohkava et al. \[[@B51]\]. Nitrates were measured according to the method of Navarro-Gonzales et al. \[[@B52]\]. Plasma uric acid and ALT were measured using the Synchron analyzer. Statistics ---------- Statistical analysis was made with the software SPSS. The effect of treatments was firstly evaluated by one-way ANOVA. If there was a significant effect, experimental data sets were compared against the control group by the Dunnett post hoc test. Significance level was set at α = 0.05. Data were normally distributed with equal variances among groups. Authors\' contributions ======================= GK carried out the *in vivo*and *in vitro*experiments, culture experiments and wrote the paper. RP and GN carried out DNA spectral analysis. TC performed measurement of SH groups and lipid peroxides. IS performed the measurement of nitrates and nitrites. TJ assisted during *in vivo*and *in vitro*experiments and did the graphical presentation. DS performed statistical analysis and assisted during *in vivo*experiments. DP and RK assisted during *in vitro*experiments and participated in the design of the study. All the authors read and approved the final manuscript.

PubMed Central

2024-06-05T03:55:47.599440

2004-8-31

PMC516786

I. Imaging principles ===================== Micro-bubbles ------------- Two aspects of micro-bubbles are important -- their gas content and the nature of their shell. Recently-approved micro-bubbles almost universally involve encapsulation of a high molecular weight gas, which improves the persistence of the bubble, optimising the number available in the left heart chambers. Air has a greater propensity to dissolve into solution and although currently unattractive because of loss of gas before arrival on the right side of the heart, better encapsulation may allow its resurgence -- the benefit would be more rapid disappearance when the bubble bursts. The nature of the shell or surface modifying agent, which improves stability and prevents dissolution, may become important for new targeted imaging approaches. Interaction of micro-bubbles and ultrasound ------------------------------------------- Even when optimal microbubble delivery to the myocardium is achieved with invasive coronary \[[@B1]\] or aortic root \[[@B2]\] injections, detection of reliable myocardial opacification using standard 2D imaging is difficult. Whilst this partly reflects difficulty distinguishing bright, grey scale echo signals from the myocardial tissue from those of micro-bubbles within the myocardial micro-circulation, the problem is multi-factorial and has been overcome by the development of contrast specific imaging modalities, which exploit the unique interaction between the ultrasound field and micro bubbles\[[@B3]\] to maximise the received contrast backscatter and minimise myocardial tissue backscatter. Micro-bubbles oscillate (expand and contract) in the ultrasound field. The pattern and nature of their oscillation, and thus the nature of the backscatter signal, differs, depending on the acoustic power of the transmitted ultrasound field, which is expressed on modern ultrasound machines as the mechanical index (MI). In general echocardiography, the amplitude of returning backscatter depends on the nature of the insonated structure, and is represented by brightness on the formed image. Most backscatter returns at the same frequency as the transmitted ultrasound (fundamental frequency). With **very low**mechanical index imaging (MI \< 0.1), micro bubbles demonstrate linear oscillation, where the contraction and expansion of the micro bubble are equal. All returning micro bubble backscatter remains within the range of the frequency transmitted by the transducer (the fundamental frequency) -- as for surrounding structures (Figure [1a](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Interaction of micro-bubbles and ultrasound. :::  ::: **Low**mechanical index imaging (0.1 -- 0.3), generates **nonlinear oscillation**of the micro bubble whereby expansion is greater than contraction. In addition to the usual backscatter of a particular amplitude within the range of the fundamental frequency, the bubbles also produce backscatter of (lesser) amplitude at **harmonic**frequencies \[[@B4],[@B5]\]. For example scatter from a bubble interrogated by a 4 MHz pulse will be composed of 4 MHz and 8 MHz sound (Figure [1b](#F1){ref-type="fig"}). Tissues and surrounding structures do not produce harmonic backscatter when imaged under low power. When exposed to **high**mechanical index imaging (MI \> 0.6, ie the MI used for standard imaging) the bubbles oscillate wildly and burst. Upon destruction, micro-bubbles produce a brief, high amplitude signal, with good resolution, which is rich in harmonic signals, containing backscatter at the second third and fourth harmonics etc (Figure [1c](#F1){ref-type="fig"}). The bubble dissipates gradually following destruction, depending upon the gas composition of the bubble, with air-based micro-bubbles dissipating much more rapidly. Importantly at high MI, tissues also produce harmonics. With standard fundamental imaging (high MI), myocardial micro-bubbles are destroyed continuously, causing apical swirling on LVO images and never replenishing the myocardium within the beam, and thus not returning significant myocardial backscatter. This exacerbates the underlying difficulty of differentiating on grey scale, between tissue backscatter and micro-bubble backscatter. Changing to standard harmonic imaging (processing only signals returned at the second harmonic of the fundamental frequency) significantly improves LVO, but offers little benefit for myocardial perfusion, with ongoing bubble destruction and sub-optimal differentiation between contrast and tissue, because both produce harmonics at high power. Contrast specific imaging modalities, with the aim of enhancing the contrast to tissue backscatter signal ratio (CTR) are required for optimal performance of a myocardial contrast study. Contrast specific imaging technology ------------------------------------ ### High Power Techniques -- Intermittent Imaging #### Triggered harmonic imaging Porter demonstrated that intermittent high power imaging demonstrated significantly better myocardial opacification than continuous imaging, with the best opacification obtained using intermittent harmonic imaging \[[@B6]\]. During intermittent high power imaging, high energy ultrasound is transmitted at specified intermittent intervals, triggered to the ECG (eg once every 4 cardiac cycles; 1:4 triggering). The time between destructive pulses allows the micro-bubbles to replenish the myocardium. With each destructive pulse, high amplitude backscatter rich in harmonics is returned to the transducer, enabling static images of myocardial perfusion. Moreover, by incrementally increasing the triggering intervals (continuous → 1:1 → 1:2 → 1:4 → 1:8 etc), the rate of replenishment of the ultrasound beam over time can be assessed both qualitatively and quantitatively. The qualitative assessment of perfusion using intermittent harmonic imaging can be improved by digital subtraction of the myocardial signal, and optimized by colour coding techniques to allow better extraction of bubble signals \[[@B7]\] (Figure [2](#F2){ref-type="fig"}). The first human study validating myocardial perfusion assessment by MCE with SPECT used digital subtraction \[[@B8]\]. However, this approach requires careful superimposition of each frame, and although the analysis is becoming more automated, the processing is still performed off-line. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Digital subtraction and colour coding of MCE images acquired using intermittent harmonic imaging. :::  ::: #### Harmonic Power Doppler Power Doppler technology is designed to detect motion of blood or tissue, and is used in myocardial contrast echo to overcome the need for off line digital subtraction \[[@B9]\]. As with traditional Doppler, two or more pulses are sent successively along each scan line of the image and the pairs of echo trains are compared for the presence or absence of a frequency shift (indicating movement). However, unlike traditional Doppler, power Doppler ignores the direction and velocity of the moving structure; if a frequency shift is detected, indicating motion of the structure, then colour is displayed as an overlay whose saturation is related to the amplitude of the echo which has moved. If no frequency shift (movement) is detected, no colour is displayed. This method is ideally suited to high mechanical index destructive imaging, as the first pulse destroys the myocardial micro-bubbles, generating a brief, high amplitude echo rich in harmonics. The second pulse finds that the bubbles have \'moved\', and thus colour is overlaid on the echo image over the areas of myocardium that contained micro-bubbles. In an area with no micro bubbles, there is no \'movement\' recognised and there is no colour overlay applied to that region. Note that the technique is not actually detecting movement of the bubbles in the circulation, but rather their destruction. This technique has been correlated in human subjects with SPECT\[[@B10]\] and has been used extensively in clinical trials (Figure [3](#F3){ref-type="fig"} and [additional file 1](#S1){ref-type="supplementary-material"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Harmonic power Doppler imaging \[see also additional file 1\] :::  ::: The most important limitation of this technique is motion artefact from tissue, because tissue motion will be expressed like bubble destruction, potentially showing perfusion when none is present (a false negative). If Doppler frequency is increased, pulse separation is reduced, so tissue movement between pulses can be minimized. However, if the pulses are too close, not all the gas within the bubble will have dissipated before arrival of the next pulse, so the difference between pulses is reduced, possibly leading to false positive perfusion defects. Air-filled micro-bubbles are optimal for this technique because of rapid dissipation of the gas, allowing closely spaced pulses. #### Pulse Inversion Doppler Another grey scale high MI technique is pulse-inversion imaging whereby two beam mode pulses are sent in rapid succession into the myocardium. The second pulse sent is a mirror image of the first (i.e. 180° phase shift). The scanner processes the consecutive returning pulses by adding them together. Tissue generates a linear echo, thus the addition of one pulse to the other should cancel out to zero and no signal is generated. Micro-bubbles produce non-linear echo signals and the summation of returning pulses will not equal zero and a signal will be registered (Figure [4](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Pulse inversion Doppler. :::  ::: Using this technique, processing can theoretically be limited only to signals generated by bubbles. As well as being a grey-scale technique, tissue motion artefacts are a major limitation, as movement of tissue also creates non-linear signals. Nevertheless the theory behind this technology has led to the development of successful real time imaging. #### Ultraharmonic imaging With the development of increased bandwidth transducers, another intermittent high MI technique has evolved to improve the contrast to tissue ratio of backscatter. While intermittent harmonic imaging demonstrates myocardial perfusion, it is limited by the presence of tissue signals at the second harmonic. However, bubble destruction causes backscatter at 3rd 4th and sub-harmonics. At these higher harmonics the tissue signal is negligible, thus processing only backscatter from these further harmonics enables selective enhancement of the contrast signal. Interestingly, current ultra-harmonic imaging involves processing signals from between the second and third harmonic. The strength of the high MI approaches are their sensitivity for the presence of contrast, because bubble destruction results in the highest amplitude backscatter. The disadvantages are that they lack simultaneous assessment of function, require reliable ECG triggering and image acquisition, and can be both technically challenging and time consuming (of particular importance for stress imaging). While the difficulty maintaining image position with long triggering intervals has been aided by the use of low MI localization images, respiratory movement is almost inevitable. ### Low Power Techniques -- Real Time Imaging In recent years, technologies have been developed which so successfully exploit the non-linear responses of micro-bubbles, that even low amplitude micro-bubble backscatter can be isolated from tissue signals for processing. This allows continuous low power imaging to be performed, with limited bubble destruction, enabling simultaneous assessment of wall motion and perfusion in real time (although frame-rate is minimized in order to reduce bubble destruction) (See table [1](#T1){ref-type="table"}). Hence low MI imaging is commonly known as real time imaging, The use of low MI has two major benefits; (1) The bubbles undergo stable non-linear oscillation emitting continuous fundamental and harmonic signals and (2) the tissues themselves do not generate harmonic signals at low MI. Like incremental triggered imaging, low MI imaging enables assessment of micro-bubble replenishment of the myocardium over time after bubble destruction. With low MI imaging, myocardial bubble destruction is achieved by transmission of a series of a high MI pulses (\"flashes\"), after which replenishment can be observed in real time (see figure [11](#F11){ref-type="fig"}). There are 2 major real time techniques. ::: {#F11 .fig} Figure 11 ::: {.caption} ###### Destruction replenishment imaging with real time MCE -- see text. :::  ::: #### Power pulse inversion imaging This technique combines the non-linear detection performance of pulse inversion with the motion discrimination capability of power Doppler. Multiple transmit pulses of alternating polarity are used, and Doppler signal processing techniques are applied to distinguish between bubble backscatter and backscatter from tissue. In a typical configuration, echos from a train of pulses are combined in such a way that signals from moving tissue are eliminated (see also figure [4](#F4){ref-type="fig"}). #### Power modulation imaging This method utilises the same signal subtraction principles, as PPI, with the transmitted pulses identical in phase but of different in amplitude or \'power\' -- hence power modulation, one impulse of full power, the other half that power. Echos reflected from stationery tissue are linear, thus if we subtract two times the lower power from full power, the signals should cancel out whereas a non-linear oscillation of micro-bubbles will generate a signal. In power modulation, fundamental imaging is most suited, because the tissue subtraction technique is so effective that the best signal to noise ratio from contrast to tissue is at the fundamental frequency. Both techniques have been validated in animals and utilised for qualitative assessment of perfusion in humans\[[@B11],[@B12]\]. Bubble administration --------------------- The decision regarding the use of a bolus or infusion for intravenous administration of micro-bubbles is dependant on a variety of factors, including the type of micro-bubble used, equipment and staff available and clinical indication for the test (LVO, qualitative perfusion or quantitative perfusion). For MCE, bubbles should be infused with the aim of LV opacification and adequate myocardial perfusion with minimal/no attenuation of the basal segments. The amount of contrast required for this varies from bubble to bubble, machine to machine and technique used (intermittent or continuous), as well as showing patient to patient variability. Ideally all studies, particularly those assessing perfusion would involve a continuous infusion of micro-bubbles, with a mechanically controlled infusion preferable to a manually controlled one (slow continuous injection). This enables establishment of a true steady state for optimal imaging and in particular quantification. II. Left ventricular opacification ================================== Accurate evaluation of regional and global left ventricular function by echocardiography is dependent on adequate endocardial border resolution. Using fundamental imaging, approximately 20% of resting echos demonstrate inadequate endocardial definition \[[@B13]\], defined as ≥ 2 segments not seen at baseline. While native tissue harmonic imaging enables better endocardial definition than standard fundamental imaging and reduces the number of patients with inadequate studies to 5--10%, contrast induced LVO still confers benefit over harmonic imaging \[[@B14]\]. The most challenging patients have obesity, chronic lung disease or chest wall deformities. Ventilated patients in intensive care also provide significant difficulties because of patient positioning and compliance. Techniques that enhance discrimination between myocardial tissue and the blood pool may therefore improve the clinical utility and diagnostic accuracy of echo. Left ventricular opacification (LVO) by contrast echo enhances this discrimination to better define the endocardial surface. Contemporary LVO involves administration of perflurocarbon micro-bubbles (which show superior duration of opacification and enhancement of endocardial definition compared with air filled bubbles \[[@B15]\]), and intermediate MI harmonic imaging (0.4--0.5) which allows continuous high frame rate imaging, results in reduced bubble destruction, leads to production of micro-bubble harmonic signals with minimal tissue harmonic production, enabling maximal discrimination between the opacified blood pool and myocardium (Figure [5](#F5){ref-type="fig"} and [additional files 2](#S2){ref-type="supplementary-material"}, [3](#S3){ref-type="supplementary-material"}, [4](#S4){ref-type="supplementary-material"}, and [5](#S5){ref-type="supplementary-material"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Importance of machine settings for using contrast for LV opacification. (a) In this example, endocardial border definition is probably adequate with standard tissue harmonic imaging -- \[see additional file 2.\] (b) The use of contrast for LVO with standard diagnostic harmonic imaging machine settings provides worse border definition in the lateral wall, and apical bubble destruction, illustrating the importance of appropriate machine settings -- \[see additional file 3\]. Image (c) shows machine settings for myocardial perfusion imaging -- this provides assessment of myocardial perfusion and wall motion, but the frame rate for WMA is 20--25 Hz and thus subtle WMA\'s could be missed -- \[see additional file 4\]. Therefore for optimal assessment of WMA image (d) displays specific intermediate MI imaging at high frame rate designed specifically to enhance the endocardial/cavity border. Even in this example there is some apical swirling despite the focal zone set in the mid LV -- \[see also additional file 5\]. :::  ::: LV volume measurement --------------------- While there is clear evidence that contrast LVO can improve endocardial border definition, there are theoretical reasons why this may not necessarily translate into more accurate assessment of LV cavity size, volume and function. Attenuation artefacts sometimes obscure the endocardial surface, particularly in the basal segments and myocardial contrast may confuse distinction of the border between the blood pool and the endocardium. Most importantly, the use of any 2-D echo technique to assess left ventricular volumes and ejection fraction (with or without contrast), requires a standard imaging plane. Fore-shortened views or views not oriented through the centre of the ventricular cavity will lead to an underestimation of volume no matter how good the endocardial border definition \[[@B16]\]. Notwithstanding these potential technical limitations, contrast LVO using fundamental imaging improves the correlation between LV volumes obtained with echo and MRI \[[@B17]\]. In addition, accurate classification of systolic function by calculated ejection fraction has been improved from 70 to 94% by the use of contrast, with this improved accuracy almost exclusive to patients with poor baseline images. A subsequent study confirmed that, even using harmonic imaging, the measurement of LV volumes was optimised with contrast LVO, using electron beam CT as the standard of reference \[[@B18]\]. While the role of LVO for 3D imaging is currently undefined, it seems likely that this will be an important application of contrast echo (Figure [6](#F6){ref-type="fig"} and additional files [6](#S6){ref-type="supplementary-material"}, [7](#S7){ref-type="supplementary-material"}, [8](#S8){ref-type="supplementary-material"} and [9](#S9){ref-type="supplementary-material"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Realtime 3D echocardiography without (a) -- \[additional file 6\], and with contrast enhancement (b) \[additional file 7\]. There is clear benefit for LV border detection. \[See also additional files 8 and 9 for real time 3-D movies without and with contrast respectively.\] :::  ::: LVO and wall motion analysis ---------------------------- Enhanced endocardial border definition can improve the accuracy and inter-observer agreement for assessment of regional wall motion at rest \[[@B19]\]. Compared with MRI, the number of segments visualized was clearly improved after contrast (86% visualised before contrast, 99% after contrast). In addition, identification of segments with abnormal wall motion improved 82% to 100%, with the clinical utility of the contrast being greatest at the lateral and anterior walls. Importantly, the inter-observer agreement for assessment of individual wall segments was significantly improved and contrast also improved intra-observer agreement for determination of normal versus abnormal wall motion and assessment of the severity of wall motion abnormality compared with MRI. The ability of LVO to improve scoring and inter-observer variability of regional wall motion at rest has important implications for stress echocardiography. Stress echocardiography is an established clinical tool with a high sensitivity and specificity for the diagnosis of coronary artery disease (CAD). During stress echo the diagnosis of CAD is based on detection of regional contractile dysfunction, and requires visualization of all myocardial segments to document or exclude abnormalities definitively. Reduced endocardial border definition is exacerbated during stress because of chest wall movement during hyperventilation and cardiac translational movement during tachycardia. With fundamental imaging, inadequate endocardial definition has been reported in up to 30% of stress echos \[[@B20]\]. In addition, Hoffman et al demonstrated that suboptimal studies have worse reproducibility and a poorer inter observer variability, with inter-institutional institutional observer agreement as low as 43% for studies with poor image quality\[[@B21]\] Tissue harmonic imaging, digital side by side analysis and standardised reporting criteria have alleviated but not overcome this problem\[[@B22]\]. Wall motion scoring and reproducibility during stress echo were even improved with air filled contrast agents and fundamental imaging\[[@B23]\]. Perflurocarbon filled agents demonstrated almost complete and consistent endocardial border definition \[[@B24]\], with superiority even to tissue harmonic imaging, and the greatest improvement being seen in patients with poorest image quality (26). Despite these clear advantages, the critical clinical question of whether LVO actually improves the accuracy of stress echo for diagnosis of CAD remains unanswered -- Dolan \[[@B25]\] came closest by investigating 229 patients undergoing dobutamine echo followed by angiography, the largest study of contrast stress echo (for LVO) with angiography as the gold standard. As previously documented, the endocardial border definition and inter observer variability was superior with contrast. Their important finding was that there was comparable sensitivity, specificity and accuracy for the presence of coronary disease between patients with poor resting images using contrast LVO and those in whom standard imaging gave good resting images. Moreover, our recent work shows that the improvement of LVO is an important contributor to the incremental benefit of MCE (Figure [7](#F7){ref-type="fig"} and additional files [10](#S10){ref-type="supplementary-material"},[11](#S11){ref-type="supplementary-material"},[12](#S12){ref-type="supplementary-material"},[13](#S13){ref-type="supplementary-material"},[14](#S14){ref-type="supplementary-material"},[15](#S15){ref-type="supplementary-material"},[16](#S16){ref-type="supplementary-material"},[17](#S17){ref-type="supplementary-material"},[18](#S18){ref-type="supplementary-material"},[19](#S19){ref-type="supplementary-material"},[20](#S20){ref-type="supplementary-material"},[21](#S21){ref-type="supplementary-material"},[22](#S22){ref-type="supplementary-material"}, and [23](#S23){ref-type="supplementary-material"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### End systolic frames of 4CV at rest (left) and post stress (right). Note there is no obvious difference in the shape of the cavity on the grey scale images. Importantly, the LVO images demonstrate a clear change in shape with the basal and mid lateral segments lagging, suggestive of LCx stenosis. In addition, the mid lateral segment has a perfusion defect which was not present at rest. Subtotal occlusion of the LCx was demonstrated at angiography. \[See also additional files 10-23 for entire study\]. :::  ::: Thus, left ventricular opacification by contrast echo: \(1) improves the visualization of myocardial endocardial border definition; \(2) improves the accuracy of ventricular volume assessment and estimation of ejection fraction compared with standard fundamental and harmonic imaging. \(3) improves the ability to identify and grade resting wall motion abnormalities \(4) provides superior endocardial visualization while imaging at peak dobutamine stress and can enable the accuracy of a dobutamine stress echo in a technically difficult patient to be at least as good as that of a patient with good resting images; \(5) reduces the inter-observer variability for all of the above. From an economic standpoint, the use of contrast agents during stress echo has been calculated to be cost effective \[[@B26]\] with the cost of the contrast agent itself more than offset by savings incurred by reducing downstream repetitive testing, improved laboratory efficiency and a lower rate of false positive and negatives. However, the calculations are based on a formulas incorporating improved accuracy of the technique for the diagnosis of CAD, compared with standard imaging, which remains un-proven. In summary, the use of contrast for LVO is justified for standard or stress imaging of technically difficult patients, and possibly, for calculation of ventricular dimensions in patients whom accurate quantitative serial follow up is critical, eg. chemotherapy or valvular heart disease. Other clinical uses of contrast for left ventricular opacification include confirming or excluding the presence of left ventricular thrombus and delineating other left ventricular structures like pseudoaneurysm, apical hypertrophic cardiomyopathy (Figure [8](#F8){ref-type="fig"} and additional files [24](#S24){ref-type="supplementary-material"},[25](#S25){ref-type="supplementary-material"}), and non-compacted left ventricles \[[@B27]-[@B29]\]. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Use of contrast echo to identify a patient with apical HCM. Note the \'spade shaped LV cavity\' on the contrast image \[See also additional files 24 and 25\] :::  ::: III. Myocardial contrast echocardiography ========================================= The ischaemic cascade --------------------- In the ischaemic cascade, hypoperfusion precedes wall motion abnormalities, which precede ECG changes and the onset of chest discomfort \[[@B30]\]. Stress echo, which provides an indirect marker of hypo-perfusion by recognition of wall motion abnormalities, is more than 80% sensitive and specific for detection of CAD \[[@B20]\]. The technique has two groups of limitations; 1\) Interpretive. Wall motion analysis is fundamentally subjective \[[@B31]\], leading to the need for specific training and discordance between observers. There are particular problems in the assessment of the inferoposterior wall. 2\) Dependence on the induction of ischaemia (increased myocardial oxygen demand) to provide diagnostic information. Many problems spring from this -- dependence on performance of adequate stress, influence by beta blockade, limited sensitivity for mild (eg single vessel) CAD, poor capacity to identify the extent of CAD (eg. 50% sensitivity for the detection of multi vessel disease)\[[@B32]\]. SPECT perfusion imaging is the best-established clinical method to address perfusion directly and theoretically should be more sensitive than stress echo because hypoperfusion occurs earliest the ischaemic cascade. However, the difference between the methods is marginal because of the technical limitations of SPECT scanning, which also relies on subjective interpretation. If technically feasible there is clear role for echocardiography to assess myocardial perfusion. Ultrasound is more widely available, portable, avoids radiation and has a better spatial resolution than SPECT scanning (up to 1 mm, compared with \>10 mm). An ideal technique would allow assessment of myocardial perfusion and wall motion simultaneously. Coronary circulatory physiology ------------------------------- Understanding the pathophysiology of normal myocardial perfusion is essential for understanding detection of CAD with MCE. Contrast micro-bubbles act as pure intravascular tracers, traversing the myocardial vasculature, and contrasting with MRI tracers (which escape into the extravascular space) and radionuclides (which enter myocytes). ### Myocardial blood volume The coronary blood volume (CBV) encompasses the entire coronary system, which includes the epicardial arteries, the arterioles, the capillary network, venules, veins and the coronary sinus. Approximately one-third of the CBV resides within the ventricular myocardium. This myocardial blood volume (MBV) includes \"microvessels\" of \< 300 microns in diameter, with approximately 90% of the total myocardial blood volume lying within the capillaries (6--7 microns in diameter)\[[@B33]\]. Assessment of myocardial perfusion with MCE involves processing backscattered signals received from micro-bubbles within the MBV, so that myocardial opacification almost entirely reflects backscatter from micro-bubbles flowing through the myocardial capillary compartment. There are 2 components of this **myocardial blood flow**appreciated with MCE i) the intensity of the backscatter signal from the micro-bubbles within the myocardium (brightness of its appearance on echo) -- related to the myocardial blood volume, ii) the rate of increase in intensity after bubble destruction reflects the red blood cell velocity. Their product, represents myocardial blood flow. The response of MBF myocardial blood flow and it\'s components to stress is central to the application of perfusion imaging. #### Normal physiology of perfusion Because of the near complete extraction of O2 from red cells by the myocardium under basal conditions, any increase in myocardial O2 demand must rapidly translate into increases in coronary blood flow. In the absence of a coronary stenosis, when myocardial O2 demand is increased, there is sufficient vasodilator reserve to allow coronary flow to increase by a factor of 4--6 fold above resting levels. The work of Kaul, Wei and Lindner at the University of Virginia has shown that the ability to increase coronary flow in response to increased demand is achieved through a process of coronary autoregulation, controlled predominantly by the arterioles (See table [2](#T2){ref-type="table"}). The coronary micro-circulation strives to maintain a minimum trans-capillary pressure of approximately 30 mmHg. In the absence of a coronary stenosis, a resting patient with a mean aortic pressure of 90 mmHg will have a pre-capillary pressure of \~45 mmHg. This natural resistance between the aorta and the capillaries (of \~45 mmHg) is provided by the arterioles\[[@B34]\] which present up to 60% of the total coronary vascular resistance. In the presence of increased myocardial oxygen demand, there is arteriolar vasodilation, reducing the resistance at the arteriolar level, which enables a higher precapillary pressure, translating into **increased red blood cell velocity**across the capillary network, and opening dormant capillary networks in order to maintain mean trans-capillary pressure, thus **increasing the overall myocardial blood volume**. Hence overall **myocardial blood flow is increased**. Importantly, pure vasodilator stress tends to increase red cell velocity without marked changes in overall myocardial blood volume. In the presence of a significant, non-critical (60--90%) epicardial artery stenosis at rest, completely **normal**resting blood flow is maintained by arteriolar vasodilation. As a result myocardial perfusion imaging techniques cannot detect defects at rest in patients with non-critical stenoses. Thus identification of perfusion defects in the setting of a non-critical coronary stenosis requires stress imaging to induce either ischaemia (increased myocardial oxygen demand) or hyperaemia (pharmacologically induced maximal arteriolar vasodilation). During stress we expect a 4--6 fold increase in flow to areas supplied by non-stenotic arteries, mediated by arteriolar vasodilation, increased red cell velocity and increased myocardial blood volume (opening of dormant capillary networks). Flow in the perfusion bed subtended by a significantly stenosed artery is not augmented, as resting arteriolar vasodilation is present, so limited augmentation of flow can be achieved with hyperaemia. In some cases the pre-capillary pressure drops significantly due to low distal coronary pressure and steal. With reduced pre-capillary pressure the only means of attempting to maintain normal trans-capillary pressure is to shut down capillary networks which were previously open (capillary de-recruitment). The net result is that direct comparison between regions subtended by a significant stenosis and normal territories reveals perfusion mismatch. In the presence of a critical stenosis, maximal arterial vasodilation maintains perfusion at rest. Any further reduction in distal coronary pressure directly translates into reduced precapillary pressure and reduced myocardial blood flow. Patients with critical lesions commonly have collateral vessels, making assessment of resting perfusion a complex phenomenon. Qualitative assessment of myocardial contrast for diagnosis of CAD ------------------------------------------------------------------ There are three aspects to myocardial contrast opacification -- signal intensity (equivalent to myocardial blood volume), pattern of filling and rate of filling. ### Signal intensity Myocardial blood volume is the easiest parameter to interpret qualitatively, and most studies have addressed differences in MBV between rest and stress in myocardial segments. Perfusion has traditionally been scored using a graded scale; 1 for homogenous perfusion, 0.5 for reduced perfusion and 0 for absent perfusion \[[@B8]\]. The presence of a stress induced perfusion defect not seen on the resting images indicates ischaemia, but in the absence of destruction-replenishment imaging, may appear normal in mild disease. The presence of a resting perfusion defect signifies infarction or artefact, with an infarction likely if there are associated regional wall motion abnormalities at rest, and if the defect conforms to the distribution of a coronary vascular territory. False positive defects often reflect technical limitations (Figure [9](#F9){ref-type="fig"}). Failure to obtain signal from a segment may be due to attenuation by overlying contrast, shadowing by rib or other structure, bubble destruction (especially in the apex) or failure to deliver sufficient contrast to enter the microcirculation. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### False positive defects with real-time MCE. Pseudo-apical defects are due to apical bubble destruction (a). Relocation of the focus from the base toward the apex (b) leads to \"resolution\" of the apical abnormality, but use of a mid-ventricular focus placement may lead to more problems with definition of the basal segments. This case also exemplifies attenuation of the basal lateral segment by contrast within the LV cavity. :::  ::: ### Perfusion distribution While most infarctions are shown as a transmural perfusion defect, stress-induced defects are often restricted to the subendocardium (Figure [10a,10b](#F10){ref-type="fig"}). The detection of this finding allows a greater level of confidence than does a transmural defect, or particularly epicardial defects (which may be artefactual). ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Contribution of regional shape changes to the identification of perfusion defects, including irregular wall contour in the apex (a) and mid-inferior segment (b), both associated with subendocardial defects. The C shape of the basal inferior segment complements the diagnosis of a perfusion defect in this segment. (c) :::  ::: The location of a defect has an important relationship to its likelihood of being a true or a false positive. Generally, defects on the left hand side of the image are most likely to be true positives and those restricted to the right side (anterior, lateral wall) should be considered very critically before they are identified as abnormal, as these are the most common sites of false positives. Fortunately, in our experience the apex is a more sensitive marker of LAD disease than the anterior wall, and is usually well seen. Likewise, a lateral segment should be matched with a posterior wall finding before circumflex disease is reported. The basal segments are also problematic -- especially with low MI imaging -- and in our experience, the basal inferior wall should be matched to an adjacent abnormal segment (basal septum, mid-inferior) to reduce false positive interpretations. The shape of the LV cavity is a clue to true positive findings. A \"C-shaped\" perfusion defect in the inferior wall (Figure [10c](#F10){ref-type="fig"}) is a common distribution with right coronary disease. Irregularities of the walls (Figure [10b](#F10){ref-type="fig"}) or apex (Figure [10a](#F10){ref-type="fig"}) or apical \"beaking\" (Figure [10c](#F10){ref-type="fig"}) may provide evidence of dyssynchrony, and are often associated with subendocardial perfusion abnormalities. ### Rate of filling The rate of replenishment after bubble destruction is dependent on coronary flow, and even if this is not quantified, rate of refill is a reliable and sensitive marker of a true positive defect. Using low MI techniques, micro-bubbles are infused until adequate LVO and myocardial opacification are achieved (Figure [11b](#F11){ref-type="fig"}). Several pulses of high mechanical index are delivered (Figure [11c](#F11){ref-type="fig"}), causing destruction of micro-bubbles in the beam elevation, such that the previously opacified myocardium is now empty (Figure [11d](#F11){ref-type="fig"}). The replenishment time of micro bubbles into the myocardium can be observed qualitatively (Figure [11e,11f,11g,11h](#F11){ref-type="fig"}). As the mean myocardial microbubble velocity is 1 mm/sec, and the beam elevation is approximately 5 mm, it would take up to 5 seconds (ie 5--6 heart beats at a resting heart rate from 60--80) for homogenous opacification of the myocardium at rest. With hyperaemia in the absence of stenosis, myocardial blood flow should increase 4--6 fold. Thus, images taken within 1--2 seconds following bubble destruction (ie 2--3) heart beats at a peak heart rate of 140) should demonstrate complete homogenous refill within this time frame, and failure to fill in a perfusion defect thus represents reduced myocardial blood flow. Perfusion defects noted on the post stress images, not evident on the resting images suggest ischaemia \[[@B35]\]. -see figures [12](#F12){ref-type="fig"}, [13](#F13){ref-type="fig"} and [14](#F14){ref-type="fig"} (and additional files [26](#S26){ref-type="supplementary-material"},[27](#S27){ref-type="supplementary-material"},[28](#S28){ref-type="supplementary-material"},[29](#S29){ref-type="supplementary-material"},[30](#S30){ref-type="supplementary-material"},[31](#S31){ref-type="supplementary-material"},[32](#S32){ref-type="supplementary-material"},[33](#S33){ref-type="supplementary-material"},[34](#S34){ref-type="supplementary-material"},[35](#S35){ref-type="supplementary-material"},[36](#S36){ref-type="supplementary-material"},[37](#S37){ref-type="supplementary-material"},[38](#S38){ref-type="supplementary-material"},[39](#S39){ref-type="supplementary-material"},[40](#S40){ref-type="supplementary-material"},[41](#S41){ref-type="supplementary-material"},[42](#S42){ref-type="supplementary-material"},[43](#S43){ref-type="supplementary-material"},[44](#S44){ref-type="supplementary-material"},[45](#S45){ref-type="supplementary-material"},[46](#S46){ref-type="supplementary-material"},[47](#S47){ref-type="supplementary-material"},[48](#S48){ref-type="supplementary-material"}, and [49](#S49){ref-type="supplementary-material"}) for evidence of LAD, RCA and multi-vessel CAD respectively. Assessment of end-systolic frames is preferable, despite the presence of more coronary flow in diastole, because the myocardium is thicker and there is less risk of contamination by the blood pool, and perhaps also because the capillary network is \'sealed\' during systolic contraction with minimal flow in or out of the capillary compartment \[[@B36]\]. ::: {#F12 .fig} Figure 12 ::: {.caption} ###### A clear apical defect is evident 2 beats post flash at peak stress (bottom line) which was not evident at rest (top line), consistent with LAD stenosis. \[See additional files 26-39 for full case movies, additional file 40 for angiogram and additional file 49 for curve fits\]. :::  ::: ::: {#F13 .fig} Figure 13 ::: {.caption} ###### A basal and mid inferior defect is evident 2 beats post flash at peak stress which was not evident at rest, consistent with RCA stenosis. \[See additional files 41-46 for movies and 47 and 48 for angiography\]. :::  ::: ::: {#F14 .fig} Figure 14 ::: {.caption} ###### On the 4CV, an apical and a subendocardial basal infero-septal defect are evident 2 beats post stress. The lateral wall is affected by artefact. On the 2CV, there is hypoperfusion of the inferior wall and subendocardial apical defect on the post stress images. This is consistent with multi-vessel disease. :::  ::: High MI techniques apply the same principles, with infusion until homogenous myocardial opacification. Imaging is then changed to a sequential intermittent mode, beginning with continuous high MI imaging (which results in bubble destruction and empties the myocardium). Images are then recorded intermittently, gated from the ECG (usually end systolic) with an incrementally lengthening triggering interval, ie 1:1 an image every cardiac cycle, then 1:2, and image every two cardiac cycles, then 1:3, 1:4 etc. The same principles are used for assessment of perfusion defects. A perfusion defect evident on 1:2 or 1:3 triggering at peak stress not seen at 1:5/6 triggering at rest is significant and reflects an ischaemic response to stress. Validation of qualitative contrast echo for diagnosis of CAD ------------------------------------------------------------ Qualitative assessment of perfusion with MCE was validated in open chest dogs using graded coronary stenosis \[[@B37]\]. The original landmark paper in humans \[[@B8]\] compared MCE (intermittent harmonic MCE with offline digital subtraction and colour coding) with sestamibi SPECT scanning in 30 patients undergoing dipyridamole stress to investigate known or suspected coronary disease. Normal segments showed a 91% concordance, and abnormal segments an 85% concordance, with 90% concordance by vascular territory. There was an overall 86% concordance for the detection of coronary disease. Subsequent studies have validated MCE following vasodilator or dobutamine stress using other imaging modalities, including power Doppler and real time power pulse inversion/power modulation in animals and in humans have produced similar results to SPECT \[[@B38]-[@B40]\]. A recent study using real time imaging was the first to report the use of MCE with exercise stress \[[@B41]\] (which is technically challenging because of cardiorespiratory movement and short duration of hyperaemia) and again demonstrated concordance between MCE and SPECT imaging. There was a 76% agreement between MCE and SPECT and an 88% agreement between the combination of wall motion and MCE with SPECT. This study incorporated the wall motion data from real time imaging, and despite the low frame rate, the results hint at the incremental benefit of combining the approaches. While numerous studies examining various stress and imaging modalities have demonstrated concordance between MCE and SPECT, there remains a paucity of data using quantitative coronary angiography (QCA) as the gold standard. The presence and extent of CAD by QCA has traditionally been used as the reference standard for the assessment of CAD, but few studies have compared MCE and angiography (Table [3](#T3){ref-type="table"}) \[[@B42]\]. In 45 patients undergoing mainly dobutamine stress echocardiography, Cwajg et al showed real time contrast-enhanced imaging was more sensitive than standard stress echocardiography (87% vs 56%), and gave a better recognition of disease extent (85% vs 39%). These results were surprisingly unfavourable for standard imaging, perhaps reflecting the limitations of lower frame-rate for real-time imaging. Moreover, no specificity data were available from this study. In the study of exercise stress (41), only 44 of the 100 patients who had MCE and SPECT proceeded to angiography, and in this group, the sensitivity and specificity of wall motion and MCE were not significantly different. In a recent study of 85 prospectively recruited patients, the largest involving quantitative coronary angiography, we demonstrated the addition of contrast (for LVO and MCE), to standard ExE significantly enhanced the sensitivity of the test for detection of CAD from 74 to 91%, with a non-significant reduction in specificity (57). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Myocardial contrast echo studies with patients undergoing coronary angiography. ::: Author CAD No CAD Sensitivity MCE Specificity MCE Frequency of angiography ------------------ ----- -------- ----------------- ----------------- -------------------------- Cwajg \[42\] 32 13 87% \_ All 45 Shimoni \[41\] 28 16 75% 100% 44 (44%) of 101 Heinle \[10\] 12 3 75% 67% 15 (12%) of 123 Wei \[43\] 15 \- 100% \- 15 (28%) of 54 Rocchi \[55\] 12 \- 89% \- 12 (48%) 25 Olszowska \[56\] 44 \- 97% \- All 44 Moir \[57\] 43 27 91% 70% 70 of 85 ::: Quantitative myocardial contrast echo for diagnosis of CAD ---------------------------------------------------------- Like stress echo and nuclear perfusion imaging, MCE is also limited by the qualitative nature of the interpretation. Subtle differences in video intensity between vascular beds may not be visually evident, potentially reducing the sensitivity for the detection of stenosis. Quantitative methods may help to alleviate this limitation and possibly reduce intra-observer and inter-observer variability in assessment. The process of quantitative myocardial contrast echo was validated in open chest dogs using intermittent imaging, and the same destruction-replenishment approach is used for contemporary qualitative assessment \[[@B43]\]. At a steady state during continuous intravenous infusion of micro bubbles, the number of bubbles entering or leaving any capillary unit is constant and depends on the flow rate of the bubbles. If the micro bubbles are destroyed at time zero, the video intensity in a selected myocardial region is close to zero dB (black). With time, bubbles will replenish the beam elevation -- the degree of replenishment into the beam elevation increases as the time after destruction is increased until eventually the entire ultrasound beam elevation is replenished and a plateau is reached, whereby no further increase in video-intensity/brightness can occur (Figures [15](#F15){ref-type="fig"} and [16](#F16){ref-type="fig"}). Because this relationship was originally described with intermittent imaging, it is usually described in terms of video-intensity vs pulsing intervals. ::: {#F15 .fig} Figure 15 ::: {.caption} ###### The raw signal intensity data from the apical septal segment is plotted against time after destruction. :::  ::: ::: {#F16 .fig} Figure 16 ::: {.caption} ###### an exponential function curve is applied to allow calculation of A, beta and A\*beta. :::  ::: The video intensity vs pulsing interval or time curve resembles an exponential growth function that can be fitted by the equation I = A (1-e-βt) where y is the video intensity at the pulsing interval or time t, A is the plateau video intensity and β represents the rate of rise of the slope. In this model the ***plateau myocardial video intensity***(A) represents the **myocardial blood volume**. The rate of rise of the slope (β) represents the mean myocardial **red blood cell velocity**and their product (A\*β) represents **myocardial blood flow**. Wei demonstrated an excellent linear relationship between absolute myocardial blood flow measured with radiolabelled microspheres and myocardial contrast echo derived myocardial blood flow. While ECG gated intermittent triggered harmonic imaging was used to validate these measurements, both power Doppler and real time techniques have subsequently demonstrated similar results in animal experiments (\[[@B44]-[@B46]\]). More recently, Wei evaluated the ability of MCE to calculate flow reserves from these measurements in humans (\[[@B47]\]), using intermittent imaging in 30 selected patients undergoing coronary angiography (11 of whom had no CAD and 19 had non-critical single vessel stenoses). Quantitative MCE and invasive measurement coronary flow (Doppler flow wire) were performed at rest and following vasodilator stress. In the normal subjects, myocardial blood flow velocity (β) and myocardial blood flow (A\*β) reserves demonstrated a linear relationship to coronary blood flow reserve measured invasively. In patients with CAD, there were significant differences in MBF velocity reserves between patients with mild moderate and severe stenoses, and a MBF velocity reserve of \< 1.8 indicated a \>70% stenosis. Thus MBF reserve appears to be a feasible non-invasive measure of CBF reserve in humans, which may allow non-invasive assessment of CAD and micro-vascular dysfunction. More recently, Dawson investigated the use of quantitatively derived MBF reserves to diagnose CAD using SPECT as the gold standard. She demonstrated moderate feasibility, with quantitative MBF reserves from both high and low MI imaging able to identify perfusion defects (\[[@B48]\]). Low MI imaging had a lower sensitivity. Contrast Echo and myocardial viability in chronic CAD ----------------------------------------------------- It is now well recognised that regional or global ventricular dysfunction does not necessarily imply irreversible necrosis. Hypokinetic, akinetic or dyskinetic, yet viable myocardium may be stunned or hibernating. Myocardial stunning occurs after a period of acute ischaemia, despite restoration of completely normal blood flow. The natural history of stunning is of spontaneous improvement in the viable myocardium over time. Hibernating myocardium is the term used to describe the presence of significant ventricular dysfunction in patients with chronic CAD, which recovers after revascularisation. Improvement in function of sufficient numbers of viable but hibernating segments is associated with symptomatic benefit and improved survival\[[@B49]\]. Sadly, many patients with chronic CAD and LV dysfunction have minimal viability, and revascularisation of these patients is associated with significant risk, minimal benefit and possibly worse outcome, hence the need for a reliable test for identification of viability. Radionuclide scanning, dobutamine echocardiography, MRI and PET scanning are currently available modalities, each with various advantages and disadvantages but similar efficacy for prediction of myocardial functional recovery after revascularization. MCE may also have an important role in this clinical setting. Viable myocardium is associated with preservation of the micro-vasculature, and as micro-bubbles act as pure intra-vascular tracers, the presence of myocardial perfusion by any MCE technique at rest implies viability (see figure [17](#F17){ref-type="fig"}). Using intra-coronary injection of bubbles, Nagueh demonstrated MCE was feasible and had similar accuracy to thallium SPECT and dobutamine echo for identification of functional recovery\[[@B50]\]. Using intravenous micro-bubble administration, MCE demonstrated comparable efficacy to SPECT and DSE. Importantly in both of these studies quantitative MCE was superior to qualitative assessment\[[@B51]\]. Unfortunately there remains a paucity of further data in this clinically important area. ::: {#F17 .fig} Figure 17 ::: {.caption} ###### Resting apical 2-chamber view, 10 beats post-flash, demonstrating absent perfusion to the anterior myocardial wall. :::  ::: Safety of contrast echocardiography ----------------------------------- The incidence of reported adverse events in human trials (mainly investigating LV opacification) has been very low. There have been particular theoretical concerns raised about histologic abnormalities and cardiac marker elevation in animals with high MI imaging\[[@B52]\], and whilst serum tropinin levels are normal in humans after high MI imaging\[[@B53]\], recent work has demonstrated troponin I and myoglobin in coronary sinus samples of humans after high MI imaging\[[@B54]\]. In recent months the agent SonoVue (a phospho-lipid shell encapsulating sulphur hexafluoride gas), approved in Europe, was withdrawn from clinical use because of adverse events including fatalities related to idiosyncratic hypersensitivity reactions. At this stage, specific details about the dose of agent and imaging modality used in these cases is not available. IV. Conclusion ============== Advances in micro-bubble development, combined with the development of contrast specific imaging modalities have enabled not only excellent LVO, but reliable qualitative and quantitative assessment of myocardial perfusion by ultrasound, following intravenous injections of micro-bubbles. Use of this technology during stress echo increases sensitivity and improves the non-invasive evaluation of CAD. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Technical details and clinical implications of contrast-specific imaging techniques ::: **[High Power Intermittent Imaging]{.underline}** Acquisition difficulty Amount of contrast used Amplitude of micro-bubble backscatter Dynamic range Greyscale or colour Processed Backscatter Myocardial perfusion Wall motion Artefacts --------------------------------------------------- ------------------------ ------------------------- --------------------------------------- -------------------------- -------------------------- ------------------------------- ----------------------- ----------------------- ----------------------- Tissue Harmonic Imaging challenging low high wide grey harmonic yes no yes Ultraharmonic challenging low high wide grey harmonic yes no yes Pulse inversion challenging low high wide grey harmonic yes no yes++ Harmonic Power Doppler challenging low high wide colour harmonic yes no yes **[Low Power Continuous Imaging]{.underline}** Power Pulse inversion **[easy]{.underline}** **[high]{.underline}** **[low]{.underline}** **[narrow]{.underline}** **[colour]{.underline}** harmonic **[yes]{.underline}** **[yes]{.underline}** **[yes]{.underline}** Power modulation **[easy]{.underline}** **[high]{.underline}** **[low]{.underline}** **[narrow]{.underline}** **[either]{.underline}** **[fundamental]{.underline}** **[yes]{.underline}** **[yes]{.underline}** **[yes]{.underline}** ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Relationship between stenosis severity and stress on capillary pressure, myocardial blood volume and red cell velocity. Arbitrary numbers given for illustrative purposes only. ::: **Distal coronary artery pressure** **Arteriolar resistance** **Pre-capillary pressure** **Trans-capillary pressure** **RBC velocity** **MBV** -------------------- ------------------------------------- --------------------------- ---------------------------- ------------------------------ ------------------ ------------ No stenosis REST 90 mm Hg Normal 45 mmHg 45 mmHg 30 mmHg Normal Normal No stenosis STRESS 90 mm Hg ↓ Maximal 25 mmHg 65 mmHg 30 mmHg ↑ ↑ Stenosis REST 75 mm Hg ↓ Sub-maximal 30 mmHg 45 mmHg 30 mmHg **Normal** **Normal** Stenosis STRESS 60 mm Hg ↓ Maximal 25 mmHg 35 mmHg 30 mmHg ↓ ↓ ::: Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Power Doppler 4CV at rest in normal subject. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 A4CV standard harmonic imaging ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Contrast LVO with standard harmonic imaging ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 Real time MCE with LVO and myocardial perfusion. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 Contrast LVO with ideal machine settings to optimize image quality and frame rate ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 6 3-D non contrast imaging ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 7 3-D imaging with contrast ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 8 Real time 3-D without contrast ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 9 Real time 3-D with contrast. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 10 Mr CC PLAX view rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 11 Mr CC PLAX view stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 12 Mr CC PSSAX view rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 13 Mr CC PSSAX view stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 14 Mr CC A4CV rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 15 Mr CC A4CV stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 16 Mr CC A2CV rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 17 Mr CC A2CV stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 18 Mr CC ALAX view rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 19 Mr CC ALAX view stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 20 Mr CC contrast 4CV rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 21 Mr CC contrast 4CV stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 22 Mr CC contrast 2CV rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 23 Mr CC contrast 2CV stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 24 Apical HCM standard 4CV ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 25 Apical HCM contrast rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 27 Mr JD Rest PSAX ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 28 Mr JD Rest A4CV ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 26 Mr JD Rest PLAX ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 29 Mr JD Rest A2CV ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 30 Mr JD Rest ALAX view ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 31 Mr JD Rest contrast 4CV ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 32 Mr JD Rest contrast A2CV ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 33 Mr JD Stress PLAX ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 34 Mr JD Stress PSSAX ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 35 Mr JD Stress A4CV ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 36 Mr JD Stress A2CV ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 37 Mr JD Stress ALAX view ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 38 Mr JD Stress contrast 4CV ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 39 Mr JD Stress contrast 2CV ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 40 Mr JD angiogram. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 41 Mr T 4CV rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 42 Mr T 4CV stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 43 Mr T 2CV rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 44 Mr T 2CV stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 45 Mr T 2CV contrast rest ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 46 Mr T 2CV contrast stress ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 47 Mr T cath LCA ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 48 Mr T cath RCA ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 49 Mr JD curve fits A4CV ::: ::: {.caption} ###### Click here for file :::

PubMed Central

2024-06-05T03:55:47.602150

2004-8-26

PMC516787

Background ========== The interest of clinical research in aging women and males increased in recent years and thereby the interest to measure health-related quality of life and symptoms. Women, as do men, experience an age-related decline of physical and mental capacity. They observe symptoms such as periodic sweating or hot flushes, impaired memory, lack of concentration, nervousness, depression, insomnia, and bone -- joint complaints. The Menopause Rating Scale (MRS) is a health-related quality of life scale (HRQoL) and was developed in response to the lack of standardized scales to measure the severity of aging-symptoms and their impact on the HRQoL in the early 1990s. Actually, the first version of the MRS was to be filled out by the treating physician but methodological critics lead to a new scale which can easily be completed by *women,*not by their physician \[[@B1],[@B2]\]. The validation of the MRS started some years ago \[[@B2]-[@B6]\] aiming at establishing an instrument to measure HRQoL that can easily be completed. The aims of the MRS were (1) to enable comparisons of the symptoms of aging between groups of women under different conditions, (2) to compare severity of symptoms over time, and (3) to measure changes pre- and post-treatment \[[@B4]-[@B6]\]. The MRS was formally standardized according to psychometric rules and initially published in German \[[@B2]\]. During the standardization of this instrument, three independent dimensions were identified explaining 59% of the total variance (factor analysis): psychological, somato-vegetative, and urogenital sub-scale. The MRS consists of a list of 11 items (symptoms or complaints). Each of the eleven symptoms contained in the scale can get 0 (no complaints) or up to 4 scoring points (severe symptoms) depending on the severity of the complaints perceived by the women completing the scale (an appropriate box is to be ticked). The scoring scheme is simple, i.e. the score increases point by point with increasing severity of subjectively perceived symptoms in each of the 11 items (severity 0 \[no complaints\]\...4 scoring points \[very severe symptoms\]). The respondent provides her personal perception by checking one of 5 possible boxes of \"severity\" for each of the items. This can be seen in the questionnaires in the additional files linked to this publication. The composite scores for each of the dimensions (sub-scales) is based on adding up the scores of each item of the respective dimensions. The composite score (total score) is the sum of the dimension scores. The three dimensions, their corresponding questions and the evaluation are detailed and summarized in an attached file linked to this publication \[see Additional file 1\]. The MRS scale became internationally well accepted. The first translation was into English \[[@B7]\]. Other translations followed \[[@B8]\], i.e. taking international methodological recommendations \[[@B9],[@B10]\] into consideration. Currently, the following versions are available: Brazilian, English, French, German, Indonesian, Italian, Mexican/Argentine, Spanish, Swedish, and Turkish language. These versions are available in a published form, and can be downloaded in PDF-format from the internet (see reference 8 and <http://www.menopause-rating-scale.info>). Like in other QoL scales, it is a challenge to satisfy the demands of a clinical utility and outcomes sensitivity, and this in addition to the conventional psychometric requirements of test reliability and validity. The aim of this paper is to present additional psychometric data to discuss the methodologically relevant characteristics of the MRS scale. Methods ======= The development of the scale, instrument characteristics (item selection, scaling), and norms and standardized scores have been published elsewhere \[[@B2]-[@B5]\]. This applies also for a few data that have been published on test-retest stability and criterion-dependent validity \[[@B3],[@B6]\]. During the last two years a number of smaller and larger investigations were made from different groups to further check methodological features of the scale. We performed recently a large, multinational survey to represent the situation across nine countries and cultures using existing and for the respective countries representative panels between November 2001 and February 2002 to get information about knowledge, attitudes and behaviour related to hormonal treatment in women aged 40--70 years: *Europe*(Germany, France, Spain, Sweden), *North America*(USA), *Latin America*(Mexico, Argentine, Brazil), and as example for *Asia*-- Indonesia. Study participants were accrued as a random sample of females aged 40 to 70 years from existing population panels. The sample size in each of the countries was about 1000 females aged 40--70 years, with exception of USA (n = 1500). The participation rates ranged between 46 and 94% across countries. The demographic details of the sample are: On average, about tertiles of the respondents were under 50 years, between 50--59, and over 60 years old in most of the countries, however, about 50% were less than 50 years in Indonesia and in Brazil. The majority of respondents reported a Christian religion in Europe (range: 74% (Germany) to 96% (Spain), 85% in USA, and in Latin America (range: 95% (Argentina) to 97% (Mexico). The use of the MRS was part of this survey, i.e. multinational data became available to reconsider methodological issues more thoroughly such as internal structure of the scale, reliability (internal consistency alpha), and reference values for different population. For the purposes of reliability assessment we performed a few preliminary studies with a test-retest approach. These small, descriptive studies of community samples of women aged 40--70 were done in summer and fall 2002 by local collaborators in the respective countries, but they were done separately and independent from the main study. These studies were done just for orientation with convenience samples -- not representative for the respective population. There is only one intervention study (before and after hormonal treatment) available to our knowledge. This study has been published \[[@B6]\] but not with regard to methodologically relevant results of the MRS. These data will be published soon. With these data available, we were able to scrutinize many methodological characteristics of the MRS scale to review most fundamental psychometric characteristics as well validity parameters. Results and Discussion ====================== Reliability ----------- The assessment of scientific measurements depends first of all on the evidence of replicability (consistency) and test-retest reliability. In contrast to systematic and random variation, reliability gives an estimate of method-related measurement error which should be low not to hide or dilute intended systematic changes -- due to treatment for example. Table [1](#T1){ref-type="table"} show the internal consistency measured with Cronbach\'s Alpha. The consistency coefficients range between 0.6 and 0.9 across countries for the total score as well the scores in the three domains. This is indicative for a very acceptable consistency of the MRS scale in our opinion. Moreover, there is no evidence that the scale works different in so many different countries in four continents. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Internal consistency coefficients (alpha) for the MRS scale across countries: total score and scores for the psychological, somatic, and urogenital domain. Data from the Nine-Country Study ::: **International** **Europe** **North America** **Latin America** **Asia** -------------------- ------------------- ------------- -------- --------- -------- ------- ------------------- ------------------- ---------- -------- ----------- --------------- **Total** **Overall** Sweden Germany France Spain **USA** **Overall** Mexico Brazil Argentina **Indonesia** N **9907** **4465** 1490 1050 941 984 **1440** **3002** 1002 1000 1000 **1000** Total score **0.83** **0.86** 0.85 0.84 0.86 0.86 **0.88** **0.86** 0.87 0.86 0.83 **0.84** Psychologic ascore **0.87** **0.88** 0.88 0.86 0.89 0.86 **0.90** **0.85** 0.86 0.87 0.81 **0.79** Somatic score **0.66** **0.64** 0.65 0.64 0.64 0.61 **0.70** **0.66** 0.65 0.69 0.64 **0.69** Urogenital score **0.65** **0.65** 0.64 0.63 0.67 0.67 **0.70** **0.60** 0.62 0.55 0.62 **0.65** ::: The test-retest correlation coefficients (Pearson\'s correlation) support the suggestion of a good temporal stability of the total scale and its three sub-scales (Table [2](#T2){ref-type="table"}), although most of the assessments across countries are based on very small numbers and convenience samples not claiming to be representative for the respective population. The intention of these pilot studies was to get a preliminary idea about retest stability. Larger sample sizes are required to permit final conclusions for individual countries / languages. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Test-retest coefficients (Pearson\'s correlation coefficient \"r\") for the MRS scale across countries: total score and scores for the psychological, somatic, and sexual sub-scale. ::: All Europe Latin America Asia --------------------- ------ ---------- --------------- ------ ------ ------ ------ ------ ------ ---------- ------ ------ ---------- ------ Total score 0,90 **0,92** 0,82 0,80 0,89 0,93 0,96 0,90 0,90 **0,81** 0,78 0,82 **0,84** 0,84 Psychological score 0,84 **0,87** 0,76 0,72 0,88 0,92 0,91 0,79 0,82 **0,72** 0,66 0,76 **0,71** 0,71 Somatic score 0,89 **0,90** 0,80 0,85 0,82 0,88 0,97 0,95 0,93 **0,85** 0,86 0,85 **0,81** 0,81 Urogen. score 0,86 **0,89** 0,77 0,82 0,94 0,98 0,95 0,87 0,89 **0,73** 0,67 0,74 **0,50** 0,50 ::: The test-retest coefficients of the total score range between 0.8 and 0.96 across Europe, North and Latin America, and Asia. When it comes to the subscales with much fewer items, the variation increased and some of the coefficients went down to 0.5 (urogenital domain in Indonesia). Altogether, the test-retest stability over a time period of two weeks aggregated at the international level supports the notion of a very acceptable test-retest reliability of the total scale and their three sub-scales. Although there is an impressive set of information currently available concerning the reliability of the MRS scale, there are also limitations: Small sample sizes prevent a final conclusion regarding test-retest reliability in some of the languages the scale has been translated in. Validity -------- Similar to reliability which assesses the consistency of measurement, the validity estimates if a scale measures what it intends to measure. But whereas reliability can be determined straight forward with very few indicators, the validity is almost always a continuous process (construct validation). It is a process of accumulating evidence for a valid measurement of what is purposed. Therefore, the currently available data are already fairly comprehensive and do pave the way for a focussed and continuing validation process. Internal structure of the MRS across countries ---------------------------------------------- The first step of validation is usually to multivariately demonstrate a similar internal structure (\"dimensions\") of a given scale through factor analysis. The first factorial analysis in 1996 was applied to identify the dimensions of the scale. Three dimensions of symptoms/complaints were identified \[[@B2]\]: a psychological, a somato-vegetative, and a urogenital factor that explained 58.8% of the total variance. The recent large, multinational survey in nine countries of four continents provided data to compare with the initial standardisation sample of the MRS. The question was: Is the internal structure of the MRS results comparable among different countries or cultures. Astonishingly similar factor loadings of the 11 items of the 3 domains of the MRS were observed (Table [3](#T3){ref-type="table"}). The same applies for the individual countries of the respective regions (data not shown). Although the prevalence of menopausal symptoms may slightly differ among regions/cultures (see later), the structure of complaints/symptoms seems to be pretty much the same. It suggests that the scale measures constantly the same phenomenon which speaks in favour of the translation/cultural adaptation of the scale. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Internal structure of the MRS scale across countries in 9 countries of four continents (2002) compared with the initial analysis of a German sample in 1996. Principal component analysis, Varimax rotation. Complaints (item number in MRS), numbers, and country groups. Only factor loadings ≥ 0.5 are shown. ::: Complaints (item number) ------------------------- ------- -------------------------- ---------- ---------- ------------ ------------ -------------- ------------ ------------ ----------- ------------ ------------- N Flushes(1) Heart(2) Sleep(3) Joints(11) Depress(4) Irritabil(5) Anxiety(6) Exhaust(7) Sexual(8) Bladder(9) Dryness(10) **Germany, 1996** 479 somatic 0.8 0.7 0.6 0.5 psychologic 0.8 0.7 0.8 0.6 urogenital 0.7 0.8 0.8 **All countries, 2002** 10297 somatic 0.7 0.8 0.5 0.5 psychologic 0.8 0.8 0.8 0.7 urogenital 0.7 0.6 0.8 **Europe, 2002** 4791 somatic 0.7 0.7 0.6 0.6 psychologic 0.8 0.9 0.8 0.6 urogenital 0.7 0.6 0.8 **N.-America (USA)** 1500 somatic 0.7 0.7 0.7 0.5 psychologic 0.8 0.9 0.8 0.6 urogenital 0.5 0.8 0.6 0.8 *Latin America, 2002* 3006 somatic 0.5 0.9 0.5 0.4 psychologic 0.5 0.8 0.8 0.8 0.7 urogenital 0.6 0.7 0.8 **Asia (Indonesia)** 1000 somatic 0.9 0.6 0.5 0.5 psychologic 0.8 0.8 0.8 0.5 urogenital 0.8 0.5 0.9 ::: However, there are indications that the domains could be somewhat overlapping and not as entirely independent as the statistical model suggests: Muscle or joint problems got a loading of 0.5 in the somatic and urogenital domain (USA), and sleep disturbances both 0.5 in somatic and psychological domain (Latin America). These two items had similar problems in Spain, Mexico, and Brazil but not in other countries (data not presented in table [3](#T3){ref-type="table"}). In clinical studies intra-individual comparisons over time (before/after treatment) will be the main criterion which might not be affected by potential slight differences in the patient reported outcome structure. Therefore the general agreement in the internal structure of the MRS scale across country groups, even accepting the possibility of slight differences in two items (cf. Table [3](#T3){ref-type="table"}), suggests that the scale can very well be used in clinical studies -- even including different countries. Sub-scores and total score correlations --------------------------------------- The relations among the sub-scales and the aggregate total scale are patterns that are important in the methodological assessment of a scale. In an ideal world, the correlations between subscales (supposed to be independent due to the statistical model) would be closer to 0 than the correlations with the construct of the aggregate total score to which all sub-scales should significantly contribute. But that is theory; Table [4](#T4){ref-type="table"} shows only somewhat lower correlations among sub-scales (0.4--0.7) as compared with correlation of sub-scales with the total score (0.7--0.9). This is less different than one would have wished. It suggests that the sub-scales are not as independent from each other as one would expect them to be -- based on a factorial analysis with orthogonal factors. The situation was similar in the four regions listed in Table [4](#T4){ref-type="table"} and in the individual countries belonging to these regions. It is important to realize how similar these correlation coefficients are among countries/aggregates. This is suggestive of pretty similar features of the MRS scale across the countries of this review. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Domain score -- total score correlations of the MRS scale across four country groups. Community samples. ::: Domains ------------------------------ --------- ----- ----- **Europe (n = 4246)** Total score 0.9 0.9 0.7 Psychological score \-- 0.6 0.5 Somatic score \-- \-- 0.5 **North America (n = 1376)** Total score 0.9 0.9 0.8 Psychological score \-- 0.7 0.5 Somatic score \-- \-- 0.5 **Latin America (n = 3001)** Total score 0.9 0.9 0.7 Psychological score \-- 0.7 0.5 Somatic score \-- \-- 0.5 **Asia (n = 1000)** Total score 0.9 0.9 0.7 Psychological score \-- 0.7 0.4 Somatic score \-- \-- 0.4 ::: Compatibility of MRS reference values for different population -------------------------------------------------------------- There are different categories of severity of complaints or problems with QoL. For the comparison of these categories across countries or cultures it is important to understand the prevalence of complaints. Currently, there is only one table with reference values and definitions published -- for the German population from the initial standardization of the MRS \[[@B2]\]. Are these reference values applicable for other countries/cultures? The data of our large multicultural survey permitted such comparisons. The mean values (SD) of the MRS total score and the three domains are depicted in Table [5](#T5){ref-type="table"}. The mean values of the total score and the 3 domain scores are not statistically significantly different between Europe and North America. Thus, there is no evidence yet to exclude direct comparisons of MRS values between these regions. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Mean values and standard deviation of MRS total score and 3 domains. Results from a large, multinational survey (see methods) ::: Total score Psychological Score Somato-vegetative Score Urogenital Score ------------------ ------------- --------------------- ------------------------- ------------------ ------ ----------- ------ ----------- Europe 4246 8.8 (7.1) 4453 3.4 (3.4) 4465 3.6 (2.9) 4465 1.9 (2.2) N.-America (USA) 1376 9.1 (7.6) 1426 3.4 (3.5) 1440 3.8 (3.1) 1437 2.0 (2.3) Lat.-America 3001 10.4 (8.8) 3002 4.9 (4.5) 3006 4.1 (3.6) 3005 1.4 (2.2) Asia 1000 7.2 (6.0) 1000 2.9 (2.9) 1000 3.3 (2.7) 1000 1.0 (1.6) ::: However, the total, psychological and somatic scores were systematically higher in Latin America, and systematically (significantly) lower in Asia (Indonesia) than in Europe/North America. The urogenital scores were significantly lower in Latin America/Indonesia than in Europe/US. Obviously the perception of the prevalent symptoms depends on cultural factors -- or the symptoms show real differences in prevalence. Thus, direct comparisons of MRS scores between Europe/North America on the one side and regions in Latin America and Asia cannot are not recommended. This does not effect intra-individual comparisons (e.g., pre/ post therapy) within these countries and it may also very little affect the comparison of relative changes (pre/post treatment) among different countries. The latter is a hypothesis and needs further evidence form research/experience. Similar mean values could still have a different distribution across the proposed four categories of \"severity of complaints\": no/little symptoms, mild, moderate, and severe complaints, i.e. for the total scale and the three domains. The prevalence of these categories across the four regions studied is seen in Table [6](#T6){ref-type="table"}. The comparison of the prevalence (and 95% confidence interval) showed that the above discussed differences between Europe/US and Latin-America or Indonesia very much depend on the severity of complaints. Whereas the differences in the psychological domain were less impressive, the dissimilarity was most pronounced in the urogenital domain and less also in the somatic domain. Whether this is due to different perception of identical symptoms (differences in the appearance of symptoms or both) remains a speculation. This however needs to be considered when direct comparisons among different cultures are intended. The prevalence of different \"degrees of severity\" of menopausal symptoms measured with the MRS was found to be almost identical in the aggregate of Europe and North America. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Comparison of \"degree of severity\" of the MRS and its domains. Prevalence in percent (%) and 95% confidence interval (in parenthesis) in the population sample studied in the respective regions (see methods) ::: **Europe** **North America** **Latin America** **Asia** ------------------------------ ---------------- ------------------- ------------------- --------------- **Total score** No, little (0--4) 28.8 (+/-1.3) 28.0 (+/-2.3) 31.0 (+/-1.7) 40.2 (+/-3.0) Mild (5--8) 21.9 (+/- 1.2) 23.9 (+/-2.2) 20.2 (+/-1.4) 27.5 (+/-2.8) Moderate (9--16) 25.1 (+/-1.2) 25.7 (+/-2.2) 26.2 (+/-1.6) 22.8 (+/-2.6) Severe (17+) 24.3 (+/- 1.2) 22.5 (+/-2.1) 22.7 (+/-1.5) 9.5 (+/-1.8) **Psychological domain** No, little (0--1) 35.4 (+/-1.4) 36.8 (+/-2.4) 36.8 (+/-1.6) 41.3 (+/-3.1) Mild (2--3) 21.8 (+/-1.2) 21.9 (+/-2.1) 21.9 (+/-1.4) 25.4 (+/-2.7) Moderate (4--6) 19.5 (+/-1.1) 18.7 (+/-2.0) 18.7 (+/-1.4) 21.3 (+/-2.6) Severe (7+) 23.4 (+/-1.2) 22.5 (+/-2.1) 22.5 (+/-1.7) 12.0 (+/-2.0) **Somato-vegetative domain** No, little (0--2) 39.5 (+/-1.4) 37.9 (+/-2.4) 42.1 (+/-1.8) 46.8 (+/-3.1) Mild (3--4) 22.6 (+/-1.2) 25.6 (+/-2.2) 19.4 (+/-1.4) 27.0 (+/-2.8) Moderate (5--8) 24.2 (+/-1.2) 24.3 (+/-2.2) 25.6 (+/-1.6) 20.8 (+/-2.5) Severe (9+) 13.7 (+/-1.0) 12.1 (+/-1.7) 12.9 (+/-1.2) 5.4 (+/-1.4) **Urogenital domain** No, little (0) 34.3 (+/-1.3) 33.4 (+/-2.4) 28.2 (+/-1.8) 55.6 (+/-3.1) Mild (1) 17.2 (+/-1.1) 17.0 (+/-2.0) 18.6 (+/-1.1) 18.6 (+/-2.4) Moderate (2--3) 23.0 (+/- 1.2) 24.2 (+/-2.2) 21.8 (+/-1.3) 17.0 (+/-2.3) Severe (4+) 25.6 (+/-1.2) 25.4 (+/-2.2) 31.4 (+/-1.3) 8.8 (+/-1.8) ::: Criterion-oriented validity: correlation with other scales ---------------------------------------------------------- In fact, the comparison with other scales of similar purpose is important. It is known from other quality of life scales that comparisons with scales with similar purposes are much more important than comparisons with so-called objective parameters such as exercise tests, physiological or chemical parameters -- in our case with hormones. Health related quality of life should be validated against quality of life measured with other generic QoL scales (e.g., SF-36), and against specific instruments to measure symptoms in aging women (e.g. Kupperman index). These data were published elsewhere \[[@B6],[@B11]\] but will be briefly summarized in the context of this review. Kupperman Index --------------- Although the Kupperman Index was not validated according to psychometric standards it is still in use in the medical practice to monitor menopausal symptoms. Therefore a comparison with the fully standardized MRS seems to be reasonable. If one divides the distribution of both scales into quartiles and compares the frequencies, both instruments were found to be closely associated: Kendall\'s tau-b coefficient 0.75 (95% CI 0.71--0.80) \[[@B6]\]. Similar was the Pearson correlation coefficient with r= 0.91(95% CI 0.89--0.93). The two scales can be regarded as measuring the same phenomena. However, some methodological problems of the Kupperman Index were identified in this comparison (see \[[@B6]\] for details). Generic QoL Scale SF-36 ----------------------- Two sub-scales of the multi-domain quality of life scale SF-36 was compared with the MRS: the somatic sum score (with somatic domain of MRS) and the psychologic sub-scales of both instruments. Both somatic domains were sufficiently well and significantly associated: Kendall\'s tau-b = 0.43 (95% CI 0.52--0.35); Pearson correlation coefficient r= 0.48 (95% CI 0.58--0.37). That means, the higher the score in the somatic dimension of the MRS, the lower the quality of life according to the somatic sum-score of the SF-36 \[[@B6],[@B11]\]. Similar was the results of the comparison of the psychological scores of both instruments: Kendall\'s tau-b = 0.49 (95% CI 0.56--0.41); Pearson correlation coefficient r= 0.73 (95% CI 0.81--0.65). ### Discriminative validity i.e., the ability of the scale to accurately measure treatment effects and to predict the clinically based assessment of physicians, was not analysed so far. At present, there is one post-marketing study that can be used to preliminary assess discriminative validity. The results will be published soon elsewhere. To this end, many clinicians understand the term \"validity\" and mean high utility for clinical work or research. Conclusions =========== The MRS scale is a standardized HRQoL scale with good psychometric characteristics. The use in many countries offered the possibility to compare the test characteristics across countries. Reliability measures (consistency and test-retest stability) were found to be good in all countries where data were obtained -- however, some samples were very small and therefore considered as preliminary information. The validity was measured in its various forms: The internal structure of the MRS across countries was sufficiently similar to conclude that the scale really measures the same phenomenon in women with complaints. The sub-sores and total score correlations showed high coefficients with the total score and less among the sub-scales. This however indicates that the subscales are not fully independent in practice. Comparisons of reference values from different populations showed that the MRS scores can easily be compared between Europe and North America/US. Direct comparisons between Europe/North America and Latin American countries and Asia (Indonesia) should be considered with caution because the severity of reported symptoms seems to differ. The reasons are not clear, further research is needed. The comparison with other scales for menopausal symptoms (Kupperman Index) showed a sufficiently close association and correlation coefficients, i.e. illustrating a good criterion-oriented validity. The same is true for the comparison with the generic QoL scale SF-36 where also high correlation coefficients have been shown. Thus, the currently available methodological evidence points towards a high quality of the MRS scale to measure and to compare HRQoL of aging males over time or intervention. It suggests a high reliability and high validity as far as the process of construct validation could be completed. Authors\' contributions ======================= KH: responsible for drafting the manuscript and running analyses. AR: responsible for designing and overseeing the multinational survey (2001/2002), contributed to writing and revising of the paper. PP: co-ordination of the field work of the multinational survey, setting up the initial database, and contributed to writing of the paper, responsibility in developing/validating the MRS scale. HPGS: Major responsibility in developing the MRS scale, contributed to writing/revision of the manuscript. FS: Provided data of a clinical study, contributed to writing of the manuscript. LAJH: responsible for the collection and evaluation of the data, and involved in writing/revising the paper. DMT: responsible for checking the integrated database, responsible for several analyses regarding validity, and contributed to writing of the paper. Acknowledgements ================ The large multinational survey was sponsored by Schering AG Berlin; the field work done by teams of NFO Worldwide (now TNS Worldwide), TNS Healthcare Munich, co-ordinated by ZEG Berlin; we thank the respective central and local teams. We are grateful to having received a part of the database for our methodological evaluation of the MRS scale. ^1^We thank the marketing research institute IM Leipzig in Leipzig (Germany) for coordinating the small test-retest investigation in several countries to get a first orientation about the magnitude of the test-retest correlations.

PubMed Central

2024-06-05T03:55:47.607430

2004-9-2

PMC516788

Background ========== Extramedullary myelomas (plasmacytoma) are malignant proliferations of plasma cells in the absence of bone involvement. When occur in the soft tissue, it usually involve the upper respiratory tract and oral cavity \[[@B1]\]. Plasmacytomas of breast are very rare. This report describes a patient with bilateral breast masses who underwent excision biopsy for suspected primary carcinoma that subsequently proved to be a recurrence from extramedullary plasmacytoma of mediastinum treated 5 years ago. To the best of our knowledge, this is the first case report of bilateral recurrence of a primary extramedullary plasmacytoma in breast tissues after a long disease-free interval. Case presentation ================= A 70 year-old woman with a one-month history of bilateral breast masses was referred to our cancer center for surgical evaluation. There was no associated breast pain, skin change or nipple discharge. There was no history of bone pain, weight loss, fatigue, fever or other systemic complaints and no family history of breast cancer. Significant past medical history included treatment for an extramedullary retrosternal plasmacytoma 5-years prior this admission. At the time of the initial work-up for the retrosternal mass, immunoelectrophoresis showed no evidence for hyperproteinemia or paraproteinemia. Whole body bone scan was negative and a bone marrow biopsy revealed less than 5% of plasma cells. Therefore, multiple myeloma was excluded by nuclear medicine, laboratory and histology studies. The patient underwent radiation therapy (40 Gy with fraction size of 200 cGy delivered over 4 weeks) followed by chemotherapy with cyclophosphamide, cisplatin and prednisolone. The patient was followed by laboratory tests, chest roentgenography, and computed tomography annually. A bone scintigraphy was carried out after 2 years and showed no uptake patient was thereafter lost to follow-up. Five years after initial diagnosis of extramedullary plasmacytoma the patient presented with bilateral breast masses. Physical examination revealed a 3.5 cm × 2.5 cm, mass in the upper inner quadrant of the right breast and a similar 5.0 cm × 4.5 cm mass in the lower inner quadrant of the left breast. No asymmetry, skin dimpling or signs of inflammation were present. There was no axillary or supraclavicular lymphadenopathy. Mammography confirmed a well-defined 3.2 cm oval-shaped mass in the upper inner quadrant of the right breast, and a lobulated 5.5 cm density in lower inner quadrant of the left breast without any tissue distortion, inflammation and fibrotic reaction.(Figure [1](#F1){ref-type="fig"}) There were no microcalcification and satellite lesions. These masses were solid and hypoechoeic with multiple septations in sonography. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Mammography of the patients\' breasts (A: mediolateral oblique, view B: craniocaudal view) :::  ::: Excisional biopsy of the masses revealed a 5.0 (left) and 3.0 (right) well-defined, capsulated gritty mass surrounded by normal breast tissue. There was no extension from the capsulated masses to pectoral muscles or chest wall. Histopathological examination showed high-grade tumors composed of immature and mature plasma cells. Mitosis, necrosis, nuclear pleomorphism and binucleated and multinucleated plasma cells were seen. (Figure [2](#F2){ref-type="fig"}) Additional studies such as serum protein electrophoresis and immunoelectrophoresis were normal. No Bence Jones or other M components were detected in the urine. Skeletal surveys (Tc^99^bone scan and skull and pelvic X-rays) did not show any pathological changes. There was no evidence of anemia, hypercalcemia or renal insufficiency. However, the patient refused a second bone marrow biopsy. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Photomicrograph showing nuclear pleomorphism, binucleated and multinucleated plasma cells with enlarged nucleoli (Hematoxylin & Eosin). :::  ::: Immunohistochemical studies were performed on the paraffin embedded tissues to determine if the infiltrate had monoclonal character. The tumor cells were diffusely and strongly positive for lambda chains but negative for kappa chains. (Figure [3](#F3){ref-type="fig"}) ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Lambda and kappa immunohistochemical stain showing strong and diffuse positivity for lambda (a) and negativity for kappa (b). :::  ::: The tumor cells were weakly positive for monoclonal mouse anti human placental V538C, and plasma cell markers (CD138). Nuclear prognostic marker (Ki67) showed 50% to 80% nuclear expression indicative of high proliferative activity and suggesting a plasmacytic tumor with anaplastic components (Figure [4](#F4){ref-type="fig"}). Other immunohistochemical stains including CD21, cytokeratin, S100, and HMB45 were negative. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Ki67 immunohistochemical stain showing 50--80% nuclear positivity :::  ::: A retrospective microscopic review of the mediastinal mass showed similar morphology to the breast tumor. Hence, the histological diagnosis of recurrent plasmacytoma was made. The patient was treated with oral Melfalan and Prednisone. The patient has been disease free for twenty months after treatment and has showed no evidence of recurrence in the mediastinum, breast or any other region. Discussion ========== Primary soft tissue extramedullary plasmacytoma (SEP) is uncommon and is defined as a malignant tumor of plasma cells arising in the soft tissue in the absence of bone involvement. It can occur in any organ as a solitary form of plasma cell neoplasm \[[@B2]\]. Although SEP can arise throughout the body, almost 90% of the cases arise in the head and neck areas, most commonly in the upper respiratory tract including the nasal cavity, para nasal sinuses, oropharynx, salivary glands and larynx \[[@B3]-[@B7]\]. Several other sites can rarely be involved, including testis, bladder, urethra, breast, ovary, lung, pleura, thyroid, orbit, brain and skin tissues (1, 8--17). Approximately forty-five cases of breast plasmacytoma have been reported in published literature since 1928 (1, 2, 18--21). More than half of the lesions were unilateral (66%), with the majority of the cases occurring in the setting of multiple myeloma (77%) (1, 2, 18--20, 22). When plasmacytoma originates from soft tissues, like the case presented here, the disease is usually associated with a relatively mild clinical behavior and long survival, suggesting that it is a truly different disease entity compared to other plasma cell tumors \[[@B23]\]. Dimopoulos *et al*(1999) reported that solitary extramedullary (soft tissue) plasmacytomas (SEP) are less common than solitary bone plasmocytoma (SBP), and have a better prognosis as the majority can be cured by local radiotherapy \[[@B24]\]. Liebross *et al*(1999) reported local recurrence rates of less than 5% after radiotherapy \[[@B6]\]. Mayr *et al*(1990) noted that the risk of distant relapse is more than 30%, which is significantly less than that seen with SBP \[[@B25]\]. Progressive disease may accrue as multiple myeloma, SBP or involvement of lymph nodes, skin or subcutaneous tissues. Its recurrence, if any, tends to be within 2--3 years of initial diagnosis. The reported ten year survival rate is at least 66% \[[@B3],[@B7]\]. Involvement of the breast with SEP is uncommon and may occur either as a solitary primary tumor or as evidence of disseminated multiple myeloma \[[@B19]\]. Our patient was referred with bilateral breast masses. In an approach to a patient with bilateral breast masses, differential diagnosis includes: fibroadenomas, complex cysts, metastasis, lymphoma, synchronous breast cancer, focal fibrosis, fat necrosis, abscess, and phyllodes tumor \[[@B20]\]. Metastatic lesions of the breast from extramammary neoplasms are rare and in larger studies have been reported to constitute 0.4 to 2% of all breast malignancies. The most common are lymphomas and other tumors of hematological origin \[[@B26]\]. The striking feature in this case was the recurrence of an isolated plasmacytoma (which was treated successfully five year prior) in both breasts. As approximately 20% of patients who present with isolated extramedullary plasmacytoma will eventually develop multiple myeloma, close follow-up is strongly recommended \[[@B1],[@B22]\]. Our patient did not have any prior breast aspiration cytology. There are many tumors, which may present with plasmacytoid appearance on aspiration cytology. Tumors such as small ductal carcinoma and lobular carcinoma of breast, metastatic carcinoid, metastatic melanoma and some lymphomas may represent with uncohesive group of cells with eccentric nuclei resembling plasma cells. Hence, a plasmacytoma (with anaplastic plasma cells) may be readily mistaken for carcinoma (or other undifferentiated neoplasm) not only clinically, but also on cytological examination. This would justify excision biopsy and the use of an extended immunohistochemical panel to include such markers as cytokeratin and S-100 in the assessments. To help the cytopathologist avoid misinterpretation, clinical history and presentation are extremely helpful. Most of the errors in histopathology and cytopathology diagnosis occur when pathologist is not aware of medical history of the patient and unusual clinical presentation. This case emphasizes the importance of distinguishing a plasmacytoma of the breast from primary mammary carcinomas and other benign lesions to avoid unnecessary surgery and provide the appropriate treatment and adjuvant therapy. Authors\' Contributions ======================= AK carried out excision biopsies and drafted the manuscript. MJZ did the histopathological examination and contributed to the pathological content of the manuscript. SKR is the pathologist who confirmed the diagnosis and prepared the immunohistochemical stains and illustrations. He also contributed to pathological content of the manuscript MN followed-up the patient and contributed to the manuscript preparation All authors read and approved the final manuscript. Acknowledgments =============== Patient\'s permission was obtained for publishing her case records. Authors also wish to thanks Dr Reza Navipour, Center for Research Development, Imam Hospital, Tehran University of Medical Sciences for his help in accessing the patient\'s medical documents.

PubMed Central

2024-06-05T03:55:47.611677

2004-8-31

PMC516789

Introduction ============ Antimicrobial proteins and peptides are widely expressed in both plants and animals. A variety of natural antibiotics belonging to different classes such as defensins, cathelicidins, cercopins and protease inhibitors \[[@B1]\] are found in epithelial tissues of organs that are most likely exposed to pathogens. Among them, the most studied in humans are the defensins, which are broadly classified into three types viz alpha, beta and theta defensins depending on their disulfide bonding, tissue distribution and genomic organization. They exhibit broad spectrum antimicrobial activity \[[@B2]-[@B5]\], thus may form an important component of the innate immune system. Antimicrobial proteins and peptides including defensins are generally cationic in nature \[[@B6]\] and are believed to exert their bactericidal effect by permeabilizing the bacterial membranes by forming pores \[[@B7]\], thinning the membrane \[[@B8]\], or by destabilizing the membrane bilayer \[[@B9]\]. In addition to membrane permeabilization, antimicrobial proteins and peptides kill bacteria by inhibition of macromolecular biosynthesis \[[@B10]-[@B12]\] and/or interacting with specific vital components inside the bacteria \[[@B13],[@B14]\]. In the epididymis, a major organ of the male reproductive tract, immature sperm released from the testis undergo sequential maturation to acquire forward motility and fertilizing ability. A wide variety of proteins including antimicrobial proteins released into the lumen of epididymis bind sperm and are thought to play an important role in epididymal immunity in addition to their role in sperm maturation \[[@B15]\]. Examples of antimicrobial proteins reported in the male reproductive tract include human cationic antimicrobial protein (hCAP18, a cathelicidin) \[[@B16]\], defensins \[[@B17]-[@B20]\], the epididymal β-defensin member Bin1b \[[@B21]\], cystatins \[[@B22],[@B23]\], lactoferrin \[[@B24]\] seminalplasmin \[[@B25]\] and seminogelin-derived peptides \[[@B26]\]. Earlier we identified and characterized the sperm binding epididymal proteins of the HE2 family \[[@B27]\], which show homology to the antimicrobial β-defensins. The HE2 gene located on chromosome 8p23 within the β-defensin gene cluster, encodes a series of isoforms containing identical proregions joined to different C-terminal peptides \[[@B27]\]. Among them, HE2β1 conserves the characteristic β-defensin-like six-cysteine motif (Figure [1](#F1){ref-type="fig"}). Furthermore, like the β-defensins, HE2 C-terminal peptides are cleaved from their proregions by a furin-like proprotein convertase and these peptides are reported to exist in the epididymal epithelium, luminal fluid and the seminal plasma \[[@B28]\]. We demonstrated the antimicrobial activity of HE2α, HE2β1 and HE2β2 proteins and their C-terminal peptides \[[@B29]\] and the epididymis specific defensin DEFB118 \[[@B30]\] against *E. coli*. Their antimicrobial activities are structure dependent and salt tolerant and their mechanism of action involves interacting with and permeabilizing bacterial membranes. However, structural evidence for the membrane changes in *E. coli*induced by these peptides is still lacking. Further, it is not still clear whether bacterial killing by HE2 peptides involves only membrane permeabilization or whether the peptides interact with specific targets inside the bacteria to inhibit metabolic processes as reported for other antimicrobial proteins is not yet demonstrated. In this study, using transmission and scanning electron microscopy, we provide further evidence that HE2 peptides induce significant structural changes in *E. coli*consistent with their membrane dependent mechanism of action as reported earlier. Further, we show that HE2 peptides inhibit *E. coli*DNA, RNA and protein synthesis suggesting that their antimicrobial action may also involve targets inside the bacteria as well as membrane permeabilization. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Amino acid sequence alignment of epididymal HE2 peptides with human β-defensins 1 and 2. Amino acid sequence shown in blue corresponds to the C-terminal peptides used in this study. The characteristic β-defensin six cysteine motif is represented in red.  represents the cleavage site where the full length proteins are cleaved to release the C-terminal peptides. :::  ::: Methods ======= Recombinant peptide preparation and synthesis --------------------------------------------- HE2α and HE2β2 C-terminal peptides were synthesized at the Peptide Synthesis Facility, University of North Carolina, Chapel Hill by standard f-moc solid phase procedures using Rainin symphony multiple peptide synthesizer (Rainin Instrument, Woburn, MA). The purified peptides eluted as single peaks upon reverse phase high performance liquid chromatography (HPLC) and were further demonstrated to have their corresponding molecular weight by MALDI-TOF mass spectrometry. HE2β1 C-terminal peptide was expressed in *E. coli*and purified as described previously \[[@B29]\]. Briefly, *E. coli*strain M15 (pREP4) was transformed with pQE30 vector (Qiagen, Valencia, CA, U.S.A) containing cDNA that codes for HE2β1 C-terminal peptide. Protein expression was induced with 1 mM isopropyl-β-D-thiogalactoside for 1 h at 37°C and the His-tagged recombinant peptide was purified using nickel-nitrilotriacetate agarose column (Qiagen, Valencia, CA, U.S.A). To avoid baseline expression of the protein prior to induction, 1% glucose was maintained in the bacterial medium and the induction time was kept to a minimum (1 h) to minimize the toxic effects of the peptide on *E. coli*. The peptide was dialyzed extensively against 10 mM sodium phosphate (pH 7.4) to remove urea. Transmission electron microscopy -------------------------------- *E. coli*resuspended in 10 mM sodium phosphate buffer (pH 7.4) were treated with 50 μg/ml HE2α, HE2β1 or HE2β2 for 30 and 60 min. After incubation, bacterial cells were washed with 10 mM sodium phosphate buffer (pH 7.4) and fixed with an equal volume of 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, followed by centrifugation at 1000 rpm for 10 minutes to concentrate the cells in a pellet. The fixed samples were stored overnight to several days at 4°C in the fixative solution. The pellet was rinsed in 0.1 M sodium cacodylate buffer several times, and post-fixed with a combination of 1.25% potassium ferrocyanide and 1% buffered osmium tetroxide for one hour at room temperature. Following dehydration with a graded series of ethanols (30--100%) and two changes of propylene oxide, the cell pellet was infiltrated and embedded in PolyBed 812 epoxy resin (Polysciences, Inc., Warrington, PA). Ultra thin sections (70 nm) were cut and mounted on copper grids followed by post staining with 4% uranyl acetate and 0.4% lead citrate. The sections were examined and photographed at an accelerating voltage of 80 kV using a LEO EM 910 transmission electron microscope (LEO Electron Microscopy, Inc., Thornwood, NY) equipped with a Gatan BioScan digital camera (Gatan, Inc., Pleasanton, CA). Scanning electron microscopy ---------------------------- The structural changes induced by HE2 peptides on *E. coli*were studied using scanning electron microscopy as described earlier \[[@B30]\]. Bacterial cells suspended in 10 mM sodium phosphate buffer (pH 7.4) after treating with 50 μg/ml of HE2 peptide were fixed with an equal volume of 4% glutaraldehyde in 0.15 M sodium phosphate buffer, pH 7.4. Immediately following the addition of the fixative solution, the sample tube was mixed by gently inverting the tube up and down for several minutes to prevent clumping of the cells. The fixed samples were stored overnight to several days at 4°C in the fixative solution. Using a microanalysis vacuum filter holder (Fisher Scientific, Suwanee, GA) and a 0.1 μm polycarbonate membrane filter (Poretics Corporation, Livermore, CA), the suspended fixed cells were vacuum-filtered onto the membrane substrate, rinsed with 0.15 M sodium phosphate buffer, and dehydrated through a graded series of ethanols (30--100%). During the entire filtration, rinsing, and dehydration process, the cells were kept covered with fluid to prevent air drying. The filters were transferred in 100% ethanol to a critical point dryer (Balzers CPD-020, Bal-Tec AG, Vaduz, Liechtenstein), and dried using carbon dioxide as the transition solvent. The filters were mounted on aluminum specimen supports with carbon adhesive tabs, and coated with a 15 nm thickness of gold-palladium metal (60:40 alloy) using a Hummer X sputter coater (Anatech, Ltd., Alexandria, VA). Samples were examined with a Cambridge Stereoscan 200 scanning electron microscope (LEO Electron Microscopy, Inc., Thornwood, NY) using an accelerating voltage of 20 kV. Macromolecular synthesis ------------------------ The effects of HE2 peptides on *E. coli*DNA, RNA and protein synthesis were studied as functions of incorporation of the radioactive precursors \[methyl-^3^H\]thymidine, \[5-^3^H\]uridine and L-\[4,5-^3^H(N)\]leucine respectively as described \[[@B30]\]. 1 × 10^6^mid-log phase *E. coli*resuspended in 10 mM sodium phosphate buffer (pH 7.4) were treated with varying concentrations of HE2 peptides and 2.5 μl/ml of either \[methyl-^3^H\]thymidine (20 Ci/mmol), \[5-^3^H\]uridine (25.5 Ci/mmol) or L-\[4,5-^3^H(N)\]leucine (59.5 Ci/mmol) for different time periods. After incubation, bacterial suspensions were added to 10% ice-cold trichloroacetic acid and allowed to stand in ice for 40 min. Samples were then collected on 2.4 cm GF/C glass microfiber filters (Fisher Scientific, Pittsburgh, PA) using vacuum filtration and washed thoroughly with 5% TCA and 70% ethanol. The filters were then dried and placed in scintillation vials containing 5 ml of EcoScint scintillation cocktail (National Diagnostics, Atlanta, GA) and counts were obtained in a LKB 1214 Rackbeta liquid scintillation counter (LKB WALLACE, Turku, Finland) for 1 min for each filter. Statistical analyses using Student\'s t-test were performed using Sigma Plot software (SPSS Inc., Chicago, IL). Values shown are Mean ± S.D. Results ======= Transmission electron microscopy -------------------------------- Transmission electron microscopy revealed striking structural alterations in *E. coli*exposed to HE2 peptides. The three images shown for each treatment were documented in different fields of view and are intended to represent the range of responses seen in the bacteria. In contrast to the smooth continuous double membrane structure clearly visible in untreated bacteria (Fig. [2A,2B,2C](#F2){ref-type="fig"}), the outer membranes of bacteria treated with 50 μg/ml HE2α peptide for 30--60 min showed thickening and the protrusion of irregular blebs. The inner membrane was indistinct in many regions after 30 min and the cytoplasm was retracting from the outer membrane (Fig. [3A,3B,3C](#F3){ref-type="fig"}). After 60 min of HE2α treatment, the inner membrane was difficult to discern and fibrous and granular material, presumably cell contents appeared to exude from the damaged membranes (Fig. [3D,3E,3F](#F3){ref-type="fig"}). Treatment with 50 μg/ml HE2β1 peptide for 30 min resulted in numerous mushroom shaped blebs and retraction of cytoplasm (Fig. [4A,4B,4C](#F4){ref-type="fig"}) and by 60 min, these bacteria appeared to lose cell contents particularly at the division septa (Fig. [4D,4E,4F](#F4){ref-type="fig"}). Similarly, the HE2β2-treated bacteria showed loss of the double membrane structure, formation of blebs and outer membrane roughening (Fig. [5A,5B,5C](#F5){ref-type="fig"}). By 60 min, numerous large vacuoles accumulated, the cytoplasm was extensively granulated and retracted from the outer membrane and cell contents appeared to escape at division septa (Fig. [5D,5E,5F](#F5){ref-type="fig"}). The peptides appeared to induce structural changes specific to each peptide besides the morphological changes that were generally observed. HE2α peptide caused membrane thickenings, which was not observed with the other two peptides. Similarly, HE2β1 peptide caused retraction of cytoplasm when treated for 30 min, whereas HE2β2 peptide induced retraction of cytoplasm after 60 min incubation. Formation of vacuoles was more evident upon treating *E. coli*with HE2β2 peptide. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Transmission electron micrographs of untreated *E. coli*showing a smooth continuous double membrane structure. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### *E. coli*treated with 50 μg/ml HE2α peptide for 30 min (A-C) and 60 min (D-F) visualized by transmission electron microscopy showed membrane thickening and blebbing with subsequent leakage of cellular contents. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Transmission electron micrographs showing cytoplasmic retraction and extensive granulation of *E. coli*treated with 50 μg/ml HE2β1 peptide for 30 min (A-C) and 60 min (D-F). :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Incubation of *E. coli*with 50 μg/ml HE2β2 peptide for 30 min (A-C) and 60 min (D-F) show discontinuous membrane structure with extensive vacuole formation. Cellular contents appear to leak at the dividing septa. :::  ::: Scanning electron microscopy ---------------------------- *E. coli*treated with HE2 peptides were observed using scanning electron microscopy to gain further insights into the membrane effects. The three images shown for each treatment were documented in different fields of view and are intended to represent the range of responses seen in the bacteria. Untreated bacterial cells had normal and smooth surface morphology (Fig. [6A,6B,6C](#F6){ref-type="fig"}). Bacteria treated with HE2α (Fig. [7A,7B,7C,7D,7E,7F](#F7){ref-type="fig"}), HE2β1 (Fig. [8A,8B,8C,8D,8E,8F](#F8){ref-type="fig"}) or HE2β2 (Fig. [9A,9B,9C,9D,9E,9F](#F9){ref-type="fig"}) peptides showed pronounced changes in their morphology consistent with the changes observed using transmission electron microscopy. *E. coli*treated with the HE2 peptides for 30--60 min showed pronounced wrinkling, surface roughening and blebbing of the membrane. A majority of the cells appeared to have lost their bacterial membrane integrity. The fibrous material and cellular debris, possibly arising due to leakage and cell lysis accumulated particularly at the dividing septa. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Scanning electron micrographs of untreated *E. coli*revealing a smooth membrane surface morphology. :::  ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### *E. coli*treated with 50 μg/ml HE2α peptide for 30 min (A-C) and 60 min (D-F) visualized by scanning electron microscopy show membrane blebbing and leakage of cellular contents. :::  ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Membrane wrinkling and blebbing were evident in *E. coli*treated with 50 μg/ml HE2β1 peptide for 30 min (A-C) and 60 min (D-F). :::  ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Scanning electron micrographs of *E. coli*treated with 50 μg/ml HE2β2 peptide for 30 min (A-C) and 60 min (D-F). Loss of bacterial membrane integrity due to surface blebbing and wrinkling was evident. :::  ::: Macromolecular synthesis ------------------------ To investigate whether HE2 peptides affect macromolecular synthesis of *E. coli*, the incorporation of radioactive precursors viz \[methyl-^3^H\]thymidine, \[5-^3^H\]uridine and L-\[4,5-^3^H(N)\]leucine into DNA, RNA and protein was studied in the presence of 2--25 μg/ml peptides. A dose and time dependent inhibition of DNA synthesis by HE2α peptide was observed (Fig. [10A](#F10){ref-type="fig"}). 2 μg/ml HE2α peptide inhibited DNA synthesis after 60 min incubation, whereas 10 and 25 μg/ml significantly inhibited DNA synthesis after 20 min incubation (Fig. [10A](#F10){ref-type="fig"}). RNA synthesis was not inhibited by 2 μg/ml HE2α peptide, whereas inhibition was observed with 10 and 25 μg/ml concentrations (Fig. [10B](#F10){ref-type="fig"}). No significant inhibition of protein synthesis by HE2α peptide was observed at any of the concentrations tested (Fig. [10C](#F10){ref-type="fig"}). ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Effect of HE2α peptide on macromolecular synthesis in *E. coli*. A, \[methyl-^3^H\]thymidine incorporation into DNA. B, \[5-^3^H\]uridine incorporation into RNA. C, L-\[4,5-^3^H(N)\]leucine incorporation into proteins. 0 μg/ml (■); 2 μg/ml (▲); 10 μg/ml (▼); 25 μg/ml (◆). Values shown are mean ± SD. \*, P \< 0.05-0.01, \*\*, P \< 0.01-0.001, \*\*\*, P \< 0.001 compared to 0 μg/ml at the corresponding time point. :::  ::: In the case of HE2β1 peptide, 2 μg/ml dose did not inhibit DNA synthesis, whereas 10 and 25 μg/ml concentrations showed significant inhibition (Fig. [11A](#F11){ref-type="fig"}) after a 20 min incubation. Similarly, significant inhibition of RNA synthesis was not observed with 2 μg/ml. However, 10 and 25 μg/ml concentrations inhibited RNA after 20 min (Fig. [11B](#F11){ref-type="fig"}) and protein synthesis (Fig. [11C](#F11){ref-type="fig"}) after a 60 min incubation. ::: {#F11 .fig} Figure 11 ::: {.caption} ###### Effect of HE2β1 peptide on macromolecular synthesis in *E. coli*. A, \[methyl-^3^H\]thymidine incorporation into DNA. B, \[5-^3^H\]uridine incorporation into RNA. C, L-\[4,5-^3^H(N)\]leucine incorporation into proteins. 0 μg/ml (■); 2 μg/ml (▲); 10 μg/ml (▼); 25 μg/ml (◆). Values shown are mean ± SD. \*, P \< 0.05-0.01, \*\*, P \< 0.01-0.001, \*\*\*, P \< 0.001 compared to 0 μg/ml at the corresponding time point. :::  ::: Inhibition of *E. coli*DNA synthesis by HE2β2 peptide was dose and time dependent. Significant inhibition of DNA synthesis was observed after a 60 min incubation with 2 μg/ml HE2β2 peptide, whereas the inhibition was observed at a earlier time point with 10 and 25 μg/ml concentrations (Fig. [12A](#F12){ref-type="fig"}). However, RNA synthesis was not inhibited by 2 μg/ml HE2β2 peptide, whereas 10 and 25 μg/ml doses were effective after 40 and 10 min incubations respectively (Fig. [12B](#F12){ref-type="fig"}). Protein synthesis was inhibited only with 10 and 25 μg/ml HE2β2 peptide after a 60 min incubation (Fig. [12C](#F12){ref-type="fig"}). It appears that HE2 peptides inhibit DNA synthesis to a greater extent than RNA and protein synthesis, suggesting that DNA synthesis may be the sensitive target for antimicrobial action after membrane permeabilization. ::: {#F12 .fig} Figure 12 ::: {.caption} ###### Effect of HE2β2 peptide on macromolecular synthesis in *E. coli*. A, \[methyl-^3^H\]thymidine incorporation into DNA. B, \[5-^3^H\]uridine incorporation into RNA. C, L-\[4,5-^3^H(N)\]leucine incorporation into proteins. 0 μg/ml (■); 2 μg/ml (▲); 10 μg/ml (▼); 25 μg/ml (◆). Values shown are mean ± SD. \*, P \< 0.05-0.01, \*\*, P \< 0.01-0.001, \*\*\*, P \< 0.001 compared to 0 μg/ml at the corresponding time point. :::  ::: Discussion ========== Earlier we demonstrated that HE2 proteins and their C-terminal peptides exhibit salt tolerant and structural dependent antimicrobial activities and their mechanism involved permeabilization of both outer and inner bacterial membranes \[[@B29]\]. In this study, structural changes induced in *E. coli*by epididymal HE2α, HE2β1 and HE2β2 peptides as visualized by transmission and scanning electron microscopy provide further evidence of the membrane dependent mechanism of bacterial killing. Such structural changes induced in *E. col*i by other antimicrobial proteins and peptides were reported previously. Membrane thickening as shown in Fig [3A,3B,3C,3D,3E,3F](#F3){ref-type="fig"} was reported in *E. coli*treated with human neutrophil peptides 1 and 2 (defensins) \[[@B14]\]. Similarly, retraction of cytoplasm and the appearance of vacuoles as shown in Fig [4A,4B,4C,4D,4E,4F](#F4){ref-type="fig"} were reported for *E. coli*treated with synthetic peptides of the antimicrobial protein apolipoprotein A-II \[[@B31]\]. Highly granular cytoplasm with discontinuous membrane was reported for *E. coli*treated with the antimicrobial peptide tigerinin-1 \[[@B32]\] similar to the changes shown in Fig. [5A,5B,5C,5D,5E,5F](#F5){ref-type="fig"}. Scanning electron micrographs of *E. coli*treated with HE2 peptides also revealed striking structural changes in their morphology. HE2 peptides caused membrane wrinkling, blebbing and leakage of fibrous material primarily at the dividing septa in *E. coli*. Such structural changes shown in Fig. [7](#F7){ref-type="fig"},[8](#F8){ref-type="fig"},[9](#F9){ref-type="fig"} were earlier reported for other antimicrobial proteins viz the cathelicidin-derived peptide SMAP-29 \[[@B33]\], temporin-L \[[@B34]\], salmon antimicrobial protein \[[@B35]\] and the epididymal proteins ESC42 (DEFB118) \[[@B30]\] and EPPIN \[[@B36]\]. An interesting observation in this study is the leakage of fibrous material primarily at the dividing septa. It is known that cell division in *E. coli*involves annular constriction of all layers of the cell envelope and synthesis and assembly of new septal materials \[[@B37]\]. It is possible that during this dynamic remodeling process, the region of division septum formation to be particularly vulnerable to attack by antibacterial proteins. The mechanism of action of antimicrobial proteins is primarily thought to be membrane dependent involving membrane permeabilization and disruption. Structural characteristics of antimicrobial peptides tend to play an important role in their mechanism of action. For example, β-defensins are cationic in nature and with β-sheet rich amphipathic structures stabilized by the three disulfide motif \[[@B38]\]. The cationic nature of β-defensins favors them to bind to and disrupt target membranes that are rich in anionic phospholipids. Similarly, HE2α, HE2β1 and HE2β2 peptides are cationic in nature with basic pIs. Our three dimensional structural analysis of HE2β1 peptide revealed that it is rich in β-sheet structure and its tertiary structure presents regional concentrations of basic and hydrophobic amino acids similar to β-defensins \[[@B29]\]. Such structural characteristics of HE2 peptides which resemble to those of β-defensins suggest that they bind to and disrupt the anionic target membranes and mediate bacterial killing similar to β-defensins. However, alternate mechanisms of antimicrobial action such as inhibition of macromolecular synthesis \[[@B10]-[@B12]\] and interaction with specific targets inside the bacterial cells \[[@B13],[@B14]\] are proposed. HE2 peptides at 10 and 25 μg/ml concentrations inhibited DNA, RNA and protein synthesis suggesting that their antimicrobial action may include interference with metabolic functions of *E. coli*. Inhibition of macromolecular synthesis was reported for bactenectins \[[@B39]\], human neutrophil peptide-1 \[[@B40]\], pleurocidin derived peptides \[[@B41]\] and the epididymal defensin DEFB118 \[[@B30]\]. In this study, it appears that HE2 peptides were more effective in inhibiting the incorporation of \[methyl-^3^H\]thymidine than \[5-^3^H\]uridine and L-\[4,5-^3^H(N)\]leucine, suggesting DNA synthesis is more sensitive to their antimicrobial action. It is possible that in bacteria that are extensively damaged by HE2 peptides, inhibition of macromolecular synthesis may result simply from the total breakdown of the cells. However the electron micrographs show that only some bacteria appear to be exuding cell contents after the 30 minute treatment. Thus during the first 10--20 minutes exposure to HE2 peptides, some peptides may be entering through pores too small for major cytoplasmic release. The early inhibition of DNA and RNA synthesis in bacteria where little loss of cell contents has occurred, may result from specific interaction of the synthetic machinery with HE2 peptides. Further studies are required to identify specific molecular targets within the bacteria and to establish whether HE2 interactions with these targets can be beneficial to the host by slowing bacterial proliferation. Increasing recognition of the ability of a number of proteins on the sperm surface to kill bacteria has led to the proposal that they may defend against microbial attack in both the male and female reproductive tracts. The cathelicidin hCAP18 on sperm is processed by the prostate-derived protease, gastricsin to release the active peptide ALL-38 and is found in the female reproductive tract after intercourse \[[@B42]\]. A member of the β-defensin family, DEFB126 also appears to have a role in fertility as a capacitation factor on sperm \[[@B43]\]. Similarly, the rat epididymis specific β-defensin Bin1b, appears to play an important role in sperm maturation \[[@B44]\]. Thus, these defense proteins may enhance the probability of successful fertilization in addition to helping prevent the spread of sexually transmitted diseases. Conclusions =========== In conclusion, we report that the epididymal antimicrobial peptides HE2α, HE2β1 and HE2β2 induce striking morphological changes in *E. coli*consistent with their membrane dependent mechanism of action \[[@B29]\]. In addition to membrane permeabilization, their antimicrobial mechanism involves inhibition of *E. coli*DNA, RNA and protein synthesis. Author\'s contributions ======================= SY performed the electron microscopy studies, radioactive incorporation assays and wrote majority of the manuscript. KGH prepared the recombinant peptides. SHH and FSF supervised and coordinated the work and the preparation of the manuscript. All authors read, commented upon and approved the final manuscript. Acknowledgements ================ We thank Victoria Madden, Microscopy Services Laboratory (MSL), University of North Carolina for her assistance with the electron microscopy. This work was supported by the Consortium for Industrial Collaboration in Contraceptive Research Program of the Contraceptive Research and Development Program, Eastern Virginia Medical School. The views expressed by the authors do not necessarily reflect the views of Contraceptive Research and Development or Consortium for Industrial Collaboration in Contraceptive Research. This work is also supported by NIH Grants R37-HD04466, by National Institute of Child Health and Human development/NIH through cooperative agreement U54-HD35041 as part of the Specialized Cooperative Centers Program in Reproduction Research, and by the Fogarty International Center Training and Research in Population and Health Grant D43TW / HD00627.

PubMed Central

2024-06-05T03:55:47.612750

2004-8-24

PMC516790

Introduction ============ Ovulation, the release of mature oocytes from the ovary, requires proteolytic degradation of the follicle wall, as well as the overlying ovarian tissues. This happens through the expression of a series of critical genes, triggered in a precise temporal and spatial pattern by the preovulatory LH surge \[[@B1],[@B2]\]. It is worthy to note that, for successful ovulation, follicle rupture has to occur just at the site of the follicle wall facing the ovarian surface, thus allowing release of the cumulus-oocyte complex (COC) to the periovarian space, while preventing proteolytic damage of the perifollicular tissues at the basolateral follicle sides. A large amount of information on the ovulatory process was accumulated during the last century (reviewed in \[[@B1]-[@B5]\]), and the involvement of crucial genes such as those encoding cyclooxygenase-2 (COX-2), and progesterone receptor (PR) has been clearly established. However, the mechanisms underlying the spatial targeting of the follicle rupture remain poorly understood. Although mechanical factors are likely involved in stigma formation and rupture \[[@B6]\], the mechanisms responsible for the specific location of proteolytic breakdown of the theca layers and perifollicular connective tissue at the apex of the follicle are not known. In recent studies \[[@B7]-[@B9]\] we have proposed that both prostaglandins (PG) and progesterone (P), classically recognized as essential ovulatory factors \[[@B1],[@B2]\], play complementary roles in the spatial targeting of follicle rupture. This was supported by detailed morphological studies in cycling rats treated with indomethacin (INDO), a strong inhibitor of PG synthesis, and RU486 (a PR antagonist), showing antiovulatory effects \[[@B1],[@B2],[@B10]-[@B12]\]. Gonadotropin-primed immature rats (GPIR) constitute a useful model for the study of ovulation. The administration of a single dose of equine chorionic gonadotropin (eCG) to immature animals induces the growth of abundant follicles, that reach preovulatory size in two days. Ovulation is then triggered by a single dose of human chorionic gonadotropin (hCG), thus providing a large number of synchronized ovulatory follicles \[[@B13]-[@B25]\]. An additional advantage of this model is the absence of regressing corpora lutea of previous cycles. This is relevant because structural luteolysis, that is temporally coincident with ovulation in cycling rats, also involves tissue remodeling and proteolytic degradation of the extracellular matrix \[[@B5]\]. For these reasons, GPIR (ranging from 21 to 28 days of age, at the time of eCG treatment \[[@B13]-[@B25]\]), have been widely used in studies focused on the ovulatory process, and a large amount of the information in this topic is derived from studies in immature rats. However, it should be kept in mind that GPIR constitute a non-physiological model and the possible immaturity of the pathways leading to ovulation cannot be ruled out. In order to examine further the role of PG and P in follicle rupture and ovulation, we performed detailed morphological analysis of the ovulatory process in inmature rats primed with gonadotropins at different ages. Surprisingly, these animals showed age-related alterations of the follicle rupture similar to those of adult rats lacking PG and P actions. We report herein the morphological alterations of follicle rupture and ovulation in GPIR and the effects of treatment with P and/or PG of the E series, as well as the response of GPIR to PG synthesis inhibition with INDO and to a PR antagonist (RU486), whose inhibitory effects in ovulation are clearly established \[[@B1],[@B2],[@B8],[@B13],[@B14]\]. Materials and methods ===================== Animals and drugs ----------------- Wistar female rats bred in the vivarium of the University of Cordoba were used. The day the litters were born was considered as day 0. Litter size was adjusted to 8 pups. The animals were maintained under controlled conditions of light (14L:10D; lights on 0500-1900) and temperature (22°C). The animals had free access to pelleted food and tap water. Experimental procedures were approved by the Cordoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union guidelines for care and use of experimental animals. Indomethacin (INDO), Progesterone (P), equine chorionic gonadotropin (eCG), human chorionic gonadotropin (hCG) and prostaglandins were purchased from Sigma.(St Louis, MO). The progesterone antagonist RU486 was obtained from Exlegin (Paris, France). Gonadotropin-priming at different ages -------------------------------------- Wistar immature rats were injected sc with 10 IU of eCG at 1700 h at 21, 23 or 25 days of age, and 48 h later were injected sc with 10 IU of hCG, a frequently used schedule \[[@B23],[@B26],[@B27]\]. Body weights were in the recommended range \[[@B23],[@B26]\] (46.0 ± 0.34, 57.0 ± 1.30 and 61.8 ± 1.4 g, mean ± SEM for n = 5). The animals (5 per group) were sacrificed at 0900 h on the day following hCG injection (i.e. at 24, 26 and 28 days of age). The ovaries, including the ovarian bursa, oviducts and periovarian fat pad, were fixed in Bouin-Hollande\'s fluid for 24 h and processed for paraffin embedding. Gonadotropin-primed rats treated with prostaglandins and/or progesterone ------------------------------------------------------------------------ Immature rats were primed with eCG at 21 days of age and with hCG 48 h later as described above. In addition, these animals were injected with 100 μg of PGE1 or PGE2, 1 mg of P or 1 mg of P plus 100 μg of PG E1 or PGE2 or vehicles (70% ethanol in saline) at 1730 h (30 min after hCG treatment). These dosages were effective, in the same body weight basis, in previous studies in adult rats \[[@B9]\]. The animals (5 per group) were killed at 0900 h on the day after hCG treatment (i.e. at 24 days of age) and the ovaries were processed as described above. Gonadotropin.primed rats treated with indomethacin or RU486 ----------------------------------------------------------- Immature rats were primed with eCG at 23 days of age and with hCG 48 h later as described in previous experiments. Indomethacin-treated rats received a sc injection of 0.5 mg of INDO or vehicle (olive oil) at 1200 h at 25 days of age. RU486-treated rats were injected sc with 0.5 mg of RU486 or vehicle (olive oil) at 0900 h at 24 and 25 days of age.The animals (5 per group) were killed at 0900 h on the day following hCG injection (i.e. at 26 days of age) and the ovaries were processed as described above. Histological analysis of follicle rupture and ovulation ------------------------------------------------------- The right ovaries were serially sectioned (6 μm) and stained with hematoxylin and eosin. All sections were examined under the microscope. The total number of cumulus-oocyte complexes (COCs) per ovary were counted. The number of COCs trapped inside the corpus luteum, released to the ovarian interstitium, retained in the bursal cavity or found in the oviducts, was recorded. COCs released to the ovarian interstitium were clearly recognizable by the presence of the oocyte in metaphase II and the first polar body, dispersed cumulus and follicular fluid. The total number of COCs per ovary matches the total number of corpora lutea (unruptured or not). In addition to absolute values, the number of COCs in each location was expressed as the percentage with respect to the total number of COCs (or corpora lutea) per ovary, to assess ovulatory efficiency, avoiding variability in the total numbers of corpora lutea among the different groups. Unruptured follicles measuring less than 575 μm in diameter, showing signs of atresia, such as irregularities of the granulosa cell layer, presence of apoptotic granulosa cells, as well as lack of dispersion of the cumulus or resumption of meiosis, were not considered. Statistical analysis was performed by ANOVA followed by the Student-Newman-Keuls method for multiple comparison among means. Significance was considered at the 0.05 level. Results ======= Ovulatory process in immature rats primed at different ages ----------------------------------------------------------- Absolute numbers of COCs, that match the numbers of corpora lutea per ovary, are presented in Table [1](#T1){ref-type="table"}, whereas relative data (the proportion of COCs found in each specific location) are shown in Fig. [6](#F6){ref-type="fig"}. On the day following hCG treatment, the total number of COCs per ovary was equivalent for the different age-groups (Table [1](#T1){ref-type="table"}). Immature rats primed with eCG at 21 or 23 days of age showed frequent ovulatory defects (see Figs [1](#F1){ref-type="fig"},[2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"},[4](#F4){ref-type="fig"},[5](#F5){ref-type="fig"} for representative micrographs and Fig. [6](#F6){ref-type="fig"}. for quantitative data). The COC was trapped in about 60% and 40% of the luteinized follicles in rats primed with eCG at 21 and 23 days of age respectively (Figs. [1A](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}, [6](#F6){ref-type="fig"}). However, many luteinized follicles containing the COC showed rupture of the theca layers and release of follicular fluid to the ovarian interstitium (Fig. [1A](#F1){ref-type="fig"}). Furthermore, 6--10% of the COCs were released to the ovarian interstitum (Figs. [1B](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [6](#F6){ref-type="fig"}). In these cases, degradation of the ovarian stroma with invasion of the blood and lymphatic vessels and formation of emboli containing the COC and follicular fluid (Figs [1C,1D,1E,1F](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}) were observed. In some animals, massive embolism of the ovarian vein at the ovarian hilus with follicular fluid and COCs (Fig. [3](#F3){ref-type="fig"}) caused ovarian hyperemia and vascular congestion. In addition, proteolytic degradation of the ovarian bursa by follicular fluid and granulosa cells, with invasion of the periovarian fat pad (Fig. [1G,1H](#F1){ref-type="fig"}) and even release of the COC to the peritoneal cavity (Fig. [4](#F4){ref-type="fig"}), was also observed. A characteristic feature of some corpora lutea ruptured at the apex, was the presence of an unruptured ovarian surface epithelium, and the COC was trapped in lacunae of follicular fluid and blood in the tunica albuginea (Fig. [5](#F5){ref-type="fig"}). Variable numbers of COCs (from 0 to 25%) were located in the bursal cavity (Fig. [6](#F6){ref-type="fig"}). From 33% to 60% of COCs were found in the oviducts, in rats primed with eCG at 21 and 23 days of age respectively. Otherwise, in immature rats primed with eCG at 25 days of age, the vast majority of COCs were found in the oviducts and abnormally ruptured corpora lutea (with trapped COC) were only occasionally found. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Number of COCs per ovary in control rats primed with eCG at different ages. ::: **Age (days) at eCG treatment** **Total COCs** **COCs located in** --------------------------------- ---------------- --------------------- ------------- ------------- ------------- 21 42.5 ± 8.72 22.5 ± 4.32 3.17 ± 1.47 0.67 ± 0.42 16.2 ± 6.93 23 58.4 ± 4.74 18.8 ± 0.70 3.20 ± 1.74 2.40 ± 0.51 34.0 ± 6.54 25 38.6 ± 3.76 0.6 ± 0.60^a,b^ \-- \-- 37.4 ± 3.20 Values are the mean ± SEM for n = 5. ANOVA and Student-Newman-Keuls multiple range test. ^a^p \< 0.05 *vs*21 days. ^b^p \< 0.05 *vs*23 days ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Representative micrographs from the ovary of immature rats primed with eCG at 21 (*A-D*, *G, H*) or 23 (*E, F*) days of age, stained with hematoxylin and eosin. **A**, luteinized follicle showing trapped COC and release of follicular fluid (*FF*) to the ovarian interstitium. The rupture of the theca layers are indicated by *arrows*. **B**, COC released to the ovarian interstitium, in a lacunae of follicular fluid (*FF*). Clusters of granulosa cells are indicated by *open arrows*. **C, D**, COCs in the lymphatic (*A*) or blood (*D*) vessels at the ovarian hilus (*OH*). **E, F**, COC inside a blood vessel located in the periovarian fat pad (*PFP*) near the ovarian hilus (*OH*). The framed area is shown at higher magnification in *F*showing rupture of the blood vessel and escape of red blood cells (*arrows*). **G, H**, Non-consecutive serial sections showing a COC released to the bursal cavity (*BC*), adhered to the ovarian bursa. Degradation of the ovarian bursa by follicular fluid and granulosa cells (*open arrows*) and invasion (*arrows*) of the periovarian fat pad (*PFP*) can be observed. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Micrograph of the ovary of a rat primed with eCG at 21 days of age, stained with hematoxylin and eosin. Several COCs trapped inside corpora lutea, showing dispersion of the cumulus, and interstitial COCs inside a blood vessel. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Non-consecutive serial sections of the ovary of a rat primed with eCG at 21 days of age and stained with hematoxylin and eosin. The ovarian vein is embolized with folicular fluid and two COCs (in **A**and **B**). A COC released to the ovarian hilus can be also observed in **A**. The ovarian surface is indicated by *empty arrows*in the *inset*. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Micrographs from non-consecutive serial sections from the ovary of an immature rat primed with eCG at 21 days of age. The ovarian bursa (*OB*) has been degraded (**A**) allowing release of the COC to the peritoneal cavity (**B**) Clusters of granulosa cells (*arrows*) can be observed free or attached to the ovarian bursa. Hematoxylin and eosin. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Representative micrographs from the ovary of immature rats primed with eCG at 21 (*A*) or 23 (*B*) days of age, showing luteinized follicles ruptured at the apex. The ovarian surface epithelium (*OSE, arrows in A and B*), and its basement membrane (*arrowheads in B*) remain intact, whereas the underlying ovarian tissue has been degraded (*asterisk in A*). The COC is retained under the ovarian surface. *OB*, ovarian bursa. Hematoxylin and eosin. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Percentage of cumulus-oocyte complexes (COCs) trapped inside the corpus luteum, released to the interstitium, located in the bursal cavity, or in the oviducts, in immature rats primed with eCG at 21, 23 or 25 days of age and ovulating at 24, 26 or 28 days of age respectively. See Table 1 for absolute counts. Different superscripts mean significant (p \< 0.05) differences. :::  ::: Effects of P and/or PGE treatment --------------------------------- The total number of COCs per ovary was equivalent in rats primed with eCG at 21 days of age and treated with vehicles, P, PGE1, PGE2, P plus PGE1 or P plus PGE2 (Table [2](#T2){ref-type="table"}). Neither P, PG or combined P plus PG treatment, prevented ovulatory defects and these animals showed the same morphological alterations as gonadotropin and vehicle-treated rats. However, degradation of the interstitial tissue and embolism of blood vessels with follicular fluid and granulosa cells were very scarce in prostaglandin-treated animals. Although the number of effectively ovulated oocytes were apparently increased in prostaglandin-treated rats, differences were not large enough to be statistically significant (Fig. [7](#F7){ref-type="fig"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Number of COCs per ovary in rats primed with eCG at 21 days of ages. ::: **Treatment** **Total COCs** **COCs located in** --------------- ---------------- --------------------- ------------ ------------ ------------- Vehicles 35.4 ± 3.52 17.2 ± 1.47 2.6 ± 0.85 0.6 ± 0.24 15.0 ± 2.72 PGE~1~ 33.2 ± 4.20 11.0 ± 2.42 2.3 ± 1.02 1.2 ± 0.58 19.0 ± 3.40 PGE~2~ 33.2 ± 1.62 10.2 ± 2.80 0.6 ± 0.39 3.6 ± 2.59 18.8 ± 3.38 P~4~ 45.6 ± 7.77 17.2 ± 2.97 3.4 ± 1.06 5.8 ± 3.10 19.2 ± 5.45 P~4~+ PGE~1~ 34.6 ± 5.17 14.6 ± 3.37 0.6 ± 0.39 0.8 ± 0.37 18.6 ± 2.82 P~4~+ PGE~2~ 35.2 ± 6.85 15.6 ± 3.10 0.6 ± 0.39 5.7 ± 2.85 14.2 ± 1.63 Values are the mean ± SEM for n= 5. ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Percentage of cumulus-oocyte complexes (COCs) trapped inside the corpus luteum, released to the interstitium, located in the bursal cavity, or in the oviducts, in rats primed with e CG at 21 days of age and injected two days later with vehicles (Veh), prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), progesterone (P) or progesterone plus PGE1 or PGE2. See Table 2 for absolute counts. No significant differences were found. :::  ::: Effects of INDO or RU486 treatments ----------------------------------- The total number of COCs per ovary was equivalent in rats treated with vehicle, INDO or RU486 (Table [3](#T3){ref-type="table"}). Immature rats treated with INDO during the preovulatory period showed the same morphological alterations of the ovulatory process as gonadotropin and vehicle-treated rats, although a higher proportion of follicles were affected (Fig. [8](#F8){ref-type="fig"}). A significantly (p \< 0.05) higher number of COCs were released to the ovarian interstitium, whereas the number of COCs found in the oviducts was significantly (p \< 0.05) decreased. Rats treated with RU486 showed increased numbers of COCs retained inside the follicle (Fig. [8](#F8){ref-type="fig"}), whereas the number of COCs released to the interstitium or effectively ovulated were significantly decreased. Most of the luteinized follicles in which the COC was trapped, did not show evident rupture of the theca layers. The numbers of COC trapped in the bursal cavity was significantly increased (Table [3](#T3){ref-type="table"} and Fig. [8](#F8){ref-type="fig"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Number of COCs per ovary in rats primed with eCG at 23 days of ages. ::: **Treatment** **Total COCs** **COCs located in** --------------- ---------------- --------------------- ---------------- ----------------- ------------------ Vehicle 39.6 ± 1.60 12.8 ± 1.0 2.6 ± 1.73 1.0 ± 0.62 23.2 ± 1.53 INDO 34.4 ± 3.76 19.2 ± 2.55 10.0 ± 1.35^a^ 0.4 ± 0.39 5.0 ± 0.05^a^ RU486 41.8 ± 3.86 22.8 ± 4.80 0.6 ± 0.39^b^ 3.2 ± 0.57^a,b^ 15.2 ± 3.10^a,b^ Values are the mean ± SEM for n = 5. ANOVA and Student-Newman-Keuls multiple range test. ^a^p \< 0.05 *vs*vehicle, ^b^p \< 0.05 *vs*INDO. ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Percentage of cumulus-oocyte complexes (COCs) trapped inside the corpus luteum, released to the interstitium, located in the bursal cavity, or in the oviducts, in rats primed with eCG at 23 days of age and treated with indomethacin (INDO) or RU486. see Table 3 for absolute counts. Different superscripts mean significant (p \< 0.05) differences. :::  ::: Discussion ========== Gonadotropin-primed immature rats (GPIR) is a popular model for the study of ovulation. In this model, eCG stimulates the growth of a large cohort of follicles, that reach preovulatory size in about 48 h and are then induced to ovulate by hCG \[[@B1],[@B2],[@B28]\]. Early data indicate that the ovulation rate in immature rats was age-dependent, and that the number of ova found in the oviducts increases, although not linearly, with age \[[@B29],[@B30]\]. However, these early studies do not provide information on the number of ova that were not effectively ovulated, and the marked variability in the rate of ovulation at different ages could be due to differences in the number of recruited growing follicles, in the responsiveness of preovulatory follicles to the ovulatory stimulus or both. In the present study, we evaluated the ovulatory process in immature rats primed with gonadotropins during the 21--25 day-old period, commonly used in studies focused on ovulation \[[@B13],[@B17],[@B18],[@B20]-[@B23],[@B25],[@B26]\]. In agreement with previous data \[[@B29],[@B30]\], the proportion of COCs found in the oviducts (i.e. effectively ovulated) increases in parallel to age. Detailed morphological evaluation indicated that ovulation was unsuccessful in 30--60% of preovulatory follicles in rats primed with eCG before 25 days of age. The nearly normal ovulatory process in immature rats primed with eCG at 25 days of age indicates that ovulatory defects were not due to gonadotropin treatment itself, but to ovarian immaturity at earlier ages. The ovulation failure described herein (in rats primed with eCG at 21 or 23 days of age) was not due to decreased recruitment of growing follicles, as indicated by the equivalent numbers of follicles that reach preovulatory size at all ages tested. The data of this study strongly suggest that the relative ovulatory incompetence of young immature rats was due to a defective capacity of preovulatory follicles to undergo the coordinate network of interactions that leads, in reponse to hCG, to COC release to the periovarian space. Defective ovulation in GPIR was related to abnormal follicle rupture. Whereas some LH-driven morphological changes in preovulatory follicles, such as cumulus expansion, resumption of the meiotic process and initial luteinization were present and showed normal features in GPIR, aberrant follicle ruptures were frequently observed. This was indicated by the presence of COCs released to the ovarian interstitium, and of corpora lutea showing rupture of the theca layers at any site of the follicle wall with release of follicular fluid to the ovarian stroma. The presence of follicle ruptures at the basolateral follicle sides suggests that the mechanisms underlying the spatial targeting of follicle rupture at the apex are not fully established in immature rats primed with eCG before 25 days of age. Almost identical ovulatory defects have been previously reported in INDO-treated adult cycling rats that also show abnormally ruptured follicles and proteolytic degradation of perifollicular tissues \[[@B7]-[@B9]\]. Furthermore, INDO-treated GPIR in the present study showed similar morphological alterations of the ovulatory process as vehicle-treated GPIR, even though the drug increased the number of affected follicles. The similarity of the ovulatory alterations found in GPIR and INDO-treated adult rats raises the question of whether the COX-2-prostaglandin pathway is fully established or not in GPIR. In this context, treatment of GPIR with PG of the E series was carried out in order to analyse whether ovulatory defects in GPIR were due to defective PG synthesis. Treatment with prostaglandin E resulted in a decrease in the invasion of the ovarian stroma and blood vessels by follicular fluid and granulosa cells, but ovulation was not improved significantly. In contrast, supplementation with exogenous PG of the E series inhibits abnormal follilce rupture and restores ovulation in INDO-treated adult rats \[[@B2],[@B8],[@B31]\]. This suggests that the possible immaturity of the COX-2-prostaglandin pathway in GPIR would be beyond prostaglandin synthesis, in agreement with previous studies reporting normal PG generation in GPIR in response to hCG treatment \[[@B28]\]. Otherwise, recent studies in COX-2 defective mice \[[@B32]\] indicate that the COX-2-PG pathway is not fully established in immature rodents. It has been clearly established that activation of PR plays a crucial role in ovulation \[[@B10],[@B11],[@B33]-[@B37]\]. Accordingly, adult cycling rats treated with PR antagonists \[[@B11]\] or inhibitors of the P synthesis \[[@B34]\] during the preovulatory period showed almost complete inhibition of follicular rupture \[[@B9],[@B11],[@B34]\]. Noteworthy, the response of GPIR to PR blockage (showing incomplete inhibition of follicle rupture) was similar to that of adult rats lacking both P and PG actions. In this sense, combined treatment of adult rats with RU486 and INDO resulted in follicle rupture in about 25% of preovulatory follicles \[[@B9]\]. Furthermore, some morphological features of the ovulatory process in GPIR, such as the persistence of the ovarian surface epithelium in spite of the degradation of the underlying tissues, was also a characteristic feature of adult rats treated with both INDO and RU486 \[[@B9]\]. However, exogenous P supplementation did not restore ovulation in GPIR, that suggest a defective response of preovulatory follicles to the ovulatory LH-dependent P surge. RU486-treated GPIR showed a significant increase in the number of COCs retained into the bursal cavity. Alterations in the transport of COCs to the oviduct has been previously described in adult rats treated with a PR antagonist \[[@B11]\], although the existence of a role for P in the COC transport to the oviduct cannot be ascertained from the present data. The end-point of the complex interaction network leading to follicle rupture and COC release, is the expression/activation of proteolytic enzymes, reponsible for extracellular matrix breakdown \[[@B24],[@B38],[@B39]\]. Proteolytic activity has to be closely modulated by protease inhibitors to prevent damage to perifollicular tissues. However, the characterization and regulation of ovarian proteolytic inhibitors has not been completely elucidated. The presence in GPIR of degradation of the ovarian stroma, invasion of blood and lymphatic vessels in abnormally ruptured follicles, strongly suggest that defective ovulation in GPIR was not due to decreased proteolytic activity but due to untargeted proteolysis. This was particularly suggested by the degradation of the ovarian bursa and invasion of the periovarian fat pad by cumulus cells and follicular fluid released at the follicle apex (see Figs. [1G,1H](#F1){ref-type="fig"}, [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}), permitting, in some cases, escape of the COC to the peritoneal cavity. In this sense, the use of very young immature rats to characterize proteolytic activity regulation during ovulation could provide conflicting data. In this study, we used Wistar rats. The possible existence of slight differences in the age of ovarian maturation among different rat strains cannot be ruled out. In conclusion, the presence of aberrant ovulations in very young GPIR, closely resembling those in adult rats lacking P and PG actions, strongly suggests that the capacity of the immature ovary to undergo changes leading to effective ovulation is not fully established in Wistar rats primed with eCG before 25 days of age. This should be taken into account when using the GPIR model for studies focused on ovulation. Acknowledgements ================ This work has been subsidized by Grant BFI2002-00485 from the DGI, Spain. The authors are very grateful to J Molina, P Cano and E Tarradas for their technical assistance.

PubMed Central

2024-06-05T03:55:47.614731

2004-9-3

PMC516791

Background ========== Resident brain cells including oligodendrocytes \[[@B1],[@B2]\], astrocytes, astrocytomas, microglia, glioblastomas \[[@B3]-[@B14]\], neurons \[[@B15],[@B16]\], neuroblastomas \[[@B17],[@B18]\] and endothelial cells \[[@B19],[@B20]\] express mRNAs for complement proteins. Although the role of complement expression by these cells remains unclear, local complement activation in the central nervous system (CNS) might damage these cells and contribute to the pathology in several inflammatory and neurodegenerative diseases including multiple sclerosis, Alzheimer\'s disease and progressive supranuclear palsy. For self-protection, resident brain cells also express complement inhibitors, such as membrane cofactor protein (MCP), decay-accelerating factor (DAF), CD59, and C1-esterase inhibitor (C1-inh). The human HOG oligodendroglial cell line produces MCP, DAF, CD59, C1-inh and S-protein, but not complement receptor 1 (CR1) \[[@B1]\]. Human oligodendrocytes have been reported to express CD59 \[[@B21]\] and DAF, but not MCP, CR1, homologous restriction factor (HRF: C8 bp) or clusterin \[[@B22]\]. Astrocytes \[[@B23]\], neurons and Schwann cells have been reported to express CD59 \[[@B24]\] and neuroblastoma cell lines C1-inh \[[@B18]\]. Astrocytoma cell lines have been reported to express MCP, DAF, and CD59 \[[@B25],[@B26]\]. In this study, the expression level of mRNAs for various complement inhibitors by human oligodendrocytes, astrocytes and microglia were compared by semi-quantitative PCR. We show that oligodendrocytes express extremely low levels of mRNA for C1-inh and significantly lower levels of mRNA for MCP compared with astrocytes and microglia. The expression level of mRNAs for CD59 and DAF showed no significant differences between the three cell types. Methods ======= Cell culture: microglial- and astrocyte-enriched cultures --------------------------------------------------------- Human microglial and astrocytic cells were isolated from surgically resected temporal lobe tissues. We thank Dr. J. Maguire, Department of Pathology and Laboratory Medicine, Vancouver General Hospital for providing the surgical specimens. Isolation protocols described by De Groot *et al*. \[[@B27],[@B28]\] were used with minor modifications. Tissues were placed in a sterile Petri dish, rinsed with Hank\'s balanced salt solution, and visible blood vessels were removed. After washing tissues two more times with Hank\'s balanced salt solution, tissues were chopped into small (\<2 mm^3^) pieces with a sterile scalpel. The fragments were transferred into a 50 ml centrifuge tube containing 10 ml of 0.25% trypsin solution (Gibco-BRL, Life Technologies, Burlington, ON, Canada), and incubated at 37°C for 20 min. Subsequently DNase I (from bovine pancreas, Pharmacia Biotech, Baie d\'Urfé, PQ, Canada) was added to reach a final concentration of 50 μg/ml. Tissues were incubated for an additional 10 min at 37°C. The cell suspension was diluted with 10 ml of Dulbecco\'s modified Eagle\'s medium (DMEM) and nutrient mixture F12 ham (DMEM-F12; Sigma-Aldrich, Oakville, ON, Canada) with 10% fetal bovine serum (FBS; Gibco-BRL, Life Technologies), and gently triturated by using a 10 ml pipette with a wide mouth. After centrifugation at 275 × g for 10 min, the cell pellet was resuspended in serum containing medium, triturated several times, and passed through a 100 μm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). The cell suspension was then centrifuged once more (275 × g for 10 min), resuspended into 10 ml of DMEM-F12 with 10% FBS containing gentamicin (50 μg/ml, from Sigma), and plated onto uncoated 10 cm tissue culture plates (Becton Dickinson). Plates were placed in a humidified 5% CO~2~, 95% air atmosphere at 37°C for 2 hr in order to achieve adherence of microglial cells. Non-adherent cells with myelin debris were removed from these microglia-enriched cultures and transferred into poly-L-lysine coated 10 cm tissue culture plates in order to achieve adherence of astrocytes. Plates were incubated for 48 hr, after which the culture medium containing myelin debris and non-adherent cells was removed and used to prepare oligodendroglial cell cultures as described below. Both microglial- and astrocyte-enriched cultures were grown for 6 to 7 days before their mRNAs were extracted. Immunostaining with antibodies against CD68 (Dako, Mississauga, ON, Canada) which stains microglia as well as macrophages, and glial fibrillary acidic protein (GFAP, Dako), which is a marker of astrocytes, showed that the microglia-enriched cultures contained 93.5 ± 3.6 % (N = 4) microglial cells, while astrocyte-enriched cultures contained 85.7 ± 3.4 % (N = 4) astrocytes. Cell culture: oligodendroglial cells ------------------------------------ These were prepared as described before \[[@B2]\]. Briefly, cell culture media containing myelin debris and non-adherent cells that were removed from astrocyte-enriched cultures were used to extract oligodendroglial cells. The non-adherent cells were collected by centrifugation at 275 × g for 10 min and replated onto uncoated 10 cm tissue culture plates for another 24 hr. Subsequently, the cell culture medium containing floating cells was transferred to 50 ml tubes and Lymphoprep solution (Axis-Shield, Oslo, Norway) used to reduce the amount of contaminating myelin debris. For this purpose, 10 ml of Lymphoprep solution was carefully placed under the oligodendrocyte cell suspension and the density gradient was centrifuged at 325 × g for 10 min. The interphase was collected and transferred to a 50 ml centrifuge tube. Fresh culture medium was added and the suspension was centrifuged at 275 × g for 7 min. The cell pellet was resuspended and the oligodendrocyte cultures seeded onto 60 mm plastic culture dishes. Immunostaining with anti-O4 antibody (Chemicon International, Temecula, CA), which is a marker of oligodendrocytes, showed that the oligodendrocytes-enriched cultures contained 95.3 ± 4.4 % (N = 4) oligodendrocytes. RNA isolation and cDNA synthesis by reverse transcription --------------------------------------------------------- Total RNA from oligodendroglial cells, microglia, and astrocytes were isolated by the acid guanidium thiocyanate-phenol-chloroform method. Two μg of the RNA was then used to prepare cDNA. RNA was treated with 10 U of DNase I (Gibco BRL, Life Technologies) for 60 min at 37°C in 25 μl of 1 × reverse transcriptase buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl~2~) containing 40 U of RNase inhibitor (Pharmacia Biotech) and 1 mM dithiothreitol (DTT), following by incubation at 85°C for 5 min to inactivate the enzyme. Reverse transcription was performed at 42°C for 90 min in 50 μl of the following mixture: 1 × reverse transcriptase buffer containing 2 μg of RNA, 5 mM DTT, 0.2 μg random hexamer primers (Pharmacia Biotech), 1 mM deoxynucleotides (Gibco BRL, Life Technologies), 40 units of RNase inhibitor, and 400 units of SuperScript II reverse transcriptase (Invitrogen Life Technologies, Burlington, ON, Canada). At the end of the incubation period, the enzyme was inactivated by heating at 65°C for 10 min \[[@B29]\]. Polymerase chain reaction ------------------------- PCR amplification was carried out in a 25 μl reaction mixture containing 1 × GeneAmp PCR buffer II (Perkin Elmer, Foster City, CA), 1.25 units AmpliTaq Gold DNA polymerase (Perkin Elmer), 2 mM MgCl~2~(Perkin Elmer), 200 μM dNTPs (Gibco BRL, Life Technologies) and 0.5 μM of each specific primer (Table [1](#T1){ref-type="table"}). The mixture was prepared before the addition of 1.25 μl of cDNA. PCR amplification was carried out using an MJResearch (Boston, MA) programmable thermal controller. The amplification program consisted of an initial denaturation step at 94°C, which was extended to 9 min in order to activate AmpliTaq Gold enzyme. The remaining cycles were 1 min at 94°C, 1 min at 55°C and 1 min at 72°C. The number of cycles performed was 27 for glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 30 for CD59, C1-inh and MCP, and 32 for DAF. After amplification, PCR products were separated on a 6% polyacrylamide gel and visualized by incubation for 10 min in a solution containing 10 ng/ml of ethidium bromide. Polaroid photographs of the gels were taken. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Oligonucleotide primers used for PCR, and the corresponding restriction endonucleases used for product confirmation. ::: Gene Sequence (5\' → 3\') Fragment size (introns) Genbank accession No Restriction enzymes used and the expected sizes of digestion products (bp) ---------- ----------------------------------- ------------------------- ---------------------- ---------------------------------------------------------------------------- C1 inh-F GTT GGG GGA TGC TTT GGT AGA TTT C 332 M13690 Sau 3AI (246, 86) C1 inh-R TTA GGA CTC TGG GGC TGC TGC TGT A (2 introns) CD59-F CTG CTG CTC GTC CTG GCT GTC TTC T 280 M34671 Pst I (233, 47) CD59-R TCC CAC CAT TTT CAA GCT GTT CGT T (2 introns) MCP-F CAA TTC AGT GTG GAG TCG TGC TGC 265 Y00651 Sau 3AI (193, 72) MCP-R TGA GGC ACT GGA CGC TGG AGA T (unknown) DAF-F GTA CTG TGA ATA ATG ATG AAG GAG 364 M30142 Hae III (330, 34) DAF-R TCT TAA CTC TTC TTT GGC TAA GTC (unknown) G3PDH-F CCA TGT TCG TCA TGG GTG TGA ACC A 251 X01677 Dde I (168, 83) G3PDH-R GCC AGT AGA GGC AGG GAT GAT GTT C (2 introns) ::: PCR primer design and restriction analyses ------------------------------------------ Primers were designed to span introns so that cDNA-derived PCR products would be of different sizes to those produced if genomic DNA was amplified (see Table [1](#T1){ref-type="table"}). DAF and MCP were exceptions, since only cDNA sequences were available. Primers were synthesized either by Sigma-Aldrich or ID Labs (London, ON, Canada). The primer sequences and predicted PCR fragment sizes are listed in Table [1](#T1){ref-type="table"}, along with the names of the enzymes used for restriction digest analysis of each PCR fragment. The restriction digestion reactions were carried out at 37°C for 2 hr in the presence of 1 × the appropriate buffer provided by the suppliers (Invitrogen, Life Technologies and New England Biolabs, Mississauga, ON, Canada). The digested PCR products were analyzed on a 6% polyacrylamide gel (data not shown). In all cases the restriction fragments observed were of the predicted size (see Table [1](#T1){ref-type="table"}). Statistical analysis -------------------- The data are presented as means ± s.e.m. The significance of difference between values was estimated by means of one-way analysis of variance (ANOVA) with Fisher\'s LSD post-hoc test. P \< 0.05 was considered to show statistically significant differences. Double fluorescence immunocytochemical analysis ----------------------------------------------- Oligodendrocytes, astrocytes, and microglia were harvested and air-dried on glass slides. Cells were then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 5 min. For inactivation of endogenous peroxidase, cells were incubated with 0.3% H~2~O~2~for 30 min. Blocking was performed for 1 hr at room temperature in 5% skim milk. For double fluorescence immunostaining, cells were incubated at room temperature overnight with a primary antibody in 1% normal serum. The primary antibody and the dilution used in the first cycle were as follows: O4 (Chemicon International, 1: 100) for oligodendrocytes, GFAP (Dako, 1: 10,000) for astrocytes, CD68 (DAKO, 1: 50) for microglia. Cells were then treated for 2 hr at room temperature with a biotin conjugated anti-mouse IgM (Vector Laboratories, Burlingame, CA, 1: 200) secondary antibody for O4, a biotin conjugated anti-rabbit IgG (Vector Laboratories, 1: 200) secondary antibody for GFAP and a biotin conjugated anti-mouse IgG (Vector Laboratories, 1: 200) secondary antibody for CD68. Then cells were incubated with Texas Red Avidin DCS (Vector Laboratories) for 1 hr. The primary antibody and the dilution used in the second cycle were as follows: for C1-inh, goat anti-C1-inhbitor (Quidel, San Diego, CA, 1: 50); for CD59, mouse anti-CD59 (Serotec Ltd, Oxford, UK, 1: 10) or rat anti-CD59 (Serotec, 1: 25). Cells were incubated at 4°C for 3 days with a primary antibody in 1% serum corresponding to the secondary antibody type. Cells were then treated for 2 hr at room temperature with FITC-conjugated anti-mouse IgG (Vector Laboratories, 1: 200), anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA, 1: 200), or anti-rat IgG (Cappel, Durham, NC, 1: 200). The glass slides were then rinsed with distilled water, and a drop of Vectashield mounting medium (Vector Laboratories) placed on the slide. Results ======= RT-PCR ------ RT-PCR was carried out using primers for C1-inh, CD59, DAF and MCP. The housekeeping gene G3PDH was amplified in parallel with each RT-PCR run as an internal standard. Figure [1](#F1){ref-type="fig"} illustrates the bands obtained for each of the RT-PCR products from oligodendrocytes (Fig. [1A](#F1){ref-type="fig"}), astrocytes (Fig. [1B](#F1){ref-type="fig"}) and microglia (Fig. [1C](#F1){ref-type="fig"}). Specificity of each of the products was established by endonuclease digestion (Table [1](#T1){ref-type="table"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Demonstration of RT-PCR products. Polaroid photographs of typical ethidium bromide-stained gels of RT-PCR products from oligodendrocytic (Fig. 1A), astrocytic (Fig. 1B) and microglial (Fig. 1C) RNA extracts. Lanes for individual mRNA products are indicated in the legend at the top. Size markers are in the right lanes. MCP, membrane cofactor protein (265 bp); DAF, decay-accelerating factor (364 bp); CD59 (280 bp); C1-inh, C1-esterase inhibitor (332 bp); G3PDH, glyceraldehyde-3-phosphate dehydrogenase (251 bp). :::  ::: Semi-quantitative RT-PCR analysis --------------------------------- To compare the ratio of each of the complement inhibitors to G3PDH, statistical analysis was performed by means of one-way ANOVA with Fisher\'s LSD post-hoc test (Fig. [2](#F2){ref-type="fig"}). The overall mean ± s.e.m. for C1-inh/G3PDH was 0.55 ± 0.12 (N = 5) in astrocytes, 0.58 ± 0.09 (N = 3) in microglia and 0.09 ± 0.06 (N = 12) in oligodendrocytes (Fig. [2A](#F2){ref-type="fig"}). Oligodendrocytes showed a highly significant difference from astrocytes and microglia (Fig. [2A](#F2){ref-type="fig"}; P \< 0.001 by one-way ANOVA with Fisher\'s LSD post-hoc test). For MCP/G3PDH, the ratios were 0.80 ± 0.22 (N = 5) in astrocytes, 0.93 ± 0.10 (N = 3) in microglia and 0.44 ± 0.19 (N = 12) in oligodendrocytes. Oligodendrocytes showed a significant difference from astrocytes and microglia (Fig. [2B](#F2){ref-type="fig"}; P = 0.002 vs. astrocytes and P = 0.001 vs. microglia by one-way ANOVA with Fisher\'s LSD post-hoc test). The corresponding means for CD59/G3PDH were 0.73 ± 0.10 (N = 5) in astrocytes, 0.83 ± 0.03 (N = 3) in microglia and 0.76 ± 0.09 (N = 14) in oligodendrocytes (Fig. [2C](#F2){ref-type="fig"}). The corresponding means for DAF/G3PDH were 0.67 ± 0.07 (N = 5) in astrocytes, 0.67 ± 0.07 (N = 3) in microglia and 0.66 ± 0.15 (N = 14) in oligodendrocytes (Fig. [2D](#F2){ref-type="fig"}). There were no significant differences between the three cell types for CD59 and DAF. Each N represents a different patient. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### A comparison of relative complement inhibitor expression level between oligodendrocytes, astrocytes and microglia. The data were estimated by one-way analysis of variance (ANOVA) with Fisher\'s LSD post-hoc test (A and B; P \< 0.05 was considered to show statistically significant differences). :::  ::: Double fluorescence immunohistochemistry ---------------------------------------- In order to establish identity between oligodendroglial cells, astrocytes or microglia and cells expressing the complement inhibitor proteins CD59 or C1-inh, double fluorescence immunostaining was carried out. Oligodendrocytes were detected by O4 staining with a Texas Red tagged secondary antibody (Fig. [3A](#F3){ref-type="fig"} and [3D](#F3){ref-type="fig"}) in the first cycle and CD59 (Fig [3B](#F3){ref-type="fig"}) or C1-inh staining (Fig. [3E](#F3){ref-type="fig"}) detected with a green FITC tagged antibody in the second cycle. Astrocytes were detected by GFAP staining with a Texas Red tagged secondary antibody (Fig. [3G](#F3){ref-type="fig"} and [3J](#F3){ref-type="fig"}) in the first cycle and CD59 staining (Fig [3H](#F3){ref-type="fig"}) or C1-inh staining (Fig. [3K](#F3){ref-type="fig"}) detected with a green FITC tagged antibody in the second cycle. Microglia were detected by CD68 staining with a Texas Red tagged secondary antibody (Fig. [3M](#F3){ref-type="fig"} and [3P](#F3){ref-type="fig"}) in the first cycle, and CD59 staining (Fig [3N](#F3){ref-type="fig"}) or C1-inh staining (Fig. [3Q](#F3){ref-type="fig"}) detected with a green FITC tagged antibody in the second cycle. With double fluorescent excitation, all cells fluoresced yellow (Fig. [3C,3F,3I,3L,3O,3R](#F3){ref-type="fig"}), indicating colocalization of O4 with CD59 or C1-inh, GFAP with CD59 or C1-inh, and CD68 with CD59 or C1-inh. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Double fluorescence immunohistochemistry of oligodendrocytes, astrocytes and microglia. Double fluorescence immunostaining for O4 and CD59 or C1-inh is demonstrated in A-F. In A and D, cells of typical oligodendroglial morphology were stained in the initial cycle for the specific oligodendroglial marker O4. Detection is by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (B) and C1-inh (E) are shown. The detections are by an FITC-linked green fluorescent secondary antibody. In C and F, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of O4 with CD59 or C1-inh. Double fluorescence immunostaining of astrocytes for GFAP and CD59 or C1-inh is demonstrated in G-L. In G and J, cells of typical astrocytic morphology are stained in the initial cycle for the specific astroglial marker GFAP. Detection is by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (H) and C1-inh (K) is shown with an FITC-linked green fluorescent secondary antibody. In I and L, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of GFAP with CD59 or C1-inh. Double fluorescence immunostaining for microglia using the specific marker CD68 and CD59 or C1-inh is demonstrated in M-R. In M and P, cells of typical microglial morphology are stained by CD68 with detection by a Texas Red-conjugated secondary antibody. Second cycle staining for CD59 (N) and C1-inh (Q) are shown. The detections are by an FITC-linked green fluorescent secondary antibody. In O and R, double immunofluorescences are shown in which the cells appear yellow, demonstrating colocalization of CD68 with CD59 or C1-inh. (Magnification: × 200) :::  ::: Discussion ========== This report shows that human oligodendrocytes express a much lower level of mRNA for C1-inh than astrocytes and microglia, and a significantly lower level of mRNA for MCP. The mRNA levels of CD59 and DAF were comparable in all the three cell types. Overall our data suggest that oligodendroglial cells, in common with other cell types, can produce complement inhibitors, but at a significantly lower level for C1-inh and MCP. It has already been reported that human neurons and Schwann cells \[[@B24]\], neuroblastoma cell lines \[[@B18]\], astrocytes \[[@B23]\], astrocytoma cell lines \[[@B25],[@B26]\], the HOG human oligodendroglial cell line \[[@B1]\] and oligodendrocytes \[[@B21],[@B22]\] produce some or all of the complement inhibitor proteins and their mRNAs. Activation of the complement cascade and deposition of activated complement fragments occur in non-infectious diseases such as multiple sclerosis, Pick\'s disease, Alzheimer\'s disease and other neurodegenerative conditions \[[@B15],[@B16],[@B30]-[@B34]\]. Complement inhibitors may play an important role in preventing such pathology. Full activation of the complement cascade requires overcoming a series of endogenous inhibitory factors. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis \[[@B35]-[@B37]\] and this vulnerability may be related to a deficiency of their ability to express complement regulatory proteins \[[@B22]\], particularly C1-inh. Sporadic complement attack, in the form of complement activated oligodendroglia (CAO) is also observed in a number of neurodegenerative conditions \[[@B38],[@B39]\], including Alzheimer\'s, Pick\'s, Huntington\'s and Parkinson\'s diseases, amyotrophic lateral sclerosis, progressive supranuclear palsy, Shy-Drager syndrome, argyrophilic grain dementia and pallido-nigral luysial atrophy \[[@B38],[@B39]\]. The source of the complement proteins that become activated is unknown, but the data presented here suggest that oligodendrocytes are vulnerable to complement attack because of a low expression of C1-inh and MCP. Conclusions =========== These results suggest that the lower expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system, particularly multiple sclerosis. List of abbreviations ===================== analysis of variance (ANOVA) central nervous system (CNS) complement activated oligodendroglia (CAO) complement receptor 1 (CR1) decay-accelerating factor (DAF) dithiothreitol (DTT) fluorescein isothiocyanate isomer (FITC) glyceraldehyde-3-phosphate dehydrogenase (G3PDH) glial fibrillary acidic protein (GFAP) homologous restriction factor (HRF) membrane cofactor protein (MCP) phosphate-buffered saline (PBS) Competing interests =================== None declared. Authors\' contributions ======================= MH was responsible for the majority of the experimental studies, and for writing the manuscript. AK contributed to the cell culture and the editing of the manuscript. PLM contributed to the conception, interpretation of results and the writing and editing of the manuscript. Acknowledgements ================ This work was supported by a grant from the Jack Brown and Family Alzheimer\'s Disease Research Fund, and the Pacific Parkinson\'s Research Institute.

PubMed Central

2024-06-05T03:55:47.617386

2004-8-24

PMC516792

Qadaffi\'s lesson ================= Thirty-five years ago, on September 1^st^, 1969, I was an eleven year old boy living with my family in Benghazi, Libya. That morning, I awoke to an eerie silence. The normally bustling traffic outside of our apartment in a busy suburb of Benghazi was not to be heard. In fact, there was to be little or no traffic for the next several days. History will recount that day as the first day of Mohmmar Qadaffi\'s bloodless coup d\'etat, seizing power in Libya. What I learned that September week in 1969 was that access to and distribution of information tipped the fate of a state. On that September day, there were in fact two groups of army officers vying to overthrow the then enfeebled Libyan King Idris. Qadaffi, at the time only twenty-seven years old, headed a small group of very junior officers. He, however, had a bold and swift strategy. His first targets were not the dams, the power stations, the police stations\...; but the radio stations (in 1969 television had a very minor penetrance in Libya) and newspapers. By dint of controlling the airwaves and printing presses and by freely distributing [his]{.underline} information during time of chaos, Qadaffi held sway over the minds and the sentiments of the populace. He was thus able to pre-empt the efforts of a larger competing group of more senior army officers. What does Qadaffi\'s story have to do with Open Access publishing? Imagine how the outcome might have been different in 1969 if broadcast radio was not freely and openly accessible by the masses but was an expensive subscription service limited to a few. In that setting, and perhaps in all settings, information that reaches a little audience at a steep cost is information with little effect. Retrovirology, freely and openly accessible by all ================================================== Curiously, the force that drives a coup d\'etat is the same that motivates scientists to publish (although I am not suggesting that scientists are like Qadaffi). As scientists, we want everyone who is interested to read what we have done; and ideally we want that information to get to the widest audience in the fastest, most informative, and least expensive manner. Until now, ideal and reality were two entirely different matters. Traditional publishing in print journals reaches a limited audience, is slow, bulky, and expensive. By its very nature (i.e. pages of paper are heavy to mail and expensive to print), there is a real impetus on the part of print publishers to restrict the space available to authors. We are all too familiar with the results: abbreviated presentation of methods and text, the necessity to \"inadvertently\" not cite many colleagues\' publications, and the ubiquitous use of microscopic figure panels that provoke blurred vision and headaches. I daresay that many after having read a short \"letter\" or a \"brief definitive\" report have been left scratching heads and wondering how the experiments were done, where did the vectors come from, and why many controls were definitely missing. For the retrovirology community, *Retrovirology*aims to change such \"reality\". *Retrovirology*is published by BioMed Central, an independent publisher committed to ensuring immediate free access to peer-reviewed biomedical research. *Retrovirology*is Open Access which means that it is universally and freely available online to everyone. You as the author retain copyright, and your paper is guaranteed to be archived in at least one internationally recognised repository \[[@B1]\]. More importantly, publishing in *Retrovirology*is rapid. For example, a full research article was reviewed and published in less than 20 days \[[@B2]\]; and a review article was similarly processed in 8 days \[[@B3]\]. *Retrovirology*will also give you all the space and all the colored prints (at no extra charge) to present your findings informatively and attractively. If we don\'t publish your paper, we will tell you honestly what is scientifically deficient about your work. I promise not to obfuscate myself using annoyingly belittling mantra that my editorial decision was guided by \"intense competition for space\". Judge a book by its cover? ========================== This is my second editorial for *Retrovirology*readers \[[@B4]\]. *Retrovirology*has been publishing on-line now for 6 months. While a first editorial can speak of dreams and aspirations, the burden of a second editorial is to demonstrate results and progress. Aided by an international cohort of extremely capable editors (Monsef Benkirane, Ben Berkhout, Masahiro Fujii, Michael Lairmore, Andrew Lever, and Mark Wainberg) and sixty editorial board members \[[@B5]\], I am indeed encouraged and gratified by *Retrovirology\'s*initial achievements. As of this writing, we have published 22 papers. These are good papers; and importantly, these papers are being widely-read. I can say with some pride that half (11 out of 22; see Table [1](#T1){ref-type="table"}) of our published papers have been read more than 1,000 times each. The remaining papers are not far behind; our most recently published paper, barely 1 week on-line, has already had more than 200 readers. Given that retrovirology is a relatively small community of researchers, my personal experience of twenty years in this field tells me that these are very respectable numbers. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Access statistics for the top 11 *Retrovirology*articles. ::: **Article** **Access Count** ---- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------ 1 **[Early steps of retrovirus replicative cycle]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-9.pdf> Sébastien Nisole, Ali Saïb *Retrovirology*2004, **1**: 9 1890 2 **[Establishment of a novel CCR5 and CXCR4 expressing CD4+ cell line which is highly sensitive to HIV and suitable for high-throughput evaluation of CCR5 and CXCR4 antagonists]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-2.pdf> Katrien Princen, Sigrid Hatse, Kurt Vermeire, Erik De Clercq, Dominique Schols *Retrovirology*2004, **1**: 2 1602 3 **[Evolution of the HIV-1 envelope glycoproteins with a disulfide bond between gp120 and gp41]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-3.pdf> Rogier W Sanders, Martijn M Dankers, Els Busser, Michael Caffrey, John P Moore, Ben Berkhout *Retrovirology*2004, **1**: 3 1332 4 **[Use of a multi-virus array for the study of human viral and retroviral pathogens: gene expression studies and ChIP-chip analysis]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-10.pdf> Elodie Ghedin, Anne Pumfery, Cynthia de la Fuente, Karen Yao, Naomi Miller, Vincent Lacoste, John Quackenbush, Steven Jacobson, Fatah Kashanchi *Retrovirology*2004, **1**: 10 1313 5 **[Increased mortality associated with HTLV-II infection in blood donors: a prospective cohort study]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-4.pdf> Jennie R Orland, Baoguang Wang, David J Wright, Catharie C Nass, George Garratty, James W Smith, Bruce Newman, Donna M Smith, Edward L Murphy, *Retrovirology*2004, **1**: 4 1209 6 ***Retrovirology*and young Turks\...**<http://www.retrovirology.com/content/pdf/1742-4690-1-1.pdf> Kuan-Teh Jeang *Retrovirology*2004, **1**: 1 1140 7 **[HIV-1 gene expression: lessons from provirus and non-integrated DNA]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-13.pdf> Yuntao Wu *Retrovirology*2004, **1**: 13 1103 8 **[Multi-faceted, multi-versatile microarray: simultaneous detection of many viruses and their expression profiles]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-11.pdf> Biehuoy Shieh, Ching Li *Retrovirology*2004, **1**: 11 1097 9 **[Two discrete events, human T-cell leukemia virus type I Tax oncoprotein expression and a separate stress stimulus, are required for induction of apoptosis in T-cells]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-7.pdf> Takefumi Kasai, Kuan-Teh Jeang *Retrovirology*2004, **1**: 7 1088 10 **[Apoptosis of uninfected cells induced by HIV envelope glycoproteins]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-12.pdf> Barbara Ahr, Véronique Robert-Hebmann, Christian Devaux, Martine Biard-Piechaczyk *Retrovirology*2004, **1**: 12 1076 11 **[HIV CTL escape: at what cost?]{.underline}**<http://www.retrovirology.com/content/pdf/1742-4690-1-8.pdf> Stephen M Smith *Retrovirology*2004, **1**: 8 1022 ::: Despite that many of you would argue to me that you would rather publish in *Cell*, *Science*, and *Nature*rather than *Retrovirology*. I wouldn\'t dispute you on that point. On the other hand, I would ask you to pause and think about a refrain that I am sure all of you have taught your children, \"Don\'t judge a book by its cover!\" Please, don\'t sell your own work short; and please don\'t belittle the objectivity of your colleagues. If you have a wonderful piece of work, do you truly need the cover of *Cell*to establish that point? On the other hand, if you have a horrible study, do you think that hiding it behind the cover of *Nature*would fool many of your colleagues for long? There was a time in the print age when display space on reading room shelves was limited and perhaps justifiably dominated by \"the big three\". Today, *Retrovirology*will get your paper listed in PubMed within 48 hours of acceptance for publication. Anyone in this electronic era whose reading habits are not guided by PubMed in one way or another is probably someone who you may not care to read your study. In short, *Retrovirology*will promise your immediacy of publication and visibility for your work. Your papers will be well-read by your colleagues. Beyond that, I believe, and I think you do too, the impact of your work should rest on its merits and not on our or any other journal\'s cover. Open Access is not free lunch ============================= Besides teaching about book covers, I think the other concept that we teach our children is that \"There is no free lunch!\" At the end of the day, anything worth doing has a price tag. I, my colleague editors, and all the editorial board members at *Retrovirology*work for free for the journal. We can do this because we collect a salary elsewhere doing research. On the other hand, we do have a dedicated staff based at the publisher in London. No one would question that the salaries for these professionals as well as other publication costs must be borne. To fund these costs, from October 1, 2004, authors of articles accepted for publication will be asked to pay a single all-inclusive article-processing charge (APC) of US\$600; there are no additional charges beyond this sum. *Retrovirology*will not levy additional page or colour charges on top of this fee. You are free to publish any number of colour figures and photographs, at no extra cost. I support the need for APC, because without this *Retrovirology*cannot be published. I think most of you will agree based on your experience with other publishers that US\$600 is more than modest and reasonable. As a point of comparison, the Public Library of Science has set up two new Open Access journals, and has set their APCs at US\$1500, fully two and one-half times our charge, for each accepted article \[[@B6]\]. Nonetheless, I realize that any APC fee can present an unacceptable financial hardship for some authors. These authors should feel free to contact me in confidence, and I will consider fee-waiver requests on a case-by-case basis. In closing, let me thank those of you who have read, submitted and reviewed papers for *Retrovirology*. More than anything else, *Retrovirology*is not my journal as much as it is your journal. I welcome your advice, and I look forward to hearing from you. Abbreviations ============= APC = article-processing charge.

PubMed Central

2024-06-05T03:55:47.619310

2004-9-3

PMC516793

[](#pbio-0020263-g001){ref-type="fig"} ::: {#pbio-0020263-g001 .fig}  ::: Can "race" be gleaned from our genes? Concern over the emerging trajectories of genetics research has led to ongoing debates about how to characterize and interpret genetic variation. Despite the mantra that humans share 99.9% of our genetic makeup, there is increasing interest in identifying the relatively small percentage difference that distinguishes individuals. Therein lies the paradox: if we are all the same, why do we continue to search for the ways in which we differ from one another? We can take an essential first step toward addressing this paradox by acknowledging the often conflicting stakes for individuals and groups in debates that center around genes, science, and race. Whose genes will be studied? For what purpose? And who has the authority to decide? These stakes are laid bare by Cassandra Medley in her play "Relativity," which ran at the Magic Theater in San Francisco this spring, in a production directed by Edris Cooper-Anifowoshe. The play focuses on the inner conflict of a young scientist, an African American woman who tries unsuccessfully to straddle the opposing world views of her profession and her family. In "Relativity," Kalima Davis is a postdoctoral fellow in a prestigious stem cell laboratory on the East Coast. Her impeccable academic pedigree distinguishes Kalima as a rising researcher, and she is one of the few women and African American scientists working in the field. Kalima is also the devoted daughter of the charismatic psychotherapist Claire Reid, who directs the "Leon Davis Foundation," which Kalima\'s late father dedicated to the belief that neuropeptide melanin, found in "people of color," enhances intelligence, athleticism, and emotional sensitivity. This theory also points to the lack of melanin among lighter skinned individuals as a cause of "white racism." Kalima, who has inherited co-directorship of her father\'s foundation, is asked by her mother to offer "scientific proof" of the melanin theories and to discount the assertion that all groups are genetically similar. At issue in "Relativity" is the struggle over what constitutes a valid belief. Reid suggests to Kalima that "science is not the sole province of what the 'West' defines it to be," and refers to Chinese acupuncture, Hindu Chakra, and tribal African shamanism as examples of legitimate "sciences." But the power of Western science to trump other interpretations of lived experience has become all too clear in the genomic era. Truth is excavated from the human body, where genes emerge as the iconographic oracles of our past, present, and future. As Kalima recounts, "We can\'t get around it. DNA is fact." Nonetheless, Kalima naively attempts to find a way to retain these opposing epistemologies. The futility of this dual position is made apparent by the arrival of Iris Preston, an African American senior scientist who has taken over as the new head of Kalima\'s lab. Preston, a highly vocal critic of Claire Reid and melanin theories, serves as a formidable apostle of the scientific method. Shortly after her arrival, Preston convenes members of her new lab to filter those seeking financial and other derivative rewards from the truly devoted, who are motivated solely by their "sense of wonder and amazement" and their desire to "cultivate what Einstein referred to as 'holy curiosity.'" She makes plain that science, like all ideologies, demands consummate faith and unwavering piety. Kalima\'s struggle to claim her "true lineage" is much more than a simple choice between her biological mother (Reid) and her intellectual mentor (Preston). Her plight forces her to explore the meaning of justice. The contrast between melanin theories and genomic research, initially stark, blurs as it becomes increasingly clear that both Reid and Preston seek to use their "science" to redress race-related disparities. Citing a history of racism, including the historically well-documented Tuskegee Syphilis Study, Reid asks rhetorically whether genetics research will result in a "genetically modified" white upper class and a lower, dark-skinned "natural birth class"? At stake are issues of power and trust, and the question of whether new genetic technologies will close the gaps between groups or make them wider. Some postulate that the "new genetics" will render conventional notions of race obsolete. However, it is doubtful that such a color-blind utopia will be won through the sequencing of genes without a serious engagement with the differences that lie outside cell walls. A social infrastructure of inequality that mediates race through nutritional deficits, exposure to pollutants, and the use of the emergency room as the sole venue for healthcare will not be rehabilitated through gene therapy or pharmacogenomics. Preston seeks to use her stature in science to focus on the inequities within the academy. Encouraging Kalima to appear with her on a television program about stem-cell research, Preston urges her to imagine the milestone of "not just one, but two black women scientists, holding forth among the usual cadre of white males." As one of the rare, highly scrutinized, "minority" scientists, Kalima embodies a "double jeopardy." She must prove that she is worthy of her position---that she is as good, if not better, than her "white" peers---yet she must always be "remembering from whence she came." When Dan, a white colleague in Kalima\'s lab who is also her boyfriend, hears that Preston has chosen Kalima to appear with her on television, he jealously accuses her of benefiting from preferential treatment. The play\'s depiction of this assertion of "reverse racism" questions the legitimate use of race in evaluating promotion and achievement in science. What is apparent is that race, while an ever-present subject, is often presented as the antithesis of conventional notions of a color-blind meritocracy. Left to linger is the critical question: does merely identifying differences among groups constitute an act of racism? Can statements of racial differences be neutral, unfettered by a relative hierarchy? Medley throws into stark relief the politics around race and gender in science. She illuminates how an individual\'s dilemma transcends the private realm; personal decisions are never truly "personal," but are inherently public because they always have wider social repercussions. In the fateful confrontation between Kalima and her mother, Reid challenges her daughter to "grow up" and to risk her disapproval. A similar challenge can be issued to those who have inherited the "new genetics"---to vanquish the continuing paradox of reciting a "mantra of sameness" all the while searching for meaningful differences. In "Relativity," Medley serves us a cautionary tale of the costs of our chronic ambivalence about the critical issues of race and justice in science. A good first step is to recognize that the search for meaning in human difference is inseparable from the struggle over the moral order in which we live. To underestimate the power of science to define our social agenda is to lose an opportunity to determine our future course. We only need to look to history as our proof. Sandra Soo-Jin Lee is a Senior Research Scholar in the Center for Biomedical Ethics and a Lecturer in the Department of Cultural and Social Anthropology at Stanford University. E-mail: <sandra.lee@stanford.edu>

PubMed Central

2024-06-05T03:55:47.620562

2004-9-14

PMC516794

Scientists have dedicated considerable effort to decoding the genomes of an ever-growing list of species, ranging from small viruses, whose genomes may be just a few thousand nucleotides in length, to large mammalian genomes, three billion nucleotides and larger. Many aspects of life science research have benefited by the accumulation of these data, but decoded genomes could be even more valuable if important information about the genome sequence, currently being lost, were preserved. Occasionally, questions arise about a specific position in the sequence---or a variant in the sequence is observed in a new sample. At times like these, it would be helpful to be able to go back to the experimental evidence that underlies the genome sequence at that position, to see if there is any ambiguity or uncertainty about the sequence. As things stand, that\'s almost impossible. To understand why this is the case, it is necessary to know a bit more about how a genome sequence is put together. Current sequencing technology can only generate 700--800 nucleotides at a time; genomes must therefore be shattered into many small fragments (in what is known as the "shotgun" approach), which are then sequenced. The sequences are assembled to generate a consensus sequence that, if all steps work perfectly, matches the original DNA molecule. Since the sequencing of Haemophilus influenzae in 1995 ([@pbio-0020285-Fleischmann1]), most bacterial and archaeal species have been sequenced by fragmenting the entire genome, sequencing the pieces, and assembling the result (the whole-genome shotgun, or WGS, strategy). In recent years, ever-larger sequencing projects have followed the WGS approach, requiring teams of computer experts and the use of increasingly sophisticated assembly algorithms in order to put together the huge number of sequence fragments. Without really being aware of it, the bioinformaticians who assemble genomes have for years been discarding the valuable information on how all of the individual sequence fragments align to the assembled chromosomes. This loss has gone largely unremarked because the scientific community has focused its attention primarily on the end product: the final genome sequence itself. It is only natural to regard the genome sequence, which is the basis for gene discovery and for functional understanding of the biology of the organism, as the primary result of a WGS project. In reality, though, a WGS project is an experiment in which large numbers of sequencing reactions are run, followed by a combination of computational work and additional sequencing to complete the genome. Three years ago, the Trace Archive (at The National Center for Biotechnology Information and The Wellcome Trust Genome Campus in Hinxton, United Kingdom) was developed to store the raw sequence data and to facilitate dissemination of this data, but currently there is no database that captures the alignment of these reads to the published genome sequence. Many scientists would be surprised to hear that genome assemblies are unavailable. One might infer that the assembly of a genome could be reconstructed from the genome sequence and the associated traces. However, aligning the traces to the genome will generally not reproduce the assembly, both because many of the traces will have alternate possible alignments and because, in some cases, parts of the assembly are manually refined based on additional experimental data. Furthermore, only a small number of large-scale centers have the computing hardware, software, and bioinformatics expertise to allow them to assemble a large genome. To bridge this gap, we have developed the Assembly Archive (<http://www.ncbi.nlm.nih.gov/projects/assembly>). The archive has been developed to store both an archival record of how a particular assembly was constructed and the alignments of any set of traces to a reference genome. Assemblies contained in this archive will be available in the GenBank (<http://www.ncbi.nlm.nih.gov/Genbank/index.html>), DDBJ (<http://www.ddbj.nig.ac.jp/>), and EMBL (<http://www.ebi.ac.uk/embl>) databases, and all underlying traces are required to be deposited in the Trace Archive. The Assembly Archive\'s first entries are a set of seven closely related strains of Bacillus anthracis (the causative agent of anthrax), which have been sequenced as part of an effort to understand the detailed variation of that species. This includes the completed reference genome of the Ames strain, sequenced from a sample kept frozen since 1981, when it was originally isolated in West Texas (J. Ravel, personal communication). For the first time, the evidence behind each polymorphism in these assembled genomes will be directly accessible to the scientific community. Microbial Forensics {#s2} =================== Recently, heightened awareness of the threat of bioterrorism has spurred efforts to sequence genomes of multiple strains and isolates of a number of microbial pathogens, with the goal of cataloging all sequence differences between genomes. These efforts began with the study of the B. anthracis bacterium (the bacterium sent through the United States mail in late 2001) in order to determine if there were any differences between it and a reference laboratory sample ([@pbio-0020285-Read1]). This and subsequent studies have prompted many scientists to focus much greater attention on the assembly of a genome, and to regard the assembly rather than the genome as the object of greatest interest. In these forensic studies, we sequence whole genomes in order to discover every possible genetic difference between two bacteria or viruses. These genomes may differ in just one or two nucleotides out of millions that are identical; for example, the study referenced above uncovered just four single nucleotide polymorphisms (SNPs) in a chromosome of 5.23 million base pairs. The close similarity between the sequences forces us to consider all the facts behind each individual nucleotide that appears different. For studies that might be used as evidence in criminal investigations, it is essential to produce this information, and furthermore to quantify our confidence in each nucleotide in the genome. Regions of a genome with deep coverage are much more accurate than those with light coverage (i.e., regions with just one or two sequence reads). [Figure 1](#pbio-0020285-g001){ref-type="fig"} shows one of the interfaces in the Assembly Archive, covering a small region of the multiple alignment of sequences and traces to one of the newly deposited anthrax genomes. It also shows how it is possible to examine the evidence underlying a specific base in the DNA sequence. ::: {#pbio-0020285-g001 .fig} Figure 1 ::: {.caption} ###### Snapshot of the Underlying Sequences and Traces from an Assembly of B. anthracis :::  ::: The consensus sequence shown across the top of the figure contains multiple sequences that validate each nucleotide in the window. Runs of a single base (monomer runs) are common causes of base-calling errors, because the peaks in the underlying trace data sometimes merge together. The sequence shown includes several monomer runs; several of the underlying traces are shown as well. For example, the run of six As at the far left of the figure is supported by several reads in which all six peaks are distinct, as well as other reads in which the six nucleotides appear as one broad peak. By examining data such as these, one can easily verify (or disprove) putative SNPs in this genome. Human SNP Research {#s3} ================== Human polymorphism studies (e.g., [@pbio-0020285-Sachidanandam1]) are a tremendously active and important area of research today. SNPs are directly implicated in a large number of diseases and inherited traits ([@pbio-0020285-Risch1], [@pbio-0020285-Chakravarti1]). Within "haplotypes," they describe individual variation for drug response ([@pbio-0020285-McLeod1]) and provide a genetic framework for understanding disease phenotype ([@pbio-0020285-Hoehe1]). In contrast with prokaryotic genomes, the human genome (as well as other animals, plants, and a broad range of eukaryotes) is diploid, and as a result many SNPs can be discovered within a single assembly, which contains the chromosomes representing the two parent organisms. SNPs can also be found through population studies in which the same locus is sampled from multiple individuals. In either case, the evidence for a SNP begins with the alignment of two different genomes. Despite the clear need for it, the original evidence for the genome itself---the assembly---is not available, and is not linked to the evidence in the Trace Archive. If it were available, many of the polymorphisms already reported could be validated, and many more SNPs might be discovered. Assemblies will also allow centers to better coordinate their gap-closing and finishing efforts, as has been recently noted ([@pbio-0020285-Schmutz1]). We hope that the availability of the Assembly Archive will encourage human genome sequencers, and sequencers of other genomes, to begin depositing their assemblies into this public resource, where it can be shared by all. Thanks to Martin Shumway, Mihai Pop, Steve Sherry, and David J. Lipman for help in defining the specifications and interface to the Assembly Archive, and for other valuable suggestions. SLS is supported in part by National Institutes of Health grant R01-LM06845. Steven L. Salzberg is with the Institute for Genomic Research (Rockville, Maryland, United States of America) and the Departments of Computer Science and Biology at Johns Hopkins University (Baltimore, Maryland, United States of America). Deanna Church, Michael DiCuccio, Eugene Yaschenko, and James Ostell are with the National Center for Biotechnology Information at the National Institutes of Health (Bethesda, Maryland, United States of America). E-mail: <salzberg@tigr.org> (SLS). SNP : single nucleotide polymorphism WGS : whole-genome shotgun

PubMed Central

2024-06-05T03:55:47.621365

2004-9-14

PMC516795

"*"It was not only difficult for him to understand that the generic term dog embraced so many unlike specimens of differing sizes and different forms; he was disturbed by the fact that a dog at three-fourteen (seen in profile) should have the same name as the dog at three-fifteen (seen from the front)....Without effort, he had learned English, French, Portuguese, Latin. I suspect, nevertheless, that he was not very capable of thought. To think is to forget a difference, to generalize, to abstract. In the overly replete world of Funes there were nothing but details, almost contiguous details." ---Jorge Luis Borges, "Funes the Memorius"*" We are told scientists are divided into experimentalists and theoreticians. The dialectic description of the dynamics of science, with one tribe gathering data and collecting evidence and another tribe providing form to these observations, has striking examples that argue for the importance of synthesis. The 16th century revolution, which settled the way in which we see the sky today, is probably one of the best examples of how comparatively ineffective each of these tribes can be in isolation. Tycho Brahe, the exquisite observer, who built, calibrated, and refined instruments to see in the sky what no one else could, collected the evidence to prove a theory that Copernicus had already stated years before (in a book he dedicated to the Pope). It was only many years later that Galileo established the bridge between theory and observation; he understood the data in terms of the theory and thereby cemented the revolution. Copernicus\'s statements, showed Galileo, were not only figments of his imagination; they were an adequate description of the universe as he and Brahe had observed it. Since my first steps in biology, after a prompt departure from physics and mathematics, I have looked for such encounters between theory and experiment. I began studying the visual system and the series of fundamental works by [@pbio-0020297-Attneave1], [@pbio-0020297-Barlow1], and [@pbio-0020297-Atick1] on the relationship between our visual world and the way the brain processes it. Their research was based on a simple hypothesis: (1) the images that we see are highly redundant and (2) the optic nerve is a limited channel, thus the retina has to compress information. Compression, redundancy? How do such concepts relate to the biology of the brain? In the middle of the last century, working on the problem of communications, languages, codes, and channels, Claude Shannon proposed a very elegant theory that formalized intuitive ideas on the essence (and the limits) of communications ([@pbio-0020297-Weaver1]). One of its key aspects was that, depending on the code and on the input, channels are not used optimally and thus do not carry all the information they potentially could. When we compress (zip) a file, we are actually rewriting it in a more efficient (though not necessarily more intelligible) language in which we can store the same amount of information in less space. This compression has, of course, a limit (we cannot convey the universe in a single point), and the mere existence of an optimal code is central to Shannon\'s idea. Attneave was probably the first to think that these ideas could help unravel how the brain worked, and in a long series of works relating these ideas to experimental data, it was shown that the retina\'s business is mainly to get rid of the redundancies in the visual world. About four years ago, Jacob Feldman published a paper, similar in spirit, proposing a simple explanation for a long-standing problem in cognitive science about why some concepts are inherently more difficult to learn than others ([@pbio-0020297-Feldman1]). An article whose first reference is to work carried out 50 years previously makes us suspect that an important gap is being filled. As in the previous experiments, Feldman borrowed a theory---he did not invent it---to explain longstanding and previously unexplained research in this area. Feldman\'s idea was based on a theory developed by Kolmogorov that established formal mathematical grounds to define and measure complexity. Kolmogorov\'s theory was focused on categories, which are just subsets of a universe, a bunch of exemplars that constitute a group. "Dogs" is a category in the universe of animals. Different statements can define the same category, for example, "big dogs" and "dogs that are not small" are the same group, and some information in the statement may be irrelevant because it does not aid in determining which elements belong or not. In the same way that Shannon spoke of a non-redundant code, Kolmogorov showed that categories could be defined with an optimal (non-redundant) statement. The length of this statement defines a measure of complexity termed Kolmogorov complexity. To visualize the intuitive nature of his theory it helps to do a thought experiment. Imagine a set of objects defined by, say, three features: form, color, and size. And imagine, for simplicity, that each feature is binary, that is, there are only two possible cases. Size is big or small, color is yellow or red, and shape can be either a triangle or a circle. This defines, of course, a universe of eight objects. We can now define categories within this universe: for example, all the circles (a category of four exemplars), or all the big and yellow objects (two exemplars), or all the triangles that are not red (again two) (see Figure 1). We can also define a category by enumeration, for example, the small red triangle, the big yellow circle, and the small yellow circle (three exemplars). Some rules (and thus the groups defined by these rules) are intuitively easier to define than others. "All the circles" is an easier statement to make (and probably to remember) than "small circles and yellow big objects." This notion of difficulty is what Kolmogorov\'s theory formalized, stating that complexity was the length of the shortest definition (among all the possible definitions) of a given set. From this thought experiment, we can understand the logic of Feldman\'s paper, which showed that. Kolmogrov complexity is very closely related to our intuitive notion of conceptual difficulty. Feldman presented subjects with all possible categories (of a fixed number of exemplars) in different universes and showed that the critical parameter to rank the difficulty of a given subset was its Kolmogrov complexity. Moreover by explicitly presenting all the members and the nonmembers of a category to naïve subjects, he showed that we can spontaneously reduce a category to its minimal form and remember it without any explicit instruction. Thus, what Feldman found, following the original ideas of Shepard, was that our psychological measure of complexity---our difficulty in defining and remembering a category or concept---is also determined by the Kolmogorov complexity that describes it.[](#pbio-0020297-g001){ref-type="fig"} ::: {#pbio-0020297-g001 .fig} Figure 1 ::: {.caption} ###### Visualizing Kolmogorov´s Complexity Intuitive categories can be defined by short statements. The universe: circles and triangles, red and yellow, big and small (A). Examples of easy categories: red objects (B); triangles (C). Example of a difficult category: yellow circles and small red circles (D). :::  ::: This essay is, in a way, about how we avoid becoming Borges\'s character Funes, who could not understand repeated observations as exemplars of a common rule and thus could not synthesize and categorize. Simply, he could not think. Probably the most disappointing moment of Feldman\'s paper comes at the very end, where it deals with its (somehow unavoidable) recursive quest. Understanding why some concepts are difficult to learn may itself be difficult to learn. Modern mathematics, together with Kolmogorov complexity and information theory, has taught us another fundamental concept that may be relevant when trying to understand the logic of the mind. In a long series of paradoxes enumerated by Bertrand Russell, Kurt Goedel, and others, we learn that a formal system that looks at itself is bound to fail. At the very end of his paper, Feldman writes, "In a sense, this final conclusion \[that psychological complexity is Boolean complexity\] may seem negative: human conceptual difficulty reflects intrinsic mathematical complexity after all, rather than some idiosyncratic and uniquely human bias." Who invented mathematics? The Martians? On the contrary, I believe this result supports a more naturalistic and less Platonic conception of mathematics. Formal ideas in mathematics are not arbitrary constructions of an arbitrary architecture; rather, they reflect the workings of the brain like a massive collective cognitive experiment. Mathematics does not only serve to help us understand biology; mathematics is biology. We are not less original if our thoughts resemble our mental constructions, we are just consistent. It is within this loop, this unavoidable recursion---mathematics understanding the logic of the brain---that we will have an opportunity to test, as some conspire, whether among all the wonders evolution has come out with, the ultimate might be a brain good enough to avoid the risk of understanding itself. Mariano Sigman is in the Cognitive Neuroimaging Research Unit of l\'Institut National de la Santé et de la recherche Médicale, Orsay, France, and a fellow of the Human Frontiers Science Program. E-mail: <mariano@rna.rockefeller.edu>

PubMed Central

2024-06-05T03:55:47.622637

2004-9-14

PMC516796

I need a "tricorder"---the convenient, hand-held device featured on *Star Trek* that can detect life forms even from orbit. Unfortunately, we don\'t have a clue how a tricorder might work, since life forms don\'t seem to have any observable property that distinguishes them from inanimate matter. Furthermore, we lack a definition of life that can guide a search for life outside Earth. How can we find what we can\'t define? An answer may lie in the observation that life uses a small, discrete set of organic molecules as basic building blocks. On the surface of Europa and in the subsurface of Mars, we can search for alien but analogous patterns in the organics. Life As We Know It {#s2} ================== The obvious diversity of life on Earth overlies a fundamental biochemical and genetic similarity. The three main polymers of biology---the nucleic acids, the proteins, and the polysaccarides---are built from 20 amino acids, five nucleotide bases, and a few sugars, respectively. Together with lipids and fatty acids, these are the main constituents of biomass: the hardware of life ([@pbio-0020302-Lehninger1], p 21). The DNA and RNA software of life is also common, indicating shared descent ([@pbio-0020302-Woese1]). But with only one example of life---life on Earth---it is not all that surprising that we do not have a fundamental understanding of what life is. We don\'t know which features of Earth life are essential and which are just accidents of history. Our lack of data is reflected in our attempts to define life. [@pbio-0020302-Koshland1] lists seven features of life: (1) program (DNA), (2) improvisation (response to environment), (3) compartmentalization, (4) energy, (5) regeneration, (6) adaptability, and (7) seclusion (chemical control and selectivity). A simpler definition is that life is a material system that undergoes reproduction, mutation, and natural selection ([@pbio-0020302-McKay1]). [@pbio-0020302-Cleland1] have suggested that life might be like water, hard to define phenomenologically, but easy to define at the fundamental level. But life is like fire, not water---it is a process, not a pure substance. Such definitions are grist for philosophical discussion, but they neither inform biological research nor provide a basis for the search for life on other worlds. The simplest, but not the only, proof of life is to find something that is alive. There are only two properties that can determine if an object is alive: metabolism and motion. (Metabolism is used here to include an organism\'s life functions, biomass increase, and reproduction.) All living things require some level of metabolism to remain viable against entropy. Movement (either microscopic or macroscopic) in response to stimuli or in the presence of food can be a convincing indicator of a living thing. But both metabolism (fire) and motion (wind) occur in nature in the absence of biology. The practical approach to the search for life is to determine what life needs. The simplest list is probably: energy, carbon, liquid water, and a few other elements such as nitrogen, sulfur, and phosphorus ([@pbio-0020302-McKay1]). Life requires energy to maintain itself against entropy, as does any self-organizing open system. In the memorable words of Erwin [@pbio-0020302-Schrodinger1], "It feeds on negative entropy." On Earth, the vast majority of life forms ultimately derive their energy from sunlight. The only other source of primary productivity known is chemical energy, and there are only two ecosystems known, both methanogen-based ([@pbio-0020302-Stevens1]; [@pbio-0020302-Chapelle1]), that rely exclusively on chemical energy (that is, they do not use sunlight or its product, oxygen). Photosynthetic organisms can use sunlight at levels below the level of sunlight at the orbit of Pluto ([@pbio-0020302-Raven1]); therefore, energy is not the limitation for life. Carbon, nitrogen, sulfur, and phosphorus are the elements of life, and they are abundant in the Solar System. Indeed, the Sun and the outer Solar System have more than 10,000 times the carbon content of the bulk of Earth ([@pbio-0020302-McKay1]). When we scan the other worlds of our Solar System, the missing ecological ingredient for life is liquid water. It makes sense, then, that the search for liquid water is currently the first step in the search for life on other worlds. The presence of liquid water is a powerful indication that the ecological prerequisites for life are satisfied. Orbital images, such as the canyon in [Figure 1](#pbio-0020302-g001){ref-type="fig"}, show clear evidence of the stable and repeated, if not persistent, flow of a low-viscosity fluid on Mars at certain times in its past history. The fluid was probably water, but the images could also suggest wind, ice, lava, even carbon dioxide or sulfur dioxide. Recently, results from the Mars Exploration Rover missions have shown that this liquid carried salts and precipitated hematite in concretions. The case for water, we could say, is tight. ::: {#pbio-0020302-g001 .fig} Figure 1 ::: {.caption} ###### Water on Another World A Mars Global Surveyor image showing Nanedi Vallis in the Xanthe Terra region of Mars. The image covers an area 9.8 km ×18.5 km; the canyon is about 2.5 km wide. This image is the best evidence we have of liquid water anywhere outside the Earth. Photo credit: NASA/Malin Space Sciences. :::  ::: On Jupiter\'s moon Europa, the cracks and icebergs on the surface of the ice indicate water beneath the ice, but not necessarily at the present time. Present water on Europa is indicated by the magnetic disturbance Europa makes as it moves through Jupiter\'s magnetic field, not unlike the way coins in the pocket of a passenger will set off an airport metal detector. Europa has a large conductor, and this is most likely a global, salty layer of water. Viking on Mars: Been There, Tried That {#s3} ====================================== The Viking missions to Mars in the late 1970s were the first (and as yet, the only) search for life outside Earth. Each Viking conducted three incubation experiments to detect the presence of metabolism in the Martian soil. Each lander also carried a sophisticated Gas Chromatograph Mass Spectrometer for characterizing organic molecules. The results were unexpected ([@pbio-0020302-Klein1], [@pbio-0020302-Klein2]). There was a detectable reaction in two of the incubation experiments. In the "Gas Exchange" experiment, a burst of oxygen was released when the soil was exposed to water. The "Labeled Release" experiment showed that organic material was consumed, and that carbon dioxide was released concomitantly. In the Labeled Release experiment, this reaction ceased if the soil was first heated to sterilizing temperatures, but the reaction of the Gas Exchange Experiment persisted. If considered alone, the Labeled Release results would be a plausible indication for life on Mars. However, the Gas Chromatograph Mass Spectrometer did not detect the presence of any organic molecules in the soil at level of one part per billion ([@pbio-0020302-Biemann1]). It is difficult to imagine life without associated organic material, and this is the main argument against a biological interpretation of the Viking results ([@pbio-0020302-Klein2]; but cf. [@pbio-0020302-Levin1]). It is also unlikely that the oxygen release in the Gas Exchange experiment had a biological explanation, because the reaction was so rapid and persisted after heating. It is generally thought that the reactivity observed by the Viking biology experiments was caused by one or more inorganic oxidants present in the soil, and was ultimately produced by ultraviolet light in the atmosphere. Consistent with the apparently negative results of the Viking biology experiments, the surface of Mars also appears to be too dry for life. Indeed, conditions are such that liquid water is rare and transient, if it occurs at all (e.g., [@pbio-0020302-Hecht1]). It\'s Life, Jim, but Not As We Know It {#s4} ====================================== [Table 1](#pbio-0020302-t001){ref-type="table"} shows a categorization of life as we have observed it. Using this diagram, we can speculate about how life might be different on Mars or Europa. At the bottom of the table, life is composed of matter---a reasonable assumption for now. Carbon and liquid water are the next level; this makes Mars and Europa likely candidates, because they have carbon and have, or have had, liquid water. Other worlds may have a different chemical baseline for life. The usual speculation in this area is that the presence of ammonia and silicon, rather than water and carbon, might be preconditions for life on other planets. Such speculation has yet to lead to any specific suggestions for experiments, or to new ways to search for such life, but this may just reflect a failure of human imagination rather than a fundamental limitation on the nature of life. ::: {#pbio-0020302-t001 .table-wrap} Table 1 ::: {.caption} ###### A Categorization of Structures That Comprise Terrestrial Life :::  ::: Life on Mars is also likely to be the same at the top of the table: at the ecological level. Primary production in a Martian ecosystem is likely to be phototrophic, using carbon dioxide and water. Heterotrophs are likely to be present to consume the phototrophs and in turn to be consumed by predators. Darwinian evolution would result in many of the same patterns we see in ecosystems on Earth. While it may be similar at the top (ecological) and bottom (chemical) levels, life on Mars could be quite alien in the middle, in the realm of biochemistry. [@pbio-0020302-Pace1] has argued that alien biochemistry will turn out to be the same as biochemistry on Earth, because there is one best way to do things and that natural selection will ensure that life everywhere discovers that way. Only observation will tell if there is one possible biochemistry, or many. Future missions to Mars might find microfossils in sedimentary rocks such as those at Meridiani Planum. Microbes don\'t readily form convincing fossils; the one exception may be the strings of magnetite formed by magnetotactic bacteria ([@pbio-0020302-Friedmann1]). As interesting as fossils might be, we could not be sure that a fossil found on Mars was not merely another example of Earth life. We know that rocks have come to Earth from Mars, and it is possible that such rocks could have carried life between the planets ([@pbio-0020302-Mileikowsky1]; [@pbio-0020302-Weiss1]). Finding fossil evidence for life on Mars does not demonstrate a second genesis in our Solar System. Finding a Way to Search for Alien Life {#s5} ====================================== If we were to find organic material in the subsurface of Mars or on the ice of Europa, how could we determine whether it was the product of a system of biology or merely abiotic, organic material from meteorites or photochemistry? If this life were in fact related to Earth life, this should be easy to determine. We now have very sensitive methods, such as PCR and fluorescent antibody markers, for detecting life like us. This case would be the simplest to determine, but it would also be the least interesting. If the life turned out to be truly alien, then the probes specific to our biology would be unlikely to work. What, then, could we do to determine a biological origin? The question is open and possibly urgent. On space missions already being planned, we may have the opportunity to analyze the remains of alien organics on the surface of Europa or frozen below ground on Mars. The instruments that will make this analysis must be designed in the next couple of years. One approach appears promising. I call it the "Lego Principle." It is based on the patterns of the molecules of life. Biological processes, in contrast to abiotic mechanisms, do not make use of the range of possible organic molecules. Instead, biology is built from a selected set. Thus, organic molecules that are chemically very similar to each other may have widely different concentrations in a sample of biological organics. An example of this on Earth is the 20 amino acids used in proteins and the use of the left-handed version of these amino acids. The selectivity of biological processes is shown schematically in [Figure 2](#pbio-0020302-g002){ref-type="fig"} by the distribution of spikes in contrast to a smooth, nonbiological distribution. General arguments of thermodynamic efficiency and specificity of enzymatic reactions suggest that this selectivity is required for biological function and is a general result of natural selection. Different life forms are likely to have different patterns, and at the very least we might find the mirror symmetry of life on Earth, with [d]{.smallcaps}- instead of [l]{.smallcaps}-amino acids. ::: {#pbio-0020302-g002 .fig} Figure 2 ::: {.caption} ###### Comparison of Biogenic with Nonbiogenic Distributions of Organic Material Nonbiological processes produce smooth distributions of organic material, illustrated here by the curve. Biology, in contrast selects and uses only a few distinct molecules, shown here as spikes (e.g., the 20 l-amino acids on Earth). Analysis of a sample of organic material from Mars or Europa may indicate a biological origin if it shows such selectivity. :::  ::: This approach has immediate practical benefit in the search for biochemistry in the Solar System. Samples of organic material collected from Mars and Europa can be easily tested for the prevalence of one chirality of amino acid over the other. More generally, a complete analysis of the relative concentration of different types of organic molecules might reveal a pattern that is biological even if that pattern does not involve any of the familiar biomolecules. Interestingly, if a sample of organics from Mars or Europa shows a preponderance of D-amino acids, this would be evidence of life, and at the same time would show that this life was distinct from Earth life. This same conclusion would apply to any clearly biological pattern that was distinct from that of Earth life. Organic material of biological origin will eventually lose its distinctive pattern when exposed to heat and other types of radiation, (examples of this include the thermal racemization of amino acids), but at the low temperatures in the Martian permafrost, calculations suggest that there has been no thermal alteration ([@pbio-0020302-Kanavarioti1]). An interesting question, as yet unanswered, is how long organic material frozen into the surface ice of Europa would retain a biological signature in the strong radiation environment. On Europa, the organic material for our tests might be collected right from the dark regions on the surface. On Mars, there is ice-rich ground in the cratered southern polar regions ([@pbio-0020302-Feldman1]), which presumably overlies deeper, older ice. The surprise discovery of strong magnetic fields in the southern hemisphere of Mars ([@pbio-0020302-Acuna1]; [@pbio-0020302-Connerney1]) indicates that the area may be the oldest undisturbed permafrost on that planet. Like the mammoths extracted from the ice in Siberia, any Martian microbes found in this ice would be dead, but their biochemistry would be preserved. From these biological remains, it would then be possible to determine the biochemical composition of, and the phylogenetic relationship between, Earth life and Martian life. We may then have, for the first time, a second example of life. Chris P. McKay is with the NASA Ames Research Center. E-mail: <cmckay@mail.arc.nasa.gov>

PubMed Central

2024-06-05T03:55:47.623854

2004-9-14

PMC516797

The ongoing battle between hosts and pathogens has long been of interest to evolutionary biologists. Because hosts and pathogens act as environments for each other, their intertwined struggle for existence is both continual and rapid. At the molecular level, this cycle of environmental change and evolutionary response means that mutations are continually being tried out by natural selection. It is therefore little wonder that the host and pathogen genes that control infection and immunity frequently show high levels of genetic diversity and present some of the best examples of positive selection (adaptive evolution) reported to date ([@pbio-0020307-Yang1]). In particular, rates of nonsynonymous substitution per site (resulting in an amino acid change; d~N~) often greatly exceed those of synonymous substitution per site (silent change; d~S~), as expected if most mutations are fixed because they increase fitness ([Figure 1](#pbio-0020307-g001){ref-type="fig"}). ::: {#pbio-0020307-g001 .fig} Figure 1 ::: {.caption} ###### Measuring Selection Pressures by Comparing the Ratio of Nonsynonymous to Synonymous Substitutions Per Site \(A) Classification of substitutions. Nonsynonymous substitutions (red) are those that change the amino acid sequence of the protein encoded by the gene, while the degeneracy of the genetic code ensures that synonymous substitutions (yellow) result in the same amino acid sequence. \(B) Calculation of d~N~/d~S~. By assuming that synonymous mutations are neutral and fixed by random genetic drift, it is possible to determine the mode of selection acting on nonsynonymous mutations. If all nonsynonymous substitutions were neutral, then their rate of occurrence per site (d~N~) would be the same as that of synonymous substitutions per site (d~S~), so that d~N~/d~S~ equals one. A lower ratio of nonsynonymous to synonymous substitutions per site (d~N~/d~S~ \< 1) means that some proportion of the nonsynonymous mutations are deleterious and removed by purifying selection. Conversely, positive selection fixes advantageous nonsynonymous mutations faster than genetic drift fixes synonymous mutations (d~N~/d~S~ \> 1), although this is usually restricted to a small proportion of amino acid sites within any gene. In the hypothetical example of five gene sequences shown here, with d~S~ given above the diagonal and d~N~ below the diagonal, there is no evidence for positive selection because mean d~N~/d~S~ the (0.577) is less than one. :::  ::: At the host level, most studies of the selection pressures acting on immune system genes have concentrated on genes implicated in the adaptive immune response against microbial pathogens, particularly those producing antibodies ([@pbio-0020307-Sitnikova1]; [@pbio-0020307-Sumiyama1]), or genes encoding reconnaissance molecules known as the major histocompatibility complex (MHC), which control the action of T-cells ([@pbio-0020307-Hughes2]; [@pbio-0020307-Yeager1]) ([Box 1](#box1){ref-type="boxed-text"}). As its name suggests, the role of the adaptive immune response is to stimulate and 'memorise' immunity to specific pathogens. As microbial pathogens such as viruses are both abundant and rapidly evolving, positive selection on components of the adaptive immune response is often very strong ([@pbio-0020307-Yeager1]). Far less attention has been directed toward the less specific innate ('nonadaptive') immune response, even though this response requires a wide array of genes and acts as the front line of immune defence ([Box 1](#box1){ref-type="boxed-text"}). Would we expect the same strength of positive selection on a generalized pathogen control system? This is a question of fundamental importance because the luxury of adaptive immunity is not available to most organisms, having probably evolved along with the vertebrates ([@pbio-0020307-Bartl1]), whereas the more widespread innate immune system is often depicted as a primitive characteristic. Molecular Evolution of the Innate Immune System {#s2} =============================================== The genes involved in innate immunity have recently come under the molecular evolutionists\' gaze. One important group are the defensins, a large class of short antimicrobial peptides that constitute an effective immune response team in organisms as diverse as plants and primates ([@pbio-0020307-Boman1]). Because defensins are cationic (positively charged), they are able to interact with negatively charged molecules on the surface of microbes and permeate their membranes. Sequence analyses of defensins and similar antimicrobial peptides have revealed the telltale signatures of positive selection, with d~N~ greater than d~S~ in many comparisons ([@pbio-0020307-Hughes1]; [@pbio-0020307-Duda1]; [@pbio-0020307-Maxwell1]). Other genes of the innate immune system also seem to be subject to powerful positive selection. One dramatic example described in this issue of *PLoS Biology* is the APOBEC3G gene of primates ([@pbio-0020307-Sawyer1]). This case is especially striking because rather than killing pathogens through protein or cellular interactions, like most immune genes, APOBEC3G works by manipulating the genome sequence of the invading microbe. The genomes of primates contain a family of nine APOBEC genes that encode enzymes involved in the editing of RNA and/or DNA through the deamination of cytosine (C), so that this nucleotide mutates to uracil (U). This is essential for various aspects of cellular function. APOBEC1, for example, is involved in the C→U editing of apolipoprotein B mRNA (therein christening the family), while another family member, the activation-induced deaminase, has a vital role in adaptive immunity in that it assists in the diversification of antibodies. Two more enzymes, APOBEC3G and APOBEC3F, form part of the innate immune system; they function as antiviral agents and are being intensively studied in the context of infection with the human immunodeficiency virus (HIV), the cause of AIDS. In particular, APOBEC3G targets the reverse transcription step of the HIV life cycle, in which the viral genomic RNA is converted into proviral DNA, which is then integrated into the host genome ([@pbio-0020307-Mangeat1]). APOBEC3G-induced deamination at this stage results in monotonous guanine-to-adenine (G→A) nucleotide changes, a phenomenon called G→A hypermutation that had long been noted by HIV researchers without a clear understanding of its cause. We now know that G→A hypermutation is part of the innate immune response to retroviral infections. Although there is still some debate over exactly how APOBEC3G leads to viral eradication, the most likely scenario is that G3A hypermutation results in the generation and incorporation of a multitude of deleterious mutations that fatally disrupt viral functions. This strategy is likely to work well for retroviruses like HIV because their genomes are so compact that individual sequence regions often perform multiple functions. Under these cramped conditions, most mutations are likely to severely disrupt some aspect of viral function and thereby reduce fitness ([@pbio-0020307-Holmes1]). Indeed, it has been estimated that the deleterious mutation rate in viruses that replicate using RNA polymerases (either reverse transcriptase in the case of retroviruses or RNA-dependent RNA polymerase for other RNA viruses) is on the order of one error per replication cycle, so that many of the viral progeny produced by replication are defective ([@pbio-0020307-Elena1]). HIV is normally able to overcome this burden of deleterious mutation because of its remarkable reproductive power; each day, on the order of 10^10^ virions are produced in a single infected individual ([@pbio-0020307-Perelson1]), so that enough fit and able recruits will make it through to the next generation. Treating RNA Virus Infections Through Lethal Mutagenesis {#s3} ======================================================== The high mutation rates of RNA viruses mean that adaptively useful genetic variation is produced frequently. The rub, however, is that fitness-enhancing mutations are a small minority, and the preponderance of deleterious mutations means that RNA viruses live on the edge of survival ([@pbio-0020307-Domingo1]). By increasing the rate at which deleterious mutations appear, APOBEC3G pushes viruses over this edge, causing a form of 'lethal mutagenesis' that results in their destruction; the rate of mutation becomes so high that no genome can reproduce itself faithfully, and the population crashes. Intriguingly, researchers designing new antiviral drugs have also begun to realise that forcing viruses into this sort of 'error catastrophe' might be an effective way to treat them ([Figure 2](#pbio-0020307-g002){ref-type="fig"}). There are a growing number of studies in which mutagens, such as ribavirin and 5-fluorouracil, are applied to viral infections in vitro and in vivo, including HIV, in the hope that these will induce so many deleterious mutations that the virus suffers an error catastrophe and is cleared ([@pbio-0020307-Loeb1]; [@pbio-0020307-Sierra1]; [@pbio-0020307-Crotty1]; [@pbio-0020307-Ruiz-Jarabo1]). The results produced to date are highly encouraging, particularly when these error-inducing drugs are combined with more conventional treatment strategies that aim to reduce the rate of viral replication ([@pbio-0020307-Pariente1]). The discovery that a natural antiviral agent, APOBEC3G, probably works in much the same way should provide even more encouragement. ::: {#pbio-0020307-g002 .fig} Figure 2 ::: {.caption} ###### Lethal Mutagenesis As a Means of Controlling RNA Virus Infections \(A) In a viral population prior to the application of mutagens, the mean error rate (white) is on the order of one per genome per replication (mutations marked by asterisks). (B) If a mutagen such as ribavirin is then applied to an infected patient, the mean error rate of the virus (black) is increased so that the population crosses a threshold of 'error catastrophe'; after this point fitness declines dramatically and the population crashes. This drug-induced lethal mutagenesis seems to work more efficiently when it is used in combination with drugs that reduce the rate of viral replication. :::  ::: Sadly, however, the pathogens have fought back. The anti-HIV properties of APOBEC3G were discovered because most viral strains escape its neutralising properties. Lentiviruses like HIV possess a gene that encodes an protein called Vif ('viral infectivity factor') that counters APOBEC3G ([@pbio-0020307-Sheehy1]). Hence, it is probably only in naturally occurring Vif-defective mutants that APOBEC3G is effective against HIV. Furthermore, because positive selection on APOBEC3G has operated for at least 30 million years and lentiviruses in general, and HIV in particular, are likely to be more recently evolved than this, it is clear that a broad range of retroviral pathogens have been responsible for the adaptive evolution of this particular immune gene ([@pbio-0020307-Sawyer1]). Given the frequency with which the remnants of past retroviral infections are found in the mammalian genome ([@pbio-0020307-Smit1]), in the form of usually defunct endogenous retroviruses ([Box 1](#box1){ref-type="boxed-text"}), it is likely that our genomes are continually bombarded with retroviruses like HIV but that the majority are cleared by innate immune mechanisms like APOBEC3G. It is possible that the retroviruses that successfully infect us are those, like HIV, that have managed to evolve strategies to avoid the destructive capacities of APOBEC3G. The intense selective pressure on the defensins and APOBEC3G illustrates that although the innate immune response is generalist in its action, it is as highly and intricately evolved as its better-studied ally, the adaptive immune system. Rather than being an evolutionary remnant, it is a dynamic and continually adapting system. Less clear is whether other host proteins act in the same manner as APOBEC3G. In particular, the most common and destructive pathogens faced by humans and other mammals are RNA viruses, such as influenza A, yellow fever, and hepatitis C. In most cases, our ability to survive these viral infections is simply a combination of good luck and good breeding; with the right combination of MHC alleles, itself a function of population history and what we by chance inherit from our parents, some individuals may be more able to fight off viral infections than others. The ubiquity of RNA viruses hints that our genomes might also contain an innate, yet highly adapted, defence system that targets this abundant class of pathogens by manipulating their mutation rate. Although utilizing lethal mutagenesis might one day be an important way to design new drugs against a variety of viral pathogens, it would come as no surprise if nature got there first. ::: {.caption} ###### Box 1. Glossary ::: **Adaptive immune system.** The pathogen-specific part of the vertebrate immune system. It is comprised of two major arms, antibodies (the humoral response) and T-cells (the cellular response), both of which lay down an immunological memory for future defence. **Innate immune system.** The nonspecific part of the vertebrate immune system. It has a wide variety of components, ranging from lysozymes in saliva to cytokines, defensins, interferons, and natural killer cells in a variety of tissues. **Endogenous retroviruses.** The (usually) dead remnants of functional retroviruses that are now passed on through the germ line like normal genes. It has been estimated that approximately 5% of the human genome is composed of endogenous retroviruses. Eddie C. Holmes is in the Department of Zoology at the University of Oxford, Oxford, United Kingdom. E-mail: <edward.holmes@zoo.ox.ac.uk> C : cytosine d~N~ : rate of nonsynonymous substitution per site d~S~ : rate of synonymous substitution per site G→A : guanine-to-adenine HIV : human immunodeficiency virus MHC : major histocompatibility complex U : uracil

PubMed Central

2024-06-05T03:55:47.625979

2004-9-14

PMC516798

The language of conservation is changing: protecting biodiversity is no longer just about ethics and aesthetics; the latest buzzwords are commodities and consumers. Traditionally, conservation initiatives have talked up the benefits they will bring to the global community---saving species, habitats, ecosystems, and ultimately the planet. But conservation also has its costs, and these are usually borne by local people prevented from exploiting the resources around them in other ways. It is unfair to expect a localised minority to pick up costs that ultimately benefit a dispersed majority, argue conservation biologists. There has to be more money made available by concerned individuals, non-governmental organisations, national governments, and international bodies, and there need to be better ways to spend this money if conservation is to be effective, they say. Biodiversity is a commodity that can be bought and sold. We are consumers and must pay. Costs and Benefits {#s2} ================== Kenya boasts one of the world\'s most spectacular networks of national parks and reserves covering around 60,000 km^2^ of the country ([Figure 1](#pbio-0020310-g001){ref-type="fig"}). But devoting such a vast area to conservation has its drawbacks. It has been estimated that were this land developed it would be worth around \$270 million to the Kenyan people every year. Similarly, two national parks in Madagascar are estimated to have reduced the annual income of local villagers by around 10%. Of course, protected areas do bring some benefits to neighbouring communities, most notably through tourism. But in many cases the rewards are not great, they are rarely distributed evenly among individuals, and do not necessarily outweigh the costs. ::: {#pbio-0020310-g001 .fig} Figure 1 ::: {.caption} ###### The Masai Mara National Park in Kenya Courtesy of Charlotte Stirling. :::  ::: 'The costs of conservation fall disproportionately on local people, whereas the benefits are dispersed,' says Andrew Balmford, a conservation biologist at the University of Cambridge in the United Kingdom. National and global communities stand to benefit from conservation of tropical biodiversity, but they must pay if they want to realise that benefit, he says. Conservation expenditure in the developed world is only about a third of what is needed for effective protection of 15% of the earth\'s terrestrial habitats, an area just large enough to preserve a representative sample of species, habitats, and ecosystems in the medium to long term ([@pbio-0020310-Balmford2]). The developed world must make up this funding shortfall, argues Balmford. What\'s more, there need to be smarter ways to spend the money that\'s available, he says. Conservation by Distraction {#s3} =========================== In recent years, many funding bodies have taken an indirect approach to conservation, investing in projects that encourage people to take up alternative practices that are compatible with conservation rather than investing in conservation itself. Perhaps the best example of this 'conservation by distraction' is ploughing money into community-based ecotourism projects. Such initiatives aim to bring the benefits of tourism to local people, thereby encouraging them to preserve the biodiversity they have. It\'s an attractive idea. In the mid 1990s, the United States Agency for International Development was investing more than \$2 billion a year in 105 conservation projects with an ecotourism component. Similarly, between 1988 and 2003, the World Bank funded 55 development projects that supported protected areas in Africa, 32 of which placed an emphasis on ecotourism. However, an absence of quantitative data and analysis has made it hard to judge whether these projects actually achieve their dual purpose of preserving biodiversity and simultaneously reducing rural poverty. 'Much of the information about community-based ecotourism is anecdotal and subjective,' says Agnes Kiss of the Environment and Social Development Unit at the World Bank. The real contribution of these initiatives to biodiversity conservation is debatable, she says. 'Many community-based ecotourism projects cited as success stories actually involve little change in existing local land- and resource-use practices, provide only modest supplement to local livelihoods, and remain dependent on external support for long periods, if not indefinitely' ([@pbio-0020310-Kiss1]). For example, communities involved in the Infierno Community Ecotourism Project in Peru have received nearly \$120,000 from their share in a tourist lodge and wages for providing services to visitors. This may have increased the income for a minority that are lodge employees, but only one family, whose adult members were all employed by the lodge, could afford to live solely on tourism. In the community as a whole, the average annual income from tourism was only \$735 compared with nearly \$2,000 earned elsewhere. Most of the community was still heavily dependent on other activities, and most of those activities are somewhat disruptive of conservation goals, says Kiss. Johan du Toit of the Mammal Research Institute at the University of Pretoria in South Africa is also critical of this kind of indirect approach to conservation. At the heart of the argument for community-based ecotourism is the idea of the 'ecologically noble savage', he says---the notion that those living closest to nature will know what\'s best for it. 'It\'s a wonderful idea, but it just doesn\'t work. Nowhere in the history of evolution has sustainability ever been naturally selected for,' says du Toit. 'The AK47 automatic assault rifle has replaced the bow and arrow....Every individual in a rural community that\'s out hunting will shoot what he sees when he sees it, because if he doesn\'t somebody else will.' Nowhere is this problem more evident than in the ecotourist paradise of the Galápagos Islands ([Figure 2](#pbio-0020310-g002){ref-type="fig"}), where a small minority of fishermen is coming into conflict with conservation aims with increasing regularity ([Box 1](#box1){ref-type="boxed-text"}). 'Things are going down very quickly,' says one Galápagos guide. 'The iceberg is starting to tip over, and we are going to lose everything.' If it still pays locals to exploit the environment rather than take part in one of the world\'s most buoyant ecotourism industries, it is clear that ecotourism alone cannot solve the world\'s conservation problems. Many think that 'direct payment' could be a useful tool. 'Direct payment, very boldly speaking, is paying people in rural areas not to bugger up their environment,' says du Toit. 'It\'s just like if we want exclusive artworks to be looked after in the Louvre Gallery in Paris. Somebody\'s got to pay for it,' he says. 'You can\'t expect the Parisians who live in that arrondissement to cover the costs.' ::: {#pbio-0020310-g002 .fig} Figure 2 ::: {.caption} ###### Ecotourist Paradise in the Galápagos Courtesy of Catriona MacCallum. :::  ::: You Get What You Pay for---You Should Pay for What You Want to Get {#s4} ================================================================== For people living in developing countries, where most of the world\'s biodiversity exists, the short-term rewards of exploiting these natural resources are significant. Replacing indirect conservation measures, such as community-based ecotourism, with payments directly into the pockets of local people could turn out to be a much more effective way to stem this exploitation, argues Paul Ferraro, an economist at Georgia State University in Atlanta ([@pbio-0020310-Ferraro1]). It could also bring far greater development benefits than indirect financial support, he says ([Box 2](#box2){ref-type="boxed-text"}). An additional spin-off is that direct payments force conservation biologists to quantify and hence clarify their objectives, says John Hough, principal technical advisor on biodiversity for the United Nations Development Programme. 'We know what we don\'t want,' he says, 'but we\'re not very good at saying what we do want.' A hypothetical model simulating how Madagascar should distribute an annual conservation budget of \$4 million reveals that direct payments would have protected some 80% of original forest compared with only 12% protected through a system of indirect incentives. What\'s more, the annual income of rural residents would have been twice that generated through indirect investment ([@pbio-0020310-Conrad1]). For Ferraro, the logic of direct payment is simple. He draws an analogy with a car journey from A to B. There are two routes that will bring you to B, one circuitous and the other direct. If you only have a single tank of fuel, opting for the direct route improves the likelihood you will arrive at your destination. An indirect approach to conservation is like taking the circuitous route, he says, and the chances are that you will run out of fuel. But if it\'s that simple, why are governments, non-governmental organisations, private bodies, and international organisations not jumping at the chance to experiment with this approach? Paying in Perpetuity {#s5} ==================== There are those that have reservations about direct payments. The distinction between indirect and direct interventions is artificial, says Thomas Lovejoy, president of the Heinz Center, a nonprofit institution dedicated to improving the scientific and economic foundation for environmental policy. 'In some cases, direct payment is the only way conservation can happen,' he says. 'In others, the indirect is important to reinforce a situation where there already is conservation. In yet others both are needed.' Sjaak Swart of the Section of Science and Society at Groningen University in The Netherlands argues that if conservation is to succeed, it must be rooted in the hearts and minds of those involved. Direct payments create a vision of nature dominated by calculable, monetary concerns, he says. This approach can only work in the short term, he argues, and indirect tools like debate and education are needed to involve communities in the long term. 'You need the commitment of the local people to save the biodiversity of our world,' he says. Marine biologist Steve Trott agrees. He is project coordinator for the Local Ocean Trust, a charity-based conservation organisation operating in the Watamu and Malindi Marine Parks and Reserve in Kenya (<http://www.watamuturtles.com>), and is using direct payments to help reduce the slaughter of turtles by local fishermen. The Watamu Turtle Watch Program is currently paying fishermen just over \$3 a turtle to release the animals from their nets rather than kill them. Before the scheme started in 2000, only around 50 turtles were being released from nets each year. By 2003, more than 500 a year were making it back into the sea. Elsewhere along the Kenyan coast, where fishermen do not get these payments, turtles continue to be killed, says Trott. However, the financial incentives are only part of a grander program of education and support to sensitise people to the conservation message, he says. Eventually, the plan is to stop payments altogether. 'Payment will be reduced as education and awareness is increased to the point where it\'s phased out,' he says. Reducing or stopping the payment could work, says Ferraro, but it is more likely that the turtles will begin to suffer once more. 'If I had to wager, I\'d bet people would go back to their old patterns eventually.' This means that direct payments require an ongoing financial commitment, and many people don\'t like this idea, he says. To the Test {#s6} =========== The idea of direct payments needs empirical testing before it can be embraced with confidence, admits Ferraro. Funding bodies should demand experimental and control data to allow the success of an intervention to be gauged. Conservation biologists must therefore be trained in the skills needed to collect and evaluate these data. 'Without adequate data and controls you\'re only going to be left with guesses and vague anecdotes about the effects of a program intervention,' he says. Decision makers should begin to design controlled experiments from which they can make inferences about the effectiveness of these different interventions, he suggests. There are other drawbacks of direct payments. One concern is that they might just shift the pressure from one site to another that was not previously being exploited. Furthermore, in developing countries, land tenure is often ambiguous, which can make investment an unattractive prospect for funding agencies---they want to be sure they know where their money is going. But, notes Ferraro, such objections also apply to indirect interventions. 'I don\'t necessarily believe that conservation payments will be successful,' he says. 'It\'s more I believe that of all the ideas out there for protecting biodiversity, this is the least bad.' All this talk of cost, benefit, and efficiency is creeping into conservation speak. For some, these cold and calculating terms are an odd way to describe the world\'s wonderfully unpredictable wildlife. But, increasingly, there are calls for conservation biology to cast aside its sentimental demons: biodiversity is a commodity that can be bought and sold; conservation is business. ::: {.caption} ###### Box 1. The Cucumber Conflict ::: At the beginning of the 1990s, fishermen in the Galápagos began collecting sea cucumbers from the waters around their islands to meet ongoing demand for these aphrodisiac 'earthworms of the sea' in Southeast Asia ([Figure 3](#pbio-0020310-g003){ref-type="fig"}). Others intent on taking advantage of this commercial opportunity began to arrive from the Ecuadorian mainland in their hundreds. In 1998, Ecuador\'s president signed the Special Law of the Galápagos, which created the Galápagos Marine Reserve, protecting its waters from commercial fishing and imposed restrictions on domestic immigration. But by then, too many were already intent on reaping the financial rewards the sea cucumber promised them---by the end of the decade, a single sea cucumber could fetch nearly \$2. Conservation biologists at the Charles Darwin Research Station on the central island of Santa Cruz worked out levels of fishing that might be sustainable. In 1999---the first season in which sea cucumber fisheries were monitored and regulated---nearly 800 fishermen collected more than 4 million animals worth more than \$3.4 million in a short two-month window. In January 2000, fishermen protesting the closure of the fishery took over offices of the Galápagos National Park Service and Charles Darwin Research Station, holding humans and animals hostage. ::: {#pbio-0020310-g003 .fig} Figure 3 ::: {.caption} ###### The Prized Galápagos Sea Cucumber, Stichopus fuscus Courtesy of Henry Nicholls. :::  ::: ::: {.caption} ###### Box 2. Paying for Forests ::: The longest-running and best-known example of a direct-payment initiative is the Programa de Pago de Servicios Ambientales (PSA) in Costa Rica ([Figure 4](#pbio-0020310-g004){ref-type="fig"}). In the second half of the 20th century, forest cover in Costa Rica fell from around 50% to 25%, and more than half of what remained was on unprotected, privately owned land. In 1996, the PSA was set up to make direct payments to individual landowners, associations of landowners, or indigenous reserves in exchange for 'environmental services'---anything from forest conservation to providing a supply of water. Some 85% of contracts have been for forest conservation. By 2001, more than 2,800 km^2^ were protected by payments of \$4,000 per km^2^ every year, and contracts covering a further 8,000 km^2^ were being processed. Most of the money for these payments comes from a petrol tax on Costa Rican citizens, although the Global Environmental Facility has put up money for biodiversity conservation, Costa Rica\'s Office of Joint Implementation has paid for carbon sequestration, and domestic hydroelectricity and municipal water providers pay for water services. ::: {#pbio-0020310-g004 .fig} Figure 4 ::: {.caption} ###### Forest Protected by Costa Rica\'s PSA Courtesy of Subhrendu Pattanayak. :::  ::: Henry Nicholls is a freelance science writer based in London, United Kingdom. E-mail: <henry.nicholls@absw.org.uk> PSA : Programa de Pago de Servicios Ambientales

PubMed Central

2024-06-05T03:55:47.627477

2004-9-14

PMC516799

Phytohormones: What Are they? {#s1} ============================= Plant growth and development involves the integration of many environmental and endogenous signals that, together with the intrinsic genetic program, determine plant form. Fundamental to this process are several growth regulators collectively called the plant hormones or phytohormones. This group includes auxin, cytokinin, the gibberellins (GAs), abscisic acid (ABA), ethylene, the brassinosteroids (BRs), and jasmonic acid (JA), each of which acts at low concentrations to regulate many aspects of plant growth and development. With the notable exception of the steroidal hormones of the BR group, plant hormones bear little resemblance to their animal counterparts ([Figure 1](#pbio-0020311-g001){ref-type="fig"}). Rather, they are relatively simple, small molecules such as ethylene gas and indole-3-acetic acid (IAA), the primary auxin in the majority of plant species. The concept of plant hormones originates from a classical experiment on phototropism, the bending of plants toward light, carried out by Charles Darwin and his son Francis in 1880. The Darwins were able to demonstrate that when oat seedlings were exposed to a lateral light source, a transported signal originating from the plant apex promoted differential cell elongation in the lower parts of the seedling that resulted in it bending toward the light source. This signal was subsequently shown to be IAA, the first known plant hormone. ::: {#pbio-0020311-g001 .fig} Figure 1 ::: {.caption} ###### Chemical Structures of the Plant Hormones A partial list of the responses elicited by each hormone is provided below. Ethylene gas promotes fruit ripening, senescence, and responses to pathogens and abiotic stresses. IAA (an auxin) regulates cell division and expansion, vascular differentiation, lateral root development, and apical dominance. Cytokinins are adenine derivatives first identified by their ability to promote cytokinesis. JA is a volatile signal that modulates pollen development and responses to pathogen infection. The BRs regulate cell expansion and photomorphogenesis (light-regulated development). GAs are diterpenoid compounds that promote germination, stem elongation, and the induction of flowering. ABA promotes seed dormancy and is involved in several stress signaling pathways. :::  ::: What Do They Do? {#s2} ================ Virtually every aspect of plant growth and development is under hormonal control to some degree. A single hormone can regulate an amazingly diverse array of cellular and developmental processes, while at the same time multiple hormones often influence a single process. Well-studied examples include the promotion of fruit ripening by ethylene, regulation of the cell cycle by auxin and cytokinin, induction of seed germination and stem elongation by GA, and the maintenance of seed dormancy by ABA. Historically, the effects of each hormone have been defined largely by the application of exogenous hormone. More recently, the isolation of hormone biosynthetic and response mutants has provided powerful new tools for painting a clearer picture of the roles of the various phytohormones in plant growth and development. How Do They Work? {#s3} ================= Plant biologists have been fascinated by the regulatory capacity of phytohormones since the time of their discovery, and the notion that hormone levels or responses could be manipulated to improve desired plant traits has long been an area of intense interest. Perhaps the best-known example of this is the isolation of dwarf varieties of wheat and rice that led to the "green revolution" in the second half of the 20th century, which is credited with saving millions of people around the globe from starvation. These dwarf varieties have shorter stems than wild-type, making these plants less susceptible to damage by wind and rain. The molecular isolation of these "dwarfing genes" has revealed that they encode components of the GA biosynthesis and response pathways ([@pbio-0020311-Peng1]; [@pbio-0020311-Sasaki1]). To elucidate the molecular mechanisms underlying phytohormone action, several researchers have utilized the genetically facile model plant Arabidopsis thaliana to isolate mutations that confer altered response to applied hormone. Molecular and biochemical analysis of the gene products defined by these mutations, coupled with expression studies aimed at identifying the downstream target genes that mediate hormonal changes in growth and development, has begun to unlock some of the mysteries behind phytohormone action. While no hormone transduction pathway is completely understood, we now have a rudimentary understanding of many of the molecular events underlying hormone action. Several reviews covering the individual hormone pathways in greater detail have recently been published ([@pbio-0020311-Turner1]; [@pbio-0020311-Gomi1]; [@pbio-0020311-Himmelbach1]; [@pbio-0020311-Kakimoto1]; [@pbio-0020311-Dharmasiri1]; [@pbio-0020311-Guo2]; [@pbio-0020311-Wang1]). Common Themes {#s4} ============= Regulation by proteolysis has emerged as a resounding theme in plant hormone signaling. The ubiquitin-mediated degradation of key regulatory proteins has been demonstrated, or is at least likely, for all of the phytohormone response pathways ([@pbio-0020311-Smalle1]). In the case of auxin, the response pathway is normally subject to repression by a large family of transcriptional regulators called the Aux/IAA proteins ([Figure 2](#pbio-0020311-g002){ref-type="fig"}). These proteins dimerize with members of the auxin response factor (ARF) family of transcription factors, thus preventing ARFs from activating auxin-responsive genes ([@pbio-0020311-Tiwari1]). Upon an auxin stimulus, an SCF (SKP1/Cullin/F-box protein) ubiquitin ligase ([@pbio-0020311-Deshaies1]) containing the TIR1 F-box protein ubiquitinates the Aux/IAA proteins, marking them for degradation by the 26S proteasome thereby de-repressing the response pathway ([@pbio-0020311-Gray1]). The hormone promotes the Aux/IAA--TIR1 interaction; however, the molecular mechanisms behind this regulation are unclear. Most yeast and animal SCF substrates must be post-translationally modified, usually by phosphorylation, before they are recognized by their cognate F-box protein. Despite numerous efforts to identify auxin-induced modification of Aux/IAA proteins, no such signal has been discovered, raising the distinct possibility that auxin uses a novel mechanism to regulate SCF--substrate interactions. ::: {#pbio-0020311-g002 .fig} Figure 2 ::: {.caption} ###### The Ubiquitin-Mediated Proteolysis of Aux/IAA Proteins Regulates Auxin Response \(A) Wild-type Arabidopsis thaliana and the *axr2-1* mutant. *axr2-1* is a dominant gain-of-function mutation in an Aux/IAA gene that confers reduced auxin response. The mutant *axr2-1* protein constitutively represses auxin response because it cannot be targeted for proteolysis by the SCF^TIR1^ ubiquitin ligase. The effect of the mutation on AXR2 stability is shown in a pulse-chase experiment (inset). Wild-type and *axr2-1* seedlings were labeled with ^35^S-methionine and AXR2/axr2-1 protein was immunoprecipitated either immediately after the labeling period (t = 0) or following a 15-minute chase with unlabeled methionine (t = 15). \(B) A simplified model for auxin response. In the absence of an auxin stimulus, Aux/ IAA proteins inhibit ARF transcriptional activity by forming heterodimers. Auxin perception (by an unknown receptor) targets the Aux/IAA proteins to the SCF^TIR1^ complex, resulting in their ubiquitination and degradation, thereby de-repressing the ARF transcription factors. Among the ARF targets are the *Aux/IAA* genes themselves, which produce nascent Aux/IAA proteins that restore repression upon the pathway in a negative feedback loop. :::  ::: Ethylene and cytokinin are both perceived by receptors sharing similarity to bacterial two-component regulators. Common in prokaryotes, but apparently restricted to plants and fungi in eukaryotes, these modular signaling systems involve a membrane-bound receptor containing an intracellular histidine kinase (HK) domain ([@pbio-0020311-Wolanin1]). Ligand binding activates the kinase, resulting in autophosphorylation and initiation of a series of phosphotransfer reactions that culminates with the activation of a response regulator protein that functions as the effector component of the pathway. Cytokinin signaling appears to largely follow this paradigm ([@pbio-0020311-Kakimoto1]). Ethylene response, however, appears more complex ([@pbio-0020311-Guo2]). Ethylene is perceived by a family of five receptors. ETR1 and ERS1 contain a consensus HK domain, however, the HK domains of ETR2, ERS2, and EIN4 are degenerate and lack elements necessary for catalytic activity. This fact, together with studies of "kinase-dead" mutants of *ETR1*, suggests that HK activity is not required for ethylene response. Mutations that abolish ethylene binding in any of the five receptor genes are dominant and confer ethylene insensitivity, indicating that the receptors function as negative regulators of the ethylene pathway. Genetic and molecular studies have positioned these receptors upstream of the Raf-like MAP kinase kinase kinase, CTR1, which interacts with the receptors and also acts as a negative regulator ([Figure 3](#pbio-0020311-g003){ref-type="fig"}). The integral membrane protein, EIN2, and the transcription factors EIN3 and EIL1 are positive regulators of ethylene signaling downstream of CTR1. Current models propose that hormone binding inactivates the receptors, thus resulting in down-regulation of CTR1 activity. Since the identification of CTR1, biologists have speculated that a MAP kinase cascade may be involved. Only recently, however, have putative MAP kinase kinase and MAP kinase components of the ethylene pathway been identified ([@pbio-0020311-Chang1]). Interestingly, these kinases appear to positively regulate ethylene response, suggesting that CTR1 must inhibit their function. If so, this would represent a novel twist on the traditional MAP kinase signaling paradigm. Precisely how the ethylene signal is transduced to the EIN3 and EIL1 transcription factors remains unclear. However, the recent finding that ethylene stabilizes these transcription factors, which are targeted for degradation by an SCF complex in the absence of ethylene, clearly indicates a role for the ubiquitin pathway ([@pbio-0020311-Guo1]; [@pbio-0020311-Potuschak1]). One of the known targets for EIN3 is the ERF1 transcription factor, which activates several genes involved in a subset of ethylene responses. ::: {#pbio-0020311-g003 .fig} Figure 3 ::: {.caption} ###### A Model for the Arabidopsis Ethylene Response Pathway Ethylene is perceived by a family of two-component receptors containing a consensus (unshaded) or degenerate (shaded) HK domain (H). Three of the receptors also contain a C-terminal receiver domain (R). The receptors negatively regulate ethylene response together with CTR1 in a complex on the endoplasmic reticulum membrane. Perception results in reduced receptor and CTR1 activities and activation of a MAP kinase kinase, which transmits the signal through the EIN2 membrane protein, ultimately resulting in the activation of a transcriptional cascade in the nucleus. The EIN3 and EIL1 transcription factors regulate primary response genes including *ERF1*, which activates a subset of secondary ethylene-induced genes involved in defense responses. EIN3/EIL1 abundance is regulated in an ethylene-dependent manner by SCF complexes containing F-box proteins encoded by the ethylene-induced genes *EBF1* and *EBF2*. Positive- and negative-acting components of the pathway are indicated in green and red, respectively. Solid lines indicate regulation that is likely to be through direct interactions. Dotted lines indicate speculative interactions based on genetic studies. :::  ::: Signal Integration and Combinatorial Control {#s5} ============================================ Long ago, plant physiologists noted the apparent antagonistic interactions between some of the phytohormones, such as between auxin and cytokinin in the regulation of root--shoot differentiation and between GA and ABA in germination. Other processes are synergistically regulated by multiple hormones. While it has long been obvious that hormones do not function in discrete pathways, but rather exhibit extensive cross-talk and signal integration with each other and with environmental and developmental signaling pathways, the molecular basis for such coordinated regulation has been unclear. Several recent findings have begun to elucidate the molecular details of some of these events. One example of such signal integration was recently described for the ethylene and JA pathways ([@pbio-0020311-Lorenzo1]). Genetic studies had previously implicated both hormones as important regulators of pathogen defense responses, as well as of the wounding response and other stress-related pathways. Additionally, microarray analysis has identified a large number of genes that are responsive to both hormones. The ERF1 transcription factor was recently found to be an intersection point for these two signaling pathways ([@pbio-0020311-Lorenzo1]). Like ethylene, JA rapidly induces *ERF1* expression, and treatment with both hormones synergistically activates *ERF1*. Induction of *ERF1* by both hormones alone or in combination is dependent upon both signaling pathways, and constitutive overexpression of *ERF1* rescues the defense-response defects of both ethylene- and JA-insensitive mutants. These findings suggest that *ERF1* represents one of the first signaling nodes identified in the complex web of hormonal cross-talk. The auxin and BR pathways also appear to converge and mutually regulate some developmental processes. Both hormones promote cell expansion, and microarray studies have revealed that as many as 40% of all BR-induced genes are also up-regulated by auxin ([@pbio-0020311-Goda1]; [@pbio-0020311-Nemhauser1]). BR is perceived by the cell surface receptor kinase BRI1 ([@pbio-0020311-Wang1]). The SHAGGY/GSK3-type kinase BIN2 acts as a negative regulator of the pathway downstream of the receptor. In the absence of a BR signal, BIN2 phosphorylates the transcription factors BES1 and BZR1, targeting them for proteolysis by the 26S proteasome. Upon a BR stimulus, BIN2 is inactivated, allowing BES1 and BZR1to accumulate in the nucleus, where they are presumably involved in regulating BR-responsive genes. Using combined genetic, physiological, and genomic approaches, [@pbio-0020311-Nemhauser1] were able to demonstrate that auxin and BR regulate Arabidopsis hypocotyl (embryonic stem) elongation in a synergistic and interdependent fashion. Elevating endogenous auxin levels rendered plants more sensitive to BR application in hypocotyl elongation assays, and this response was dependent upon both the auxin and BR signaling pathways. Genetic studies suggest that the convergence of these two pathways occurs at a late point in hormone signaling, perhaps at the promoters of the many genes responsive to both hormones. In support of this notion, bioinformatic analysis identified distinct sequence elements that were enriched specifically in the promoters of auxin-induced, BR-induced, and auxin/BR-induced genes. Many Unanswered Questions {#s6} ========================= While great strides have been made in recent years in understanding the molecular basis of phytohormone action, many fundamental questions remain. Receptors and other upstream signaling components remain to be identified for the majority of the phytohormones. Equally important are the elucidation of hormonal networks and the integration of these networks with the morphogenetic program, such that our understanding of hormone action can be placed in a developmental context. The author wishes to thank members of his lab for helpful comments on this manuscript. Work in the author\'s laboratory on auxin response is supported by National Institutes of Health grant GM067203 and the Mcknight Foundation. William M. Gray is in Plant Biology at the University of Minnesota, St. Paul, Minnesota, United States of America. E-mail: <grayx051@tc.umn.edu> ABA : abscisic acid ARF : auxin response factor BR : brassinosteroid GA : gibberellin HK : histidine kinase IAA : indole-3-acetic acid JA : jasmonic acid SCF : SKP1/Cullin/F-box protein

PubMed Central

2024-06-05T03:55:47.629164

2004-9-14

PMC516800

Background ========== The evaluation of the complex regulatory networks underlying molecular processes poses a major challenge to current research. With modern experimental methods in the field of gene expression, it is possible to monitor mRNA abundance for whole genomes \[[@B1],[@B2]\]. To elucidate the functional relationships inherent in this data, a commonly used approach is the clustering of co-expressed genes \[[@B3]\]. In this context, the choice of the similarity measure used for clustering, as well as the clustering method itself, is crucial for the results obtained. Often, linear similarity measures such as the Euclidean distance or Pearson correlation are used in an ad-hoc manner. By doing so, it is possible that subsets of non-linear correlations contained in a given dataset are missed. Therefore, information theoretic concepts, such as mutual information, are being used to extend more conventional methods in various contexts ranging from expression \[[@B4]-[@B8]\] and DNA sequence analysis \[[@B9],[@B10]\], to reverse engineering \[[@B11]\] and independent component analysis \[[@B12],[@B13]\]. Also aside the bioinformatics field, mutual information is widely utilised in diverse disciplines, such as physics \[[@B14]\], image recognition \[[@B15]\], speech recognition \[[@B16]\], and various others. In extension to other similarity measures, mutual information provides a general measure of statistical dependence between variables. It is thereby able to detect any type of functional relationship, extending the potentialities of linear measures as illustrated in Figure [1](#F1){ref-type="fig"}. In this work, we discuss mutual information as a measure of similarity between variables. In the first section, we give a short introduction into the basic concepts including a brief description of the commonly used approaches for numerical estimation from continuous data. In the following section, we then present an algorithm for estimating mutual information from finite data. The properties arising from this approach are compared to previously existing algorithms. In subsequent sections, we then apply our concept to large-scale cDNA abundance datasets and determine if these datasets can be sufficiently described using linear measurements or if a significant amount of non-linear correlations are missed. Mutual information ------------------ Mutual information represents a general information theoretic approach to determine the statistical dependence between variables. The concept was initially developed for discrete data. For a system, *A*, with a finite set of *M*possible states {*a*~1~, *a*~2~, \... , }, the Shannon entropy *H*(*A*) is defined as \[[@B17]\]  where *p*(*a~i~*) denotes the probability of the state *a~i~*. The Shannon entropy is a measure for how evenly the states of *A*are distributed. The entropy of system *A*becomes zero if the outcome of a measurement of *A*is completely determined to be *a~j~*, thus if *p*(*a~j~*) = 1 and *p*(*a~i~*) = 0 for all *i*≠ *j*, whereas the entropy becomes maximal if all probabilities are equal. The joint entropy *H*(*A, B*) of two systems *A*and *B*is defined analogously  This leads to the relation *H*(*A, B*) ≤ *H*(*A*) + *H*(*B*)     (3) which fulfils equality only in the case of statistical independence of *A*and *B*. Mutual information *MI*(*A, B*) can be defined as \[[@B17]\] *MI*(*A, B*) = *H*(*A*) + *H*(*B*) - *H*(*A, B*) ≥ 0     (4) It is zero if *A*and *B*are statistically independent and increases the less statistically independent *A*and *B*are. If mutual information is indeed to be used for the analysis of gene-expression data, the continuous experimental data need to be partitioned into discrete intervals, or bins. In the following section, we briefly review the established procedures; a description of how we have extended the basic approach will be provided in the subsequent section. Estimates from continuous data ------------------------------ In the case of discrete data the estimation of the probabilities *p*(*a~i~*) is straightforward. Many practical applications, however, supply continuous data for which the probability distributions are unknown and have to be estimated. In a widely used approach \[[@B7]\], the calculation of mutual information is based on the binning of data into *M*discrete intervals *a~i~*, *i*= 1\... *M~A~*. For experimental data consisting of *N*measurements of a variable *x~u~*, *u*= 1\... *N*, an indicator function Θ~*i*~counts the number of data points within each bin. The probabilities are then estimated based on the relative frequencies of occurrence  with  For two variables the joint probabilities  are calculated analogously from a multivariate histogram. Additionally it has been suggested \[[@B14]\] to adaptively choose the sizes of the bins, so that each bin constructed nearly has a uniform distribution of points. In a different approach, kernel methods are used for the estimation of the probability density of Eq. (5) \[[@B18]-[@B20]\]. Entropies are then calculated by integration of the estimated densities. Recently, an entropy estimator  was suggested \[[@B21]\] and showed in an extensive comparison to other commonly used estimators to be superior. Results ======= Fuzzy mutual information ------------------------ In the classical binning approach, described above, each data point is assigned to one, and only one, bin. For data points near to the border of a bin, small fluctuations due to biological or measurement noise might shift these points to neighbouring bins. Especially for datasets of moderate size, the positions of the borders of the bins can thereby strongly affect the resulting mutual information \[[@B18]\]. In a manner analogous to kernel density estimators (KDE), we now present a generalisation to the classical binning in which we aim to overcome some of the drawbacks associated with the simple approach. Within our algorithm, we allow the data points to be assigned to several bins simultaneously. For this, we extended the indicator function Θ(*x*) to the set of polynomial B-spline functions. Here, we do not provide the mathematical details for these functions since they have been discussed extensively in the literature \[[@B22]-[@B24]\], but rather focus on the practical applicability. Within the B-spline approach, each measurement is assigned to more than one bin, *i*, with weights given by the B-spline functions *B*~*i,k*~. The spline order *k*determines the shape of the weight functions and thereby the number of bins each of the data points is assigned to. A spline order *k*= 1 corresponds to the simple binning, as described in the previous section: Each data point is assigned to exactly one bin (Figure [2](#F2){ref-type="fig"}, left). For *k*= 3, each data point is assigned to three bins, with the respective weights given by the values of the B-spline functions at the data point (Figure [2](#F2){ref-type="fig"}, right). B-spline functions ------------------ The first step in the definition of the B-spline functions is the definition of a knot vector *t*~*i*~for a number of bins *i*= 1\... *M*and one given spline order *k*= 1\... *M*- 1 \[[@B22]\]  where the spline order determines the degree of the polynomial functions. The domain of the B-spline functions lies in the interval *z*∈ \[0, *M*- *k*+ 1\]. To cover the range of the variables, the new indicator function based on the B-spline functions needs to be linearly transformed to map their range. The recursive definition of the B-spline functions are as follows \[[@B22]\]  An important property of B-spline functions is the implicit standardisation of coefficients: All weights belonging to one data point sum up to unity. Algorithm --------- ### Input • Variables *x*and *y*with values *x*~*u*~and *y*~*u*~, *u*= 1\... *N* • Bins *a*~*i*~, *i*= 1\... *M*~*x*~and *b*~*j*~, *j*= 1\... *M*~*y*~ • Spline order *k* ### Output • Mutual information between variable *x*and *y* ### Algorithm 1\. Calculation of marginal entropy for variable *x* \(a) Determine  with  \(b) Determine *M*~*x*~weighting coefficients for each *x*~*u*~from  \(c) Sum over all *x*~*u*~and determine *p*(*a*~*i*~) for each bin *a*~*i*~from  \(d) Determine entropy *H*(*x*) according to Eq. (1) 2\. Calculation of joint entropy of two variables *x*and *y* \(a) Apply steps 1 (a) and (b) to both variables *x*and *y*, independently \(b) Calculate joint probabilities *p*(*a*~*i*~, *b*~*j*~) for all *M*~*x*~× *M*~*y*~bins according to  \(c) Calculate the joint entropy *H*(*x,y*) according to Eq. (2) 3\. Calculate the mutual information *MI*(*x,y*) according to Eq. (4) Example ------- We show the estimation with the standard binning and our approach ex-emplarily on two artificial variables *x*= 0.0,0.2,0.4,0.6,0.8,1.0 and *y*= 0.8,1.0,0.6,0.4,0.0,0.2 for *M*= 3 bins, spline order *k*= 2, and the logarithm to basis two. ### Simple binning For both variables, each of the three histogram bins contains two values *p*(*a*~1~) = *p*(*a*~2~) = *p*(*a*~3~) = , analogously for *p*(*b~i~*) due to the symmetry of data *H*(*x*) = *H*(*y*) =  = log~2~3 ≈ 1.58. For the calculation of the joint probability, three of the nine two dimensional bins contain two values each *p*(*a*~1~, *b*~3~) = *p*(*a*~2~, *b*~2~) = *p*(*a*~3~, *b*~1~) =  resulting in *H*(*x, y*) = log~2~3 and *MI*(*x, y*) = log~2~3. ### B-spline approach The modified indicator function  is determined to *B*~*i,k*~(2*x*) according to Eq. (9) (rule 1(a)). For each value *x*~*u*~three weighting coefficients are determined (rule 1(c)) and probabilities are calculated (rule 1(d)) (Table [1](#T1){ref-type="table"}). The analogous procedure is applied to variable *y*and the single entropies are calculated to *H*(*x*) = *H*(*y*) = Iog~2~(10) - 0.61og~2~(3) - 0.41og~2~(4) ≈ 1.57. Both, *H*(*A*) and *H*(*B*), are slightly smaller than the entropies calculated from the simple binning. The joint probabilities are *p*(*a*~1~, *b*~1~) = *p*(*a*~3~, *b*~3~) = 0, *p*(*a*~1~, *b*~2~) = *p*(*a*~2~, *b*~1~) = *p*(*a*~2~, *b*~3~) = *p*(*a*~3~, *b*~2~) = 0.56/6, *p*(*a*~1~, *b*~3~) = *p*(*a*~3~, *b*~1~) = 1.24/6, *p*(*a*~2~, *b*~2~) = 1.28/6 (rule 2 (b)) resulting in *H*(*x,y*) = 2.7 and *MI*(*x,y*) = 0.45. In the next sections, we discuss some of the properties arising from the utilisation of B-spline functions for the estimation of mutual information and compare our approach to other commonly used estimators. We support this discussion using examples for which the underlying distributions and thereby the true mutual information is known. Size of data ------------ It has been discussed elsewhere \[[@B25]-[@B28],[@B20]\] that the estimated mutual information is systematically overestimated for a finite size of *N*data points. For the simple binning approach, the mean observed mutual information can be calculated explicitly as the deviation from the true mutual information  As can be seen for an example of artificially generated equidistributed random numbers (Figure [3](#F3){ref-type="fig"}, left), mutual information calculated from the simple binning scales linearly with 1/*N*, with the slope depending on the number of bins *M*in accordance with Eq. (12). Figure [3](#F3){ref-type="fig"} shows that this scaling is preserved for the extension to B-spline functions, while the slope is significantly decreased for *k*= 3, compared to the estimation with the simple binning (*k*= 1). Mutual information calculated from KDE does not show a linear behaviour but rather an asymptotic one with a linear tail for large datasets. The values are slightly increased compared to the ones from the B-spline approach. The entropy estimator  gives values comparable to the ones obtained from the B-spline approach. More importantly, a similar result also holds for the standard deviation of mutual information. As shown in Figure [3](#F3){ref-type="fig"} (right), the standard deviation of the mutual information estimated with the simple binning (*k*= 1) scales with 1/*N*for statistically independent events \[[@B26],[@B29]\]. For the B-spline approach (*k*= 3), this scaling still holds, but the average values are decreased significantly. For the KDE approach, an asymptotic run above the values from the B-spline approach is observed, again with linear tail for large datasets.  shows a linear scaling slightly below the simple binning. The spline order ---------------- The interpretation of any results obtained from the application of mutual information to experimental data is based on testing to see if the calculated results are consistent with a previously chosen null hypothesis. By following the intuitive approach that the null hypothesis assumes the statistical independence of variables, mutual information is tested against a surrogate dataset, which is consistent with this null hypothesis. As discussed previously in more detail \[[@B20]\], one way of generating such a surrogate dataset is by random permutations of the original data. From the mutual information of the original dataset *MI*(*X,Y*)^data^, the average value obtained from surrogate data \<*MI*(*X*^surr^, *Y*^surr^) \>, and its standard deviation σ^surr^, the significance *S*can be formulated as  For each *S*the null hypothesis can be rejected to a certain level α depending on the underlying distribution. With increasing significance the probability of false positive associations drops. In the following, we address the influence of the spline order and the number of bins on the estimation of mutual information. Based on 300 data points of an artificially-generated dataset drawn from the distribution shown in Figure [1](#F1){ref-type="fig"}, we calculate the mutual information for *M*= 6 bins and different spline orders *k*= 1\... 5 (Figure [4](#F4){ref-type="fig"}, left). From 300 shuffled realisations of this dataset, the mean and maximum mutual information are shown with the standard deviation as error-bars. For all spline orders the null hypothesis can be rejected, in accordance with the dataset shown in Figure [1](#F1){ref-type="fig"}. To estimate the strength of the rejection, we calculate the significance according to Eq. (13) (Figure [4](#F4){ref-type="fig"}, right). It can be observed that the largest change in the significance of the mutual information occurs in the transition from *k*= 1 (simple boxes) to *k*= 2 with an increase by roughly two-fold. Using more sophisticated functions (*k*≥ 3) does not further improve the significance. Similar findings have been reported in the context of kernel density estimators \[[@B19]\]. The major contribution leading to this increase of the significance is given by the distribution of surrogate data which becomes more narrow for *k*\> 1 leading to smaller standard deviations σ^surr^. The same dataset is used to show the dependency of mutual information on the number of bins for two spline orders *k*= 1 and *k*= 3 (Figure [5](#F5){ref-type="fig"}). Mutual information estimated from data as well as from surrogate data shows a robust run without strong fluctuations within the range of bins shown. From this we can conclude that the choice of the number of bins does not affect the resulting mutual information notably as long as it is chosen to be within a reasonable range. Again, the significance is calculated (Figure [6](#F6){ref-type="fig"}) and compared to the significances obtained from the KDE approach and the  estimator. It can be observed that the significance of the mutual information calculated with B-spline functions increased roughly by two-fold compared to the simple binning. The significance obtained from KDE is not depending on *M*and was determined to be similar to the significance estimated from the B-spline approach. The numerically expensive integration of KDE, however, limits the size of utilisable datasets. The KDE run time requirements were (10^4^) times higher than the ones from the B-spline approach. Strategies to simplify the integration step were proposed \[[@B20]\] but have to be used with caution since they assume particular properties of the distribution of experimental data that are in general not fulfilled. The recently introduced entropy estimator  produces intermediate significances between the ones from the binning and the B-spline approach for higher bin numbers. For low bin numbers, the significances are relatively poor. Application on data ------------------- We now turn to the analysis of experimentally measured gene expression data. As shown previously, the application of mutual information to large-scale expression data reveals biologically-relevant clusters of genes \[[@B7],[@B30]\]. In this section, we will not repeat these analyses, but determine if the correlations detected using mutual information are missed using the established linear measures. Among the most frequently used measures of similarity for clustering co-expressed genes are the Euclidean distance and the Pearson correlation coefficient *R*\[[@B3]\]. If correlations are well described by the Pearson correlation and the distribution of data is approximately Gaussian like, the relationship between the mutual information and the Pearson correlation given by \[[@B32]\]  is expected to be fulfilled. Therefore, we calculated both, the mutual information and the Pearson correlation, for two large-scale gene expression datasets (Figure [7](#F7){ref-type="fig"}). For each pair of genes *X*and *Y*we plot the tuple (*MI*(*X,Y*), *R*(*X,Y*)). In order to address significance, we additionally calculate all tuples from shuffled data. The first dataset contains cDNA measurements for *S. cerevisiae*for up to *E*~1~= 300 experiments \[[@B31]\]. To avoid numerical effects arising from different numbers of defined expression values (missing data points) for each gene, we exclusively utilised genes that are fully defined for all experimental conditions resulting in *G*~1~= 5345 genes. Analysis on this dataset using mutual information has been done before \[[@B20],[@B32]\] on rank-ordered data. The rank-ordering lead to homogeneously distributed data and thereby enabled the application of a simplified algorithm for the numerical estimation from kernel density estimators. The utilisation of our B-spline approach allows us to extend this analysis to non rank-ordered data thereby keeping the original distribution of experimental data. In contrast to the previous studies we find for non rank-ordered data that the theoretical prediction of Eq. 14 is no longer a lower bound for the comparison. Many tuples with high Pearson correlation but low mutual information can be detected arising from outlying expression values (Figure [8A](#F8){ref-type="fig"}). However, pairs of genes with high mutual information and low Pearson correlation, thus indicating a non-linear correlation, are not observed. The only remarkable tuple (marked with an arrow in Figure [7](#F7){ref-type="fig"} and shown in Figure [8B](#F8){ref-type="fig"}) also arises from outlying values. The second dataset contains cDNA measurements for *E*~2~= 102 experiments on G~2~= 22608 genes derived from 20 different human tissues \[[@B33]\]. In contrast to the first dataset, tuples with low Pearson correlation but high mutual information are indeed detected. For two exemplary chosen tuples (Figure [8C](#F8){ref-type="fig"} and [8D](#F8){ref-type="fig"}), clusters of experimental conditions can be clearly detected by eye. Such type of correlations are missed by analyses based exclusively on linear measures, such as the the analysis done in the original publication of this dataset. For both datasets, tuples calculated from shuffled data (Figure [7](#F7){ref-type="fig"}, blue data points) result in small values for both similarity measures. Thereby, they indicate a high significance of the original associations. Peaks with high Pearson correlation in the first dataset arise from gene-gene associations with outlying values. Significance values for the exemplarily chosen pairs of genes of the second dataset (Figure [8C](#F8){ref-type="fig"}, and [8D](#F8){ref-type="fig"}) were explicitly calculated (Figure [9](#F9){ref-type="fig"}). They show high significance values for the two examples of observed non-linear correlations on the basis of the mutual information. Compared to this, the significances calculated from the Pearson correlation are poor. In summary, our analysis confirms for the first dataset that the Pearson correlation does not miss any non-linear correlations. As a side effect we are able to detect gene-gene pairs containing outlying values. For the second dataset, however, a substantial amount of non-linear correlations was detected. Gene-gene pairs exemplarily chosen from this fraction show a clustering of data points (experiments) with a high significance. Even though such patterns can be easily found by eye, computational methods need to be applied for the inspection of several hundred million comparisons. Discussion and conclusion ========================= After a brief introduction into the information theoretic concept of mutual information, we proposed a method for its estimation from continuous data. Within our approach, we extend the bins of the classical algorithm to polynomial B-spline functions: Data points are no longer assigned to exactly one bin but to several bins simultaneously, with weights given by the B-spline functions. By definition, the weighting coefficients for each data point automatically sum up to unity. Though our algorithm is reminiscent of kernel density estimators \[[@B18]\], it keeps the basic idea to associate data points to discrete bins. In this way, we are able to avoid time-consuming numerical integration steps usually intrinsic to estimates of mutual information using kernel density estimators \[[@B20]\]. To show that our approach improves the simple binning method and to compare it to KDE and the recently reported estimator , we provided a systematic comparison between all these algorithms for artificially generated datasets, drawn from a known distribution. We found that mutual information, as well as its standard deviation, scales linearly with the inverse size of a dataset for the standard binning method, for the B-spline approach, and for . For the KDE approach we find an asymptotic behaviour with a linear tail for large datasets. Moreover, the discrimination of correlations from the hypothesis of statistical independence is significantly improved by extending the standard binning method to B-spline functions, as shown by a two-fold increase of the significance. Compared to KDE, the B-spline functions produce similar significances. However, due to the numerical expenses of the KDE, an application of this algorithm is limited to datasets of mod-erate size. The application of  leads to significances in-between the standard binning and the B-spline approach for reasonable bin numbers. Linear correlation measures are among the most applied measures of similarity in the literature. Often, they are used on an ad-hoc basis and it is unclear whether a considerable number of non-linear correlations are missed. Here, we asked the question whether previous analyses, based on linear correlations, sufficiently described the correlations within gene expression datasets or whether mutual information detects additional correlations that are not detected by linear measures, such as the Pearson correlation. For data that is well described by the Pearson correlation, we can give the relation of the Pearson correlation to the mutual information explicitly \[[@B32]\]. Both measures were then applied to publicly available large-scale gene expression datasets \[[@B31],[@B33]\]. We aimed to verify whether non-linear correlations shown as deviations from this relation can be detected. Our findings show that the first dataset is fairly well described by the given relation of the Pearson correlation to the mutual information. No data points with high mutual information and low Pearson correlation are detected. Comparisons of genes containing outlying values, however, result in deviations with low mutual information and high Pearson correlation. From this, it follows that previous analyses on this dataset, based on Pearson correlation, did not miss any non-linear correlations. This presents an important finding since it is by all means supposable that the regulations inherent in the genetic network under consideration might show more complex behaviour than the observed linear ones. Even for one of the largest expression datasets at hand, insufficient data might complicate the detection of such complex patterns of regulation. Alternatively, the biological mechanisms which underlay the regulatory networks might not lead to non-linear correlations. It also has to be considered that the experimental methods applied for the generation of this dataset may make non-linear correlations difficult to detect. The second dataset, in contrast, reveals highly significant tuples with high mutual information and low Pearson correlation. Detailed gene-gene plots of such tuples show that the expression values of the contributing genes fall into groups of experimental conditions. Without attempting to draw conclusions about the biological context of such clusters here, they might reflect interesting situations worth to be analysed in detail. Authors\' contributions ======================= Most of the manuscript text was written by CD and edited by all authors. CD carried out the calculations and produced the figures. RS strongly contributed to the theoretical background of entropy and mutual information. The implementation of the C++ program was carried out by SK. JS and SK supervised this work. All authors read and approved the final manuscript. Acknowledgements ================ The authors would like to thank Joachim Kopka and Janko Weise for stimulating discussions and Megan McKenzie for editing the manuscript (all of the MPI-MP). RS acknowledges financial support by the HSP-N grant of the the state of Brandenburg. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Two datasets *X*and *Y*(100 data points) show a hypothetical dependency *f*(*x*) = 4*x*(1 - *x*) (top). The Pearson correlation coefficient is not able to detect a significant correlation as shown in the histogram plot of the dataset compared to 300 realisations of shuffled data (left). Mutual information clearly shows that the two datasets are not statistically independent (right). :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The continuous experimental data for the variable *x*needs to be binned for the calculation of mutual information. The indicator function of Eq. (5) counts the number of data points within each bin (example with *M*~*x*~= 5 bins, left). The generalised indicator function based on B-spline functions of Eq. (8) extends the bins to polynomial functions (example with *M*~*x*~= 5 bins and spline order *k*= 3, right). The bins now overlap and the weight of each data point to each of the bins is given by the value of the respective B-spline functions at the data point. By definition, all weights contributing to one data point sum up to unity. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Mutual information is estimated for artificially generated equidis-tributed random numbers from the simple binning (*k*= 1), the B-spline approach (*k*= 3), and the entropy estimator  using *M*= 6 bins, and additionally from the kernel density estimator. The average over an ensemble of 600 trials is shown as a function of the size of the dataset (left) together with the standard deviation (right). :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Mutual information calculated for a dataset of 300 data points drawn from the distribution shown in Figure 1 (crosses). The number of bins was fixed to *M*= 6. The average mutual information for 300 shuffled realisations of the dataset is shown (circles) together with the standard deviation as error-bars. The largest value found within the ensemble of shuffled data is drawn as a dotted line (left). The significance was calculated from Eq. (13) (right). :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Based on the distribution of Figure 1, the mutual information for 300 data points and two spline orders *k*= 1 and *k*= 3 is shown as a function of the number of bins *M*(crosses) together with mean (circles) and standard deviations (error-bars) of 300 surrogates. The dotted lines indicate the largest mutual information found within the ensemble of surrogate data. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### The significance, *S*, as a function of the number of bins, *M*, for the two examples of Figure 5, and for the entropy estimator . For kernel density estimators (KDE), the significance, which is not depending on *M*, is calculated to *S*= 92. :::  ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### The Pearson correlation coefficient and the mutual information for all pairwise comparisons of genes for two large-scale gene expression datasets are shown (black points) overlayed by the same measures obtained from shuffled data (blue points). The expected mutual information calculated from Eq. (14) is shown as read curve. For the first dataset (left) genes containing undefined values were omitted resulting in 5345 genes measured under 300 experimental conditions \[31\]. For the second dataset (right) containing 22608 genes measured under 102 experimental conditions \[33\], a representative fraction is shown. :::  ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Examples of gene-gene plots for genes *X*and *Y*are shown for characteristic tuples (*MI*(*X,Y*), *R*(*X,Y*)) detected in Figure (7). For the first gene expression dataset under consideration \[31\], no non-linear correlations are detected. Moreover, tuples with high Pearson correlation and low mutual information, examples A and B, resulting from outlying values are detected. For the second dataset \[33\], however, tuples with low Pearson correlation and high mutual information are observed, see examples C and D. Such non-linear correlations are missed by solely using linear correlation measures. :::  ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Significance values for the two gene-gene comparisons shown in Figure 8, C and D (top and bottom, respectively) are calculated from 300 shuffled realisations based on the Pearson correlation coefficient (left) and the mutual information (right) as distance measures. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### For the calculation of probabilities *p*(*a~i~*) according to the B-spline approach, *M*~*x*~weighting coefficients are determined for each value *x*~*u*~of variable *x*. ::: *B*~*i*~=1,*k*=2(*x*~*u*~) *B*~*i*~=2,*k*=2(*x*~*u*~) *B*~*i*~=3,*k*=2(*x*~*u*~) --------------- ---------------------------- ---------------------------- ---------------------------- *x*~1~ 1.0 0.0 0.0 *x*~2~ 0.6 0.4 0.0 *x*~3~ 0.2 0.8 0.0 *x*~4~ 0.0 0.8 0.2 *x*~5~ 0.0 0.4 0.6 *x*~6~ 0.0 0.0 1.0 *p*(*a*~*i*~) 1.8/6 2.4/6 1.8/6 :::

PubMed Central

2024-06-05T03:55:47.630792

2004-8-31

PMC517399

Background ========== Hippocampal synapses display a variety of activity dependent changes that may represent basic elements of memory. Of foremost interest are long-term potentiation (LTP) and depression (LTD), especially forms that depend on N-methyl-D-aspartate (NMDA) receptor activation and therefore can attain \"associative\" properties \[[@B1]-[@B3]\]. The selective induction of LTP versus LTD has been attributed to differing amounts of Ca^2+^ions entering via postsynaptic NMDA receptor channels \[[@B4]\]. Depending on type of stimulation, enzymes with different sensitivities to Ca^2+^may be engaged and change the balance between kinase and phosphatase activities, leading to either phosphorylation or dephosphorylation of postsynaptic target proteins, such as ionotropic receptors \[[@B2]\]. It has been shown that afferent stimulation by frequencies in the range 0.5 to 5 Hz reliably produces LTD whereas higher frequencies, 50--100 Hz, lead to LTP \[[@B5]\]. Several studies suggest that temporal factors are also important, implying that LTD requires a longer time to be induced than LTP \[[@B6]\]. We have previously demonstrated that under conditions of facilitated activation of NMDA receptors by low extracellular Mg^2+^synaptic plasticity can be induced by frequencies as low as 0.1--0.2 Hz when applied for prolonged periods of time \[[@B7]\]. Following an initial phase of transient potentiation there was a substantial depression that developed gradually during several hours and that remained stable after termination of NMDA receptor activation. Although the relation to \"standard LTD\" was not fully clarified, such slowly developing depression in low Mg^2+^solution may provide a useful model for studying certain forms of NMDA receptor dependent depression. In the present study, we will further develop the concept of gradually decaying responses. One critical issue regarding LTP, LTD as well as other forms of glutamatergic synaptic plasticity, is the relative contribution of different glutamate receptor subtypes in creating the synaptic modification. Knowledge about this matter may be helpful in elucidating the underlying modification. While a selective change of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors has been cherished \[[@B8]-[@B10]\], especially in the case of LTP, several studies also observed NMDA receptor mediated changes in both LTP and LTD \[[@B11]-[@B14]\]. Previous work on LTD in our lab described an equal change of AMPA and NMDA responses \[[@B15]\]. However, it was reported by others that the relative contributions of AMPA and NMDA responses during LTD depend on experimental conditions, an equal change being one possible outcome \[[@B12]\]. In our recent examination of a slowly developing depression using composite AMPA-NMDA excitatory postsynaptic potentials (EPSPs) \[[@B7]\], the two responses declined in close parallel, indicating a common factor. Such an equal change is compatible with both a coordinated change of receptors and a presynaptic one via a decrease of glutamate release. However, in view of other studies reporting a coupling between responses via AMPA and NMDA receptors \[[@B16],[@B17]\], one may ask whether our observation of declining NMDA responses could be secondary to the change of AMPA. In the present study, isolated NMDA receptor mediated EPSPs were shown to decline progressively during prolonged low frequency activation (0.1 Hz). Moreover, following sudden start of stimulation there was an initial, transient potentiation. Our findings also resolved some questions regarding input specificity and durability of the slow decay, which were previously addressed only for AMPA EPSPs. Results ======= Isolated NMDA EPSPs show a progressive decay -------------------------------------------- AMPA EPSPs were initially recorded in low Mg^2+^solution in the presence of NMDA receptor antagonist AP5 to allow for pathway equalization (see Methods) without evoking NMDA EPSPs. Synaptic transmission was then entirely blocked by adding AMPA receptor antagonist CNQX, followed by unblocking of NMDA receptors by wash out of AP5. During this time, only one pathway was stimulated, keeping the other one silent for later use. As illustrated in Fig. [1A](#F1){ref-type="fig"} (upper part), an NMDA receptor mediated EPSP appeared within 10 min and reached maximum about 30 min after switching to AP5-free solution. During the following recording period of nearly 2 h, the NMDA EPSP decayed substantially, on average down to 58 ± 6 % of peak value (n = 8). Control experiments showed that isolated AMPA EPSPs recorded in low Mg^2+^remained stable for several hours (changing to 96 ± 5 % of baseline after 2 h, n = 5, not illustrated). Reinduction in a naive pathway ------------------------------ To exclude the possibility that the observed decay of NMDA responses were due to deterioration of slices, implying a general decrease of essential physiological processes, the experiment was repeated for the other pathway, i.e. the one that had not previously expressed NMDA responses (continuation in the same set of 8 slices). As seen in Fig. [1A](#F1){ref-type="fig"} (lower part), a similar result was obtained as above. The NMDA EPSP peaked at 98 ± 7 % and declined to 63 ± 8 % relative to the peak in the first experimental session. As illustrated in Fig. [1B](#F1){ref-type="fig"}, the curve obtained for a naive pathway during the second session of NMDA unblocking was similar to the one obtained for the pathway activated during the first session, the two curves overlapping closely for the entire recording period. Comparison between pathways --------------------------- ### Specificity and persistency The same experiment was used to address the question of persistency as well as input specificity of the slowly developing depression of NMDA EPSPs. As can be seen in Fig. [1A](#F1){ref-type="fig"}, the pathway that was active during the first experimental session was retested during the second one together with the pathway receiving novel NMDA receptor activation. This allowed for a comparison between the pathways (for convenience, the peak in the first session is still used as reference for the values in the following). During the second session of NMDA receptor unblocking, the previously treated pathway displayed a substantially smaller peak than the naive one (48 ± 3 % vs. 98 ± 7 % p \< 0.05, n = 8), and the two curves were still different by the end of the session (44 ± 3 % vs. 63 ± 8 %; difference 19 ± 4 %, p \< 0.05). Since the latter time point was located at 3 h after the end of the first session it is evident that the depression of NMDA EPSPs lasted for at least 3 h. It is noteworthy that the previously depressed pathway showed no significant decay during the second session, passing from 48 to 44 %, i.e. a relative change by 92 % (p \> 0.05), as if it had already been saturated. For a comparison, the naive pathway changed by a factor 63/98 = 64 % (p \< 0.05) (see curves in Fig. [1C](#F1){ref-type="fig"}). A graphic summary of all \"peak\" and \"end-of-session\" values is given in Fig. [3B](#F3){ref-type="fig"}. It can also be noted that NMDA EPSPs recorded for contiguous time intervals up to 4 h reached a saturation level after 2--3 h (n = 3, not illustrated). ### Instantaneous versus persistent depression The above results suggest that the progressive decay observed in a single pathway as an instant event actually represents a long-term, pathway-specific change that can be assessed long later by comparison across pathways. To further examine the relation between the instantaneously recorded depression and the one measured about 90 min later, the relation between the two was plotted as illustrated in Fig. [3C](#F3){ref-type="fig"}. The two variables were found to be positively correlated (r = 0.71, p \< 0.05, n = 8), implying that depression to a lower level in one pathway led to smaller responses in that pathway at later times compared to another pathway. The regression line, passing below rather than through the point of no depression (100 %, 100 %), indicates that even a slight instantaneous decay may be coupled to a noticeable change in the long term. Possibly, the declining trend was partially masked by recovery from AP5 leading to an underestimation of it. Effect of NMDA receptor blockade on subsequent NMDA EPSP decay -------------------------------------------------------------- To pinpoint the induction mechanism, in terms of a pre- versus postsynaptic location, experiments were carried out in a similar way as above except for keeping AP5 in the solution during the first session (see Fig. [2A](#F2){ref-type="fig"}). Hence, the stimulus pattern included a 3 h long interval with no stimulation in one of the pathways. The other pathway was stimulated during that time, and most likely releasing glutamate, but no postsynaptic response was expressed due to blockade of NMDA receptors. It can be argued that successful blockade of depression would predict a postsynaptic mechanism whereas a failure to do so would predict a presynaptic one. Fig. [2B](#F2){ref-type="fig"} shows that the depression was actually blocked, demonstrating the importance of NMDA receptor activation in the induction process. As illustrated, the two curves obtained during unblocking of NMDA receptors in the second session were quite similar. The continuously stimulated pathway, being depressed in the standard case, peaked at a level of 105 ± 5 % relative to the control pathway (n = 5; see also graphic summary of values in Fig. [3B](#F3){ref-type="fig"}). Phase trajectories as indicators of waveform change --------------------------------------------------- Depending on the type of synaptic modification, EPSP waveforms may change in different ways, and previous work in our lab has demonstrated that NMDA EPSPs are more prone than AMPA EPSPs to show these changes \[[@B18]\]. Taking advantage of such waveform analysis, which might shed light on the underlying mechanism, we examined phase plots based on measures of the initial part and the later part of the NMDA EPSP (see Methods) on the X- and Y-axis, respectively. The curve in Fig. [3D](#F3){ref-type="fig"} is based on a total of 20 experiments (pathways), including some with only a single session. As shown, the phase trajectory displayed a loop indicating a difference between the effect of AP5 and the gradual decay of NMDA EPSPs. On the average the encircled area was 15 ± 2 % (p \< 0.05, n = 20; scaling peak × peak as 100 %, clockwise being positive). While our data show that the time window matters for measuring NMDA EPSPs, all the above results were qualitatively similar regardless of which window was used. It can be noted, however, that the depression of NMDA EPSPs by the end of a recording session was less pronounced using early measurements than late ones (responses attaining 72 ± 3 % vs. 61 ± 3 % of the peak value, p \< 0.05, n = 20). In 12 out of the 20 experiments, the fiber volley was well separated from the stimulus artifact allowing it to be properly measured. No significant change was detected, the end-of session value amounting 103 ± 2 % of the value at the EPSP peak (p \> 0.05, not illustrated). Short-term effects induced by sudden onset stimulation ------------------------------------------------------ In the above, NMDA receptor activation occurred gradually while the antagonist AP5 was washed out. This is in line with the experimental protocol used in our previous work on composite EPSPs containing both AMPA and NMDA components \[[@B7]\]. However, a natural question is whether sudden, novel activation of NMDA receptors is equivalent in producing the results observed here. We therefore pursued experiments in which stimulation was silenced until washout of AP5 was complete. One pathway, receiving such sudden stimulation, was compared to a control pathway subjected to gradual NMDA receptor activation during a single recording session. Fig. [4A](#F4){ref-type="fig"} shows an essential difference in behavior between the pathways, the sudden start of activation leading to substantially larger responses for about 5 min. Fig. [4B](#F4){ref-type="fig"} reveals additional complexity, the initial responses showing actual growth of responses for about a minute before they started to decay, implying an early potentiation process. The total range of responses was substantial, from a peak above 200 % to about 70 % by the end of the recording session (relative to the peak of the control pathway), i.e. about 3 times. In order to determine whether the transient potentiation had any obvious relation to the slow depression of NMDA EPSPs, the relation between the two was examined. Thus, the degree of initial potentiation was calculated by comparing the pathways just after stimulation was started and the depression was determined, as before, by comparing the end-of-session value with the peak value (see legend of Fig. [4](#F4){ref-type="fig"} for further details). The two variables, illustrated by the XY-plot in Fig. [4C](#F4){ref-type="fig"}, were found to have no significant correlation (r = 0.35, p \> 0.05). Discussion ========== Our study revealed a progressive decline of pharmacologically isolated NMDA EPSPs, as observed for several hours in response to low rate (0.1 Hz) activation of afferents. The decline was found to be a form of long-term synaptic depression with an induction linked to NMDA receptor activation and with an expression that was maintained through periods without such activation. Several of its basic characteristics were similar to those of conventional LTP and LTD, suggesting a possible relation to these phenomena. Synapse specificity and NMDA-dependent induction ------------------------------------------------ Decaying responses is a potential side effect in long-term, electric recording *in vitro*due to declining viability of biological tissue or other experimental imperfections. Such unspecific \"run down\" can not account for the present findings since the gradual depression of responses could be repeated in the same slice, using a previously undepressed pathway. On the other hand, if the experiment was repeated twice in the same pathway, the second occasion revealed a diminished NMDA EPSP that showed little further decay. Together, these results show that the depression is input specific and long lasting and that it can saturate. Moreover, the lack of associated changes of the fiber volley speaks against a failure of axon conductance \[[@B19]\], favoring a synaptic localization of the process. While both pre- and postsynaptic expression mechanisms appear feasible, certain mechanisms of induction can be excluded. For instance, a decrease in probability of glutamate release due to a direct depletion of the vesicle pool is unlikely since AMPA EPSPs could be evoked for several hours without significant decay (see also \[[@B7],[@B20]\]). Even so, a use-dependent reduction of vesicle content may affect NMDA responses selectively under certain conditions by restricting \"glutamate spillover\" \[[@B21]\]. The most critical data with respect to the induction mechanism is that a period of conditioning stimulation, normally leading to reduction of NMDA EPSPs in the same pathway later on, was ineffective if delivered during blockade of NMDA receptors. This implies that the induction of the depression requires activation of NMDA receptors, most likely postsynaptically. Other observations of decaying NMDA EPSPs ----------------------------------------- The input specificity and NMDA dependent induction of the current depression conform with basic properties of conventional LTP and LTD \[[@B22]\]. The depression might then be a case of LTD, although induced by an alternative protocol. In fact, LTD was shown to be associated with changes involving both AMPA and NMDA receptors, although the linkage between the two contributions is controversial \[[@B12],[@B15]\]. Moreover, both of the cited studies demonstrated LTD of isolated NMDA EPSPs induced by 1--2 Hz stimulation. In contrast, experiments in cultures, inducing LTD by field stimulation at a higher frequency (5 Hz), reported only AMPA receptor mediated changes \[[@B10]\]. Direct interaction tests may further clarify the relation between the present depression and LTD. Gradually decaying, NMDA receptor mediated responses were observed previously in our lab during recording of composite AMPA-NMDA EPSPs for several hours \[[@B7],[@B23]\]. Attempts to relate the decay to LTD demonstrated a weak reduction of subsequent LTD of AMPA responses suggesting at least some elements in common \[[@B7]\]. In view of studies reporting forms of AMPA-NMDA coupling \[[@B16],[@B17]\], it is arguable that the studies demonstrating a decay of both components could have been influenced by the use of composite responses. In one of our studies \[[@B7]\], the observed depression of the AMPA component of composite EPSPs was verified by additional comparison between isolated AMPA EPSPs obtained under blockade of NMDA receptors. A similar verification was lacking for the depression of the NMDA response. By recording of isolated NMDA EPSPs, the present study ascertains that NMDA receptor mediated responses undergo a use-dependent depression, which is manifested in the absence of AMPA receptor activation. However, the decay was less pronounced than that reported previously for the NMDA component of composite EPSPs (average reduction to 60 % of peak as compared to 40 % in the previous study \[[@B7]\]). While we observed that isolated NMDA EPSPs decay \"spontaneously\", most prior studies employing such EPSPs did not report a decay. It might be that limitations of recording time concealed the effect and cell dialysis during whole cell recording could also be a limiting factor. Actually, a recent study, recording \"novel\" responses under whole cell conditions, reported on decaying AMPA EPSPs but constant NMDA EPSPs \[[@B24]\]. The possibility of AMPA receptor LTD under the present conditions could not be excluded as the blockade of the receptors may just conceal the effect. Further studies may help to reveal this matter. Persistency and saturability ---------------------------- Standard LTP/LTD experiments compare relatively stable periods of recording before and after induction of the synaptic modification. This was not possible in the present case, since merely test stimulation evoked the decay. Therefore, comparisons were generally made between synaptic pathways subjected to different stimulus paradigms. The induction of depression in a single pathway during an initial 2 h period caused a subsequent difference between NMDA responses of the two pathways throughout a subsequent test period. The degree of initial decay was closely related to the later difference between pathways, suggesting that once depression occurred it could be maintained through periods of receptor blockade until testing was performed. Our data suggest a duration of the depression of more than 3 h after the initial induction period. This is in the range commonly referred to as \"late\", which is believed to involve special biochemistry such as gene expression and protein synthesis \[[@B25],[@B26]\]. Whether, the presently studied depression involves such changes remains to be determined. The gradual depression of NMDA EPSPs was found to saturate after 2--3 h as evidenced by both single and double session experiments. This is in line with several other forms of NMDA-dependent plasticity, including LTP, LTD and chemically induced variants, which are shown to be saturable \[[@B27]-[@B29]\]. Whether, the saturation observed here is a \"true one\" at the level of expression is not known. Alternatively it could be a phenomenon at the induction level, related to weaker induction due to the diminished NMDA response. Possible expression mechanisms ------------------------------ Previous work on conventionally induced LTD revealed an essential role for protein phosphatases in mediating the synaptic modification \[[@B30],[@B31]\]. Consistent with the idea that changes of AMPA receptors mediate NMDA-dependent synaptic plasticity \[[@B32],[@B33]\] it was demonstrated that certain sites of the GluR1 subunit were targeted in LTP/de-potentiation and other ones in LTD/de-depression \[[@B2],[@B34]\]. Less is known about mechanisms underlying NMDA receptor changes in LTP/LTD as well as in the current depression. A previous study in our lab recording composite EPSPs reported that LTD of the NMDA component was blocked by a phosphatase inhibitor in a similar manner as \"standard LTD\" \[[@B15]\]. Hence, one can envisage that NMDA receptors would be controlled via dephosphorylation in a similar manner as inferred for AMPA receptors. NMDA receptors also have a number of other regulatory sites, allowing for modulation by glycine, polyamines, calcium, and redox agents \[[@B35]\] and they have shown to be mobile as well \[[@B36]-[@B38]\], in keeping with the idea of mobile AMPA receptors in LTP/LTD \[[@B39],[@B40]\]. Regardless of details, additional factors are needed to stabilize the synaptic modification in the long term, perhaps via synthesis of new proteins as previously demonstrated for LTP and LTD lasting longer than about 3 h \[[@B25],[@B28],[@B41]\]. Changes in synaptic morphology and altered subunit composition of receptors are examples of protein synthesis dependent mechanisms that have been implied in late forms of plasticity \[[@B32],[@B42]\]. Although a postsynaptic modification appears to be the primary choice, a presynaptic one that is initiated postsynaptically is also conceivable. In previous attempts to distinguish between pre- and postsynaptic mechanisms, LTD was compared with depression caused by various pharmacological agents with respect to the ability to influence the waveform of EPSPs \[[@B18]\]. While LTD in that study was found to affect isolated NMDA EPSPs in a uniform manner, i.e. no waveform change, the present data appeared to be less clear-cut. Nevertheless, the relation between early and late EPSP measurements differed for the initial AP5 washout period and the following period of actively induced depression, indicating a change in EPSP waveform. The depression therefore appeared to be distinct from a postsynaptic modification via modulation of channel gating. However, a clear test of the pre-post issue still remains. Unfortunately, the MK-801 test of release probability \[[@B43]\] does not appear useful when dealing with decaying responses as in the present case. Short-term changes and their possible mechanisms ------------------------------------------------ While the main line of experiments employed a smooth start of NMDA receptor activation following the gradual washout of AP5, another set of experiments made use of sudden activation by awaiting full washout until stimulation was started. Compared to smooth activation, there was an additional, transient potentiation that largely decayed within 20--30 stimuli. This is in accord with a previous study in hippocampal slices showing that stopping stimulation of composite AMPA-NMDA EPSPs for 10--60 min (and one case of isolated NMDA EPSP for 10 min) resulted in a transient potentiation when stimulation was resumed \[[@B23]\]. Several other studies describe decaying NMDA responses in relation to inactivation or desensitization of receptors \[[@B44],[@B45]\]. Accordingly, synaptically evoked NMDA responses in cell cultures were found to inactivate (i.e. decay) within a few minutes in much the same manner as observed here \[[@B45]\], a process shown to be triggered by postsynaptic influx of Ca^2+^via the NMDA channels. Similar mechanisms of receptor desensitization/inactivation might be responsible in the present case in forming the transient phase after starting stimulation. Some details remain unexplained by this simple model, such as the biphasic character of the transient phase in terms of initial growth and subsequent decay. One can speculate that a minor LTP, or short-term potentiation, might be induced by the sudden activation of NMDA receptors and so would contribute to the initial growth, although the underlying cause is not addressed in this kind of explanation. Conclusions =========== The above results emphasize that NMDA receptor mediated responses are highly plastic and that mere test stimulation can induce a short-term potentiation as well as a slowly developing depression that persists for several hours. The depression was input specific and saturable, and its induction required NMDA but not AMPA receptor activation in conformity with conventionally induced LTP and LTD, suggesting a relation to these phenomena. While a low Mg^2+^solution was used in our case to unblock NMDA receptors, similar unblocking may occur naturally in response to depolarization. Several important issues are still not settled. Is the saturation of the NMDA EPSP depression an absolute matter or can it be overcome, leading to further down regulation and possibly silencing of synapses? Conversely, is it possible to reverse, i.e. de-depress, the change by LTP or similar processes, allowing for bidirectional control? Further research is needed to resolve these questions. Methods ======= Experiments were performed on 12 to 18 day old Sprague-Dawley rats. The animals were decapitated after isoflurane (Forene) anesthesia in accordance with the guidelines of the Swedish Council for Laboratory Animals. All animal procedures were approved by the Local Ethics Committee at Göteborg University. The brain was removed and placed in an ice-cold artificial cerebrospinal fluid solution containing (in mM) NaCl 119, KCl 2.5, CaCl~2~2, MgCl~2~2, NaHCO~3~26, NaH~2~PO~4~1 and glucose 10, oxygenated by 95% O~2~, 5% CO~2~. The hippocampus was dissected out and transverse 400 μm thick slices were prepared by a vibratome or tissue chopper. The slices were initially kept in the same solution at room temperature for at least 60 min. As required, slices were then transferred to one or several \"submerged type\" recording chambers. During the experiment, slices were perfused at 30°C by a solution similar to that above except that the concentration of Mg^2+^was 0.1 mM. The usage of low Mg^2+^allowed for expression of NMDA receptor mediated responses. Stimulation was delivered as 0.1 ms negative constant current pulses via monopolar tungsten electrodes. For each slice, two stimulating electrodes were placed in the apical dendritic layer of CA1 pyramidal cells on either side of the recording electrode to provide for stimulation of two separate sets of afferents. Field EPSPs were recorded by using a glass micropipette filled with 3 M NaCl (4--10 MΩ resistance). The basal test stimulus frequency was 0.1 Hz with stimuli delivered alternately to the two electrodes, successive stimuli being separated by 5 s. To test the effect of stimulus interruption, one of the two electrodes was given no stimulation during a certain time, the other one remaining stimulated at 0.1 Hz. Recording commenced by monitoring isolated AMPA EPSPs in the presence of AP5 (50 μM) to block NMDA responses. A low concentration of CNQX (1 μM) was used to partially suppress the AMPA responses. In this way, somewhat larger stimulus strengths could be applied, suitable for evoking isolated NMDA EPSPs in the later part of the experiment. During the time of AMPA EPSP recording, the stimulus strengths were adjusted for each slice to equalize the synaptic inputs of the two pathways. This was essential for later comparison of NMDA EPSP across pathways. After obtaining a baseline of equal AMPA responses, the concentration of CNQX was raised to 10 μM which entirely blocked synaptic responces. The remaining non-synaptic response, consisting of stimulus artifact and presynaptic volley, was used to define \"true zero\". To study NMDA receptor mediated responses, CNQX (10 μM) was maintained in the solution while AP5 was washed out for one or several 2 h periods, referred to as sessions in the following. In between the sessions as well as afterwards, synaptic transmission was again blocked by applying AP5 (50 μM), framing in the sessions by periods of recording non-synaptic responses. Under the sessions, various tests were made depending on the purpose of investigation. Usually one input remained silent during the first session and stimulation was not resumed until after synaptic transmission was reblocked. In another kind of experiment, the initially silent pathway was reactivated in the early part of the first session after NMDA receptors were unblocked, providing a means for sudden start of NMDA receptor activation. Signals were amplified, filtered and transferred to a PC clone computer for on-line and off-line analysis by specially designed electronic equipment (based on an Eagle Instruments multifunction board) and own developed computer software. AMPA EPSPs were measured using an early time window (first 1.5 ms after the fiber volley) while NMDA EPSPs were measured using both an early (first 5 ms after volley) and a late (35--45 ms after artifact) time window. The late measurement was used in presenting most of the results, allowing easy comparison with previous work in our lab that estimated the NMDA component of composite EPSPs via a late measurement \[[@B7]\]. Similar albeit not identical results were obtained with early and late measurements (see illustration in Fig. [3D](#F3){ref-type="fig"}). Measurements were calculated by integrating the curve along the specified time window after substraction of the prestimulus baseline. All values were corrected by substracting the corresponding measurements of the non-synaptic potential obtained after total blockage of the EPSPs (except when measuring the fiber volley). The final data were quantified as relative values compared to a reference level defining 100 %. While the initial baseline formed a natural reference for AMPA responses, the choice was less obvious for NMDA responses, leading us to use the highest level of responses for one of the pathways in one of the experimental sessions (selected to make sense). Results are expressed as mean ± S.E.M. Statistical comparisons were made using Student\'s t-test. Drugs were obtained from Tocris Cookson, UK; prefabricated stimulating electrodes were obtained from World Precision Instruments, FL USA, type TM33B. Abbreviations ============= AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; AP5, D(-)-2-amino-5-phosphonopentanoic acid; CA, cornu ammonis; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; EPSP, excitatory postsynaptic potential; LTD, long-term depression; LTP, long-term potentiation; NMDA, N-methyl-D-aspartate; Authors\' contributions ======================= MD planned and carried out most of the experiments including data analysis, and compiled the manuscript. RL carried out experiments, participated in the planning process and helped in shaping the final manuscript. HPX carried out the initial experiments establishing the effect of NMDA EPSP depression. BJ was responsible for logistics planning and participated in experiments. HW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements ================ This study was supported by the Swedish R.C./medicine 05954 and the foundations of Adlerbert, Åhlen, Märtha & Gustaf Ågren, Herbert & Karin Jacobsson, Sigurd & Elsa Golje, Wilhelm & Martina Lundgren, Svenska Läkaresällskapet, Göteborgs Läkaresällskap, and Alzheimerfonden. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Decay of NMDA receptor mediated EPSPs in low Mg^2+^solution.**(A) Experimental design: Measurements of field EPSPs from a representative experiment are plotted for two independent pathways, referred to as input 1 and input 2. By appropriate use of specific blockers CNQX and AP5, either isolated AMPA EPSPs (used for initial pathway equalization) or isolated NMDA EPSPs (used in testing sessions N1 and N2) were recorded with periods of fully blocked responses in between. The pathways were stimulated alternately, each at 0.1 Hz, except for silencing input 2 for a 3 h period that contained session N1. Each point represents the average of measurements within 1 min. As seen, during N1 the responses to input 1 decayed. During session N2 both inputs were stimulated, revealing a novel decay for input 2 and occluded depression for input 1. Samples of recorded potentials of both inputs are given for the indicated time points a-e. (B) Superimposed, averaged time courses of NMDA EPSPs for input 1 during session N1, and input 2 during session N2. (C) Superimposed averaged time courses of NMDA EPSPs for input 1 and input 2, both during session N2. For B and C, each point represents the average of measurements within 3 min intervals. The peak during session N1 was used as 100 %. Values are expressed as mean ± S.E.M (n = 8 experiments). :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Activation of NMDA receptors is necessary for inducing persistent depression.**(A) Experimental design: Measurements of field EPSPs are plotted for a representative experiment as in Fig. 1A. The experiment conformed with the previous one except that AP5 was present in the solution during session N1. Accordingly, synaptic transmission was blocked for the entire period when input 2 was stopped. In session N2, AP5 was eventually washed out and both inputs displayed decaying NMDA EPSPs. Samples of recorded potentials of both inputs are given for the indicated time points a-e. (B) Superimposed averaged time courses of NMDA EPSPs from both inputs during session N2. Each point represents the average of measurements within 3 min intervals. The peak of input 2 was chosen as 100 %. The equal appearance of the curves shows that no pathway specific depression remained during N2 due to the differential treatment during N1 (compare such a depression in the reference case in Fig. 1C). Values are expressed as mean ± S.E.M (n = 5 experiments). :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Summary of NMDA EPSP depressions during unblocking of NMDA receptors.**(A1) Schematic representation of the experiment described in Fig. 1. (A2) Schematic representation of the experiment described in Fig. 2. Pathways are defined as \"treated\" or \"naive\" depending on whether stimulation was \"on\" (solid) or \"off\" (dashed) during session N1. Each number (1--10) in A1 and A2 represents a portion where measurements were taken for analysis. (B) Comparison between NMDA EPSP measurements, each bar representing an average obtained for 30 consecutive responses (5 min). Value no. 1 was chosen as 100 % for experiments depicted in A1. Since a corresponding value was lacking for experiments depicted in A2, value no. 9 was chosen as 100 %. All values are expressed as mean and S.E.M (n = 8 and n = 5, respectively, for A1 and A2 type experiments). (C) Relation between instantaneous depression in session N1 (abscissa, final value relative to peak) and persistent depression in session N2 (ordinate, interpathway comparison of peaks). Each dot represents a single experiment. The analysis revealed a significant correlation between the variables as illustrated by the superimposed regression line (n = 8). (D) Relation between early and late measurements obtained with two different time windows (initial 5 ms and 35--45 ms, respectively) plotted as a \"phase trajectory\" (abscissa, early measure; ordinate, late measure). The curve represents an average of 20 experiments with additional smoothing to reduce noise (5 min moving average). Samples of recorded NMDA EPSPs corresponding to locations a, b of the trajectory are overlaid. Bars above EPSPs curves indicate time windows used for early and late measurements, respectively. The curve at b is shown superimposed on a dotted copy of the curve at a. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Transient potentiation induced by sudden activation of NMDA receptors.**(A) Superimposed averaged time courses of NMDA EPSPs for two pathways subjected to different stimulation protocols. Each point represents the average of measurements within 3 min intervals. Stimulation of one pathway (input 2) was stopped for a period of 30 min and then restarted at a time point when NMDA receptors were fully unblocked as judged by responses for the other pathway (input 1) that was exposed to the standard protocol. The peak of input 1 was taken as 100 %. (B) Time course of the newly evoked NMDA responses. Expanded portion of A immediately following restart of stimulation, each point representing measurement of a single EPSP. It can be noted that following the initial response there was additional growth for about a minute before responses started to decay. Values in both A and B are expressed as mean ± S.E.M (n = 8 experiments). (C) Examining the relation between the slow depression and the transient potentiation of NMDA EPSPs. Depression was quantified as end-of-session value relative to peak value (5 min averages) for input 1 that received the standard protocol. Transient potentiation was estimated as the average of the first 18 responses (3 min) of input 2 relative to the average of the corresponding interleaved responses of input 1. The latter value was nearly the same as the peak value. Each dot represents a single experiment (same set of 8 experiments as in A, B). The analysis revealed no significant correlation between the data points. :::  :::

PubMed Central

2024-06-05T03:55:47.633691

2004-8-3

PMC517492

Background ========== The development of DNA microarrays has revolutionized high throughput gene expression analysis. The two main platforms are: cDNA microarrays, where PCR products of individual cDNA fragments are immobilized on glass slides \[[@B1]\], and oligonucleotide microarrays, where oligonucleotides *in situ*synthesized or spotted on glass slides are used \[[@B2],[@B3]\]. In a typical cDNA microarray experiment, total cellular RNA from two sources, a reference (or control) and an experimental sample are converted to cDNA by reverse transcription, labeled with two different fluorescent colors, and hybridized to an array of cDNA probes. One of the major concerns of cDNA microarrays is cross-hybridization of the labeled RNA (or cDNA) to non-target homologous probe sequences on the array \[[@B4],[@B5]\]. Cross-hybridizations that may arise due to poly(A)-tail of mRNA or repetitive elements may be reduced or eliminated using hybridization blocking reagents like poly(A) oligonucleotides \[[@B6]\] or Cot1 DNA. Another major source of cross-hybridization may come from conserved sequences shared by two or more cDNAs, such as gene family members \[[@B4],[@B7]\]. Analysis of sequenced multicellular eukaryotic genomes suggests that a large percentage of genes belong to gene families, some of which have high sequence similarities \[[@B8]\]. A recent study reported that approximately 17% of human transcripts in the UniGene database contain perfect match repeats of 20 bp minimum lengths \[[@B9]\]. From this study, however, the frequency of long, non-perfect match sequences such as those shared among paralogs, was not clear. Some studies have suggested that cross-hybridization can be a source of errors in cDNA microarray experiments \[[@B3],[@B4],[@B7],[@B9],[@B10]\]. In general, these studies indicate that cDNAs with nucleotide sequence identities higher than 70--80% over a certain length show significant levels of cross-hybridization. Analysis of chemokines, cytochrome P450 isozymes, G proteins and protease homologous gene families showed that cross-hybridization signals of 0.6--12% and 26--57% could arise from shared nucleotide identities of 55--80% and \>80%, respectively \[[@B4]\]. Using synthetic 50 mer oligonucleotides, it was also shown that non-target sequences with \>75--80% identity to 50 mer probes in microarrays could result in cross-hybridization \[[@B3]\]. In addition, cross-hybridization was also observed if a non-target sequence included stretches of ≥ 15 continuous bases identical to a 50 mer probe sequence \[[@B3]\]. Cross-hybridization is thought to contribute to discrepancies of results observed between oligonucleotide arrays and cDNA arrays \[[@B11]\]. Cross-hybridization may occur for both oligonucleotide and cDNA microarrays. An advantage with oligonucleotide arrays is that the complete sequences of the probes are known. Because cDNA arrays are constructed from PCR products of cDNA clones sequenced from 3\'- or 5\'-ends, the complete sequences of the spotted probes may not be available. We have not been able to identify programs or web servers capable of doing flexible cross-hybridization analysis. Programs with related functionality have been described, including ProbeWiz \[[@B12]\], OligoWiz \[[@B13]\] and PROBEmer \[[@B14]\], but these are for designing specific probes by avoiding regions of the cDNAs where there may be a cross-hybridization problem. For the assessment of the cross-hybridization potential in DNA microarray analysis, a more specific tool is required. In the present study we limit ourselves to the following problem: Given a probe (or a set of probes), identify the targets to which it can hybridize given user-defined criteria. The exact criteria to be used to decide whether two genes with high sequence similarity can cross-hybridize will depend on a number of factors, including the set of probes used and the experimental protocols -- in particular hybridization and washing stringencies. We propose to use generic forms of criteria allowing the user to choose parameters in order to define the criteria for cross-hybridization. We investigate whether BLAST can be used to detect targets satisfying a set of criteria for cross-hybridization, based on previous experimental findings \[[@B3],[@B4]\], and assess different (transcript) databases in order to evaluate their suitability for our purpose. A web tool is developed that allows the user to query precomputed results from several probe sets, or to analyze own probes with respect to a database representing transcripts in the organism where the microarray experiments are performed. The tool is applicable to analysis of both oligonucleotide and cDNA probes. Below we describe the developed XHM (cross hybridization on microarrays) system. We also include analysis performed in order to identify appropriate analysis methods and databases. Furthermore we describe the web server for XHM. In the final section we report some example results using our system on three cDNA probe sets for human, mouse and rat, and one oligonucleotide probe set for mouse. Implementation ============== The XHM system queries the given nucleotide sequences (for example, a list of differentially regulated genes from DNA microarray experiments) against a database representing all possible targets (transcripts) in the organism being studied, and produces a list of all targets that can hybridize to each input sequence under the defined criteria. In this section we evaluate databases for use in the XHM system and we evaluate whether BLAST can be used to identify potential cross-hybridizing genes. Definitions ----------- For the calculation of melting temperature, the following formula is used \[[@B15]\]: *Tm =*81.5 + 16.6 \* *log*\[*Na*^+^\] + 41 \* (*numG + numC*)/*n*- 500/*n* where \[*Na*^+^\] is the *Na*^+^concentration, *numG*and *numC*is the number of Gs and Cs in the given alignment, and *n*is the total number of nucleotides aligned. We differentiate between two kinds of sequence similarity cutoffs for cross-hybridization, based on the situations described for oligonucleotide microarrays \[[@B3]\]: • *Type A*similarities for sequence segments of a certain minimum length with a defined minimum percentage identity (long alignments with mismatch). • *Type B*similarities for identical segments of some minimum length (short perfect match). To perform the searches in the different databases, NCBI BLAST version 2.2.1 (Mon Jul 9 14:02:00 EDT 2001) \[[@B16]\] was used. The default settings of *blastn*are used unless specified otherwise. This includes using the DUST filter for masking lowcomplexity regions. Databases and sequences ----------------------- In this study we used three cDNA clone sets (corresponding to three cDNA sets spotted on microarrays). The 40 k *Homo sapiens*(Hs) and the 14 k *Rattus norvegicus*(Rn) clone sets are I.M.A.G.E. Consortium \[LLNL\] cDNA clones \[[@B17],[@B18]\] obtained from Research Genetics (Huntsville, AL). The 15 k *Mus musculus*(Mm) clone set is from NIA \[[@B19]\]. In addition, oligonucleotide sequences (Mouse Genome Array Ready Oligonucleotide Set) from QIAGEN Operon \[[@B20]\] have been used to test the application. The details of the databases used to search for possible cross-hybridizations are as follows: • RefSeq Version 2 -- RefSeq mRNA collection \[[@B21]\]. Number of sequences: 25377/21200/21724 (mouse/rat/human). • UniGene -- UniGene Unique, build 129/123/164 (mouse/rat/human) \[[@B22]\]. Number of sequences: 87495/50137/118326 (mouse/rat/human). • TIGR gene index \[[@B23]\] -- Only tentative consensus sequences (TCs), version 020403/042503/020503 (mouse/rat/human). Number of sequences 58129/51330/187287 (mouse/rat/human). • BeGIn -- Bergen Gene Index version 1.0. Number of sequences: 100654/49285 (mouse/rat). BeGIn is an alternative database we have developed (using tools described in \[[@B24]\]), by producing consensus sequences from each cluster in UniGene (build 129/123 for mouse/rat). In order to achieve high specificity and sensitivity, we favored the databases where the highest number of probes were represented (completeness) and multiple matches per probe were minimized (minimum redundancy). Our criteria for choosing an appropriate database for a given organism was based on the results obtained for our probe sets. The presence of alternative splice forms in either the probe or the target set may complicate analysis. We do not consider this explicitly in the current study. Overview of the XHM system -------------------------- The basis of the XHM system is a BLAST search (see Figure [1](#F1){ref-type="fig"}). The BLAST search provides one or several alignments between the probe sequence and each of the possible cross-hybridizing target sequences. The alignments are analyzed with respect to whether they fulfill the user-defined criteria for *type A*or *type B*matches. The XHM system presents to the user a list of matches satisfying the criteria, and in addition information on GC-content in similar regions, estimated melting temperature (Tm) and position on the sequence (proximity to 3\' or 5\' end). Although the estimated Tm may not be completely accurate under microarray hybridization conditions, particularly on solid surfaces, it can give a relative measure of the impact of the potential cross-hybridization detected. An expert user may use the information to assess the likelihood and importance of the cross-hybridization effect. Appropriateness of BLAST ------------------------ Experiments were performed to assess whether BLAST is able to identify potentially cross-hybridizing targets. The analysis was done by generating simulated targets (*T*~1~\...*T~n~*) for a number of probes (*P*~1~\...*P~n~*). The simulated targets were ensured to satisfy the criteria described in \[[@B3]\] (50 bp sequences with at least 75% identity or 15 bp identical sequence). BLAST was then used to query the original probes against a large database containing the designed targets. It was then checked whether or not *T~i~*was contained in *P~i~*\'s hitlist. We found that *type A*matches can be missed if they do not contain any one stretch identical to the probe of length larger than BLAST\'s initial word length. Our experiments also showed that, depending on the probe type and the criteria used, we sometimes had to allow BLAST to generate very long lists of hits and using very high (permissive) cutoffs on E-value in order to cover the intended targets. Part of the reason for this is that BLAST is designed to identify homologous sequences and the sequences satisfying the cross-hybridization criteria need not necessarily receive significant scores or E-values. Our conclusion was that BLAST can be used, but to achieve optimal results, non-default parameters for initial word size and mismatch penalty should be applied. This was taken into consideration in the web-server. A web server ------------ A web interface has been designed <http://www.bioinfo.no/tools/xhm/> with two entry points for accessing cross-hybridization information (see Figure [1](#F1){ref-type="fig"}). The user may either query a database of precomputed alignments, or compute new alignments using a real-time BLAST search. Both versions have parameters for the different thresholds and output alternatives. For ease of use, it is possible to run queries with no parameters explicitly specified. When querying with a large number of clones against a large database, running BLAST may be time consuming. Therefore, we pre-run BLAST with different clone-sets against different databases, and store the results. In this way the user may experiment with different thresholds for a set of clones repeatedly, and get the results within seconds. Nucleotide sequences or GenBank accession numbers may be used as input to the system when running the real-time BLAST searches. Searching using the precomputed BLAST alignments does not accept nucleotide sequences as input. ### User-adjustable input parameters Some of the parameters are shared among the two main versions of the XHM system. These include minimum length and minimum percentage similarity for *type A*hits (defaults are 75% over 50 bp) and minimum length for *type B*hits (default is 15 bp), based on the findings described in \[[@B3]\], Na^+^concentration (default is 0.1 M), and size of GC-clamp (default is 10). The Na^+^concentration is used to calculate melting temperature, and the size of the GC-clamp is used to plot the GC-content throughout the query sequence. In addition, the real-time BLAST version also contains BLAST specific user-adjustable parameters. Main parameters include threshold on E-value (default is 10), number of alignments in the output from BLAST and whether DUST (low complexity) filter should be used. The user can adjust other BLAST specific parameters including gap opening and extension penalties, initial word size, mismatch penalty and whether or not to allow gapped alignments. ### Output The output from the XHM system consists of different parts (see Figure [2](#F2){ref-type="fig"}). If there are several input sequences (batch query), the results are given for each sequence individually. For each probe the system generates a plot of GC-content along the sequence. This GC-plot may help the user to identify areas in the sequence with high GC content in order to evaluate the importance of the potential cross-hybridization detected. For each hit (possible hybridization), XHM presents the name and identifier of the hit, identity tuples (from BLAST -- number of identical bases and number of bases in total in the BLAST alignment, or in a sub-alignment), start and end position on the probe, calculated Tm, percentage GC in alignment and type of hit (A or B). Results ======= Choice of database ------------------ Experiments using the three cDNA sets and the oligonucleotide set as input to the XHM system yielded the results shown in Table [1](#T1){ref-type="table"}. Using a 70% identity threshold for the *type A*similarities, in the rat clone set the Rat Gene Index from TIGR appears to be the best choice. For the mouse clone set it appears that the BeGIn database is the best choice, and for the human clone set it seems that RefSeq or UniGene Unique may be two good choices. These evaluations were based on completeness (high number of probes represented) and minimum redundancy of the database. We used only the tentative consensuses (TCs) of the Gene Indices databases from TIGR. Using the \"full\" versions decreased the number of probes having zero hits, but substantially increased the number of probes having two or more hits (results not shown). Application to microarray probes -------------------------------- As a practical experiment, we tested the XHM system on the cDNA probe sets for human, mouse and rat, and the mouse oligonucleotide set from QIAGEN. For the cDNA sets we used the following thresholds: 70% over 200 nucleotides for *type A*similarities, and 25 nucleotides for the *type B*similarities. For the oligonucleotides we used 70% identity over the whole oligonucleotide sequence (*type A*) and 20 nucleotides perfect identity (*type B*). Choosing the parameters is not trivial, and these thresholds, which are in the lower range of % similarities leading to cross-hybridizations, are meant as examples of a possible configuration. The XHM tool allows for full flexibility to experiment with the settings. The reason why we chose to consider a 200 nucleotides stretch for the cDNA probes is that sometimes these sequences contain errors, especially toward the ends. Results are shown in Table [1](#T1){ref-type="table"}. Using RefSeq, we observed that even though a substantial number of the 14 k rat cDNA probes (63.3%) had no *type A*hit, 809 (about 16.3% of the probes actually represented in RefSeq) had two or more hits, indicating potential cross-hybridization. In most cases, a single hit represents the probe sequence itself, and does not represent a potential cross-hybridization. Looking at a more complete database, the TIGR Rat Gene Index, only 700 (5.2%) probes had no hits, and 2712 (21.3% of the probes represented in the database) had two or more hits. A conclusion from this was that the number of rat probes having potential cross-hybridizing partners was between 16% and 21%. For the mouse cDNA probes the cross-hybridization numbers appeared to be higher. Using RefSeq, the number of probes having two or more *type A*hits was 3101 or 34.2% of the probes represented in the database. RefSeq is relatively incomplete, so this number may be a conservative estimate on the cross-hybridization occurrence. For the BeGIn database only 725 probes had no hits (5.3%) and 5211 probes (about 40% of the probes in the database) had two or more hits. Even though as many as 47.3% of the mouse probes were not found in the TIGR Gene Index, the proportion of probes found in the database that had two or more hits was 59%. This number is most likely an artifact caused by redundancy in the database. The difference in number of possible hybridizations found in mouse versus rat is most likely due to the fact that more mouse sequences are available. The human cDNA probes seems to have a lower cross-hybridization potential than the mouse probes. In UniGene Unique and RefSeq, looking only at the human probes that were found in the databases, just above 30% of them had two or more hits. The results using TIGR Human Gene Index suggested that almost 70% of the probes found in the database could cross-hybridize, but this is most likely an artifact caused by redundancy. The mouse oligonucleotide set is clearly less prone to cross-hybridization. Using the UniGene Unique database, we observed that 14% of the probes found in the database had two or more hits, whereas the corresponding number for the 15 k mouse cDNA probe set using UniGene Unique was 45%. Discussion ========== A flexible tool for assessing the cross-hybridization potential of microarray probes has been developed and made available. Several transcriptome databases can be used for searching, and more may be added upon request. Using the XHM tool, analysis of three cDNA microarray probe sets and one oligonucleotide probe set revealed that a high proportion of the cDNA probes can potentially cross-hybridize with one or more other transcripts in the organism. As expected, compared to the cDNA probes, a smaller percentage of the mouse oligonucleotide probes showed a potential for cross-hybridization. This is because the oligonucleotide probe sequences are much shorter (69-mers), designed from specific regions of cDNAs to minimize cross-hybridizations. Despite increasing use of the more specific oligonucleotide arrays, cDNA sequences including full-length clone sets \[[@B25],[@B26]\] are widely used in production of microarrays. Depending on the completeness and redundancy levels of the transcriptome databases used, with the chosen cutoff for *type A*similarities (at least 70% identity), 15--45% of the cDNA probes showed hybridization with two or more apparently different transcripts (disregarding the results using the TIGR Gene Indices). This high percentage of potentially cross-hybridizing genes suggests that it is essential to carefully validate results from microarray experiments, particularly where cDNA clones are used to prepare arrays. Although cross-hybridization is known as one of the main sources of errors of cDNA microarrays, the high proportions of cross-hybridizing genes detected in our test are likely to be overstated by the possible redundancies in the databases (not all entries represent unique genes). Also one may argue that the 70% identity threshold is somewhat low. Whether two different genes that are candidates for cross-hybridization actually lead to erroneous results in hybridization experiments will depend on factors such as the level of sequence identity, the stringency of the hybridization and the relative abundances of the transcripts. For example, two potentially cross-hybridizing genes do not necessarily pose a problem unless both are expressed in the tissue or cell-line analyzed. Quick inspection of the hit-list produced by the XHM tool for a typical input of differentially regulated genes will help in identifying significant noise from cross-hybridizations. A main advantage of the XHM tool is that it allows the user to perform searches at various stringencies to detect potential cross-hybridizations. Candidate genes from microarray analysis that show potential cross-hybridizations using the database search may then be further checked using other methods such as RT-PCR and Northern blot. Conclusions =========== We have shown that a significant proportion of probes used in cDNA microarray analysis may show cross-hybridization with non-target sequences. We have developed a flexible tool, XHM, suitable for detecting potential cross-hybridization artifacts during microarray data analysis. The tool may also be used to select specific probes for preparation of microarrays. Availability ============ The XHM system is freely available at <http://www.bioinfo.no/tools/xhm/>. Program code for academic use can be supplied upon request to the author. Author\'s contributions ======================= KF implemented the system and made a draft of the manuscript. FY and AL contributed with ideas and proofread the manuscript. IJ has formulated and supervised the work, and edited the manuscript. All authors have read and approved the final manuscript. Acknowledgments =============== We would like to thank Per Winge at Department of Biology, Norwegian University of Science and Technology (NTNU) for useful discussions on DNA hybridization. Ketil Malde at Department of Informatics, University of Bergen is the main developer of the BeGIn databases for Mouse and Rat. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Main components of the XHM system.**One or several query sequences (or identifiers) are entered, a BLAST search is performed, the resulting hits are filtered based on length and similarity, and the results are displayed. The dashed arrow symbolizes the possibility to use a sequence id whose hits to the database have been stored. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Screenshot of the XHM web output.**The screenshot shows an example output from the XHM system. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Extent of potential cross-hybridizations in DNA microarray probes. ::: Clone set DB **Type A (%)** **Type B (%)** CHS(A,B) ----------- --------------- ---------------- ---------------- ---------- ------- ------ ------ ------ ----------- = 0 = 1 ≥ 2 ≥ 2\* = 0 = 1 ≥ 2 Rn 14 RefSeq 63.3 30.7 6.0 16.3 91.3 6.6 1.7 70/200,25 Rn 14 TIGR RGI 5.2 74.6 20.2 21.3 87.7 8.5 3.8 70/200,25 Rn 14 UniGeneUnique 15.9 63.0 21.2 25.2 84.7 11.1 4.2 70/200,25 Rn 14 BeGIn 26.2 62.8 11.1 15.0 82.3 15.4 2.3 70/200,25 Mm 15 RefSeq 34.3 43.2 22.5 34.2 84.5 5.8 9.7 70/200,25 Mm 15 TIGR MGI 47.3 21.5 31.3 59.0 79.5 8.5 12.1 70/200,25 Mm 15 UniGeneUnique 9.9 49.1 40.1 45.0 76.2 11.6 12.3 70/200,25 Mm 15 BeGIn 5.3 57.0 37.8 40.0 78.7 11.5 9.8 70/200,25 Hs 40 RefSeq 13.1 59.5 27.4 31.5 93.3 4.0 2.7 70/200,25 Hs 40 TIGR HGI 1.0 30.8 68.2 68.9 72.5 18.2 9.3 70/200,25 Hs 40 UniGeneUnique 10.9 60.7 28.4 31.8 87.4 7.7 4.9 70/200,25 Mm Oligo RefSeq 32.5 64.4 3.2 4.7 97.7 1.9 0.3 70/69,20 Mm Oligo TIGR MGI 59.6 26.2 14.2 35.1 95.8 3.2 1.0 70/69,20 Mm Oligo UniGeneUnique 9.6 77.7 12.7 14.0 94.8 4.3 0.9 70/69,20 Mm Oligo BeGIn 15.6 77.1 7.3 8.6 95.7 3.6 0.6 70/69,20 Three cDNA probe sets, human (Hs 40), rat (Rn 14) and mouse (Mm 15), and one mouse oligonucleotide set (Mm Oligo) were checked against cDNA databases (DB). In most cases, a single hit shows that the probe is represented in the DB. Two or more hits indicate potential cross-hybridization. If a hit qualifies as a *type A*hit, it is not considered to be *type B*, even if it satisfies the criteria. The percentages represent the number of probes having the given number of hits, relative to the total number of probes in the probe set. The column ≥ 2\* shows the number of probes with two or more hits, relative to the total number of the probes that were found in the database. Default BLAST settings were used, except for word-size (9), and mismatch penalty (-1). The column CHS(A,B) shows the Cross-Hybridization Settings used for *type A*and *type B*search. Total number of probes: Hs 40: 40000, Rn 14:13447, Mm 15:13797 and Mm Oligo:16463. :::

PubMed Central

2024-06-05T03:55:47.637213

2004-8-27

PMC517493

Background ========== With the completion of many genome sequencing projects and advances in the methods of automatic discovery of sequence patterns (see Brazma \[[@B1]\] and Brejova et al \[[@B2]\] for reviews), it is now possible to search or discover protein sequence motifs at the genome level. If one regards protein sequences as \"sentences\" of the biological language with amino acids as the alphabet, then protein motifs can be considered as words or phrases of that language and determining the function of a motif is equivalent to determining the sense of a word. Identifying biological sequence motifs has been a fundamental task of bioinformatics, which has led to the development of several motif (pattern) databases, such as PROSITE, BLOCKS, SMART and Pfam \[[@B3]-[@B6]\]. These databases are usually constructed by studying the set of protein sequences that are known to have certain functions and extracting the conserved sequence motifs that are believed to be responsible for their functions. However, the number of motifs that can be extracted in this way is quite limited, and it has been a major challenge to discover new motifs. With the advent of algorithms and programs that can automatically discover sequence motifs from any given set of sequences \[[@B1],[@B2],[@B7]-[@B9]\], it is possible to mine a large number of sequences to find novel motifs without necessarily knowing their functions and to compile a dictionary of biological language accordingly. An essential task involved in the compilation of such a dictionary is to determine the function (the meaning) of newly identified protein motifs. Here, we report development of general methods that can be used to predict the function of protein motifs by mining the knowledge in the Gene Ontology. The Gene Ontology™ (GO) project \[[@B10]\] is a concerted effort by the bioinformatics community to develop a controlled vocabulary (GO terms) and to annotate biological sequences with the vocabulary. A biological sequence is described in three different aspects, namely, biological process, cellular component, and molecular function. The standardized annotation with a controlled vocabulary is the main advantage of Gene Ontology, which facilitates both communications among scientists and information management. Both the number of annotated sequences and the number of GO terms associated with individual sequences in the Gene Ontology database are increasing very rapidly. Moreover, natural language processing techniques are also being used to automatically annotate gene products with GO terms \[[@B11],[@B12]\]. Thus, it can be foreseen that the annotations of protein sequences in the Gene Ontology database will become more and more detailed, and have a great potential to be used as an enriched knowledge base of proteins. The basic approach for determining the function of a motif is to study all the sequences that contain the motif (pattern). Intuitively, if all the functional aspects of the sequences matching a motif are known, we should be able to learn which function is most likely encoded by the motif, based on the assumption that every protein function is encoded by an underlying motif. This means that we would need a knowledge base of protein sequences, in which the functions of a sequence are annotated as detailed as possible. In addition, we would also need prediction methods that can work on a given set of protein sequences and their functional descriptions to reliably attribute one of the functions to the motif that matches these sequences. To determine the function of any novel motif, we would first search the protein knowledge base to retrieve all the functional descriptions of the proteins containing the motif, and then use such prediction methods to decide which function is encoded by the motif. In this research, we use the Gene Ontology database as our protein knowledge base and explore statistical methods that can learn to automatically assign biological functions (in the form of GO terms) to a protein motif. Our approach is based on the observation that the Gene Ontology database contains protein sequences and the GO terms associated with the sequences. In addition, the database also contains information of known protein motifs, e.g. the PROSITE patterns that match the sequences. Thus, the protein sequences in the database provide a sample of potential associations of GO term with motifs, among which some are correct (i.e., the GO term definition matches the functional description of the motif) and some are not. This provides us an opportunity to perform supervised learning to identify discriminative features and use these features to predict whether a new association is correct or not. Current Gene Ontology database is implemented with relational database system, which allows one to perform queries like \"retrieve all GO terms associated with the sequences that matches a given motif\" and *vice versa*. However, the database usually returns more than one GO terms that may or may not describe the function of the motif in the query. Thus, we need methods to disambiguate which GO term describe the function of the motif (assign a GO term to a motif) and determine how confident we are as the assignment is concerned. We use statistical approaches to learn from known examples and cast disambiguation task into a classification problem. Furthermore, the probability output by the classifier can be used to represent its confidence for the assignment. Recently, Schug et al \[[@B13]\] published their result of automatically associating GO terms with protein domains from two motif databases -- ProDom and CDD \[[@B14],[@B15]\]. Their approach is to use protein domains to BLAST \[[@B16]\] search against GO database and assign the molecular functional GO term from the sequence matching the domains with most significant *p*-value. They found that, in the database they worked with, most sequences only had one functional GO term. Therefore, they could assign the GO term of a sequence to the motif that matched with highest score with fairly good accuracy. However, due to restrictive assumption that each sequence has only one GO term, their approach can not address the potential problem that a sequence matching a motif has multiple associated GO terms, which is common case now, and how to resolve such ambiguity. Results ======= The data set ------------ We use the May 2002 release of the Gene Ontology sequence database (available online \[[@B17]\]), which contains 37,331 protein sequences. For each sequence, a set of GO terms assigned to the sequence is identified, and a set of PROSITE patterns that match the same sequence is also retrieved. If both sets are nonempty, all the possible pattern-term combinations formed by the two sets are produced. Table [1](#T1){ref-type="table"} shows an example association of GO terms with PROSITE motifs. The protein MGI\|MGI:97380 from the database is assigned seven GO terms and the sequence also matches two PROSITE patterns. Thus, as cross product of two sets, 14 distinct associations are produced. Note that the same pattern-term association may be observed multiple times within the database. A total of 4,135 GO terms, 1,282 PROSITE motifs, and 2,249 distinct PROSITE-GO associations have been obtained from this database. Using the information stored in the Gene Ontology and PROSITE, we manually judged a set of 1,602 cases of distinct PROSITE-GO associations to determine whether the association is correct or not. The PROSITE-GO association set has been judged in two different ways. One way is to label an association as correct if and only if the definition of the GO term and the PROSITE motif match perfectly according to the annotator. Gene Ontology has the structure of a directed acyclic graph (DAG) to reflect the relations among the terms. Most terms (nodes in the graph) have parent, sibling and child terms to reflect the relation of \"belonging to\" or \"subfamily\". The second way of judging GO-PROSITE association is to label an association as correct if the GO term and the PROSITE motif are either exact match or the definitions of GO term and PROSITE motif are within one level difference in the tree, i.e., the definition of GO term and the PROSITE motif have either a parent-child relation or a sibling relation according to the GO structure. Thus we have two sets of labeled PROSITE-GO associations, the perfect match set and the relaxed match set (with neighbors). Both sets are further randomly divided into training (1128 distinct associations) and test (474 distinct associations) sets. Since the test sample size is fairly large, the variance of the prediction accuracy can be expected to be small. Thus we have not considered any alternative split of training and test sets. Measuring term-motif associations --------------------------------- Intuitively, we may think of the GO terms assigned to a protein as one description of the function of a protein in one language (human understandable) while the motifs contained in the protein sequence as another description of the same function in a different language (biological). We would like to discover the \"translation rules\" between these two languages. Looking at a large number of annotated sequences, we hope to find which terms tend to co-occur with a given motif pattern. Imagine that, if the sequences that match a motif are all assigned a term *T*, and none of the sequences that do not match the motif is assigned the term *T*, then it is very likely that the motif pattern is encoding the function described by term *T*. Of course, this is only an ideal situation; in reality, we may see that most of, but not all of the proteins matching a motif pattern would be assigned the same pattern, and also some proteins that do not match the motif may also have the same term. Thus, we want to have a quantitative measure of such correlation between GO terms and motif patterns. A commonly used association measure is mutual information (M.I.), which measures the correlation between two discrete random variables *X*and *Y*\[[@B18]\]. It basically compares the observed joint distribution *p*(*X*= *x*, *Y*= *y*) with the expected joint distribution under the hypothesis that *X*and *Y*are independent, which is given by *p*(*X*= *x*)*p*(*Y*= *y*). A larger mutual information indicates a stronger association between *X*and *Y*, and *I*(*X;Y*) = 0 if and only if *X*and *Y*are independent. For our purpose, we regard the assignment of a term *T*to a sequence and the matching of a sequence with a motif *M*as two binary random variables. The involved probabilities can then be empirically estimated based on the number of sequences matching motif *M*(*NM*), the number of sequences assigned term *T*(*NT*), the number of sequences both matching *M*and assigned *T*(*NT*-*M*), and the total number of sequences in the database. Table [2](#T2){ref-type="table"} shows the top five terms that have the highest mutual information with PROSITE motif PS00109, which is the specific active-site signature of protein tyrosine kinases, along with the related counts. We set out to test whether we can use mutual information as a criterion to assign a GO term to a PROSITE motif. One approach is to use a mutual information cutoff value *c*to define a simple decision rule: assign term *T*to motif *M*, if and only if *I*(*T;M*) ≥ *c*. For a given cutoff *c*, the precision of term assignment is defined as the ratio of the number of correct assignments to that of the total assignments according to the cutoff *c*. In Figure [1](#F1){ref-type="fig"}, we plot the precision at different mutual information cutoff values. It is easy to see that, in general, using a higher (i.e., stricter) cutoff, the precision is higher; indeed, the Pearson correlation coefficient between the precision and the cutoff is 0.837. This suggests that mutual information is indeed a good indicator of the correlation However, a drawback of such an approach is that, given a motif, sometimes, many observed motif-term associations can have mutual information above the cutoff value, making it difficult to decide which pair is correct. While in other cases, the mutual information of the observed motif-term pairs may all be below the cutoff value, but we still would like to predict what terms are *most*likely to be appropriate for the motif. To address this problem, we can use a different cutoff strategy, and adopt a decision rule that assigns a GO term to a motif based on the ranking of mutual information, which is a common technique used in information retrieval text categorization \[[@B19]\]. More specifically, for each PROSITE motif *M*in the annotated data set, all observed motif-term associations containing *M*are retrieved and ranked according to mutual information, then the term that has highest mutual information is assigned to *M*. Alternatively, if we use this approach to facilitate human annotation, we can relax the rule to include GO terms that have lower ranks, thus allowing multiple potential GO terms to be assigned to a motif, assuming that a human annotator would be able to further decide which is correct. In this method, the key in making a decision is to select a cutoff rank that covers as many correct associations as possible (high sensitivity) while also retrieves as fewer incorrect associations as possible (high specificity). The optimal cutoff can be determined by the desired utility function. Figure [2](#F2){ref-type="fig"} shows the Receiver Operating Characteristic (ROC) curve \[[@B20]\] of assigning GO terms to PROSITE motifs in our data set according to the rank of motif-term associations. The two curves are for the two different labeled association sets (i.e., perfect match and relaxed match) respectively. The areas under the two curves are 0.782 and 0.735 respectively, which can be considered as fairly good. We also plot the precision, also referred to as positive predictive value, in panel B. The precision is calculated as the percent of predicted assignments that are truly correct. As shown in panel B, if we assign the GO terms at the top rank for all PROSITE motifs, 50--70% of the cases will be predicted correctly. As we loosen the threshold to include lower ranked terms, we would assign more terms to a motif, and as expected, precision would decline. But even at rank 5, we still have a precision of about 50%. Also shown in Table [2](#T2){ref-type="table"}, with respect to the PROSITE pattern of tyrosine kinase (PS00109), most of the top five associated GO terms are related to kinase activity and the term with the highest rank is the most specific. Predicting motif functions using logistic regression ---------------------------------------------------- While the mutual information measure appears to give reasonable results, there are three motivations for exploring more sophisticated methods. First, the mutual information value is only meaningful when we compare two candidate terms for a given motif pattern; it is hard to interpret the absolute value. While a user can empirically tune the cutoff based on some utility preferences, it would be highly desirable to attach some kind of confidence value or probability of correctness to all the potential candidate motif-term associations. Second, there may be other features that can also help predict the function (term) for a motif. We hope that the additional features may help a classifier to further separate correct motif-term assignment from wrong ones. Third, there exist many motifs with known functions (e.g., those in the PROSITE database), and it is desirable to take advantage of such information to help predict the functions of unknown motifs. This means that we need methods that can learn from such information. In this section, we show that the use of logistic regression can help achieve all three goals. Specifically, we use logistic regression to combine the mutual information with other features, and produce a probability of correct assignment. The motifs with known functions serve as training examples that are needed for estimating the parameters of the regression function. Feature extraction and parameter estimation ------------------------------------------- We now discuss the features to be used in logistic regression, in addition to the mutual information discussed in the previous section. The goal is to identify a set of features that is helpful to determine whether association of any pair of a GO term and a motif is correct or not, without requiring specific information regarding the function of GO term and motif. For a distinct motif-term pair, we collect following frequency-based features: (1) The number of sequences in which the GO term (*T*) and PROSITE motif (*M*) co-occur (*NT-M*). (2) The number of sequences in which *T*occurs (*NT*). (3) The number of sequences in which M occurs (*NM*). (4) The number of distinct GO terms (*G*) seen associated with *M*(*NG\|M*). (5) The number of distinct PROSITE patterns (*P*) seen associated with *T*(*NP\|T*). In addition, we also consider, as a feature, the similarity of the sequences that support a motif-term pair. Intuitively, if a motif is conserved among a set of diverse sequences, it is more likely that the motif is used as a building block in proteins with different functions. Thus, the average pair-wise sequence similarity of the sequence set can potentially be used as a heuristic feature in the logistic regression classifier. Given a set of sequences, we use a BLAST search engine to perform pair-wise sequence comparisons. We devised a metric *AvgS*to measure the averaged pair-wise sequence similarity per 100 amino acids (see methods) and use it as an input feature for classifier. To cast the prediction problem as a binary classification problem, we augment our data set of motif-term pairs with a class label variable *Y*, so that *Y*= 1 means correct assignment and 0 means incorrect. We represent a motif-term pair by a vector of features ***X***= (*X*~1~,\..., *X*~*k*~), where *k*is the number of features. The seven features/variables used in our experiments are *NT-M, NT, NM, NG\|M, NP\|T, AvgS*, and *M.I.*. Suppose we have observed *n*motif-term pairs, then we have *n*samples of (*y*~*i*~, ***x***~*i*~), *i*= 1, 2, \..., *n*, where, *y*~*i*~is the correctness label and ***x***~*i*~is the feature vector for the corresponding motif-term pair. Our goal is to train a classifier which, when given a motif-term pair and feature vector ***X***, would output a label *Y*with value 1 or 0. Alternatively, we can also consider building a classifier which outputs a probability that *Y*= 1 instead of a deterministic label. Thus, our task is now precisely a typical supervised learning problem; many supervised learning techniques can potentially be applied. Here, we choose to use logistic regression as our classification model because it has a sound statistical foundation, gives us a probability of correct assignment, and can combine our features naturally without any further transformation. In order to build a model only with the truly discriminative features, it is a common practice to perform feature selection for logistic regression. We use a combined forward and backward feature selection algorithm. Starting from the intercept, we sequentially add features into the model and test if the log-likelihood increases significantly; we keep the current feature if it does. After the forward selection, we sequentially drop features from the model, to see if dropping a feature would significantly reduce the log-likelihood of the model; if it does, we exclude the feature from the model, otherwise continue. When testing the significance, we use the likelihood ratio statistic *G*, given by 2*l*(*D\|β*~*f*~)/*l*(*D\|β*~-*f*~), where, *l*(*D\|β*~*f*~) and *l*(*D\|β*~-*f*~) are the log-likelihood of the model with feature *f*and the model without feature *f*, respectively. Since we add or drop one feature at a time, *G*follows χ^2^distribution with degree of freedom of 1 \[[@B21]\]. We use the *p*-value of 0.1 as a significant threshold. Figure [3](#F3){ref-type="fig"} illustrates the procedure of feature selection. We found that the average pair-wise similarity of supporting sequence set does not contribute to the model significantly and so excluded it; all other variables contribute to the model significantly. The results of parameters estimation are show in the Table [3](#T3){ref-type="table"}. Logistic regression classification ---------------------------------- After fitting the model using the training set, we tested the model on the test set, i.e., we used the model to compute an output *p*(*Y*~*i*~= 1\|***X***~*i*~) for each test case. Table [4](#T4){ref-type="table"} shows an example of computed conditional probability of correct assignment for the GO terms associated with the protein motif possible the motif \"PS00383\", which is the \"tyrosine specific protein phosphatases signature and profiles\". The table [4](#T4){ref-type="table"} lists top 5 GO terms, which are observed to be associated with the motif and ranked according to the conditional probability returned by logistic regression. As the results from the logistic regression are the conditional probability that an association of a GO term with a given motif is correct, we need to decide the cut off threshold for making decision. We calculate the sensitivity and specificity for a different threshold from 0.1 to 0.9 with a step of 0.1 and plotted the ROC curves as shown in Figure [4](#F4){ref-type="fig"}. The areas under the logistic regression ROC curves are 0.875 and 0.871 for perfect match and relaxed match test set respectively. The precision of the rules is plotted in panel B, where we see that, as the rule becomes more stringent (using a higher threshold), predictions generally become more accurate. We noticed that the precision on the perfect match test set is more variable. This is probably due to the fact that this data set has fewer cases with *Y*= 1, thus, a small change in the number of cases introduces a large change in percentage. For example, when the threshold is set at 0.9, only three cases are covered by the rule and two of them are correct, thus percent correct drop to 66%. To see whether the additional features are useful, we also performed ROC analysis using different mutual information cutoff threshold on the perfect match test set. The result is shown in Figure [4](#F4){ref-type="fig"} panels **C**and **D**. We see that using mutual information alone performs almost as well as logistic regression with additional features. However, the area under the curve (0.816) is smaller than that of logistic regression (0.875), indicating that logistic regression does take advantage of other features and has more discriminative power than mutual information alone. The coefficients *β*~1~, *β*~2~and *β*~3~for the three features *NT-M*, *NT*and *NM*, which are also involved in the calculation of mutual information, have a very interesting interpretation -- they indicate that the roles of these three variables in the logistic regression model actually are to compromise the effect of mutual information! Indeed, according to the formula of the mutual information, a strong correlation corresponds to a high *NT-M*, low *NT*, and low *NM*, but the coefficients shown in Table [3](#T3){ref-type="table"} clearly suggest the opposite. We believe that this actually corrects one drawback of mutual information -- over-emphasizing the correlation but ignoring the support or the strength of evidence. For example, if a term is rare, say occurs only once in the data set, then it would have a very high mutual information value (due to an extremely low *NT*) with respect to any pattern matched by the sequence to which the term is assigned. But, intuitively, one occurrence is very weak evidence, and at least should be regarded as weaker than when we have a term occurring 10 times in total and co-occurring 9 times with the same motif. The key issue here is that mutual information only reflects the correlation between variables, but does not take into account the strength of evidence, therefore, tends to over-favor the situation where there is a perfect correlation but very little evidence. However, the number of sequences in which the co-occurrence happens, which is called the \"support\" for the association, is also very important. The coefficients for the other two parameters, *NG\|M*and *NP\|T*, are also meaningful. Their negative signs indicate that the more terms a motif co-occurs with or the more motifs a term co-occurs with, the less likely a particular association is correct. This also makes sense intuitively, since all those co-occurring terms can be regarded as \"competing\" for a candidate description of the motif\'s function, so the more terms a motif is associated with, the competition is stronger, and thus the chance that any particular term is a correct description of function should be smaller. Thus, the logistic regression model not only performs well in terms of prediction accuracy but also gives meaningful and logically plausible coefficient values. Discussion ========== In this paper, we explore the use of the Gene Ontology knowledge base to predict the functions of protein motifs. We find that the mutual information can be used as an important feature to capture the association between a motif and a GO term. Evaluation indicates that, even used alone, the mutual information could be useful for ranking terms for any given motif. We further use logistic regression to combine mutual information with several other statistical features and to learn a probabilistic classifier from a set of motifs with known functions. Our evaluation shows that, with the addition of new features and with the extra information provided by the motifs with known functions, logistic regression can perform better than using the mutual information alone. This is encouraging, as it shows that we can potentially learn from the motifs with known functions to better predict the functions of unknown motifs. This means that our prediction algorithm can be expected to further improve, as we accumulate more and more known motifs. Although we have so far only tested our methods on the known motifs, which is necessary for the purpose of evaluation, the method is most useful for predicting the functions of new and unknown motifs. For the future work, we can build a motif function prediction system and apply our algorithm to many candidate *new*motifs e.g., those discovered using TEIRESIAS, SPLASH or other programs. This would further enable us to perform data mining from the Gene Ontology database in several ways. For example, we can hypothesize the functions of a large number of novel motifs probabilistically, then we will be able to answer a query, such as \"finding the five patterns that are most likely associated with the GO term tyrosine kinase\". This is potentially very useful because it is not uncommon that substantial knowledge about the functions and sub-cellular location of a given protein is available even though a structural explanation for the functions remains obscure. On the other hand, we believe that our methods will facilitate identifying potentially biological meaningful patterns among the millions of patterns returned by pattern searching programs. A sequence pattern that associates with certain GO term with high M.I. or probability is more like to be a meaningful pattern that that with low scores. Furthermore, our methods can also be used in automatic annotation of novel protein sequences as suggested in Schug et al and Rigoutsos et al \[[@B9],[@B13],[@B22]\]. Our methods provide different approaches to associate sequence patterns with functional descriptions. After associating functional descriptions (in the form of GO term) to motifs, we can determine what motifs a novel protein sequence matches and correspondingly transfer the functional descriptions associated with motifs to the sequence. One key advantage of our methods is that the probability of correctness for a GO-motif association can be considered as confidence or uncertainty. This enables one to optimize the automatic annotation according to Bayesian decision theory and minimize the risk of incorrect annotation. Having stated the potential uses of our approaches, we also realize that there exist some limitations for our methods. For example, in order to predict the function of a newly identified sequence pattern correctly, we would require functional annotations of the sequences of GO database be complete and accurate, which may not always be the case. In this paper, we mainly used the motifs with known function to evaluate the capability of the methods developed in this research. Our result shows that the methods work well with known sequences patterns. Currently, the annotation of motif function with GO term is carried out manually at the European Bioinformatics Institute (the GOA project). Such approach is warranted because human annotation is more accurate than automatic ones. However, as the amount of information regarding protein functions accumulates and a large number of new potential motifs are discovered, it will be very labor intensive to annotate the potential association of protein function and protein patterns. By then, the methods studied in this research will potentially prove to be useful to discover the underlying protein motifs that are responsible for the newly annotated function. For example, the methods can be used as prescreening to narrow down to the most possible associations of protein function and motifs, thus facilitate human annotation. Conclusions =========== In summary, we have developed methods that disambiguate the associations between of Gene Ontology terms and protein motifs. These methods can be used to mine the knowledge contained in the Gene Ontology database to predict the function of novel motifs, discover the basis of a molecular function at primary sequence level and automatically annotated the function of novel proteins. Methods ======= Mutual information ------------------ Mutual information is defined as follows  In which the probabilities *p*(*X*= *x*, *Y*= *y*), *p*(*X*= *x*) and *p*(*Y*= *y*) can be empirically estimated from the data by counting occurrence/co-occurrence followed by normalization. Sensitivity and specificity --------------------------- The sensitivity and specificity of the rules are calculated as   where *TP*(True Positive) is the number of associations labeled as correct among the retrieved motif-term pairs meeting the ranking cutoff criteria, *FN*(False Negative) is the number of associations labeled as correct but not retrieved, *TN*(True Negative) is the number of associations labeled as incorrect and not retrieved, and *FP*(False Positive) is the number of associations labeled incorrect but are retrieved. Averaged sequence similarity ---------------------------- Calculation of the average pair-wise sequence similarity per 100 amino acids (*AvgS*) of a sequence set is as follows  Where S~*ij*~is raw BLAST pair-wise similarity scores between the sequence *i*and sequence *j*; *L*~*i*~and *L*~*j*~are the lengths of sequences *i*and *j*, respectively; *n*is the number of sequences in the set; and *δ*(*i*, *j*) is a delta function which equals 1 if *i*= *j*and 0 otherwise. Logistic regression ------------------- The logistic regression model is a conditional model that assumes the following linear relationship between *p*(*Y*= 1\|***X***) and *X*~1~, \..., *X*~*k*~:  where, *β*= (*β*~0~, *β*~1~, \..., *β*~*k*~) is the parameter vector. We can fit the logistic regression model (i.e., estimate the parameters) using the Maximum Likelihood method -- essentially setting the parameters to values at which the likelihood of the observed data is maximized (Hosmer and Lemeshow 1989, Hastie et al 2001). In our experiments, we use iteratively reweighted least squares (IRLS) algorithm \[[@B23]\] to fit the logistic regression model. All features are normalized to zero mean and unit variance before training. Acknowledgments =============== This research is partially supported by National Library of Medicine (NLM) training grant to Lu, X (No. 3 T15 LM07059-15S1), Gopalakrishnan, V (No. 5 T15 LM07059-15) and NLM grant to Buchanan, B.G. (No. LM06759). We would like to thank Drs. Roger Day, Milos Hauskrecht and Gregory Cooper for insightful discussions. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Correlation of mutual information cutoff and term assignment precision**. Different M.I. cutoff value is used to assign GO terms to motifs. The precision of assignment is plotted vs M.I. cutoff value. The Pearson correlation coefficient between the precision and the cutoff is 0.837. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Assigning GO term to motif according to rank of M.I.A**. ROC curves of assigning GO terms to motifs according to rank of mutual information. The filled circle is for the perfect match data set, and the area under the curve is 0.782. The empty triangle is for the relaxed match data set, and the area under the curve is 0.735. The numbers next to data points indicate cut-off ranks of decision rules. Diagonal line corresponds to random model. **B**. Precision of rules based on different mutual information cutoff ranks. Filled bars are results on the perfect match data set. Empty bars are the results on the relaxed match data set. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The algorithm for feature selection. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Comparison of results with probability and M.I.A**. ROC curves for classifying motif-term associations at different probability threshold. Filled circles are the results on the perfect match test set with an area under curve of 0.8715. Empty triangles are on the relaxed match test set with an area under curve of 0.871. Data points correspond to thresholds of p(*Y*= 1\|***X***) from 0.9 to 0.1 (from left to right) with a step of 0.1. **B**. Precision (positive predictive value) at different probability cutoffs, where solid bars are the result on the perfect match test set and the open bars for the relaxed match test set. **C**. ROC curve for decision rules based on different M.I. cutoff thresholds with an area under curve of 0.816. **D**. Precision at different M.I. cutoffs. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### GO terms and PROSITE patterns for the protein MGI\|MGI:97380 ::: **GO Terms and Descriptions** **PROSITE Motifs and Descriptions** ------------------------------- ------------------------------------------- --------- ------------------------------------------------------ GO:0005576 Extracellular PS00248 Nerve growth factor family signature and profile (1) GO:0005515: Protein-binding PS50270 Nerve growth factor family signature and profile (1) GO:0005166: neurotrophin p75 receptor ligand GO:0008544 epidermal differentiation GO:0007422 peripheral nervous system development GO:0007420 brain development GO:0007403 [determination of glial fate]{.underline} ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Five GO terms associated with PROSITE pattern PS00109 (tyrosine kinase signature) ::: GO Term GO Definition NM NT NT-M M.I. ------------ ------------------------------------------- ----- ------ ------ --------- GO:0004713 Protein tyrosine kinase 246 68 51 0.00599 GO:0006468 Protein amino acid phosphorylation 246 409 69 0.00464 GO:0004714 Transmembrane receptor protein kinase 246 33 29 0.00362 GO:0004715 Non-transmembrane protein tyrosine kinase 246 17 14 0.00168 GO:0005887 Integral membrane protein 246 1162 44 0.00118 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Result of logistic regression parameters estimation ::: Estimated Coefficients -------------------- ------------------------ --------- Intercept (*β*~0~) -1.7549 -0.6263 NT-M(*β*~1~) -0.3845 -0.4546 T (*β*~2~) 1.3652 1.6827 NM (*β*~3~) 1.0497 0.4735 NG\|M (*β*~4~) -1.9792 -1.1113 NP\|T (*β*~5~) -1.7883 -2.5494 M.I. (*β*~6~) 1.0002 1.1598 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Top 5 GO terms associated with the motif PS000383 ranked according the conditional probability of correctness of association. The column 2\~7 consist of the feature vector for motif-GO association is listed, the conditional probability p(*Y*= 1\|***X***) is calculated with trained model and the true classes are list in right two columns of the table. The definition of the GO terms is listed at the bottom of the table. ::: GO Terms Input Features (***X***) p(*Y*= 1\|***X***) True Class ------------ -------------------------- -------------------- ------------ ------- ------- ---------- --------- --- NT-M NT NM NG\|M NP\|T M.I GO:0006470 65 122 191 95 27 0.007268 0.97577 1 GO:0005001 23 24 191 95 5 0.003177 0.87681 1 GO:0004726 10 11 191 95 10 0.001331 0.68682 1 GO:0005634 11 1785 191 95 281 5.08E-06 0.27882 0 GO:0005887 17 1162 191 95 225 0.000193 0.15536 0 GO:0006470: Protein amino acid dephosphorylation; Cellular Processes GO:0005001: transmembrane receptor protein tyrosine phosphatase activity: Molecular Function GO:0004726: non-membrane spanning protein tyrosine phosphatase activity: Molecular Function GO:0005634: Nucleus; Cellular Component GO:0005887: Integral to plasma memberane; Cellular Component :::

PubMed Central

2024-06-05T03:55:47.639506

2004-9-2

PMC517494

Background ========== Viruses as obligate intracellular parasites rely upon the host cell for different steps in their life cycle, including attachment, disassembly, transcription, translation, reassembly, and egress. Consequently, characterization of these cellular processes will be essential for any understanding of viral life cycles, and may provide cellular targets for new antiviral therapies. The susceptibility to virus infection varies greatly among different cell types, and virus-resistant cells frequently emerge post-infection \[[@B1]-[@B4]\]. This suggests that host cell contributions to the virus life cycle, although complex, have genetic determinants. We therefore used a genetic approach to identify cellular genes required for infection by reovirus, a small cytolytic RNA virus that replicates in the cytoplasm. A gene trap retrovirus was use to create libraries of rat intestinal epithelial (RIE-1) cell clones in which each clone contained a single gene disrupted by an integrated retrovirus. The mutant libraries were then infected with reovirus, and resistant clones were selected. We hypothesized that genes mutated by gene entrapment may confer reovirus resistance as a result of either haploinsufficiency or loss of heterozygosity and could be identified by characterizing the genes disrupted by the entrapment vector. From these experiments we have isolated 152 clones and have characterized mutations in 111 different genes, providing potential candidates required for reovirus infection and/or cell killing. Many of the disrupted genes have known or imputed functions, and several are known to function in the same or related pathways. For example, four mutations affected genes in the insulin growth factor-2 (IGF-II) pathway, including genes encoding the IGF-ll/manose-6-phosphate receptor \[[@B5],[@B6]\] (*Igf2r*, locus ID 25151), the IGF binding protein 5 protease \[[@B7]\] (*Prss11*, locus ID 65164, 2 clones), and CTCF (*Ctcf*, locus ID 83726), a transcriptional repressor of the IGF-II gene (*Igf2*) involved in paternal imprinting. The frequency of mutations involving the IGF-II pathway led us to investigate the role of IGF-II in reovirus infection. Clone 6B72, which contains a mutation in *Ctcf*\[[@B8]\], was found to over express *Igf2*transcripts, consistent with the known role of CTCF as a transcriptional repressor of the *Igf2*gene. Moreover, forced expression of IGF-II in RIE-1 cells was sufficient to confer cellular resistance to lytic reovirus infection. Enforced IGF-II expression also transformed RIE-1 cells to anchorage independent growth, a phenotype associated with malignant change. These results represent the first use of gene entrapment to identify components of host cell metabolism required for virus infection and illustrate a potential mechanism of carcinogenesis associated with chronic virus infection. Results ======= Disruption of the CTCF gene results in cells resistant to lytic infection by reovirus ------------------------------------------------------------------------------------- Gene entrapment strategies to identify host genes required virus replication depend on methods to select for virus resistant clones present at about one in 10^4^--10^5^mutagenized cells. Unfortunately, cells persistently infected with reovirus (PI) can emerge at high frequencies (one in 10^2^--10^3^) and are intrinsically resistant to the virus \[[@B9],[@B10]\]. In preliminary studies, we found that hepa 1/a cells were not suitable for genetic studies due to the emergence of PI clones. However, persistently infected rat intestinal epithelial cells (RIE-1) \[[@B11],[@B12]\] were found to require a serum survival factor and die when placed in serum free media (Figure [1](#F1){ref-type="fig"}). *In vitro*infection of RIE-1 cells with reovirus also appears to mirror virus replication in the rodent host \[[@B13]-[@B15]\]. Consequently, RIE-1 cells were used in the present study, and reovirus-resistant clones were selected in serum-free medium to remove PI survivors. RIE-1 cells were mutagenized by using the U3NeoSV1 gene trap shuttle vector \[[@B16]\] (see Methods) and the resulting libraries of mutagenized cells were infected with reovirus serotype 1/Lang at a multiplicity of infection (MOI) of 35, to select for clones resistant to lytic infection. The isolated clones did not express reoviral antigens, and did not produce infectious virus as assessed by plaque assay, suggesting these reovirus resistant clones were not PI. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Persistently infected RIE-1 cells fail to survive in serum-free media.**RIE-1 parental cells and cells persistently infected with reovirus type 1 were plated in complete medium (FBS^+^) or in media in which the serum was omitted (FBS^-^). Surviving cells were stained with gentian violet after 7 days. Darkly staining wells represent cell survival. :::  ::: Regions of genomic DNA adjacent to the U3NeoSV1 provirus in each virus resistant clone were isolated by plasmid rescue and sequenced. Altogether, of the 151 isolated clones, 62% of flanking sequences matched known or presumptive genes, and an additional 23% were represented in the public databases of expressed sequence tags (dbEST) or non-redundant sequences (nr). From the 111 clones matching known or presumptive genes, 10 genes were represented more than once. Many of the disrupted genes have known or imputed functions, and several are known to function in the same or related pathways. For example, the library included 4 independent mutations involving three genes that encoded proteins associated with the insulin growth factor-2 (IGF-II) signalling pathway, namely, IGF-ll/manose-6-phosphate receptor \[[@B5],[@B6]\] (*Igf2r*, locus ID 25151), the IGF binding protein 5 protease \[[@B7]\] (*Prss11*, locus ID 65164, 2 clones), and CTCF (*Ctcf*, locus ID 83726), a transcriptional repressor of IGF-II involved in paternal imprinting \[[@B17],[@B18]\]. The position of the provirus (clone 6B72) in the first intron of the *Ctcf*gene is shown in Figure [2](#F2){ref-type="fig"}. CTCF differentially represses maternal *Igf2*gene expression, whereas the imprinted paternal gene escapes repression due to methylation of CTCF binding sites \[[@B17],[@B18]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Disruption of the *Ctcf*gene in 6B72 cells.**DNA sequences flanking the U3NeoSV1 provirus in 6B72 cells were cloned and sequenced. The flanking sequences were identical to sequences in the rat genome, placing the provirus in the first intron of the *Ctcf*gene (A). Filled and open boxes indicate coding and non-coding exons, respectively. Flanking sequences 5\' of the U3NeoSV1 provirus (B) include the first exon (shaded) of the gene. :::  ::: Igf2 transcripts induced by CTCF disruption ------------------------------------------- Considering that four clones had mutations in the IGF-II pathway, the relationship between the *Ctcf*mutation in 6B72 cells and the virus resistant phenotype was investigated further. Levels of CTCF protein in 6B72 cells were reduced by about 50% as assessed by western blot analysis (Figure [3A](#F3){ref-type="fig"}), consistent with the disruption of one allele. Diminished CTCF expression was associated with an increase in *Igf2*transcripts as assessed by Northern blot hybridization (Figure [3B](#F3){ref-type="fig"}). In addition, two products were amplified from 6B72 cells by RT-PCR using primers flanking the IGF-II pro-hormone coding sequence (Figure [3C](#F3){ref-type="fig"}). The first product was identical to the rat IGF-II pro-hormone (pro-IGF-II) coding sequence (Accession X17012), whereas, the second contained 14 additional nucleotides generated by splicing of exon 2 to an alternative splice acceptor located 14 nucleotides upstream of the normal *Igf2*exon 3 splice acceptor site. The alternative transcript is expected to encode a pro-IGF-II protein extending 11 amino acids into the E-peptide sequence ending in a stretch of 60 amino acids lacking homology with any known protein (Figure [4](#F4){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **CTCF and *Igf2*expression in RIE-1 and 6B72 cells.**Levels of CTCF protein were assessed by Western blot analysis (A), normalized to a β-actin control. Levels of *Igf2*transcripts were assessed by Northern blot analysis (B) normalized to GAPDH control. Protein content, as assayed by western blot analysis and standardized to β-actin was decreased in the 6B72 cell clone to 30% of control. Reverse transcriptase PCR analysis of *Igf2*transcripts (C). The RT-PCR products (arrows) were separated on a 2% agarose gel, revealing an additional transcript in the 6B72 cells. The DNA sequence of the larger RT-PCR product (D) revealed an alternatively spliced transcript (*Igf2*^*sv*^) generated by splicing of exon 2 to a cryptic 3\' splice site located 14 nucleotides upstream of exon 3. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Alternatively spliced product contains a single nucleotide polymorphism in the igf2 coding sequence.**Intron sequences incorporated into the alternatively spliced transcript (highlighted in black) alter the translational reading frame of the pro-homone downstream of the coding sequence of the processed IGF-II protein (italics and bold). The *Igf2*^*sv*^PCR product also contained a G to A base substitution (underlined) that replaces alanine with threonine at codon 62 (boxed) of the mature hormone. :::  ::: Resistance to reovirus lytic infection results from increased Igf2 expression ----------------------------------------------------------------------------- The stability of virus resistance in the 6B72 cell was tested by infecting RIE-1 and 6B72 cells with reovirus type 1 at a MOI of 10. There was approximately a 10 fold lower titre of reovirus obtained following infection of 6B72 cells as compared to RIE-1 cells at 24 hrs. post-infection (4.5 × 10^5^versus 5.1 × 10^6^), and the difference was also maintained at 48 hours post-infection (data not shown). Additionally, there was a dramatic difference in the survival of 6B72 cells after being exposed to high titres of reovirus type 1 (Figure [5](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **IGF-II modulates reovirus resistance in RIE-1 and 6B72 cells.**RIE-1 and 6B72 cells expressing either the IGF-II pro-hormone (proIGF2) or the alternatively spliced transcript (IGF2^SV^, Figure 3D) were challenged with serial dilutions of reovirus type 1 (upper panel), and the surviving cells were stained with gentian violet 4 days post-infection. The multiplicity of infection (MOI) for each row is indicated. pro-IGF-II converted RIE-I cells to a reovirus resistant phenotype (RIE-1/proIGF2) but had little if any effect on already-resistant 6B72 cells (6B72/proIGF2). Plasmids expressing the alternatively spliced Igf2 transcript had no effect on RIE-1 cells (RIE-1/IGF2^SV^) but abrogated virus resistance in 6B72 cells (6B72/IGF2^SV^). The experiment was repeated 3 times. Expression of I*gf2*transgenes (lower panel) was monitored by Northern blot hybridization, and the expression of Igf2 in RIE-1 cells is shown for comparison. Expression of *Igf2*in 6B72 is not shown. :::  ::: To determine whether *Igf2*confers resistance to reovirus infections, clones of RIE-1 cells over-expressing the full-length *Igf2*transcript or the splice variant (*Igf2*^*sv*^) were generated and examined for their capacity to resist lytic infection. As shown in Figure [5](#F5){ref-type="fig"} expression of wild type (*Igf2*), but not the splice variant (*Igf2*^*sv*^) increased the resistance of RIE-1 cells to reovirus infection by over 100 fold. However, when the *Igf2*^*sv*^was transfected into 6B72 cells, the ability of 6B72 cells to survive infection was abolished. Expression of the *Igf2*gene in an anti-sense orientation caused no significant difference in the capacity of 6B72 cells to resist infection (data not shown). These studies suggest that increased *Igf2*expression in 6B72 cells is associated with their capacity to resist reovirus infection, and that the *Igf2*^*sv*^encodes a trans-dominant isoform that blocks the activity of *Igf2*. The IGF-II hormone confers resistance to lytic infection -------------------------------------------------------- *Igf2*transcripts encode a pro-hormone of 180 amino acids that is processed to generate the 67-residue IGF-II protein \[[@B19]\]. Other proteolytic products including the 89-residue carboxy-terminal E peptide may also be biologically active \[[@B20]\]. The *Igf2*^*sv*^PCR product also contained a single nucleotide substitution (G1393A) that results in the substitution of a threonine for alanine at position 62 of IGF-II, raising questions about *Igf2*sequences that influence reovirus resistance. Vectors expressing only the 68-residue IGF-II protein both with and without the A62T change were compared for their ability to confer resistance to lytic infection by reovirus type 1 (Figure [6](#F6){ref-type="fig"}). The IGF-II expression vector converted RIE-1 cells to a virus resistant phenotype; whereas, the IGF-II^62T^expression plasmid was inactive (Figure [6](#F6){ref-type="fig"}) and did not suppress the resistance of 6B72 cells (data not shown). These results indicate that virus resistance can be affected by mutations in the IGF-II coding sequence and downstream sequences, including the E-peptide, are not required. However, the trans-dominant effects of *Igf2*^*sv*^apparently require alterations to the carboxy-terminus of the IGF-II pro-hormone. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **IGF-II sequences lacking the E-peptide can convert RIE-1 cells to a reovirus-resistant phenotype.**Two independent clones of RIE-1 cells transfected with plasmids expressing native (IGF-II) and mutant (IGF-II^A62T^) proteins without the carboxyl terminal extension-peptide (E-peptide) were challenged with serial dilutions of reovirus type 1 as described in Figure 4. Native IGF-II protected RIE-1 cells from reovirus infection (IGF-II^(a)^and IGF-II^(b)^), while the IGF-II^A62T^mutant (IGF-II^A62T(a)^and IGF-II^A62T(b)^did not. Non-infected RIE-1 cells (C), and infected 6B72, and RIE-1 cells were included as controls. Expression of *Igf2*transgenes (lower panel) was monitored by Northern blot hybridization, and the expression of Igf2 in RIE-1 cells is shown for comparison. The native IGF-II (small arrow) is slightly larger than the cDNA constructs (larger arrow), whereas the double-sided arrow marks the constitutively expressed *GAPDH*, as shown. Expression of *Igf2*in 6B72 is not shown. :::  ::: Over expression of *igf2*confers resistance to other reovirus sertotypes ------------------------------------------------------------------------ RIE-1 cells are intrinsically resistant to infection by reovirus type 3 \[[@B13]\]; therefore, experiments to assess the effects of IGF-II expression on lytic infection by reovirus type 3, were performed using murine L-cells. As shown in Figure [7](#F7){ref-type="fig"}, L-cells expressing *Igf2*were significantly more resistant to both reovirus serotypes than the parental cells, indicating that the ability of IGF-II to confer resistance to reovirus infection is not limited to a single cell or virus type. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Decreased lytic infection of L-cell clones over expressing the IGF-II gene.**Constitutively expressed GAPDH was used to assess loading of RNA in lanes. Survival of lytic infection was determined by infecting 10^5^L-cells or L-cells expressing the pro-IGF2 transgene with varying concentrations of reovirus type 1 (A) or type 3 (B). The multiplicity of infectious virus particles per cell (MOI) is indicated for each virus serotype. Surviving cells were visualized at 4 (A) or 5 days (B) following infection with gentian violet. Transgene expression was determined by northern blot (C). Experiments were repeated three times and a representative experiment is shown. :::  ::: IGF-II expressing RIE-1 cell has delayed disassembly of virus particle ---------------------------------------------------------------------- Previous studies have indicated that proteoytic disassembly of virus particles occurs in the lysosome and requires the participation of cathepsin B and L that are transported by the *Igf2r*gene \[[@B21]-[@B23]\]. *Igf2*binds to *Igf2r*resulting in alteration in cathepsin trafficking \[[@B24]\]. Georgi and colleagues showed that directly fluorescentated reovirus particles dissipated their fluorescent signal coincident with disassembly of the outer capsid \[[@B25]\]. To determine whether over expression of *igf2*would affect disassembly of virions, we examined the fate of virus during the first hours of infection. Purified reovirus type 1 virions were directly labeled with fluorescein and adsorbed to RIE-1 or 6B72 cells, and the persistence of a fluorescein signal determined at 2 hrs following virus absorption (fig [8A,8B](#F8){ref-type="fig"}). The binding of virions to RIE-1 cells and 6B72 cells and initial accumulation of virus particles within cells was similar, however fluorescence was almost non-detectable in RIE-1 cells (Figure [8A](#F8){ref-type="fig"}) whereas it was still present in 6B72 cells at 2 hrs (Figure [8B](#F8){ref-type="fig"}). In addition, western blot analysis of virion proteins 2 hrs following attachment to cells confirmed the persistence of the σ3 protein in the 6B72 cell clone, but not RIE-1 cells (fig [8C,8D](#F8){ref-type="fig"}). Therefore, disassembly of virions is altered in the 6B72 cell clone. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **6B72 cells have delayed disassembly of reovirus type 1.**Fluorescein-labelled reovirus particles were absorbed to RIE-1 (A) or 6B72 cells (B). Persistent fluorescence at 2 hours was found in 6B72 cells, but not in RIE-1 cells. Non-replicating reovirus type 1, at 3 × 10^4^particles per cell, was adsorbed to RIE-1 (C) or 6B72 (D) cells at 4°C, washed and incubated at 37°C for 2 and 4 hours. Cells were lysed and the state of virus particles determined by western blot. The outer capsid proteins μ1 and σ3 are present in the 6B72 cell preparations at 2 and 4 hours, but not in the RIE-1 cells. :::  ::: CTCF deficient RIE cells grow in soft agar, a consequence of increased Igf2 expression -------------------------------------------------------------------------------------- Accumulating evidence suggests that enhanced IGF-II expression associated with loss of *Igf2*genomic imprinting may promote tumour formation \[[@B26],[@B27]\]. Although RIE-1 and 6B72 cells appeared to proliferate at similar rates as assessed by MTS/PMS incorporation (Data not shown), 6B72 cells that have a disrupted *Ctcf*gene were capable of forming colonies in soft agar (Figure [9b](#F9){ref-type="fig"}). To assess the role of IGF-II in anchorage-independence, cells transfected with *Igf2*and *Igf2*^*sv*^were tested for their ability to grow in soft agar. RIE-1 cells transfected with *Igf2*, but not *Igf2*^*sv*^formed colonies in soft agar (Figure [9a](#F9){ref-type="fig"}), whereas 6B72 cells transfected with *Igf2*^*sv*^lost their capacity to proliferate in soft agar as did cells transfected with an anti-sense *Igf2*construct (Figure [9b](#F9){ref-type="fig"}). The IGF-II^62T^expression plasmid had no transforming activity (Figure [9d](#F9){ref-type="fig"}). On balance, transforming activities of the different *Igf2*plasmids on RIE-1 and 6B72 cells as assessed by the soft agar assay were similar to their effects on reovirus resistance, although the vector expressing only IGF-II was less active in transforming RIE-1 cells to anchorage independence than vectors expressing the entire pro-hormone. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Anchorage-independent growth phenotypes of RIE-1 cell clones.**10^5^cells were suspended in media containing 1% agarose and plated in 6 well culture dishes. RIE-1 cells acquire the ability to grow in soft agar after being transfected with a vector expressing pro-IGF2 but not the IGF2^SV^splice-variant (a). The vector inserted in the *Ctcf*gene (6B72) confers the ability to grow in soft agar, but the phenotype is suppressed by expression of IGF2^SV^(b). Clones selected for reovirus resistance with gene trap vectors inserted into the *Prss11*, *Igf2r*and *Anxa2*genes failed to grow in soft agar (c). RIE-1 cells expressing native IGF-II protein without the E-peptide grew in soft agar but the colonies were smaller (d) than produced by pro-IGF-2 (a), while the corresponding IGF-II^A62T^protein (E-peptide) did not transform RIE-1 cells to anchorage independence (d). Colonies were photographed (20×) after 7 days except (d) where the cells were photographed after 10 days. :::  ::: The capacity to proliferate in soft agar was not a property of other reovirus resistant cell clones that contained mutations in the IGF-II pathway. Figure [9](#F9){ref-type="fig"} shows that cell clones with disrupted *Igfr*or *Prss11*genes did not form colonies in soft agar (Figure [9c](#F9){ref-type="fig"}). An additional reovirus resistant-clone with an insert in the *Anxa2*(annexin 2) gene, associated with cytomegalovirus infection \[[@B28]\] and recognized to bind to the insulin and insulin growth factor receptor-1 \[[@B29]\], also failed to grow in soft agar (Figure [9c](#F9){ref-type="fig"}). Therefore, the capacity to proliferate in soft agar was not a general property of reovirus-resistant cells, even in clones that contain mutations in the IGF-II pathway. However, L-cells also displayed enhanced ability to grow in soft agar in addition to virus resistance, following enforced *Igf2*expression (Figure [10](#F10){ref-type="fig"}). ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **IGF-II increases colony formation of L-cells in soft agar.**Forced expression of the rat pro-Igf2 gene (pro-Igf2^c^) in L-cells increases the number of soft-agar colonies by 4 to 5 fold as compared to the L-cell parent, as shown in this representative photomicrograph at 7 days (10×). :::  ::: Discussion ========== Insertional mutagenesis provides an approach to identify genes associated with selectable cellular phenotypes. We have isolated over 100 potential clones with mutations in genes that may play roles in the life cycle of reovirus. In the present study, one clone resistant to reovirus lytic infection contained a provirus inserted into the gene encoding the CTCF transcriptional regulator. CTCF binding motifs are present in many genes, including *Igf2*\[[@B26]\], *H19*\[[@B27]\], and *Myc*\[[@B8]\]. However, since 3 other clones selected for reovirus resistance contained mutations in the IGF-II pathway, the role of IGF-II in virus-resistance was investigated further. Reduced expression of the *Ctcf*gene was associated with enhanced *Igf2*expression in virus-resistant cells, while forced expression of the *Igf2*gene in the parental RIE-1 line was sufficient to confer resistance to lytic reovirus infection. By inference, the recovery of inserts affecting other genes in the IGF-II signalling pathway suggests that mutations in multiple genes may affect the same phenotype by acting on a common pathway. The insert in *Igf2r*was found to decrease the expression of the gene as assayed by northern blot analysis (data not shown). As IGF-II targets the *igf2r*to lysosomal degradation, mutations in the genes encoding either the receptor (*Igf2r*) or its ligand (*Igf2*) will affect the activity of the other, and result in a reduced endosomal trafficking of hydrolases necessary for reovirus disassembly \[[@B24]\]. Our data indicates there is a decrease in virus disassembly in 6B72 cells, consistent with a block at this step in morphogenesis. Further studies will be required to assess if and how inserts in the *Prss11*and *Igf2r*genes influence reovirus resistance. As the entry, disassembly, transcription, translation, and repacking of viruses share common features; we anticipate that common cellular pathways will influence infection by other virus families. Indeed, *Igf2r*has been implicated in herpes simplex and zoster virus infection \[[@B30],[@B31]\]. However, the present study is the first to show a direct connection between *Igf2*gene expression and resistance to lytic virus infection. By using constructs that encode the mature hormone without the E-peptide, we were able to show that forced IGF-II expression is sufficient to confer a reovirus resistant phenotype. These results differ from other studies in which reovirus replication was enhanced by treatment of RIE-1 cells with insulin. The latter effect is presumably caused by enhanced virus replication associated with cell proliferation \[[@B13]\]. Expression of the *Igf2*gene is frequently elevated in common childhood and adult neoplasms \[[@B27],[@B32]-[@B38]\] and has been associated with tumour progression and metastasis \[[@B39],[@B40]\]. *Igf2*also transformed RIE-1 cells to anchorage-independence, a phenotype that predicts the potential for malignant growth *in vivo*. Virus-resistant 6B72 cells also grew in soft agar, presumably as a result of enhanced IGF-II expression. Since plasmids expressing only IGF-II were less active in transforming RIE-1 cells to anchorage independence than vectors expressing the entire pro-hormone, further study is needed to determine whether the capacity of RIE-1 cells to proliferate in soft agar is enhanced by the E-peptide or other products derived from the carboxy terminus of the pro-hormone. In other studies, the E-peptide enhanced insulin secretion from β-cells \[[@B20]\], and may play a role in cellular transformation \[[@B31]\]. cDNA clones of an alternatively spliced *Igf2*transcript (*Igf2*^*sv*^), blocked the ability of IGF-II to promote reovirus resistance and anchorage independent growth in a trans-dominant manner. The protein coding sequence of *Igf2*^*sv*^contains a frame shift in the E-peptide region and lacks a site \[[@B20],[@B41]\] required for the proteolytic processing of the pro-hormone. The alternative splice site is used very infrequently \[only one EST (AA259833) in dbEST was similarly spliced\] and thus probably plays no physiological role. Further studies are planned to determine molecular basis for the dominant negative activity of *Igf2*^*sv*^. 6B72 cells were highly resistant to reovirus infection as determined by virus yield and cell survival at different times post-infection. Virus resistance was a genetically selected trait manifested by clonally pure cell populations, could be conferred by enforced IGF-II expression, and involved decreases in virus disassembly. Although decreased virus disassembly is sufficient to explain virus resistance, we do not exclude the possibility that other mechanisms may contribute to the resistance of 6B72 cells, since any genetic selection may generate clones with multiple, independent mutations. The fact that an early step in infection (uptake/disassembly) is defective in 6B72 cells makes it very difficult to test whether downstream steps might also be affected. While the original impetus of our studies was to understand the replication cycle of intracellular pathogens that cause acute and chronic infectious diseases, the finding of cell growth phenotypes associated with virus resistance is of some interest. It has been proposed that lytic viruses may used to treat certain malignancies \[[@B42]-[@B45]\]. However, based upon our observations, such therapy may carry a risk associated with selection of virus resistant cell clones with enhanced growth/survival potential. Additionally, chronic infections contribute to the development of a number of human cancers \[[@B46]-[@B49]\]. While the carcinogenic process is not well understood, cell proliferation associated with inflammation is also thought to contribute to tumour promotion \[[@B50],[@B51]\]. The present study illustrates how carcinogenesis could also be influenced by selection for virus-resistant cells with mutations in genes affecting cell proliferation or survival. Conclusions =========== This is the first reported use of gene entrapment to identify host genes affecting the susceptibility of cells to virus infection. These results indicate that IGF-II, and by inference other components of the IGF-II signaling pathway, can confer high levels of resistance to lytic reovirus infection. IGF-II expression specifically blocked virus disassembly. *Ctcf*disruption and forced *Igf2*expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth *in vivo*. Therefore, these results illustrate a potential indirect mechanism of viral carcinogenesis by which cells selected to virus resistance may also have enhanced oncogenic potential. Methods ======= Entrapment mutagenesis and selection of reovirus resistant clones ----------------------------------------------------------------- To identify genes required for reovirus lytic infection, a gene trap retrovirus shuttle vector, U3NeoSV1, was used to generate mutagnized rat intestinal (RIE-1) cells \[[@B52]\]. RIE-1 cells were infected with the gene trap vector at a multiplicity of infection \<0.1, and were selection in media containing G418 sulfate (0.7 mg/ml) (Clontech, Palo Alto, CA, USA)\[[@B52]\]. Twenty libraries of mutant RIE-1 cells, each consisting of 10^4^independent gene entrapment events, were generated and expanded until each mutant clone was represented by approximately 10^3^sibling cells. These cells were plated at low density and incubated in serum-free media for 3 days until they became quiescent, infected overnight with reovirus serotype 1 at a multiplicity of infection of 30 plaque forming units (pfu) per cell. The infected cells were detached with trypsin, DMEM medium containing 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, UT, USA) was added and the cells were allowed to reattach. After 4--6 hours the medium was replaced with serum-free medium and the cells were incubated for several days until only a few cells remained attached to the culture flask. Cells that survived the selection were allowed to form colonies that were expanded for further analysis. Reovirus stocks and infectivity assays -------------------------------------- Reovirus type 1 (Lang) and reovirus type 3 (Dearing) were previously described \[[@B53]\]. A stock of reovirus that was passaged twice in L cells was purified \[[@B14]\] and the purified virus band was fluorescein labelled as previously described \[[@B25]\]. For some experiments the top component, consisting of virus particles that are devoid of genome, was used to study the entry pathway \[[@B54]\]. Survival of parental L- and RIE-1 cells and RIE-1 and L-cells transfected with *Igf2*constructs was determined in 96-well plates seeded at 5 × 10^4^per well. On the following day, serial dilutions of reovirus type 1 or type 3 were added in 100 μl of media and cells were incubated at 37°C and 5%CO~2~for 1 hour. Cells were washed three times in PBS, and fresh media was added containing 0.1% anti-reovirus antibodies to inhibit secondary infection. Cells were incubated for 4 to 5 days, and surviving cells visualized with gentian violet. Studies were repeated a minimum of three times. To determine the titre of reovirus present in cells, cells were frozen and thawed three times and plaque assays were performed as previously described \[[@B14]\]. Titres of virus were repeated twice. Fluorescence microscopy ----------------------- For experiments involving fluorescein labeled reovirus, cells were grown on glass slides and fixed following appropriate times with 4% paraformaldehyde, dehydrated, and mounting in cytoseal acrylic resin (Stephens Scientific, Cornwall NJ, USA) to improve clarity and prevent bleaching. Fluorescence microscopy was performed using an Axiophot microscope (Carl Zeiss, Inc., Thornwood, NY, USA), with a 40×/1.3 plan Neofluar objective and fluorescein filter set. Images were captured with a low-light, cooled CCD camera (Micromax, Photometries, Inc., Tucson, AZ, USA). Identification of genes disrupted by gene entrapment ---------------------------------------------------- To identify the gene disrupted by the vector in clones surviving reovirus infection, the shuttle-vector property of U3NeoSV1 was utilized. Regions of genomic DNA adjacent to the U3NeoSV1 provirus were cloned by plasmid rescue, and sequenced \[[@B52]\]. Sequencing was done using an automated sequencer (ABI 3700 DNA Analyzer, Applied Biosystems, Foster City, CA, USA), and the results obtained were compared to databases available in the public domain (BLAST nr, est, and hgts). The probability of a match to sequences in the databases occurring by chance alone varies due to interspecies conservation and the length of the match. Matches to characterized genes were considered significant if the interspecies matches had a probability score p \<10^-5^and involved non-repetitive sequences. As indicated, virtually all of the genes identified had matches to murine or human gene sequences with p \< 10^-10^and rat with p \< 10^-20^. Igf2 expression was assessed by northern blot hybridization ----------------------------------------------------------- Total RNA was isolated from cultured cells using Trizole reagent (Gibco BRL, Gaithersburg, MD, USA). 5 μg of RNA was separated on 1.2% agarose gel, and transferred to a nitrocellose membrane. Membranes were hybridized with random prime-labeled (Strategene, Cedar Creek, TX, USA) probes corresponding to a full length of *Igf2*cDNA and either *glyceraldehyde dehydrogenase (GAPDH*) or β-actin cDNA. Igf2 cDNA isolation and expression ---------------------------------- Rat *Igf2*cDNAs were obtained using reverse transcriptase PCR (RT-PCR). Total RNA was extracted from RIE and *6B72*cells using Trizole reagent (Life Technologies, Rockville, MD, USA). RT was performed on 1 μg of total RNA (PTC-100 programmable Thermal Controller, MJ Research. Inc, Watertown, MA, USA). A pair of primers was designed according to rat sequences: CTTCCAGGTACCAATGGGGATC (forward) and TTTGGTTCACTGATGGTTGCTG (reverse). A 500 bp DNA was amplified under following conditions: 95°C, 1 min; 40 cycles of 95°C 30 seconds, 60°C 30 seconds and 68°C 3 minutes; 68°C 10 min; 4°C. Immunoblotting analysis ----------------------- Cells were washed with PBS and lysed in SDS Lamelli buffer. Protein concentration was determined using the bicinchoninc acid protein assay (Sigma-Aldrich Corp., St. Louis, MO, USA). 20 μg of protein extract was loaded in each lane of a 10% SDS-PAGE and run at 100 V. Protein was transferred to a nitrocellulose membrane at 22 V overnight at 4°C. The membrane was washed three times with TBST (50 mM Tris pH 7.5, 150 mM NaCI, 0.05 % Tween 20) and then incubated in blocking buffer (TBST and 5% non fat dry milk, pH 7.5) for 1 hour at room temperature. The membrane was then incubated with anti-mouse, CTCF (1:500, BD Transduction laboratories) and β-actin (1:3000, Sigma-Aldrich Corp., St. Louis, MO, USA) in blocking buffer overnight at 4°C. Following 3 washes, the membranes were incubated with goat anti-mouse secondary antibody (1:20,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour at room temperature, followed by three 15-min washings. Immune complexes were visualized by addition of chemiluminescence reagent (Renaissance, DuPont NEN, Boston, MA, USA) and the membrane was exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY, USA). Transfection ------------ Cells were cultured to semi-confluence and plasmids expressing wild type and variant IGF-II transcripts were transfected into RIE-1 and 6B72 or L-cells using SuperFect Reagent (Qiagen, Inc. Valencia, CA, USA) according to the manufacturer\'s protocol. After 48 hours, transfected cells were passaged, 1:10, into medium containing hygromicin B (selective medium) at a concentration determined to kill 100% of non-transfected cells (150 mg/ml for RIE-1 and 6B72 cells, 650 mg/ml for L-cells). Cells were maintained in selective medium until clones appeared. Soft agar colony forming assay ------------------------------ Dual layers of sea plaque agarose were made with the bottom layer consisting of a 50:50 mixture of 1.6% agarose solution 1:1 and 2X medium. The bottom layer was allowed to set for 4 hours, and then a 50:25:25 solution consisting of 2X medium, 1.6% stock agarose, and 1X medium containing cells, at a final concentration of 5000 cells/ml, was vortexed in a conical tube, and 2 ml was added to each well. Following 30 minutes at room temperature to allow the upper layer to set, plates were incubated at 37°C, 5% CO~2~incubator for 7--10 days and checked for colony formation by microscopy. Analysis of viral protein expression in infected cells ------------------------------------------------------ Cells were plated at 1.5 × 10^6^per well in 2 ml of medium in 6-well plates and allowed to sit over night. Cells were washed with phosphate-buffered saline (PBS), pH 7.4, and then infected with reovirus type 1 at the specified MOI. Virus was allowed to adsorb to cells for 1.5 hours at 4°C, washed twice with serum-free medium and incubated at 37°C and 5% CO~2~. At the indicated times, cells were scraped and lysed in Tris lysis buffer (10 mM Tris \[pH 7.5\], 2.5 mM MgCI~2~, 100 mM NaCI, 0.5% Triton x-100, 1 tablet Protease Inhibitor Cocktail Tablets \[Roche Applied Science, Indianapolis, IN, USA\] per 10 ml). After 30 min on ice, Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) were added to cell lysate samples (1:1). Protein samples were loaded in a 12% SDS-PAGE gel and run at 100 V. Protein was transferred to a nitrocellulose membrane at 100 V for 1 hour on ice. The membrane was washed three times with TBST (50 mM Tris pH 7.5, 150 mM NaCI, 0.05 % Tween 20) and then incubated in blocking buffer (TBST and 5% non fat dry milk, pH 7.5) for 1 hour at room temperature. The membranes were then incubated with rabbit anti-reovirus type 1 (1:50) and β-actin (1:3000, Sigma-Aldrich Corp., St. Louis, MO, USA) antibodies in blocking buffer overnight at 4°C. Following 3 washes in TBST, the membranes were incubated with goat anti-rabbit (for reovirus) or goat anti-mouse (β-actin) secondary antibodies (1:20,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour at room temperature, followed by three 15-min washes. Immune complexes were visualized by addition of chemiluminescence reagent (Renaissance, DuPont NEN, Boston, MA, USA) and the membrane was exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY, USA). Cell Proliferation Assay ------------------------ RIE-1, 6B72, or *Igf2*transfected RIE-1 or 6B72 cells were seeded at 5 × 10^4^per well in 96-well plates, incubated at 37°C and 5% CO~2~. At 4, 6, 18, 48 hours post plating, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium \[MTS\], and an electron coupling reagent (phenazine methosulfate, \[PMS\]) were added at 20 μl per well (CellTiter 96 Aqueous Non-Radioactiver Cell Proliferation Assay, Promega, Madison, Wl). Plates were incubated for 2 hours, and then the absorbance was determined at 490 nm. Each set of conditions was repeated in triplicate. Authors\' contributions ======================= JS, ELO, and CH conducted most of the laboratory work. KSW assisted in the analysis of fluorescein-labelled virus preparations. HER provided the vectors and advice on their use. DHR discovered that persistently infected cells require serum to survive, allowing the selection of genetically resistant cell clones. HER and DHR provided funding and supervision for the research, and prepared the manuscript. All authors have read and approved the final manuscript. Acknowledgements ================ This work was supported by Public Health Service Grants (R01HG00684 to HER, RO1CA682383 to DHR), by a grant from the Kleberg Foundation, partially supported by Cancer Centre (Core) grant P30CA42014, and Avatar BioSci, Inc. Fluorescence microscopy was performed, in part, through the use of the VUMC Cell Imaging Shared Resource, supported by NIH grants CA68485, DK20593, DK58404 and HD15052. We would like to thank J. Hawiger, T. Hodge, and E. Eisenberg for review of the manuscript. B. Mooneyhan provided expert secretarial assistance.

PubMed Central

2024-06-05T03:55:47.643227

2004-8-27

PMC517495

Background ========== Kimura has argued that the majority of nucleotide substitutions that accumulate in genes over time are selectively neutral, and go to fixation purely by chance \[[@B1]\]. One major prediction of Kimura\'s neutral theory is that the substitution process should be a Poisson process, with the mean number of substitutions per unit time equal to the variance. In contrast to this theory, empirical studies often find the variance to be significantly larger than the mean \[[@B2]-[@B8]\]. This observation has been termed the \"overdispersed molecular clock\". (For an excellent review of both empirical evidence and mathematical theories, see Ref. \[[@B9]\].) It is possible to reconcile Kimura\'s theory with the overdispersed molecular clock via Takahata\'s fluctuating neutral space model \[[@B10]-[@B12]\]. If the neutral mutation rate fluctuates slowly, then the substitution process ceases to be Poissonian, and becomes indeed overdispersed. However, the problem with the fluctuating neutral space model is that it does not offer any argument for why the neutral mutation rate should fluctuate, and thus ultimately fails to explain the observed substitution patterns. An explanation for fluctuations in neutral mutation rate was recently proposed by Bastolla et al. \[[@B13]-[@B16]\]. Different proteins with identical structure naturally vary in their neutrality, that is, in the fraction of single-point mutants that are viable. Therefore, as a gene slowly drifts through sequence space, the neutral mutation rate will fluctuate in correlation to the changing neutrality, and this fluctuation alone could be sufficient to explain the overdispersed molecular clock. Bastolla et al. studied the substitution process in a variety of models of neutral protein evolution *in silico*, and found significant overdispersion in all cases they considered. However, the simulations that Bastolla et al. carried out were limited to cases in which the product of mutation rate *μ*and population size *N*~e~is small (because Bastolla et al. used only a single sequence as the representative of the whole population, an approach that is justified for *μN*~e~≲ 1). Since population size and mutation rate can be substantial in some species (most notably in RNA viruses), it is justified to ask how general this result is for arbitrary values of *μN*~e~. It is known that large populations and populations evolving under high mutation pressure experience a strong selective pressure to avoid regions of low neutrality, an effect that has been termed \"evolution of mutational robustness\" \[[@B17]-[@B20]\]. In equilibrium, such populations settle in areas of sequence space that have above-average neutrality. As a result, regions of low neutrality are not represented in the population, and the distribution of neutralities in the population is much narrower than the total distribution of neutralities in sequence space. Therefore, we should expect that the neutral mutation rate does not fluctuate strongly under these conditions, and that the molecular clock will not be significantly overdispersed. For the present paper, I have studied the behavior of the substitution process under neutral protein evolution as a function of mutation rate *μ*and population size *N*~e~. I have found that the accumulation of non-synonymous mutations is substantially overdispersed for small *μN*~e~, in agreement with the results of Bastolla et al., but approaches a Poisson process when *μN*~e~≫ 1. The accumulation of synonymous substitutions is always Poissonian, regardless of the value of *μN*~e~. Results ======= I carried out simulations with DNA sequences of length *L*= 75. I determined the fitness of a DNA sequence by translating it into the corresponding amino-acid sequence, and determining its native fold within the framework of a lattice-protein model. (A sequence would have fitness 1 if it folded into a pre-determined target structure, and fitness 0 otherwise.) I used a simple model of maximally compact proteins on a 5 × 5 lattice. This protein-folding model is much simpler than the ones used by Bastolla et al. \[[@B13],[@B14]\], but has been shown to produce realistic distributions of folding free energies and neutralities \[[@B21]-[@B23]\]. The advantage of the simpler model is that entire populations of evolving sequences can be simulated, instead of just individual sequences. First, I have found that my model produces overdispersion (that is, an index of dispersion *R*substantially above 1) for non-synonymous substitutions, but not for synonymous substitutions. The finding for synonymous mutations is not surprising, because changes in the protein\'s neutrality do not affect the probability with which a synonymous mutation is neutral (which is always one). Neutral evolution could produce overdispersion in the synonymous substitutions only if the number of synonymous sites in the sequence were undergoing significant fluctuations. While these fluctuations do occur, they are apparently not large enough to affect the index of dispersion. Second, I have found that for non-synonymous substitutions, *R*decays quickly with increasing population size *N*~e~at fixed *μ*(Fig. [1](#F1){ref-type="fig"}). Since one reason for a decaying index of dispersion could be a reduced number of accumulated mutations, I have studied how the mean number of accumulated mutations behaves as a function of population size. Instead of staying constant or decreasing, the mean increases with increasing *N*~e~, while the variance decreases (Fig. [2](#F2){ref-type="fig"}). This result shows that the reduction in *R*is not caused by a mere reduction in the accumulated mutations, and that the substitution process does indeed shift from overdispersed to Poissonian as the population size increases. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Index of dispersion as a function of population size *N*~e~for synonymous and non-synonymous substitutions (*τ*= 1000, *μ*= 0.075). :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Mean,  and variance,  of lineage-adjusted number of non-synonymous substitutions as a function of population size *N*~e~(*τ*= 1000, *μ*= 0.075). Quantities were calculated from all 500 replicates at each population size. :::  ::: For non-synonymous substitutions, *R*decays with *N*~e~because of evolution of mutational robustness. However, mutational robustness is caused by large *μN*~e~, rather than large *N*~e~alone, and the parameter region in which mutational robustness becomes relevant is *μN*~e~≳ 10 \[[@B17]\]. Therefore, it is more instructive to plot *R*as a function of *μN*~e~. The only problem with a naive plot of that sort is that *R*increases as a function of *μτ*, where *τ*is the length of the time window during which mutations accumulate \[[@B9]\]. Thus, in Fig. [3](#F3){ref-type="fig"}, I show *R*for constant *μτ*as a function of *μN*~e~. Note that in this figure, instead of the sequence-wide mutation rate *μ*, I use the non-synonymous mutation rate *μ*~n~= 0.76 *μ*, which is corrected for the fact that only approximately 76% of mutations hit non-synonymous sites. (76% is the expected fraction of non-synonymous sites in a random DNA sequence.) Figure [3](#F3){ref-type="fig"} shows that the transition from an overdispersed to a Poissonian substitution process occurs for *μN*~e~between approximately 10 and 100, in agreement with Ref. \[[@B17]\], and that the transition region seems to be largely independent of the value of *μτ*. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Index of dispersion for non-synonymous mutations as a function of the product of non-synonymous mutation rate *μ*~n~(= 0.76 *μ*) and population size *N*~e~. :::  ::: Figure [3](#F3){ref-type="fig"} also shows that *R*increases with *μτ*. This dependency becomes clearer in Fig. [4](#F4){ref-type="fig"}, where I display *R*as a function of *μτ*for fixed *μN*~e~. The figure shows substantial increase in *R*with increasing *μτ*for small to moderate *μN*~e~. However, even for *μN*~e~well above 50, there is still a slight increase in *R*with *μτ*. Therefore, my results do not settle the question of whether the substitution process becomes truly Poissonian for sufficiently large *μN*~e~, or whether it just approaches a Poisson process but always remains slightly overdispersed. To settle this question, one would have to carry out simulations with much larger *τ*and *N*~e~. Unfortunately, the protein folding model I use is still too computationally intensive to permit such simulations with current computational resources. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Index of dispersion for non-synonymous mutations as a function of the product of non-synonymous mutation rate *μ*~n~(= 0.76 *μ*) and divergence time *τ*. :::  ::: Discussion ========== My results show that the size of the product *μN*~e~has a substantial effect on the index of dispersion under neutral evolution. The substitution process is strongly overdispersed for small *μN*e, but approaches a Poisson process as *μN*~e~grows large. Therefore, the next question is which of the two regimes has more biological relevance. As discussed by Cutler \[[@B9]\], the biggest problem in explaining the overdispersed molecular clock is not to come up with mechanisms that produce overdispersion, but to find a general mechanism that does not depend on special conditions or finely-tuned parameters. To assess the likelihood that fluctuations in neutrality contribute to the overdispersed molecular clock, we have to know the mutation rate and population size for the species of interest. It is notoriously difficult to obtain accurate data for these parameters, and only a few species have been studied in depth. One of the best data sets available is probably the one for *Drosophila*. Keightley and Eyre-Walker estimated the per-nucleotide substitution rate in *Drosophila*to be *u*= 2.2 × 10^-9^\[[@B24]\]. If we assume that the average gene in *Drosophila*is 1770 bp long \[[@B24]\], and that 76% of the nucleotides are non-synonymous (this number stems from averaging the number of non-synonymous sites over all codons with equal weight), then the average number of non-synonymous sites per gene is 1345 bp. Thus, the average rate of non-synonymous mutations per gene is *μ*~n~= 3.0 × 10^-6^. With an effective population size of approximately 3 × 10^5^\[[@B25]\], we get a product of population size and per-gene-non-synonymous mutation rate of approximately 1. Since selection for mutational robustness starts to take effect when this product is substantially larger than 1, *Drosophila*lies well within the parameter region in which we expect overdispersion to be caused by neutral evolution. For other higher organisms, in particular mammals, which tend to have comparatively small population sizes, we can expect that the product *μ*~n~*N*~e~falls into the same parameter region. On the other hand, for microorganisms, which can have very large population sizes, mutational robustness may play a role in their evolution. In particular, RNA viruses have genomic mutation rates on the order of one \[[@B26],[@B27]\] and their genomes consist typically of only a handful of genes. Because RNA viruses undergo severe bottlenecks on a regular basis, their effective population size *N*~e~is much smaller than the number of virus particles in infected individuals (which can exceed 10^12^), and is more closely related to the number of infected individuals. For HIV-1, *N*~e~has been estimated to be approximately 10^2^for subtype A, and 10^5^for subtype B \[[@B28]\]. The preceeding paragraph shows that neutral evolution of proteins is probably one source of overdispersed non-synonymous substitutions in *Drosophila*and other organisms. However, overdispersion has been observed in synonymous substitutions as well. For example, Zeng et al. \[[@B29]\] found an index of dispersion *R*significantly above one for synonymous, but not for non-synonymous substitutions in *Drosophila*. For mammals, some studies found *R*significantly above one for both synonymous and non-synonymous substitutions \[[@B8]\], while others found only the non-synonymous substitution process to be overdispersed \[[@B30]\]. Therefore, it is likely that other processes than neutral protein evolution also contribute to overdispersion. Such processes can be selection for optimal codon usage in the case of synonymous mutations, and positive selection on the amino acid level in the case of non-synonymous mutations. I have demonstrated that large *μN*~e~results in a substitution process with little overdispersion. However, I have not yet given an explanation for how overdispersion is reduced in populations with large *μN*~e~. There are two elements: First, selection for mutational robustness reduces the fraction of sequences with low neutrality, and increases the fraction of sequences with high neutrality, thus making the population more homogeneous and reducing the overall range of neutralities \[[@B17]-[@B20]\]. Second, a sequence with low neutrality will experience a real selective disadvantage in comparison to a sequence with high neutrality for large *μN*~e~, and will therefore have a reduced probability to end up on the line of descent. While this selective disadvantage is often small, it can nevertheless determine the evolutionary fate of a sequence in a large population. The larger the population, the more sensitive it becomes to small fitness differences, so that in a very large population a sequence with only a moderate reduction in neutrality will have a small probability to end up on the line of descent. (The fact that the mean substitution rate increases with the population size, as seen in Fig. [2](#F2){ref-type="fig"}, is also consistent with this reasoning. The larger the population size, the more high-neutrality sequences end up on the line of descent, which is reflected in the increase in the mean substitution rate.) Throughout this paper, I have considered only neutral or lethal mutations. It is a reasonable question to ask if and how deleterious mutations would change my results. The answer is that they probably have only a minor impact, and the less so the larger *N*~e~, unless they are very slightly deleterious. In order to affect the molecular clock, the deleterious mutations must end up on the line of descent, that is, they must go to fixation. The probability of fixation *p*~fix~of deleterious mutants drops exponentially with the population size, *p*~fix~= \[1 - exp(2*s*)\]/ \[1 - exp(2*sN*~e~)\], where *s*is the selective disadvantage of the deleterious mutation \[[@B31]\]. Therefore, for reasonable population sizes, only very slightly deleterious mutations can go to fixation and thus affect the molecular clock. This reasoning is independent of the size of *μN*~e~, as long as *N*~e~is large in comparison to *s*. Conclusions =========== The present study supports the following conclusions: • Neutral drift of proteins can lead to an overdispersed substitution process for non-synonymous mutations, but not for synonymous mutations. • The amount of overdispersion in the non-synonymous substitution process depends strongly on the product of mutation rate and population size. As this product increases, the substitution process becomes more and more Poissonian. The transition region starts at *μN*~e~≈ 10, and extends to values well above 100. • It is not clear whether there are any species that have a sufficiently large population size and mutation rate to prevent overdispersion through neutral drift. In *Drosophila*, the product of mutation rate and population size is close to one, which is well below the parameter region in which the substitution process turns Poissonian. Methods ======= Lattice protein model --------------------- I implemented a version of the 5 × 5 lattice protein model put forward *by*Goldstein and coworkers \[[@B21]-[@B23],[@B32]\]. In this model, proteins are sequences of *n*= 25 residues that fold into a maximally compact structure on a two-dimensional grid of 5 × 5 lattice points. There are 1081 distinct possible conformations in this model, and the partition function can be evaluated exactly by summing over the contact energies of all distinct conformations. The contact energy of a conformation *i*is  where  is the contact energy between amino acids  at location *j*and  at location *k*in the sequence, and  is 1 if the two amino acids are in contact in conformation *k*, and 0 otherwise. The partition function is  where the sum runs over all 1081 conformations. A sequence folds into conformation *f*if the contact energy for that conformation is lower than the contact energies of all other formations, *E*~*f*~\<*E*~*i*~for all *i ≠ f*, and if the free energy of folding, which is defined as  is smaller than some cutoff Δ*G*~cut~. Throughout this study, I used *kT*= 0.6 and Δ*G*~cut~= 0. The contact energies  where taken from Table VI in Ref. \[[@B33]\]. Sequence evolution ------------------ I simulated the evolution of populations of DNA sequences in discrete, non-overlapping generations. Population size is denoted by *N*~e~. The fitness of a sequence was 1 if the DNA sequence translated into a peptide sequence that could fold into a chosen target structure, and had a free energy of folding smaller than *G*~cut~. Otherwise, the fitness of the sequence was 0. All sequences had length *L*= 75. In each successive generation, sequences with fitness 1 were randomly chosen to reproduce, until the new generation had *N*~e~members. At reproduction, the sequences were mutated, with an average of *μ*base pair substitutions per sequence. I let each population evolve for several thousand generations, and kept track of the full genealogic information of all sequences in the population. In order to measure the molecular clock of fixed mutations only, I studied the pattern of base substitutions in a window of *τ*generations along the line of descent backwards in time, starting from the most recent common ancestor of the final population. I varied the parameters *N*~e~(10, 33, 100, 330, 1000, 3300), *μ* (0.0075, 0.075, 0.75), and *τ*(500, 1000). For each set of parameters, I carried out 500 replicates (each with a different, randomly chosen target structure), to obtain a distribution for the number of synonymous and non-synonymous substitutions *S*~d~and *N*~d~. Since there was some variation in the number of synonymous and non-synonymous sites across different target structures (on the order of approximately ± 5% variation from the mean), I then applied a correction factor to *S*~d~and *N*~d~to bring them into comparable units: I calculated the corrected number of synonymous substitutions  as  Here, *S*is the mean number of synonymous sites for the given replicate, and (*S*) is the average of *S*over all 500 replicates. Likewise, I calculated  (Indices of dispersion calculated without this correction factor are slightly larger than the ones reported here, because the variation in *S*and *N*creates additional variance in *S*~d~and *N*~d~). Similar correction factors have been used in sequence analysis \[[@B7]\], and are generally referred to as lineage adjustments. They control for differences among lineages that are primarily related to the expected number of substitutions in a lineage, and thus should not enter the index of dispersion. To obtain an estimate for mean and standard error of the index of dispersion, I subdivided the 500 results into 10 blocks of 50 each, and calculated mean and variance of the number of substitutions for each block. The ratio of variance to mean for a given set of substitutions (synonymous or non-synonymous) in a block is the index of dispersion for this data set. I then calculated mean and standard error for the index of dispersion from the individual results of the 10 blocks. The total CPU time needed to carry out all simulations was several months on a small cluster of Pentium II 500 MHz machines. Calculation of synonymous and non-synonymous substitutions and sites -------------------------------------------------------------------- I calculated the number of synonymous and non-synonymous sites *S*and *N*and the number of synonymous and non-synonymous substitutions *S*~d~and *N*~d~according to the method proposed by Nei and Gojobori \[[@B34]\]. In short, under this method the number of synonymous sites *s*~*i*~of a codon *i*is the fraction of possible substitutions to that codon that leave the residue unchanged. The number of non-synonymous sites *n*~*i*~for the same codon is *n*~*i*~= 3 - *s*~*i*~. For the complete sequence, *S*and *N*are calculated as  and  where *i*runs over all codons in the sequence. The number of synonymous or non-synonymous substitutions *s*~d,*i*~or *n*~d,*i*~between two codons is the average number of such substitutions, where the average is taken over all paths that lead from one codon to the other. The total number of synonymous or non-synonymous substitutions between two sequences is the sum over all individual constributions,  and  (again, *i*runs over all codons in the sequence). To calculate the number of synonymous or non-synonymous substitutions along the line of descent, I simply summed up all synonymous or non-synonymous substitutions that occurred from generation to generation. Because the full evolutionary history was known, a correction for multiple mutations such as the Jukes-Cantor correction \[[@B35]\] was not necessary. I also averaged the number of synonymous and non-synonymous sites over all sequences along the line of descent, to get the mean number of synonymous and non-synonymous sites for the given evolutionary trajectory. Authors\' contributions ======================= COW carried out all aspects of this study. Acknowledgements ================ This work was supported in part by the NSF under Contract No. DEB-9981397. I thank Ugo Bastolla for helpful comments on an earlier version of this paper.

PubMed Central

2024-06-05T03:55:47.646599

2004-8-27

PMC517496

Background ========== In the sequential model of transcriptional regulation including viral *trans*-activation, coactivators CBP/p300 require concerted action of multiple protein factors provided the nucleosomal structures allow access to the DNA template \[[@B1]-[@B3]\]. The factors that interact with coactivators encompass sequence-specific DNA binding activators, non-DNA binding coactivators, and essential components of the basal transcriptional machinery. Two large, closely related human proteins, p300 and CBP, were identified and shown to function as versatile signal integrators of many transcription factors to facilitate transcriptional activation or repression, and, in addition, as connectors of multiple transduction pathways. Both proteins contain several conserved domains that include three Cys/His-rich (C/H1, C/H2, and C/H3) domains, the histone acetyltransferase (HAT), KIX, and Gln-rich (Q) domains among others (Fig. [1](#F1){ref-type="fig"}) \[[@B2],[@B4],[@B5]\]. It is mainly due to these domains that a plethora of transcriptional activators interact with p300/CBP. Thus, p300/CBP coactivators act as a physical and functional scaffold or bridge between various cellular or viral *trans*-activators and the basal transcriptional machinery. Both proteins function by mediating positive or negative cross talk between different signaling pathways and participate in fundamental cellular processes that include embryonic development, cell growth, differentiation, and apoptosis. In addition, they can act as tumor suppressors and, last but not least, directly interact with diverse viral *trans*-activators to facilitate virus replication or viral activator-mediated transformation \[[@B6],[@B7]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Coactivator p300 enhances Bel1/Tas-mediated *trans*-activation.**Simplified schematic drawing of p300 domains and structure of deletion mutants (not drawn to scale). Characterized domains of p300 \[2, 5\] relevant for this report are highlighted by differently marked boxes: the FLAG epitope upstream of the N-terminus of p300 is marked by shaded boxes in constructs no. 1 through 8, the C/H1 and KIX domains by black and striped boxes, respectively. Numbers above the rectangles indicate the number of amino acids of p300 and its derivatives. The intrinsic HAT domain of p300 is depicted between the C/H2 and C/H3 domains; additional HATs PCAF and SRC-1 reported to associate with the distinct p300 regions \[8, 24, 25\] are indicated as rectangles above p300. The domain marked \"Q\" is the glutamine-rich domain of p300. Construct no. 5 has a deletion between the C/H1 and KIX domain (broken line). Constructs no. 9 and 10 contain the GST-C/H1 and GST-ΔC/H1-KIX fusion proteins; partial shadings mark GST regions. :::  ::: Several models have been proposed to explain transcriptional activation. Coordinated recruitment of coactivators by diverse transcriptional activators to specific promoter target sites has been shown by a collective effort of many groups \[[@B8]\]. According to the models, different coactivators either modify chromatin structure by altering the nucleosomal DNA thereby affecting its accessibility to DNA-binding proteins or, alternatively, form complexes with HAT activities that by acetylation of specific Lysines in histone N-terminal tails weaken interactions between DNA and the histone octamer. Moreover, the HAT activities of some coactivators acetylate non-histone substrates such as viral and cellular *trans*-activators, for instance p53 \[[@B9]\]. Diverse viral *trans*-activator proteins were found to interact with distinct domains of p300/CBP and PCAF. Prominent among them are the early adenoviral E1A antigen \[[@B10],[@B11]\], Epstein-Barr virus protein EBNA-2 \[[@B12]\], and human T-cell leukemia virus oncoprotein Tax \[[@B13],[@B14]\]. Several other modifications including methylation, phosphorylation, and ubiquitination lead to either diminished or increased DNA binding of the activators that, in turn, will result in either a repression or activation of gene expression. In addition, both coactivators were reported to interact with additional HAT enzymes, namely PCAF and SRC-1 \[[@B15],[@B16]\]. The apparently nonpathogenic primate foamy viruses (PFV) show a wide host range and tissue tropism and have been developed into vectors that efficiently transduce SCID-repopulating cells \[[@B17]\]. The PFV Bel1/Tas protein has been characterized as a transcriptional *trans*-activator of the acidic class and is known to directly interact with its responsive elements (BRE) \[[@B18],[@B19]\]. Bel1/Tas is a nuclear protein and acts as the key regulator absolutely required for virus replication. The minimal Bel1-specific DNA target site is 27 base-pairs long and located within the internal promoter (IP.BRE) upstream of the second cap site that is part and parcel of the second PFV transcription unit \[[@B20]\]. Additional Bel1/Tas DNA target sites in the LTR region of the PFV DNA genome were not analyzed in this study. The acidic *trans*-activation domain (TAD) was mapped to the C-terminus of Bel1 with little if any protein homology to other FV Bel1/Tas proteins from different species. Previously, we identified the nuclear factor 1 (NF1) as a repressor of Bel1/Tas-mediated *trans*-activation \[[@B21]\]. This repression was due to the fact that the specific family members NF1-C and -X interacted with parts of the IP.BRE and its flanking sequences. Since the NF1-mediated repression of the promoter of mouse mammary tumor virus was abrogated by distinct coactivators \[[@B22]\], we investigated which of the known coactivators and HAT proteins were capable of interacting with the PFV Bel1/Tas activator in the context of the IP.BRE promoter that is absolutely required for virus replication \[[@B20]\]. Here we report that the Bel1/Tas DNA binding protein functionally interacted with p300 and with the well-known HAT factor PCAF. In addition, SRC-1 enhanced Bel1/Tas *trans*-activation. This is the first time that these cellular coactivators have been shown to interact with the Bel/Tas1 trans-activator protein. Furthermore, Bel1/Tas binding to the C/H1 domain of p300 and coactivator-driven *trans*-activation seem to follow a unique pathway. Results ======= Coactivator p300 enhances Bel1/Tas-mediated activation ------------------------------------------------------ To examine whether p300 (Fig. [1](#F1){ref-type="fig"}, first line) enhanced the ability of Bel1 to *trans*-activate the Bel1 internal promoter (IP.BRE), transient reporter gene assays were performed. The IP.BRE that extends from -1 to -192 of the second cap site of the PFV genome was cloned into the pGL3-pro-luc reporter plasmid \[[@B21]\]. The results of the luc assays showed that full-length p300FL enhanced Bel1/Tas-mediated activation in an apparently nonlinear fashion (Fig. [2](#F2){ref-type="fig"}, upper left panel). To monitor the expression level of the p300 protein, Western blot analysis was carried out in parallel with increasing concentrations of the coactivator at fixed concentrations of the pbel1s expression plasmid that carries the retroviral *trans*-activator under the control of the CMV-IE promoter. The results of immunoblotting shown in the lower left panel of Figure [2](#F2){ref-type="fig"} revealed that Bel1/Tas was expressed at similar levels, as expected, and p300FL expression levels proportional to the input. In parallel experiments, truncated p300 forms, p300N and p300M, were also assayed and yielded moderate levels of enhancement of Bel1/Tas-mediated activation lower than those of p300FL (Fig. [3](#F3){ref-type="fig"}). Unexpectedly, the most extensive level of enhancement of Bel1/Tas-induced activation was reached with the C-terminal region p300C (Fig. [2](#F2){ref-type="fig"}, upper right hand panel). Again, Western blot analysis of p300C showed that expression levels of p300C protein increased proportionately to the transfected plasmid DNA while Bel1/Tas expression levels were unchanged (Fig. [2](#F2){ref-type="fig"}, lower right panel). The precise boundaries of the three p300 versions used are shown in Figure [1](#F1){ref-type="fig"}. The p300 bands marked by arrows are likely due to modified p300 proteins that are known to be modified by phosphorylation, acetylation and sumoylation (2, 5, 26). We next sought to determine whether the enhancing effect of p300 on Bel1/Tas-mediated activation was due to a physical interaction with the Bel1/Tas protein. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Coactivator p300 enhances Bel1/Tas-mediated activation**. Transient expression assays were performed with pGL3-luc plasmids containing the internal PFV BRE promoter (-1 to -192) after transfection of the pCMV-bel1s expression plasmid alone or separate cotransfection with p300FL and p300C expression plasmids \[4\]. Normalized luc activities are shown as fold activation in upper panels (for details, see Methods). Expression levels of Bel1/Tas and p300 proteins after cotransfection of 293T cells with increasing p300FL and p300C DNA concentrations and pbel1s DNA of 1.0 μg. Aliquots of the cellular lysates used for luc assays shown in upper panel were in parallel subjected to immunoblot analysis with monoclonal antibody against the FLAG epitope of the p300FL protein (lower left panel), and against the p300C protein (lower right hand panel). Polyclonal antibody against Bel1 was used for Bel1/Tas expression (bottom lanes in lower panels). :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Reporter gene assays of shortened p300 expression plasmids**. Transient expression assays were done as described under Fig. 2 except for that cotransfections were carried out with p300N and p300M plasmids; for boundaries of p300, see Fig. 1. :::  ::: Bel1/Tas interacts with p300 *in vivo* -------------------------------------- To examine whether the coactivator p300 interacts with the retroviral activator Bel1/Tas, binding of p300FL to the Bel1/Tas protein was analyzed by immune precipitation. 293T cells were cotransfected with the full-length p300FL and pbel1s expression plasmids and metabolically labeled with \[^35^S\]-Methionine and -Cysteine. Cellular lysates were precleared and subjected to immunoprecipitation with an antibody directed against the Bel1/Tas protein except for that in lane 1 (Fig. [4](#F4){ref-type="fig"}). After separation by SDS-PAGE and exposure, the resulting autoradiogram showed that the Bel1-specific antibody had effectively precipitated the p300FL-Bel1/Tas protein complex at both p300FL DNA concentrations of 10 μg (Fig. [4](#F4){ref-type="fig"}, lane 3) and 20 μg (lane 6). In the control where pbel1s was omitted the immune precipitation did not reveal any band comparable in size to p300 (lane 4). In the immunoprecipitation shown in lane 1 instead of an antibody against Bel1/Tas, an antibody against p300 was used and showed that the bands marked in Fig. [4](#F4){ref-type="fig"} was p300 (lane 1). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Direct physical interaction of Bel1/Tas with p300 determined by co-immunoprecipitation**. Bel1/Tas binds to p300 *in vitro*and *in vivo*. After transfection of 293T cells with expression plasmids pbel1s and p300FL, cells were labeled with \[^35^S\]-Methionine plus \[^35^S\]-Cysteine. Cellular extracts were precipitated, separated by SDS-PAGE and exposed. Protein p300FL is marked by bold arrowhead (lanes 1, 3, and 6). In the control, pbel1s was omitted (lane 4). Arrowheads indicate the following protein size markers (M, lanes 2 and 5): myosin of apparent molecular mass of 236, phosphorylase b of 97, and BSA of 66 kDa. :::  ::: To check the data obtained, co-immunoprecipitations were performed with non-labeled 293T cells followed by Western blot analysis. Cellular lysates were prepared from 293T cells separately cotransfected with pbel1s and each one of the three truncated p300 expression plasmids, p300N, p300M, or p300C and analyzed as described above. A polyclonal antibody directed against the Bel1/Tas protein was used in the co-immunoprecipitation followed by immunoblotting with a monoclonal antibody directed against the FLAG epitope fused in-frame with the N-terminus of the three truncated p300 versions. The results shown in Figure [5](#F5){ref-type="fig"} indicate that p300N protein specifically interacted with Bel1/Tas (lane 1) whereas the middle and C-terminal regions, p300M and p300C, respectively, do not seem to bind to Bel1/Tas under the conditions used (Fig. [5](#F5){ref-type="fig"}, lanes 3 and 5). This experiment was repeated several times and yielded the same result. In the controls, pbel1s was omitted in the cotransfections (lanes 2, 4, and 6). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Analysis of physical interaction of Bel1/Tas with truncated forms of p300.**After cotransfection of 293T cells with 2 μg pbel1s, and, separately with 2 μg each of p300N, p300M, and p300C expression plasmids, cellular extracts were prepared in parallel, divided into equal parts, and analyzed by co-immunoprecipitation and Western blotting. Lysates were precipitated with anti Bel1/Tas antibody and the Western blot was developed with a monoclonal anti-FLAG antiserum. The three arrows in the middle panel mark the correct sizes of the p300N, p300M, and p300C proteins (two blots pasted together). Controls for each p300 plasmid without pbel1s are in lanes 2, 4, and 6. Lower part confirms that the p300N protein specifically binds the Bel1/Tas protein (lane1, marked by arrow); two blots pasted together. :::  ::: Mapping of the p300-Bel1/Tas interaction domain ----------------------------------------------- We next determined which N-terminal p300 domain was responsible for the specific interaction with the Bel1/Tas protein. Different truncated versions of p300N (Fig. [1](#F1){ref-type="fig"}) were prepared, cloned, and subjected to separate immunoprecipitations and Western blot analyses as described above for p300N. In addition, two different GST fusion proteins that contained either the C/H1 or KIX domain were bacterially expressed, purified, and analyzed (Fig. [1](#F1){ref-type="fig"}). Two different expression plasmids p300N-C/H1-ΔKIX that lacked the KIX but still expressed the C/H1 domain were capable of binding Bel1/Tas (Fig. [6](#F6){ref-type="fig"}, right panel, lanes 2 and 3). In contrast, the results shown in Fig. [6](#F6){ref-type="fig"}, lanes 4 and 5, revealed that the plasmid p300-ΔC/H1-ΔKIX that lacks both the C/H1 and KIX domains but retains the short N-terminal region of 196 amino acids did not bind Bel1/Tas. The expression levels of the five constructs were monitored by immunoblot analysis and exhibited the expected bands of p300-derived proteins (Fig. [6](#F6){ref-type="fig"}, left panel). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Mapping of the p300 Bel1/Tas-interaction domain**. The p300 expression plasmids, constructs no. 2, and 5 to 8 shown in Fig. 1 were analyzed by immunoprecipitation as described above for p300N. The right hand panel shows the results of immunoblotting obtained after reaction with the monoclonal antibody against the FLAG epitope; arrows and asterisks mark the Bel1/Tas-interacting protein bands from cellular extracts of pbel1s-cotransfected cells (lanes 1--3); intentionally overexposed to visualize the marked bands in lanes 1 and 2. The left panel presents the Western blots of the five recombinant p300N plasmids used for separate co-immunoprecipitations. Numbers in brackets refer to Fig 1. :::  ::: To unambiguously demonstrate that the C/H1 domain of p300 is the Bel1/Tas-interacting region, we performed pull-down assays with two pGST-C/H1 and pGST-ΔC/H1-KIX fusion proteins (Fig. [1](#F1){ref-type="fig"}, constructs no. 9 and 10). The results revealed that purified pGST-C/H1 clearly interacted with Bel1/Tas as shown in Fig. [7](#F7){ref-type="fig"}, lane 2 whereas the pGST-ΔC/H1-KIX and a control GST plasmid did not (lanes 1 and 3). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Bel1/Tas interacts with GST-C/H1 fusion proteins detected by GST pull-down assays**. The recombinant GST-C/H1 and GST-ΔC/H1-KIX proteins were expressed in *E. coli*BL21 cells and purified by binding to glutathione Sepharose 4B. Each GST-fusion protein bound to glutathione Sepharose 4B was separately mixed with lysates obtained from pbel1s-transfected 293T cells. After incubation and extensive washing with the binding buffer, bound proteins were eluted, separated by SDS-PAGE, and visualized by staining (upper panel); immunoblotting was carried out with an antibody against Bel1/Tas protein (lower panel). :::  ::: To summarize this part, our data show that p300 physically interacted with Bel1/Tas *in vivo*, and that the C/H1 domain of p300 was responsible for this interaction at least *in vitro*. Effect of the Bel1-C/H1 domain on Bel1/Tas-mediated activation by p300C ----------------------------------------------------------------------- To gain more insight into the mechanism of the C/H1-Bel1 complex that affects3 p300C-mediated activation, cotransfections of pbel1s and p300C with expression plasmid p300N-C/H1-ΔKIX were carried out (Fig. [1](#F1){ref-type="fig"}, construct 6). Cellular lysates of 293T cells were prepared and luciferase assays performed. Cotransfections of fixed concentrations of the pbel1s, 0.5 μg, with the pC/H1-ΔKIX expression plasmid did not enhance p300C-mediated *trans*-activation (Fig. [8](#F8){ref-type="fig"}, lanes 5, 7, 9, 11, and 13). In contrast, the expression of the C/H1 domain resulted in a partial suppression of the p300C-driven activation at higher C/H1 domain concentrations (Fig. [8](#F8){ref-type="fig"}). Similar degrees of inhibition were obtained when lower Bel1/Tas concentrations were used. Western blot analysis was carried out in parallel with increasing concentrations of the coactivator to ascertain Bel1/Tas expression (data not shown). The inhibition by the C/H1 domain explains why the level of *trans*-activation of p300N and p300FL did not reach the full extent of p300C-driven activation. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Effect of p300-C/H1 domain expression on enhancement of p300C-mediated *trans*-activation.**Transient expression assays with pGL3-luc containing the internal BRE promoter after cotransfection of the p300C and pC/H1 expression plasmids with pbel1s. :::  ::: PCAF interacts with Bel1/Tas ---------------------------- We next analyzed whether different HAT-expressing genes such as GCN5, PCAF, and SRC-1 were able to enhance and interact with Bel1/Tas. Transient luciferase gene assays with pGCN5 expression plasmids did not affect Bel1/Tas-mediated activation (data not shown). In contrast, when the HAT PCAF expression plasmid was used for co-expression, an enhancement of Bel1/Tas-induced activation was detectable at a concentration of 0.02 μg PCAF DNA and 0.5 μg pbel1s (Fig. [9](#F9){ref-type="fig"}, upper panel). At higher PCAF DNA concentrations, repression of Bel1/Tas-mediated activation was observed. When the levels of PCAF and Bel1/Tas protein expression was determined by Western blot analysis, a decreased level of the Bel1/Tas expression was detected that was likely due to degradation (Fig. [9](#F9){ref-type="fig"}, lower panel). The band of the PCAF protein corresponded to the correct size of about 95 kDa (marked by arrow). ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Functional and physical interaction of Bel1/Tas with PCAF**. *A*, Transient expression assays with pGL3-luc containing the internal BRE promoter after cotransfection of the pCI-FLAG-PCAF with 0.5 μg pbel1s expression plasmid (upper panel). Aliquots were subjected to Western blot analysis (lower panel) :::  ::: We next analyzed the potential interaction between the Bel1/Tas and PCAF proteins by carrying out co-immunoprecipitation with 293T cellular lysates that had been cotransfected with 2.0 μg pbel1s and pCI-PCAF expression plasmids, followed by immunoreaction with an antibody against Bel1 and subsequent immunoblotting with an anti-FLAG antibody to detect PCAF. The results showed that PCAF did indeed interact with the PFV Bel1/Tas activator (Fig. [10](#F10){ref-type="fig"}, lane 2). As control, an immunoprecipitation and Western blot analysis was performed with the PCAF plasmid in the absence of pbel1s (Fig. [10](#F10){ref-type="fig"}, lane 1). The result showed that a PCAF band was not detectable under the conditions used. ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **Physical interaction of PCAF with Bel1/Tas.**Co-immunoprecipitation and immunoblot analysis of pCI-FLAG-PCAF-cotransfected 293T cells with pbel1s DNA (lane 2). The cellular lysates were incubated with an anti Bel1/Tas antibody and treated as described in the legend to Fig. 5 except for that an antibody directed against the FLAG epitope of PCAF was used for immunoblotting. In the control, cotransfection was done without pbel1s (lane 1). :::  ::: To determine if an additional HAT protein, SRC-1, affected Bel1/Tas-mediated activation, transient reporter gene expression assays were carried out after cotransfection of 293T cells with the coactivator SRC-1a and 0.5 μg pbel1s. Surprisingly, a relatively strong enhancement of Bel1/Tas-mediated activation was detected (Fig. [11](#F11){ref-type="fig"}, upper panel). The level of expression of both SRC-1a and Bel1/Tas proteins was determined and found to be approximately proportional to the input (Fig. [11](#F11){ref-type="fig"}, lower panel). ::: {#F11 .fig} Figure 11 ::: {.caption} ###### **Coactivator SRC-1 enhances Bel1/Tas-mediated *trans*-activation**. Reporter luc gene expression assays with pGL3-luc plasmids containing the internal PFV BRE promoter after cotransfection of pCR3.1-FLAG-SRC-1a with the pbel1s expression plasmid (upper panel). Immunoblotting of 293T cellular extracts cotransfected with the SRC-1a and pbel1s expression plasmids (lower panel). :::  ::: To assess whether SRC-1a was able to physically interact with the Bel1/Tas protein, immunoprecipitations and Western blot analysis were carried out under different conditions. The specificity of SRC-1a was ascertained by using monoclonal antibody directed against SRC-1a in control reaction. However, evidence for an interaction between the SRC-1a protein and Bel1/Tas was not obtained. Discussion ========== It was previously reported that Bel1/Tas is capable of inducing the expression of many cellular genes \[[@B23]\]. While it is known that Bel1/Tas binds directly and to a large number of DNA target sites \[[@B18]-[@B21]\], the mechanism of activation and the identity of the cellular partner molecules of Bel1/Tas remained unknown. As a first step, we have sought to identify the cellular proteins that interact with the PFV retroviral *trans*-activator and mediate its activating potential. The data presented here show that the coactivators p300 and HAT PCAF physically bound Bel1/Tas *in vitro*and both enhanced Bel1/Tas-mediated activation whereas SRC-1 enhanced with Bel1/Tas activation without direct binding. According to our data, Bel1/Tas specifically interacted with the C/H1 domain of p300, although we cannot rule out binding to other p300 domains with much lower affinity not detectable under the rather harsh conditions of co-immunoprecipitation used here. When the levels of the relative luciferase activity of p300FL and its three shortened versions are compared, it is noteworthy that p300C reached the highest level of enhancement of Bel1/Tas-mediated activation (Fig. [2](#F2){ref-type="fig"}). Besides its intrinsic HAT activity, p300C contains both the intact C/H3 and Q domains that interact with the HAT enzymes PCAF and SRC-1, respectively \[[@B10],[@B24],[@B25]\]. These three HAT enzymes are likely responsible for the large enhancement of the observed *trans*-activation either directly by acetylation of Bel1/Tas or indirectly by histone acetylation, or both. In contrast to p300C, p300N and p300M do not possess any HAT activities nor do they bind to HAT-containing interaction partners. On the other hand, it is well known that p300 and its three subregions bind a plethora of various partner molecules leading to either activation or repression of transcription. Since the high level of p300C-mediated activation was partially inhibited in cotransfections with C/H1 and pbel1s, competition for Bel1/Tas between the C/H1 and the p300C-terminal interaction partners PCAF and SRC-1 cannot be ruled out so that both direct and indirect mechanisms might be responsible for the relative increase in Bel1/Tas-mediated activation by p300C. When Bel1/Tas binds to the C/H1 domain, the degree of Bel1/Tas acetylation may be much lower, since the protein surface of Bel1/Tas may be occluded and, hence less accessible. Some residual Bel1 acetylation might still occur by endogenous p300 and PCAF. Other factors might play additional roles. The ability of full-length p300 to *trans*-activate Bel1/Tas was relatively low for two reasons. First, the transfection efficiency of the full-length p300 is very low because of its large plasmid size, and the concentration of endogenous p300 is limiting. Secondly, the activation loop of p300 HAT is not fully activated by auto-acetylation as required for full *trans*-activation \[[@B26]\]. Since the relative activation by SRC-1 was not as high as that of p300C, we consider the HAT activity of PCAF as one of the major players of Bel1/Tas-mediated *trans*-activation. This result is supported by the observed enhancement of Bel1/Tas activation after cotransfection with PCAF that resulted in higher levels of Bel1 acetylation thereby leading to increased binding to the IP.BRE \[J. Bodem, personal communication\]. Thus, the observed high level of enhancement of p300C might be due to the synergistic effects brought about by formation of ternary p300C-PCAF-Bel1 and binary p300C-SRC complexes, respectively. In these multimeric protein complexes, Bel1/Tas binds p300C indirectly through PCAF. Consistent with the HAT activities of PCAF and p300, we detected acetylated Bel1/Tas in pbel1s-transfected 293T cells using monoclonal antibody against acetyl-Lysine after immunoblotting (our unpublished data). It is intriguing that the distribution of the closely spaced Lysines of Bel1/Tas apparently mimics the correspondingly spaced Lysines in histones. This observation is further complicated by our observation that cotransfection with higher levels of PCAF led to a reduced stability of Bel1/Tas (Fig. [9](#F9){ref-type="fig"}). We assume that PCAF acetylates or even hyper-acetylates the Bel1/Tas protein at closely spaced Lysines in analogy to other activators reported previously \[[@B9]\]. The decreased stability of acetylated Bel1/Tas might indicate that modified Bel1/Tas is less stable than the unmodified form. This observation adds an additional layer of combinatorial regulation to Bel1/Tas-mediated *trans*-activation. It is intriguing that some viral *trans*-activators interact with more than a single p300 domain \[[@B10],[@B14]\]. However, Bel1/Tas might recruit a second interacting region of p300 through binding PCAF (Fig. [10](#F10){ref-type="fig"}) that is known to interact with a p300 domain different from the C/H1 domain (Fig. [1](#F1){ref-type="fig"}) \[[@B10]\]. The complex nature of p300-Bel1/Tas interactions reported here might serve to strengthen the overall binding affinity between Bel1/Tas and the PCAF interaction domain of p300 within a larger transcriptional complex \[[@B27]\]. PCAF is known to specifically acetylate distinct Lysine residues of a subset of core histones and thereby regulate the transcriptional activity of many genes depending on the genetic context. It is well documented that acetylation, methylation and other covalent histone modifications are essential signals for the regulation of transcription \[[@B28]\]. It remains to be seen whether the stronger level of enhancement of p300C is a special if not unique feature of Bel1/Tas activation and due to over-expression or to repressive effects of other p300-interacting protein factors that cannot bind to the truncated p300 protein. Alternatively, many other factors were reported to bind to the C-terminal domains of p300 that also encompass general transcription factors TBP and TFIIB proteins that might also be responsible for the enhancement observed here \[[@B1]-[@B3]\]. In search of viral and cellular activators that are comparable with the ability of Bel1/Tas to interact with the C/H1 domain of p300, we found one case. A report indicates that EBNA-2 protein shares many features with Bel1/Tas that include the C-terminal acidic activation domain as well as the abilities to bind both the C/H1 domain and PCAF \[[@B12]\]. There remain two differences, however. First, EBNA-2 binds to both the C/H1 and the C/H3 domain, and, secondly, PCAF does not coactivate EBNA-2 in strong contrast to Bel1/Tas \[[@B12]\]. Of note, an additional HAT enzyme, GCN5, did not interact with Bel1/Tas when tested in reporter genes assays indicating that only distinct HAT sets such as those identified in this report specifically interact with Bel1/Tas in *trans*-activation. The precise roles of the HAT activities of PCAF, p300, and SRC-1 during Bel1/Tas-mediated *trans*-activation remain to be addressed in future studies. Conclusions =========== Coactivators PCAF and p300 were identified to physically and functionally interact with the spumaviral Bel1/Tas *trans*-activator. Coactivator SRC-1 was found to strongly enhance Bel1/Tas-mediated *trans*-activation. The C/H1 domain of p300 was responsible for binding the retroviral activator and found to partially inhibit the p300-driven *trans*-activation. Methods ======= Antibodies ---------- Mouse monoclonal antibodies directed against the FLAG epitopes of p300FL, p300N, p300M, p300C, and PCAF were purchased from Sigma, rabbit polyclonal antibodies against the N-terminal p300FL, the C-terminal p300C, and SRC-1a from Santa Cruz Biotechnology. The polyclonal serum direct against Bel1/Tas was used as described previously \[[@B23]\]. Typically, 5 μl of each antiserum was used for each immunoprecipitation. Monoclonal antibody against acetyl-Lysine was purchased from Sigma. Plasmids, cells, transfections, and metabolic labeling ------------------------------------------------------ Plasmids pUC18, pCMVβ-gal, pbel1s \[[@B29]\], pGL3-pro-IP. BRE (-1 to -192), pCI-FLAG-p300FL, pCI-FLAG-p300N, pCI-FLAG-p300M, pCI-FLAG-p300C \[[@B4]\], and pCI-FLAG-PCAF \[[@B29]\] were separately, or in the combinations indicated, transfected into 293T cells using Lipofectamine 2000 (Invitrogen). In general, unless otherwise indicated, 0.1 -- 10 μg plasmid DNA were transfected into 293T cells grown in Petri dishes with a diameter of six cm. Full-length pCI-FLAG-300FL plasmids and three different shortened versions (Fig. [1](#F1){ref-type="fig"}, constructs no. 1 to 4) were grown in *E. coli*, DH5α cells \[[@B4]\]. The PFV internal promoter was constructed by PCR-mediated amplification of the defined promoter fragments as reported previously \[[@B19],[@B21]\]. Recombinant clone p300N-C/H1-ΔKIX\* (construct no. 5, Fig. [1](#F1){ref-type="fig"}), was constructed by first generating two PCR products with pCI-p300FL as template using the sense (s) and antisense (as) primers s1: 5\'-CTTATGGTTCACCATATACTCAGAATCC-3\', as1: AAACTGGAACCATGCCTGCATTTCTCTTATCACC-3\', s2: 5\'-GAAATGCAGGCATGGTTCCAGTTTCCAT-3\', and as2: 5\'-GGAAGGAACTGGCCCTGGTTGGAAGGCTGTTG-3\' to amplify the sequence from nucleotide 755 to 1275 and nucleotides 1984 to 2297 fused in-frame. The resulting DNA product of 833 nucleotides was cloned into the pCR2.1 Topo vector and designated as pCR2.1-ΔKIX. An *SphI-NotI*DNA fragment obtained from pCR2.1-ΔKIX and was inserted into pCI-p300N that had been predigested with *Sph*I and *NotI*. The borders of construct no. 5 are shown in Fig. [1](#F1){ref-type="fig"} and the expressed recombinant protein had the expected size. p300-C/H1-ΔKIX (no. 6) was constructed by inserting the *SphI*/*NotI*DNA fragment from pGEX-C/H1 (35) into pCI-p300N digested with *SphI*/*NotI*for expression of residues 1 to 424 of p300. Construct no. 7 (Fig. [1](#F1){ref-type="fig"}) was constructed by digesting pCI-p300N with *SphI*and re-ligating the larger fragment for the expression of residues 1 through 347 of p300. Finally, p300ΔN-ΔC/H1-ΔKIX was constructed from pCI-p300N by digesting with *MunI*and re-ligated to express residues 1 to 196 of p300. Bacterial plasmids coding for glutathion-S-transferase (GST) fusion proteins pGST-C/H1 (328--424) and pGST-KIX (436--661) (Fig. [1](#F1){ref-type="fig"}) were constructed from pGEX-6p-2GST-p300 \[[@B30]\] and grown in *E. coli*, BL21 cells. pCR3.1-FLAG-SRC-1a was grown in *E. coli*, JM109 cells \[[@B24]\]. Human 293T or HeLa cells were cultivated in DMEM medium supplemented with 1% penicillin and streptomycin, 1% Glutamine and 10 % fetal calf serum. Plasmid p300FL was transfected into 293T cells and metabolically labeled with L-\[^35^S\]-Methionine plus L-\[^35^S\]-Cysteine (spec. act. of 37 TBq/mM, PRO-MIX, Amersham) for 6 hr. Cells were harvested and used for immunoprecipitation as described above. The precipitates were analyzed by SDS-PAGE on 12% gels, dried, and exposed on KODAK Biomax MR1 films. Luc reporter gene expression assays ----------------------------------- Plasmid pCMV-βgal directing β-galactosidase expression from the CMV-IE promoter was used for normalization of transfection efficiency. Luc reporter gene assays were performed and quantified as described \[[@B31]\] using a Luminoskan TL Plus luminometer (Labsystems, Frankfurt, FRG). pUC18 vector plasmid DNA was used as carrier DNA to equalize the DNA concentration of each transfection. Cells were harvested 18 h after transfection. The results of luc assays were based upon at least triplicate experiments on multiple independent occasions. Expression levels were monitored by Western blot analysis. Co-immunoprecipitation ---------------------- Immunoprecipitation was performed as described previously with minor modifications \[[@B32]\]. Lysates of subconfluent cotransfected layer of 293T cells were prepared by first washing the cells with PBS, and subsequently lysed in lysis buffer (150 mM NaCl, 20 mM Tris-HCl \[pH 7.5\], 1 mM phenylmethylsulfonyl-fluoride containing 1% (v/v) Triton X-100. To inhibit unspecific protease activity, protease inhibitors (Biomol) were added to the lysis buffer. Lysates were precleared with protein A-SepharoseCL-4B (Amersham Bioscience AB, Uppsala). Co-immunoprecipitation of p300FL, p300N, p300M, p300C, SRC-1, and PCAF were performed with rabbit anti Bel1 antiserum \[[@B23]\]. The immune precipitates were retrieved with protein A-SepharoseCL-4B (Pharmacia) and eluted by boiling. To detect the specific precipitate, immunoblotting was performed with specific antibodies against expressed p300, shortened p300 versions, and SRC-1. For the detection of expressed PCAF, Western blot analysis with monoclonal anti-FLAG antibody was carried out. The immunoprecipitates were washed three times with the lysis buffer and analyzed by immunoblotting on 12% gels for expressed p300FL and on 14% gels for shortened p300 forms, PCAF, and SRC-1a. The experiments were repeated at least three times, specially the immunoprecipitations of lysates of p300C-transfected cells. GST pull-down assays -------------------- The recombinant GST-C/H1 and GST-KIX proteins were expressed in *E. coli*BL21/DE3 cells after transformation with the corresponding plasmids. Expressed proteins were purified by binding to glutathione Sepharose 4B resin. Each of the GST-fusion proteins bound to glutathione Sepharose 4B were mixed with lysates obtained from pbel1s-transfected 293T cells. After incubation at 4°C overnight in binding buffer \[[@B30]\] and extensive washing with the binding buffer, bound proteins were eluted, separated by SDS-PAGE, and visualized by staining with Coomassie Brilliant Blue according to Ariumi \[[@B30]\] or detected by Western blot analysis. Immunoblotting -------------- Cells were harvested two days after transfection by lysis in 1% SDS and the protein concentration was determined using the DC protein assay (BioRad). Identical amounts of proteins were separated by SDS-PAGE on 12% gels, blotted, reacted with monoclonal serum directed against the FLAG epitope of the four different FLAG-tagged p300 (Sigma), or polyclonal serum direct against Bel1 \[[@B23]\], and detected by enhanced chemoluminescence. List of abbreviations used ========================== CBP, CREB-binding protein; C/H1, 2, and 3, Cys/His-rich domains of p300; FL, full-length; HAT, histone acetyltransferase; LTR, long terminal repeat; luc, luciferase; PCAF, p300/CBP-associated factor; Q, Glutamine-rich domain of p300; SRC, steroid receptor coactivator; p300N, p300M, and p300C, amino-, middle and C-terminal regions of p300; PFV, primate foamy virus; IP.BRE, internal promoter Bel1 response element; TAD, *trans*-activation domain; NF1, nuclear factor 1; CMV, cytomegalovirus; GST, glutathione-S-transferase. Authors\' contributions ======================= HB and WM contributed equally to the manuscript. VO carried out the molecular cloning of p300 derivatives. YN participated in the design of the study. All authors read and approved the final manuscript. Acknowledgments =============== We thank Kunitada Shimotohno for critically reading the manuscript and helpful comments and our colleagues for generously providing plasmids and reagents: Shelley Berger, Don Chen, Ella Englander, Richard Gronostajski, Tasuku Honjo, Steve McMahon, Bert O\'Malley, Marc Montminy, Sharon Roth, and Kunitada Shimotohno. We are grateful to Jennifer Reed for critically reading our paper and to Gholamreza Darai and Harald zur Hausen for support. The Fond der Chemischen Industrie supported this work.

PubMed Central

2024-06-05T03:55:47.648774

2004-9-6

PMC517497

Background ========== Green tea (leaves of *Camellia sinensis*, Theaceae) is a popular beverage in East Asia, and also used as a herbal remedy in Europe and North America. Green tea is considered to be antiinflammatory, antioxidative, antimutagenic, and anticarcinogenic \[[@B1],[@B2]\], and can prevent cardiac disorders. Epidemiologically, it has been suggested that green tea consumption prevents type 2 diabetes. The amelioration of insulin resistance by green tea is associated with the increased expression level of glucose transporter IV in a fructose-fed rat \[[@B3]\]. Green tea extract contains polyphenols (e.g., catechin, epicatechin, epigallocatechin, and their gallates), teanin and caffeine. The extract also includes pyrroloquinoline quinone, a newly discovered vitamin \[[@B4]\]. Some constituent components have been shown to enhance the basal and insulin-stimulated glucose uptake of rat adipocytes \[[@B5]\], to inhibit intestinal glucose uptake by inhibiting the sodium-dependent glucose transporter of rabbit intestinal epithelial cells \[[@B6]\], and to reduce serum glucose level in alloxan-diabetic rats \[[@B7]\]. Controversially, caffeine acutely lowers insulin sensitivity in humans \[[@B8]\]. The present study was aimed at providing evidence of the improvement in glucose metabolism in humans and diabetic mice upon green tea consumption. Furthermore, to investigate whether some serum proteins mediate the effects of green tea on hyperglycemia in the diabetic state, proteomic analyses were performed using SELDI (surface enhanced laser desorption/ionization)-TOF (time-of-flight)-MS (mass spectrometry), because the SELDI-TOF-MS enables the measurement of the relative abundance of proteins of various sizes in a blood sample. Here, we compared the proteomic patterns of sera from diabetic db+/db+ and wild-type mice, and further investigated the influence of green tea administration on the proteomic patterns of serum in diabetic mice. If the diabetic modifications of serum protein levels are reversed by green tea administration, these affected proteins can be used as therapeutic targets in diabetes. Results ======= Oral glucose tolerance test (OGTT) ---------------------------------- We investigated whether oral glucose tolerance is improved in healthy volunteers by drinking a suspension of green tea powder. Blood glucose levels were measured before and 30, 60, and 120 min after drinking 1.5 g of green tea. The effects of hot water without tea powder on blood glucose level in the same participants were also investigated. Fig. [1](#F1){ref-type="fig"} shows that glucose tolerance was substantially improved with tea administration compared with hot water administration. In detail, glucose metabolism increased in 14 participants, remained unchanged in 3 participants, and worsened in 5 participants. Basal blood glucose levels (BGLs) did not significantly change in all participants (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Increase in glucose metabolism in healthy humans administered with green tea.**Oral glucose tolerance curves (OGTT, n = 22) and basal blood glucose levels (Basal, n = 25) before and 30, 60 and 120 min after administration with either a suspension of green tea powder (●) or hot water (○). All participants were fasted since the last supper before starting the experiment at 9:30 a.m. In the glucose tolerance test, 225 ml of Trelan-G75 containing 75 g glucose was perorally administered 10 min after drinking a suspension of green tea powder (1.5 g/150 ml hot water) or hot water (150 ml). Effect of green tea or hot water on basal blood glucose levels was examined without administrating Trelan-G75. Blood glucose levels (mg/dl) measured were averaged in each treatment group, and compared by cross-over test. Values represent the means ± S.E.M. \*P \< 0.05, \*\*P \< 0.01; significantly different from the vehicle control at each time-point, determined by one-way ANOVA, followed by Scheffé\'s test. :::  ::: Antihyperglycemic effects of green tea in diabetic mice ------------------------------------------------------- The crude extract of fresh tea leaves picked from either No.12 or No. 13 cultivar at 300 mg/kg significantly lowered BGL 2--6 h after peroral administration in streptozotocin (STZ)-diabetic ddY mice in the fasting state (Fig. [2](#F2){ref-type="fig"}). In the saline-treated control group (n = 5), BGL slightly changed from 273 ± 28 mg/dl to 241 ± 28 mg/dl after 6 h, whereas in the green-tea (No. 12)-treated group (n = 3), BGL markedly changed from 235 ± 15 mg/dl to 116 ± 12 mg/dl. Similarly, BGL markedly changed from 201 ± 10 mg/dl to106 ± 8 mg/dl after 6 h in the green-tea (No. 13)-treated group (n = 3). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Antihyperglycemic effects of green tea leaf extracts in the fasting STZ-diabetic ddY mice.**The dried extracts of fresh tea leaves prepared at the Taiwan Tea Experiment Station \[TTES, Cultivar No. 12 (Chinhsuan) and No.13 (Tzuiyu)\] were dissolved in saline and perorally administered at 300 mg/kg into STZ-diabetic ddY mice in the fasting state. Blood glucose levels 2 h, 4 h, and 6 h after administration of green tea leaf extracts are expressed as percentages of the glucose levels at 0 h (before the administration) in each mouse, and then averaged. Values represent the means ± S.E.M. (n = 3--5). \*P \< 0.05; significantly different from the blood glucose levels of STZ-diabetic mice administered with saline (control) at each time-point, determined by one-way ANOVA, followed by Scheffé\'s test. :::  ::: The antihyperglycemic effects of green tea powder suspension at 30, 150, and 300 mg/kg were examined 2 h after administration to the STZ-diabetic mice in the fasting state. As shown in Fig. [3](#F3){ref-type="fig"}, green tea tended to lower BGL at 150 mg/kg, and significantly lowered BGL at 300 mg/kg. Furthermore, the effect of green tea was compared among different groups of mice, i.e., STZ-diabetic ddY mice, normal ddY mice, diabetic db+/db+ mice and +m/+m mice (Fig. [4](#F4){ref-type="fig"}). BGLs were significantly lowered in both STZ-diabetic mice and db+/db+ mice 2 h after administration of green tea powder suspension (300 mg/kg), whereas no significant changes were observed in normal ddY mice and +m/+m mice. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Antihyperglycemic effects of green tea powder suspension in the fasting STZ-diabetic ddY mice.**STZ-diabetic ddY mice were perorally administered with a suspension of green tea powder at 30, 150, and 300 mg/kg in the fasting state. Blood glucose levels 2 h after the administration of green tea are expressed as percentages of the glucose level 0 h before the administration in each mouse, and then averaged. Values represent the means ± S.E.M. (n = 6 -- 20). \*\*P \< 0.01; significantly different from the blood glucose levels of STZ-diabetic mice administered with saline (control, 0 mg/kg of green tea), by one-way ANOVA, followed by Scheffé\'s test. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Blood-glucose-lowering effects of green tea in diabetic mice but not in normal mice.**STZ-diabetic ddY, normal ddY, diabetic db+/db+, and wild-type (+m/+m) mice were perorally administered with either a suspension of green tea powder at 300 mg/kg or saline in the fasting state. Blood glucose levels (mg/dl) were measured before and 2 h after the administration, and then averaged. Values represent the means ± S.E.M. (n = 5 -- 21). \*P \< 0.05, \*\*P \< 0.01; significantly different from values at 0 h (before the administration), by one-way ANOVA, followed by Scheffé\'s test. :::  ::: No elevation of blood insulin levels was observed during the decrease in BGL either by tea leaf extract or by green tea suspension in STZ-diabetic mice (data not shown). These results suggest that both fresh green tea leaf extract and green tea suspension lower BGL without changing serum insulin concentration. Serum protein profiles altered in diabetic db+/db+ mice ------------------------------------------------------- To explore the mechanisms underlying the antihyperglycemic effect of green tea, proteomic patterns of sera from diabetic (db+/db+) and wild-type (+m/+m) mice were first investigated and compared to identify peaks specific to the diabetic state using SELDI-TOF-MS. Samples of sera from fasting +m/+m mice were loaded onto CM10 (a cationic exchanger, pH4) ProteinChip arrays (Fig. [5](#F5){ref-type="fig"}). The other types of ProteinChip Array, i.e., CM10 (a cationic exchanger, pH7), Q10 (an anionic exchanger, pH8) and IMAC30-Cu^2+^(immobilized metal affinity chromatography, pH7), were also used to fractionate proteins in the serum. Then, these arrays were analyzed using a ProteinChip system, and the amount of individual serum proteins was estimated from the peak intensity of the mass spectral signal and the mass/charge ratio (m/z) equivalent to the molecular weight of each protein. All the spectral peaks of serum proteins from diabetic mice, which were significantly different (P \< 0.05, by unpaired *t*-test) in intensities from those of serum proteins from wild-type mice, are shown in Fig. [6A](#F6){ref-type="fig"}: the peak intensities of proteins at m/z 4119, 4203, 4206, 4211, 4579, 9311 and 18691 were \>3 times lower, and those at m/z 13075, 17406, 17407, 17418, 17622, 18431, 26100 were \>3 times higher than respective peak intensities of proteins of sera from +m/+m mice. Typical spectra are shown in Figs. [6B](#F6){ref-type="fig"} and [6C](#F6){ref-type="fig"} (see [additional file 1](#S1){ref-type="supplementary-material"} concerning the other peaks). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Analysis of proteomic profiles in mouse serum using SELDI-TOF-MS.**Serum samples from fasting wild-type (+m/+m) mice were loaded onto the spots of CM10 (a cationic exchanger, pH4) ProteinChip arrays. The spots were analyzed using the SELDI ProteinChip system on three different ranges: m/z 3000 -- 10000, m/z 10000 -- 20000 and m/z 20000 -- 100000. The y-axis of the spectra indicates the mass-to-charge ratio (m/z) of protonated proteins, and the x-axis indicates the relative intensities of mass spectral signals. Note: The intensity of each peak is directly proportional to the amount of protein, but the peak intensities could not be comparable among the different proteins because of the difference in ionization. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Proteomic analyses demonstrating the differences in protein profiles of sera from diabetic and wild-type mice.**Serum samples from fasting diabetic (db+/db+) mice and fasting wild-type (+m/+m) mice were loaded onto ProteinChip arrays. **(A)**List of peaks of proteins and/or peptides with indicated m/z values, the peak intensities of which were significantly changed in the diabetic state. Relative peak intensities were averaged (n = 8). The fold changes are presented as ratios of the peak intensities at indicated m/z values in db+/db+ to those in the +m/+m mice. Peaks at m/z 4203, 4576, 8515, 9291, 17406 and 18678 were obtained using CM10 ProteinChip (pH4). Peaks at 8733 and 9311 m/z were obtained using CM10 ProteinChip (pH7). Peaks at m/z 3933, 4119, 4206, 4369, 4566, 4579, 4637, 8523, 8827, 8915, 9283, 13075, 17407, 17418, 17622, 18431, 18691, 22334 and 26100 were obtained using Q10 ProteinChip. Peak at m/z 4211 was obtained using IMAC30. The chips were analyzed by SELDI-TOF-MS. **(B)**and **(C)**Typical data of relative peak intensities in +m/+m and db+/db+ mouse sera (upper, representative of 4--8 independent observations) and the peak intensity averages at m/z 4206 and 26100 (lower, n = 4 for +m/+m, n = 8 for db+/db+). The analyzed peak is indicated by arrows in the data of mass spectral signals. \*\*P \< 0.01; significantly different from the peak in wild-type mice, by unpaired *t*-test. :::  ::: Modification of serum protein profiles of diabetic db+/db+ mice by green tea ---------------------------------------------------------------------------- Serum protein profiles of db+/db+ mice were investigated 2 h after green tea administration, and compared with those administered with saline (control). Again, we confirmed a significant decrease in BGL by green tea in the blood samples used in these proteomic analyses (data not shown). All the protein signals that were significantly changed (P \< 0.05, by unpaired *t*-test) in terms of peak intensity by the green tea administration, but not by the saline administration, are described in Fig. [7A](#F7){ref-type="fig"}. The serum proteomic patterns of the green-tea-treated group demonstrate that the peak intensities of proteins at m/z 11651 and 11863 decreased to less than one third after 2 h of tea administration, whereas no significant changes were observed in the control group. Typical spectra are shown in Fig. [7B](#F7){ref-type="fig"} (see [additional file 2](#S2){ref-type="supplementary-material"} for the change at m/z 11651). When the results in Fig. [7A](#F7){ref-type="fig"} are compared with those in Fig. [6A](#F6){ref-type="fig"} to identify protein peaks specific for diabetes and which are sensitive to green tea administration, a peak at m/z 4211 (4212) was found (Fig. [8](#F8){ref-type="fig"}). The intensity of this peak was significantly lowered in the diabetic state (Fig. [8A](#F8){ref-type="fig"}), and was significantly decreased 2 h after green tea administration (Fig. [8B](#F8){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Changes in serum protein profiles in db+/db+ mice by administration of green tea.**Serum samples from fasting diabetic (db+/db+) mice 2 h after administration of green tea suspension were loaded onto ProteinChip Arrays. The chips were analyzed using SELDI-TOF-MS. **(A)**List of peaks of proteins and/or peptides with indicated m/z values, the peak intensities of which were significantly changed by the green tea administration. Relative peak intensities were averaged (n = 4). The fold changes are presented as ratios of the peak intensities at indicated m/z values 2 h after to before green tea administration. Peaks at m/z 7495, 7595, 7808, 7920, 14983, 15612 and 15614 were obtained using CM10 ProteinChip (pH4), whereas those at m/z 7503, 7611, 7823, 7926, 11651, 11664, 11863, 15004 and 15638 were obtained using CM10 ProteinChip (pH7). Peaks at m/z 4212, 4226, 7499, 11637, 11846, 13711, 13831, 14974, 15180, 31204 and 65906 were obtained using IMAC30 ProteinChip. **(B)**Typical data of relative peak intensities (upper, representative of 4 independent observations) and the peak intensity average at m/z 11863 (lower, n = 4) after green tea administration and saline control. The analyzed peak is indicated by arrows in the data of mass spectral signals. \*\*P \< 0.01; significantly different from the peak obtained before the administration, by unpaired *t*-test. :::  ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Changes in peak intensity at m/z 4211 in diabetic state and after green tea administration.(A)**Decrease in the peak intensity at m/z 4211 in serum samples from fasting diabetic (db+/db+) mice, compared with that of the fasting wild-type (+m/+m) mice. Typical data of relative peak intensities in +m/+m and db+/db+ mouse sera (upper, representative of 4--8 independent observations) and the averaged intensities of the peak at m/z 4211 (lower, n = 4 for +m/+m, n = 8 for db+/db+) indicated above by arrows. \*\*P \< 0.01; significantly different from the peak in wild-type mice, by unpaired *t*-test. **(B)**Decrease in the peak intensity at m/z 4212 in serum samples from fasting db+/db+ mice 2 h after green tea administration. Typical data of relative peak intensity (upper, representative of 4 independent observations) and the averaged intensities of the peak at m/z 4212 (lower, n = 4) after administration with either green tea or saline. \*\*P \< 0.01; significantly different from the peak obtained before the administration, by unpaired *t*-test. :::  ::: The relative peak intensities of hemoglobin-related multisignals ranging from m/z 14974 to 15638 \[[@B9]\] were increased by green tea administration (Fig. [7A](#F7){ref-type="fig"}). Parallel changes were observed at half and double these m/z values (Fig. [7A](#F7){ref-type="fig"}, [9](#F9){ref-type="fig"}) (see [additional file 2](#S2){ref-type="supplementary-material"} concerning these changes). Since multicharged protein ions are apparently observed as proteins of different sizes in the mass spectrometry, these changes seem to reflect the modification of one group of proteins (hemoglobin-related proteins) by green tea intake. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Hemoglobin-related multiple SELDI-TOF-MS signals in db+/db+ mice.**Samples of sera from nontreated db+/db+ mice in the fasting state were loaded onto ProteinChip arrays. Left panel: hemoglobin-related multi-MS signals. From the average of 8 data, the signals were detected from m/z 14974 to 15638 (+H) using CM10 ProteinChip (pH7). The double-charged m/z values appear to be observed from m/z 7495 to 7823 (+2H). These values may correspond to hemoglobin α- and β-chains, and other hemoglobin-related proteins. Right panel: multi-MS signals observed at approximately double the single-charged m/z values for the hemoglobin-related signals, using IMAC ProteinChip. These signals may correspond to the dimers of hemoglobin α- and β-chains. :::  ::: Discussion ========== Green tea is widely consumed in Asian countries, while black tea is most popular in Western countries. The manufacturing process of green tea differs from that of black tea because freshly picked young leaves of the tea are immediately steamed. This process destroys the enzymes responsible for breaking down the color pigments in the leaves and allows the tea to maintain its green color during the subsequent rolling and drying processes. The amounts of constituent compounds are slightly different from those of black tea. Pharmacological studies using constituent compounds in green tea have been recently reviewed by Kaszkin et al. \[[@B10]\]. Green tea extracts are more stable than pure epigallocatechin gallate, the major constituents of green tea, because of the presence of other antioxidant constituents in the extract \[[@B10]\]. In general, herbal medicines are complex mixtures of different compounds that often act in a synergistic fashion and exert their full beneficial effect as total extracts \[[@B11]\]. In the present study, we demonstrated that green tea produces an antihyperglycemic effect without affecting insulin secretion in STZ-diabetic mice. We therefore explore the mechanism underlying the green tea effect by investigating the serum protein profiles of db+/db+ mice, a genetic model of type 2 diabetes, using SELDI-TOF-MS. First, we performed a preliminary analysis to determine the peaks (molecular weights) of biomarker proteins that were abnormally contained in the serum of diabetic mice, without identifying individual serum proteins. As a result, we found that the levels of several serum proteins were significantly altered in the diabetic state. Secondly, we investigated which marker proteins are affected by green tea administration. Despite the changes in the levels of several serum proteins after green tea administration, none of the protein peaks specific for diabetes were sensitive to the administration, except for a peak at m/z 4211(4212). The level of this 4211(4212) Da protein was reduced both in the diabetic state and by green tea administration. Thus, no simple reversed effect of green tea on the diabetes-induced modifications of serum protein levels was observed. The 4211(4212) Da protein has not yet been identified, but only two candidate groups of vertebrate proteins are found by scanning Swiss-Prot database (molecular weight, 4211; molecular weight ranges, 0.1% according to the standard errors of the current MS analysis; pI, 6; pI ranges, 10): pancreatic polypeptide (PP) (primary accession numbers P13083, P37999, P41519, P38000, P11967) and antibacterial peptides \[β-defensin C7 precursor (018815), 4 kDa defensin (P56686), cryptidine-5 precursor (P28312), antibacterial peptide BMAP-34 precursor (P56425)\]. PP is primarily expressed in the endocrine cells of the pancreas, and the plasma PP concentrations are elevated by food intake \[[@B12]\]. Peripheral PP administration results in a reduction in food intake and an increase in energy expenditure \[[@B12]\]. Interestingly, a previous study revealed that PP-containing cell populations in the islets of Langerhans are reduced in db+/db+ mice with mild hyperinsulinemic diabetes \[[@B13]\], which is consistent with the observed reduction in the intensity of the peak at m/z 4211(4212) in the diabetic state. We speculate that the reduction of BGL by green tea causes a decrease in serum PP concentrations as a means of maintaining energy homeostasis, although more precise studies are required. The changes in serum protein profiles by green tea also demonstrate the increase in the peak intensities of hemoglobin-related multi-MS signals, suggesting the adverse side effects of green tea, although blood samples from db+/db+ mice tended to exhibit a hemolytic feature compared with those from wild-type mice (data not shown). Interestingly, the hemoglobin-related multi-MS signals shown in Fig. [9](#F9){ref-type="fig"} may include hemoglobin α-chains and β-chains \[[@B9]\], their dimers, and possibly variously glycated hemoglobins. Hemoglobin A1c is used as a marker of diabetes in clinical diagnostic tests. Until now, however, the extent of glycation per hemoglobin that practically occurred is not clear. More detailed analyses of serum protein profiles using SELDI-TOF-MS will provide a more useful clinical index of the diabetic state. We observed that green tea improved oral glucose tolerance in humans. It is therefore likely that green tea is prophylactic against diabetes and ameliorates diabetic hyperglycemia. Green tea consumption at moderate doses may be associated with a reduced risk of type 2 diabetes in apparently healthy individuals by controlling postprandial hyperglycemia. Conclusions =========== The control of postprandial hyperglycemia by green tea can help reduce the risk of type 2 diabetes. In the present study, we provide evidence showing that green tea promotes glucose metabolism in healthy humans, and produces an antihyperglycemic effect in diabetic mice. In addition, we analyzed the serum protein profiles of db+/db+ and +m/+m mice for the first time using SELDI-TOF-MS, and further investigated its association with any effects of oral green tea administration on serum proteins. Among the several proteins that were significantly lowered in the serum of diabetic mice, the 4211(4212) Da protein was significantly decreased after green tea administration. This is the first report demonstrating that a certain serum protein is involved in the antihyperglycemic effect of green tea. The contribution of this protein, therefore, should be further investigated in a future study. Moreover, we speculate that the observed effects of green tea on BGL are primarily due to the promotion of insulin action in peripheral tissues, such as skeletal muscles and adipocytes. Indeed, a recent paper showed that green tea supplementation for 12 weeks ameliorates insulin resistance and increases glucose transporter IV content in a fructose-fed rat model resembling the human type 2 diabetes mellitus \[[@B3]\]. Since the administration of green tea produced an acute antihyperglycemic effect on BGL in diabetic mice in the present study, additional mechanisms, such as changes in amelioration or enhancement of insulin action, should be clarified in a future study. To elucidate whether new protein synthesis is required for green tea action, it would be useful to examine the influence of protein synthesis inhibitors on the acute antihyperglycemic effect of green tea. Methods ======= Housing and care of animals --------------------------- C57BLKS/J db+/db+ mice (male, 9 -- 11 weeks old, 36.2 -- 46.3 g, BGL: 243--411 mg/dl) and its age-matched control C57BLKS/J +m/+m mice (male, 20.6--23.9 g, BGL: 110--185 mg/dl) were purchased from SLC (Shizuoka, Japan) and used as the type 2 diabetic mouse model. Male ddY mice (4 weeks old, purchased from SLC, Shizuoka, Japan) were singly injected with STZ (150 mg/kg, i.v.), and then used 4--6 weeks after the injection (28.8--38.5 g, BGL: 247--600 mg/dl). The age-matched normal ddY mice (34.7--38.6 g, BGL: 129--197 mg/dl) were also used. The animals were housed (3--5 per cage) under a daily cycle of 12 h light and 12 h darkness, with free access to food and water. Animals were treated as approved by the Toyama Medical and Pharmaceutical University Animal Research Committee, and according to the guidelines for animal experiments established by the Japanese Pharmacological Society. Sampling of blood ----------------- Mice were deprived of food for 11--14 h. Blood samples (20 μl) were collected from mouse tail veins under ether anesthesia. In experiments for proteomic analyses and serum insulin measurements, blood samples (100 μl) were collected from the orbital venous plexus of mice under ether anesthesia. The food deprivation was continued throughout the measurement of blood glucose levels (until 6 h after the administration with green tea). In the oral glucose tolerance test as described below, blood samples (0.3 μl) were obtained from human skin microvessels using FreeStyle (Nipro, Osaka, Japan). Oral glucose tolerance test (OGTT) ---------------------------------- Healthy human volunteers (18--24 years old) were fasted 12 h before the starting point of experiments. The participants were perorally administered with either a suspension of green tea powder or hot water at 9:20 a.m. Ten min after the administration (at 9:30 a.m.), the participants were perorally administered with 225 ml of Trelan-G75 (Shimizu Pharmaceuticals Co., Shimizu, Shizuoka) containing 75 g glucose. BGLs were measured before and 30, 60, and 120 min after the administration with Trelan-G75 (glucose). All the subjects enrolled in this study were ethnic Japanese. Before participation, the purpose and risks of the study were carefully explained, and written informed consent was obtained from all the participants. The protocol was approved by the Toyama Medical and Pharmaceutical University Ethics Committee regulating human research. Measurement of blood glucose levels and serum insulin levels ------------------------------------------------------------ BGLs were measured using ANTSENSE II (Horiba, Kyoto, Japan) in the mouse study and using FreeStyle (Nipro, Osaka, Japan) in the human study. Serum insulin levels were measured using an insulin-ELISA kit (Morinaga Seikagaku, Tokyo, Japan). To prepare the serum samples, blood samples from mice (100 μl) were kept on ice for 2 h, centrifuged at 16,000 × *g*for 1 min at 4°C, and its supernatant was immediately separated from the pellet. SELDI ProteinChip Analysis -------------------------- Quantitative serum proteomic profiles were measured with SELDI-TOF-MS (Ciphergen Biosystems, Yokohama, Japan). The mouse serum was prepared as described above. Several types of ProteinChip Array, i.e., Q10 (an anionic exchanger, pH8), CM10 (a cationic exchanger, pH4 and pH7) and IMAC30-Cu^2+^(immobilized metal affinity chromatography, pH7) (Ciphergen Biosystems), were used to fractionate proteins in serum. For the proteomic analyses with Q10 ProteinChip array, samples were 10-times diluted with denaturation buffer (7 M urea/ 2 M Thiourea/ 4% CHAPS/ 1% dithiothreitol/ 2% ampholine) and incubated on ice for 10 min, and then 10-times more diluted with buffer of 50 mM Tris-HCl (pH8). When CM10 ProteinChip array was used, samples were 10-times diluted with the denaturation buffer and incubated on ice for 10 min, and then 10-times more diluted with buffer of 100 mM NaOAc (pH4) or 50 mM HEPES (pH7). When IMAC30 ProteinChip array was used, samples were 100-times diluted with phophate-buffered saline. Spots on the different arrays were equilibrated with the buffer used for sample dilution, e.g., Tris-HCl buffer for spots on Q10 array, and each sample solution (70 μl) was loaded onto two separate spots on the arrays. After incubation for 20 min with rotation spots were rinsed with water and air-dried completely. The spots were analyzed using the SELDI ProteinChip system (PBS-IIc, Ciphergen Biosystems). In each sample, data from the two spots were averaged (duplicate assay). If a signal/noise ratio was larger than 2, the peak was considered to reflect the amount of a protein. Quantitative nature of the instrument was confirmed as previously described \[[@B14]\]. The m/z value of each protein peak (the molecular weight of each detected protein) was corrected, based on the m/z values of the external standards of peptides or proteins, the molecular weights of which are known. Standard errors in the estimated molecular weights were less than 0.1%. The axis of abscissa in spectra (Figs [6B](#F6){ref-type="fig"}, [7B](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"} and [9](#F9){ref-type="fig"}) indicates the mass-to-charge ratio (m/z) of protonated proteins. Reagents -------- Fresh raw leaves of tea (*Camellia sinensis*, Theaceae) (Cultivar No.12, Chinhsuan and No.13, Tzuiyu) were prepared at the Taiwan Tea Experiment Station, picked in May at Nan-Tou County, and immediately used without drying. The leaves were steeped in hot water (95°C) for 30 min and the filtrate was condensed under reduced pressure. The dry weight of the extracts was determined, and the extracts were dissolved in saline for administration into mice. On the other hand, green tea powder was donated by Fukuju-en (Kyoto, Japan). The particle size (median diameter) of the green tea powder was 2.9 μm, according to the measurement using a Centrifugal Particle Size Analyzer (Shimadzu, Kyoto, Japan). In the human study, the tea powder (1.5 g) was added to 150 ml of hot water (80°C) and then whipped with a bamboo whisk. In the mouse study, the tea powder was suspended in saline at room temperature using a sonicator. The contents of caffeine and catechins in these tea samples were determined by HPLC: the samples were injected into an HPLC column (TSK-GEL, ODS-80T~M~, Tosoh, Tokyo, Japan); the eluent was 10 mM phosphate buffer (pH2.6)/MeCN (gradient: 5 to 15%) at a flow rate of 1.3 ml/min. The data are shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Contents of catechins and caffeine in green tea samples (grams per 100 g) ::: Green tea powder No. 12^a^ No. 13^b^ ----------- ------------------ ----------- ----------- catechins  C 0.1 1.3 0  EC 0.6 1.9 1.0  ECg 0.6 1.6 0.2  EGC 0.3 0.9 5.8  EGCg 5.6 10.6 3.5  total 7.2 16.3 10.5 caffeine 4.3 4.5 1.7 C: (+)-catechin, EC: (-)-epicatechin, ECg: (-)-epicatechin gallate, EGC: (-)-epigallocatechin, EGCg: (-)-epigallocatechin gallate, total: total catechins. ^a,b^The dried extract of fresh raw leaves of tea (*Camellia sinensis*, Theaceae) prepared at Cultivar No.12 (a, Chinhsuan) and No. 13 (b, Tzuiyu). ::: Statistical analysis -------------------- The significance of differences between two groups was assessed by Student\'s *t*-test, and the differences between multiple groups were assessed by one-way analysis of variance (ANOVA) followed by the Scheffé\'s multiple range test. Values of P less than 0.05 were considered to be significant. Especially, to determine the significance of the time-dependent effects of green tea (Fig. [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}) on the blood glucose levels, repeated measures ANOVA was performed, and then, if the statistical significance was detected by this analysis, further statistical comparison at each measurement time between the groups was conducted by one-way ANOVA, followed by the Scheffé test. Authors\' Contributions ======================= IK had the original idea for the study, designed and coordinated the experiments, and wrote the manuscript. JW prepared fresh young leaves of green tea and the hot water extract. MT assisted with measurement of blood glucose and insulin level. MI, HT and TS were involved in study design, data analysis, data interpretation and writing of the manuscript. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 **Differences in serum protein profiles between diabetic and wild-type mice**Typical data of relative peak intensities in +m/+m and db+/db+ mouse sera (left 2 panels, representative of 4--8 independent observations) and the peak intensity averages at m/z indicated (right panels; +m/+m: open column, n = 4; db+/db+: closed column, n = 8). The analyzed peak is indicated by arrows in the data of mass spectral signals. \*\*P \< 0.01; significantly different from the peak in wild-type mice, by unpaired *t*-test. Types of ProteinChip used were described in the Fig. 6 legend. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 **Changes in serum protein profiles of db+/db+ mice after green tea administration**MS spectra shows typical changes in the serum protein profiles of db+/db+ mice administered with saline (left) or green tea (right). Graph shows the peak intensity averages at m/z indicated, before (open column) and after (closed column) administration with saline (n = 4) or green tea (n = 4). The analyzed peak is indicated by arrows in the MS spectra. \*\*P \< 0.01; significantly different from the peak obtained before the administration, by unpaired *t*-test. Types of ProteinChip used were described in the Fig. 7 legend. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The present work was supported in part by Fugaku\'s Grant by Charitable Trust (Japan). Powdered green tea was kindly donated by Fukujuen (Sourakugun, Kyoto, Japan). The donator had no role in study design, data analysis, data interpretation or writing of the manuscript. We thank Dr. Y Sugihara, Y Fukuda, and S Hara for technical assistance; Dr. R Taniguchi (CHA Research Center, Fukujuen) for measurement of particle size of green tea powder and the amount of caffeine and catechins in the tea samples; and Dr. H Tanaka and R Wakatabe (Ciphergen Biosystems KK, Yokohama Laboratory, Japan) for measurement and analysis of SELDI-TOF-MS data.

PubMed Central

2024-06-05T03:55:47.652406

2004-8-26

PMC517498

Background ========== Human fascioliasis, a commonplace infection caused by a leaf-shaped Trematode Fasciolahepatica affects a human host by chance \[[@B1]-[@B4]\]. It seems that the rate of infection is increasing in many countries worldwide \[[@B1],[@B5]\]. Human fascioliasis has to be differentially diagnosed from such hepatic and biliary diseases as acute hepatitis, neoplasm, visceral toxocariasis, biliary tract diseases, hepatic amebiasis, and infection with other liver flukes like schistosomiasis \[[@B2],[@B5]\]. Diagnosis of the disease is achieved by locating the ova either in feces or duodendal drainage or by \[[@B7]-[@B9]\]. Imaging techniques proved to be the most useful method for confirming the diagnosis and also the follow-up of fascioliasis \[[@B6],[@B8],[@B10]\]. Small, often peripheral, nonenhancing, hypo dense nodules with tortuous, linear, branching tracts in CT scans, which decrease in size after successful therapy, are highly suggestive of the disease \[[@B8]-[@B12]\]. Immature flukes can produce ectopic masses or abscesses in various locations and during the acute phase of the disease, other structures such as subcutaneous tissue, heart, lungs, pleura, abdominal wall, brain, cecum, epididymis, and stomach can be involved \[[@B2],[@B8],[@B9]\]. In a particularly unusual report, direct peritoneal involvement with granuloma formation has been reported \[[@B1]\]. Eosinophilic reactions can be exhibited in the body as pleuritis and pericarditis \[[@B7],[@B9]\]. Here, we present an extremely unusual radiologic incident of fascioliasis involving multiple organs. Case presentation ================= In Dec 2001, a 52 year old man, who was a known case of Multiple Myeloma (MM), was presented to one of our affiliated hospitals with persistent right upper quadrant and epigastric pain, and anorexia for a period of 1 month. At the time of admission, the patient had been receiving prednisolone and melphalan for his MM, which was currently in remission. His recent condition began with tongue and facial edema two weeks before appearance of the abdominal pain. Upon physical examination, mild epigastric tenderness and a palpable liver were found. Neither icterus nor any positive sign of cardiopulmonary abnormalities were noted. Additionally, the patient did not have a fever and his peripheral lymph nodes were not enlarged. Initial laboratory findings were as follows: a hemoglobin of 10.6 g/dL, white blood cell count of 10,800/mm3 with 18% eosinophils, and a sedimentation rate of 90 mm at the end of the first hour. The total bilirubin was 0.5 mg/dL (0.2--0.8 mg/dl), alanine aminotranferase (ALT) 48 IU/L (0--40 IU/L), aspartate aminotransferase (AST) 46 IU/L (0--40 IU/L), and alkaline phosphatase (Alk P) 651 IU/L (60--140 IU/L). The eosinophilia fluctuated between 12 to 55 percent in various tests performed during the time period in question, with no unique patterns noted. Serum electrophoresis showed a monoclonal spike in the gamma region. Specific enzyme-linked immunosorbant assay (ELISA) produced a positive result for Fasciola hepatica, while the test was negative for Toxocara canis. Serologic tests for the presence of hepatitis A, B, and C viruses were negative. Blood and urine cultures were found to be sterile. Other laboratory studies, including repeated stool examinations for ova and parasites, showed no abnormalities. Chest x-rays did not demonstrate any parenchymal or pleural abnormality. Abdominal ultrasonography showed a mild hepatomegaly with multiple hypoechoic lesions in the liver. A CT scan revealed multiple but poorly defined, hypodense lesions in the liver, and a completely enlarged pancreas with mild bilateral pleural reaction, suggesting metastatic cancer (Fig. 1). In the search for a potential malignancy, diagnostic laprascopy was performed, which revealed the presence of white-colored lesions ranging from 1 to 3 cm in diameter on the surface of both lobes of the liver with mild ascites. Multiple liver and peritoneal biopsy specimens revealed fibrinoid necrosis, associated with granulomatous reaction and a high concentration of eosinophils in the liver, (Fig. 2) accompanied by markedly inflamed peritoneal tissue with eosniphilic infiltration. No malignant cells were identified and no evidence of extramedulary plasmocytoma was found. Specific staining for fungal organisms and acid-fast bacilli were negative. The ascitic fluid also had a high level of eosinophils. Endoscopic retrograde cholangiopancreatography (ERCP) failed to show any filling defect within the biliary tree. Furthermore, the patient underwent bone marrow aspiration that only indicated high eosniophilic infiltration. The patient was placed on albendazole (400 mg twice daily for 1 week). The treatment was well tolerated and the abdominal pain was improved rapidly. At the time of discharge, the patient was in good clinical condition. During a follow-up visit two months later, a second CT unexpectedly showed not only an increase in the number and size of the hypodense lesions in the liver, but also the extension of lesions into the pancreas, the spleen and both kidneys (Fig. 3). No evidence of peripheral enhancement of the hepatic lesions or ascites was documented. The patient was still experiencing upper quadrant pain on the right side. Laboratory investigations produced a white blood cell count of 7200/mm3 with 16% eosinophilia. Repeated stool examinations failed to identify ova and parasites. The patient was given triclabendazole (10 mg/kg, bid for two days). As recommended, the patient had another follow-up CT scan three months later. At that time all of his symptoms were resolved. Follow-up CT scans revealed a considerable improvement in the number and size of the lesions. At the time of the CT scan, all of his symptoms were resolved (Fig. 4A, B). At this time the WBC was 6000/mm3 with 6% eosinophils. After 5 months and in the last CT, the lesions had almost disappeared completely (Fig. 4C, D). Discussion ========== While fascioliasis is a well-known human parasite, it sometimes produces unusual characteristics that may influence a clinician to misdiagnose the condition. In the vast majority of cases, the diagnosis is difficult in both acute and chronic phases and some important conditions such as liver abscesses and metastasis cannot be easily differentiated from fascioliasis \[[@B1],[@B13]\]. Interestingly, the larvae is able to migrate to a number of ectopic locations such as subcutaneous areas, intestines, pleura, lungs, abdominal wall, brain, cecum, epididymis, stomach, pericardial, or cerebral sites, producing very unique clinical manifestations \[[@B8],[@B9]\]. Serologic testing, when performed by an enzyme linked immunoabsorbent assay (ELISA) for the detection of antibodies specific to the parasite is nearly one hundred percent sensitive and specific. Thus ELISA may be used to confirm the diagnosis in acute and chronic phases \[[@B7]-[@B9]\]. Among imaging tools, ultrasonography is of little diagnostic value during the acute phase, while a contrast-enhanced CT scan can be very useful for diagnosis \[[@B9],[@B17]\]. In CT images one can find two distinct kinds of lesions: single or multiple hypodense nodular areas caused by deposition of the parasite (abscess-like lesions) and tunnel-like branching or tortuous hypodensity which is created as the result of parasite migration through the liver and is highly suggestive of the disease \[[@B9],[@B17]\]. If peripheral tortuous lesions are present, hepatic fascioliasis should be the primary diagnostic consideration \[[@B14]\]. As clinical and laboratory findings of fascioliasis may easily be confused with many other conditions, a high index of suspicion is required to establish a correct diagnosis \[[@B9],[@B17]\]. Both CT scan and ultrasonography can be helpful in evaluating the response to treatment \[[@B15]\]. The ingested metacercariae of Fasciola hepatica penetrate the intestinal wall and migrate through the peritoneal cavity to reach the liver. However, ectopic migration to other locations is one of the strangest manifestations of the infection \[[@B8],[@B9]\]. The precise route of migration toward ectopic sites is unclear but most often occurs in the acute stage of the disease \[[@B9]\]. In addition, a syndrome of eosinophilic reaction without direct parasitic involvement may accompany acute fascioliasis \[[@B9]\]. Ascites is not a common finding in fascioliasis, but it is not unheard of, as the peritoneum is the usual route of migration of the parasite towards the liver. Kabaalioglu A. et al reported mild splenomegaly and enlargement of the left rectus abdominalis muscle in hepatic fascioliasis \[[@B17]\]. In a study the authors found that among pleuro-pulmonary diseases, parenchymal infiltrates resembling the Loeffler syndrome and pleural effusion were the most common radiological features \[[@B8]\]. Radiologic manifestations of fascioliasis in a case with peritoneal involvement have been described as low-density lesions in the mesentery \[[@B18]\]. While the radiologic presentations of the disease are contradictory, reporting any new set of images related to the unusual organ involvement is of paramount importance. The case presented here is probably the first report of fascioliasis with spleen, pancreas, and kidney involvement. Migration of Fasciola hepatica to the spleen and pancreas and perhaps the kidneys was considered as the cause of the imaging findings in this patient, however hypersensitivity reaction to Fasciola antigens might also be implicated. Pathology examination to confirm the ectopic locations of the involvement was not performed, however improvement of the lesions on follow-up imaging studies after treatment defended the involvement of those regions by the parasite The compromised immunity of the patient linked to the use of immunosuppressive medicines could partly explain the extensive extra-hepatic involvement of the disease. The patient responded fully to Triclabendazole and is now healthy. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-230X/4/15/prepub> Acknowledgement =============== With special thanks to Kathleen Hekmatdoost, APRN, MSN, NP, for editing the article.

PubMed Central

2024-06-05T03:55:47.654955

2004-8-4

PMC517499

Backround ========= Oral pharyngeal dysphagia due to disordered swallowing has become increasingly recognized in children with cerebral palsy and other neurodevelopmental disorders. This has led to the increasing use of enteral tube feedings either for full or supplemental nutritional support. Symptoms of foregut dysmotility, such as vomiting, retching gagging and bloating, are often associated with tube feeding in neurologically handicapped children \[[@B1]-[@B4]\]. Previous studies have demonstrated that recurrent vomiting, aspiration and/or failure to thrive may be present in as many as 10--15% of institutionalized patients with psychomotor retardation. Antroduodenal motor function has been little studied in such children \[[@B5],[@B6]\]. In order to elucidate the mechanisms behind these symptoms we reviewed the charts of a group of children followed in the Tube Feeding Clinic at the Children\'s Hospital of Wisconsin with neurological dysfunction, who were intolerant of tube feedings and who had undergone antroduodenal motility studies as part of their evaluations. Methods ======= The charts of 18 neurologically handicapped children (mean age 4 years, range 1--10 years; 10 males) with dysphagia and symptoms of foregut motility were reviewed. The symptoms, underlying disorders, feeding route, the presence of fundoplication, and the use of prokinetic agents and H~2~b1receptor antagonists are summarized in Table [1](#T1){ref-type="table"}. All except one patient were completely or partially fed enterally. One patient ate orally but required frequent venting of his gastrostomy. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Patient population ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Patient Number** **Age (y)** **Diagnosis** **Symptoms** **Feeding Route** **UGI** **Gastric Emptying** **EGD** **PEG** **Fundoplication** **Other** **Medications** -------------------- ------------- ------------------------------------------------- ---------------- ------------------- ------------------------ ---------------------- --------------- ---------------------- -------------------- ----------------------------------------------- ----------------- --------------- **H2RA** **Cisapride** 1 9 CP GER vomiting\ oral paraesophageal hernia normal normal \+ gastric bezoar retching 2 4 feeding aversion vomiting\ gastrostomy esophageal dysmotility ND esophagitis \+ \+ \+ retching 3 3 Down\'s syndrome gagging\ gastrostomy normal delayed normal \+ retching 4 2 cerebral dysgenesis seizures Irritability jejunostomy GER delayed esophagitis \+ omeprazole 5 5 chromosome 19 deletion, GER subglotic stenosis retching gastrostomy normal normal ND \+ 6 4 hydrocephalus retching gastrostomy normal normal normal \+ \+ \+ 7 1 CP irritability gastrostomy paraesophageal hernia normal normal \+ \+ \+ 8 2 1/2 cerebral atrophy recurrent aspiration pneumonia vomiting gastrostomy GER normal normal \+ \+ \+ 9 2 diphragmatic hernia GER vomiting jejunostomy GER delayed ND \+ surgical jejunostomy \+ \+ 10 5 CP spina bifida retching\ jejunostomy normal normal normal \+ surgical jejunostomy \+ \+ bloating\ constipation 11 9 Floating Harbour syndrome retained food\ oral ND rapid retained food \+ \+ \+ \+ vomiting 12 17 mitochondrial disease vomiting\ gastrostomy esophageal dysmotility delayed normal \+ \+ \+ diarrhea\ retching\ gagging\ bloating 13 3 CP GER retching\ gastrostomy ND normal normal \+ \+ gagging 14 6 charge syndrome chromosome 13 Deletion retching\ gastrostomy normal normal esophagitis \+ \+ \+ gagging 15 2 CP hepatoblasoma vomiting gastrostomy paraesophageal hernia delayed normal \+ Nissen breakdown liver resection pyloroplasty \+ \+ 16 1 CP vomiting gastrostomy GER delayed esophagitis surgical gastrostomy \- pyloroplasty \+ \+ 17 2 feeding aversion vomiting gastrostomy normal \+ normal \+ \+ \+ 18 10 CP seizures hydrocephalus vomiting\ TPN \+ \+ \+ \+ \+ retching\ gagging\ bloating ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- GER: gastroesophageal reflux CP: cerebral palsy: NE: not done TPN: total parenteral nutiriton ::: Following an overnight fast, antroduodenal motility studies were performed using a multilumen catheter with 8 recording ports spaced 2.5--5 cm apart, passed through the gastrostomy either under fluoroscopic guidance or endoscopically, connected to a low compliance, pneumohydraulic capillary infusion system (Arndorfer Medical Specialties, Greendale, WI) and a computerized motility system (Redtech, Calabasas, CA). Fasting activity was recorded for 3--4 hours. Erythromycin (1 mg/kg) was given intravenously over 10 minutes and the recording continued for another hour. Octreotide (0.5 mcg/kg) was then given intravenously over 5 minutes. 45 minutes later a liquid meal was given followed by an additional 2--3 hour recording period. The meal varied and consisted of the usual formula and volume given at home. Patients receiving jejunal feedings or TPN were given a bolus gastrostomy feeding. In the one patient receiving TPN, the TPN was discontinued during the study. Prokinetics were stopped at least 48 hours prior to study. Phase 1 of the MMC was defined as motor quiescence. Phase 2 was defined as the time between Phases 1 and 3 and is characterized by random contractions of varied amplitude and frequency. Phase 3 of the MMC is characterized by an aborally propagating cluster of repetitive contractions with a frequency of 11--13/minute in the duodenum and 3/minute in the antrum with a duration of 3--10 minutes. The tracings were analyzed by visual inspection. This study was approved by the Research and Publications Committee/Human Rights Review Board of the Children\'s Hospital of Wisconsin and the Institutional Review Board of the Medical College of Wisconsin. Results ======= Eleven children had had recent upper GI series. Of these 5 were normal, 3 had gastroesophageal reflux and 3 had paraesophageal hernias (all following fundoplications), 1 had a bezoar and 1 had esophageal dysmotility. Fourteen patients had liquid phase gastric emptying studies. Of these 7 were normal, 6 had delayed emptying and 1 had rapid emptying. Two of 16 patients who had had recent endoscopies had esophagitis, 14 were normal. The diagnoses and clinical histories of the patients are summarized in Table [1](#T1){ref-type="table"}. Twelve of the 18 patients had had a fundoplication and 9 of the 12 had had a pyloroplasty. Indications for fundoplication were frequently poorly described in the medical records but typically included vomiting and feeding intolerance. The incidence of symptoms such as retching and sweating could not be determined No patients had a normal antroduodenal motility study (Figures [1](#F1){ref-type="fig"},[2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"}). In the fasting state 12/18 failed to have phase 3 of the MMC. Eight had predominantly phase 2 activity and did not demonstrate normal fasting phase 1. Nine had non-propagating clusters. One had MMCs that propagated in a retrograde fashion. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### An 8-year-old boy who was TPN dependent demonstrates reverse peristalsis. Note lack of antral contractions. Channel 1--2 antrum; 3--6 duodenum. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Lack of fasting phase 3 activity during a 3 hour monitoring period in an 8-year-old boy. He also had no phase 3 like activity following erythromycin. Channel 1 antrum; 2--6 duodenum. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Lack of responsiveness to erythromycin in a 3-year-old boy with feeding aversion. This patient also had postprandial hypomotility. Channels 1 stomach; 2--3 antrum; 4--7 duodenum. :::  ::: Following erythromycin eight had a normal response consisting of antral contractions with a frequency of 3/min followed by phase 3 like activity in the duodenum. Three patients had no response and seen had abnormal responses consisting of abnormal clusters in three, no antral response in three and no duodenal response in three. Fourteen patients had a normal response to octreotide consisting of cessation of antral activity and the development of phase 3 activity in the duodenum. Two patients had continued antral contractions and two did not develop phase 3 duodenal activity. These patients had non-propagating duodenal clusters. Fourteen patients had normal postprandial phase 2-like activity. Eight developed premature phase 3 activity within 30 minutes following the meal. Seven patients failed to develop phase 3 activity during the 2--3 hour postprandial monitoring period and seven had no antral contractions in the postprandial monitoring period. One patient had a retrograde MMC. Three patients developed severe pain or irritability associated with antral or duodenal contractions following erythromycin \[[@B2]\] or octreotide \[[@B1]\]. There was no correlation between any constellation of symptoms and manometric abnormalities. Conclusions =========== Foregut dysmotility is common in children with neurodevelopmental disorders such as cerebral palsy \[[@B1]-[@B4]\]. Up to 75% of institutionalized children with psychomotor retardation have GER \[[@B7]-[@B11]\]. A number of investigators have reported that neurologically handicapped children have abnormalities of lower esophageal function \[[@B12],[@B13]\]. Delayed gastric emptying is common in such patients \[[@B1],[@B2]\]. Many of these patients undergo fundoplication. Continued symptoms of GER following fundoplication or the development of new symptoms such as retching and gagging suggests that a more generalized foregut motility disorder is present in many of these patients \[[@B5]\]. The rates of complications of surgical treatment of GER that might relate to foregut dysmotility include breakdown of the wrap (0.9--13%) and the gas bloat syndrome (1.9--8%) \[[@B14]\]. Other complications not reported in enough detail to estimate complication rates include dumping, and gastroparesis. Ravelli and Milla have shown that gastric electrical activity as measured by the electrogastrogram (EGG) was abnormal in 31/50 neurologically handicapped children \[[@B2]\]. Eleven of 18 patients who were symptomatic after fundoplication had gastric dysrhythmias. Richards et al showed that neurologically impaired children with pallor, sweating, retching or forceful vomiting preoperatively were at high risk for postoperative retching and vomiting. They hypothesized that these symptoms were indicative of activation of the emetic reflex and that children with these symptoms had a more generalized disorder than those children without such symptoms \[[@B15]\]. In a previous study we compared gastric electrical activity as measured by EGG in a group of neurologically handicapped children who were tolerant of their tube feedings to a group that were intolerant or symptomatic during tube feedings \[[@B16]\]. The percentage of children in each group who had undergone fundoplication was the same. We found that although the percentage of time that normogastria, bradygastria and tachygastria were present was not different in the 2 groups, there was a significant difference in the postprandial power between the groups. This finding suggests that symptoms present in these patients such as vomiting, retching and gagging might be due to an underlying foregut motor disorder. There have been few reports of antroduodenal motility, which have focused on neurologically handicapped children with feeding intolerance. DiLorenzo and colleagues reported that 25/28 children who remained symptomatic following fundoplication had abnormal antroduodenal motility \[[@B5]\]. Similar to our patients a wide variety of abnormalities were found. These authors did not report how many of their patients that had neurological handicaps. Miki et al found that fasting antroduodenal motility was abnormal in 11 neurologically impaired children with symptoms of gastroesophageal reflux \[[@B6]\]. Due to the nature of our patients there are some limitations in the design and interpretation of our study. Because many of our patients had had multiple formula changes, we decided to use the formula, which the child was receiving at the time of the study, so that any symptoms occurring during the studies could not be attributed to a formula change. Bolus feeds were given to all patients during the study even those who had been receiving drip feeds. The fasting state could not be recorded for 4 hours in all patients, thus it is possible that absence of phase 3 of the MMC in these patients might not be abnormal. Since normal manometry data have not been published for children, non propagating clusters may or may not be normal in our patients. Since only 12 of our 18 patients had undergone fundoplication, we agree with DiLorenzo et al \[[@B5]\] that these motor abnormalities were not caused by surgery, rather we believe that the underlying motility disorder was more generalized than had been recognized at the time of fundoplication. In this study we have confirmed that the incidence of foregut dysmotility is very high in neurologically handicapped children with feeding intolerance. Prokinetics and acid suppression did not resolve the symptoms in our patients. Twelve of our 18 patients had had fundoplications and two had undergone two fundoplications in unsuccessful attempts to control what had been thought to be reflux symptoms. While there is no way to know how much abnormal antroduodenal motility contributed to our patient\'s feeding disorders, following antroduodenal manometry a number of our patients were treated successfully with jejunal feeding, suggesting that while they had foregut dysmotility, midgut motility is normal. In neurologically handicapped children foregut dysmotility may be more common than is generally recognized and can explain many of the upper gastrointestinal symptoms in neurologically handicapped children. Thus in this patient population generalized foregut dysmotility may mimic reflux and the decision to perform a fundoplication should be made very cautiously and only after a complete evaluation of foregut motility particularly in children with gagging retching and forceful vomiting. Competing Interests =================== None declared. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-230X/4/19/prepub>

PubMed Central

2024-06-05T03:55:47.656114

2004-9-1

PMC517500

Background ========== IgA nephropathy (IgAN) is the most common form of glomerulonephritis (GN) worldwide \[[@B1]\]. Approximately 20--30% of individuals with IgAN develop end-stage renal disease (ESRD) by 10--20 years following diagnosis \[[@B2]\]. Despite the increasing recognition of IgAN as a significant cause of chronic kidney disease (CKD), no specific therapies for IgAN have been developed. Glucocorticoids and fish oil have been recommended for selected patients with IgAN based on controversial results from small clinical trials \[[@B3]-[@B5]\]. Many patients with IgAN receive no specific therapy. Clinical reports suggest that individuals of Asian/Pacific Islander heritage are more likely to be affected by IgAN than whites, African Americans, and persons of Hispanic descent. Reports from U.S. centers have generally compared results of white and African American IgAN patients, with little or no available information on U.S. patients of Asian/Pacific Islander heritage. Studies from Japan and China have reported that more individuals with ESRD in these countries had IgAN, implying that IgAN may have a more severe disease course in certain Asian populations \[[@B6],[@B7]\]. To explore whether IgAN was more common and severe among Asian/Pacific Islanders in our population, we examined clinical and pathologic data on 149 patients with IgAN and a random sample of 149 patients with other primary glomerular lesions (focal segmental glomerulosclerosis, membranous nephropathy and minimal change disease) at the University of California San Francisco (UCSF) Medical Center. Methods ======= The records of 183 percutaneous native and transplant kidney biopsies with a diagnosis of IgAN received between November 1994 and May 2001 at the Renal Pathology Laboratory at UCSF were reviewed. Baseline demographic and clinical data included age, gender, race or ethnicity, history of kidney transplant, date of biopsy, and serum creatinine concentration at the approximate time of biopsy. In addition, the presence or absence of heavy proteinuria (≥ 3.0 g/day, with or without the nephrotic syndrome) and the presence or absence of hypertension at the time of biopsy were recorded. Major ethnic groups included white, Asian/Pacific Islander, Hispanic, African American, and other/unknown. Ethnicity was determined using information from patient health insurance forms and history provided at the time of biopsy. Any case in which Henoch-Schönlein purpura, systemic lupus erythematosus or chronic liver disease were considered likely diagnoses were excluded, as were cases of IgAN superimposed on a systemic disease involving the kidney (e.g., diabetic nephropathy). Two examiners unaware of the clinical data independently reviewed the biopsies. Biopsies displaying fewer than six glomeruli by light microscopy or insufficient immunofluorescence staining, as defined below, were also excluded. Fifteen biopsies were additionally excluded due to incomplete recovery of microscopic slides from files. Biopsies from 149 patients, including 25 kidney transplant recipients, satisfied the criteria for inclusion and provided the basis for the IgAN analytic sample. Aside from IgAN, the most commonly diagnosed primary glomerular lesions at our institution over the same time period were focal segmental glomerulosclerosis (N = 314), membranous nephropathy (N = 197) and minimal change disease (N = 147). To establish baseline race/ethnicity prevalences for our region and referral population, we collected demographic data on a computer-generated random sample of individuals with non-IgAN glomerular disease (N = 149), stratified by kidney transplant. Pathologic assessment --------------------- Pathologic assessment of the IgAN native kidney biopsies was performed based on Haas\' IgA nephropathy classification system \[[@B8]\]. All cases included in the study also met the following criteria: (1) immunofluorescence studies showing at least 2+ (scale 0 to 3+) mesangial deposition of IgA, with IgA comprising the dominant immunoglobulin deposited in the glomeruli, and (2) electron microscopy (EM) studies showing the presence of mesangial deposits. Statistical analysis -------------------- Demographic and clinical data are reported as mean ± standard deviation, medians with interquartile ranges, and proportions with 95% confidence limits. Inter-ethnicity comparisons were performed using the Cochrane-Mantel-Haenzsel χ^2^test for categorical variables, and analysis of variance (general linear models) or Kruskal-Wallis test for continuous variables. Two-tailed P-values \<0.05 were considered statistically significant. SAS version 8.2 was used for all statistical analyses (SAS Institute, Cary, NC, USA). Results ======= Patient clinical characteristics for the IgAN group are summarized in Table [1](#T1){ref-type="table"}. Eighty-nine (60%) patients were male and 60 (40%) were female. Fifty-seven (38%) patients were white, 53 (36%) Asian/Pacific Islander, 29 (19%) Hispanic, 4 (3%) African American and 6 (4%) were of other or unknown race/ethnicity. The mean age of the IgAN patients at the time of kidney biopsy was 37 ± 14 years. Among the three main ethnic groups (whites, Hispanics, and Asian/Pacific Islanders), Hispanic patients tended to be slightly younger at the time of biopsy compared with whites and Asian/Pacific Islanders. The distributions of age and gender, however, did not differ significantly among white, Hispanic, and Asian/Pacific Islander groups. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary characteristics: IgA nephropathy ::: Characteristic^a^ Total (N = 149) White (N = 57) Asian/PI^b^(N = 53) Hispanic (N = 29) African American (N = 4) Unknown (N = 6) *P*-value^c^ --------------------------- ----------------- ---------------- --------------------- ------------------- -------------------------- ----------------- -------------- Mean age (yr) 37 ± 14 38 ± 15 37 ± 15 34 ± 14 26 ± 22 36 ± 24 0.54 Mean SCr (mg/dL) 3.1 ± 3.8 3.0 ± 2.6 3.1 ± 4.5 2.9 ± 4.2 6.3 ± 6.2 2.7 ± 2.9 0.96 Male, N (%) 89 (60) 37 (65) 29 (55) 16 (55) 3 (75) 4 (67) 0.50 Proteinuria ≥ 3g/d, N (%) 66 (44) 28 (49) 20 (37) 13 (45) 3 (75) 2 (33) 0.08 Hypertension, N (%) 74 (50) 28 (49) 28 (53) 14 (48) 2 (50) 3 (33) 0.86 Transplant, N (%) 25 (17) 4 (7) 13 (24) 8 (28) 0 (0) 0 (0) 0.15 Haas subclass^d^, N (%) I 10 (8) 4 (8) 5 (13) 0 (0) 0 (0) 1 (17) 0.76 II 12 (10) 5 (9) 4 (10) 2 (10) 0 (0) 1 (17) III 23 (19) 10 (19) 8 (20) 2 (10) 1 (25) 2 (32) IV 30 (24) 13 (24) 8 (20) 7 (33) 1 (25) 1 (17) V 49 (40) 21 (40) 15 (37) 10 (47) 2 (50) 1 (17) ^a^Values represent mean ± standard deviation. ^b^Asian/Pacific Islander ^c^*P*-value refers to overall NOVA, Kruskal-Wallis test, or χ^2^for comparison of white, Asian/Pacific Islander and Hispanic groups. ^d^Haas subclass assessment performed on native kidney biopsies (N = 124). ::: The median serum creatinine (SCr) of the IgAN cohort was 1.7 mg/dL (interquartile range 1.1--3.4 mg/dL). The median SCr of the African American group was signficantly higher (5.0 mg/dL) than the other ethnic groups; however, these calculations were based on a small sample size (N = 4) due to the low prevalence (3%) of African Americans with in our IgAN cohort. Median serum creatinine concentrations were not significantly different among white, Hispanic, and Asian/Pacific Islander groups (*P*= 0.64). Sixty-six patients (44%) exhibited heavy (≥ 3 g/d) proteinuria, and 74 (50%) had documented hypertension (systolic blood pressure ≥ 140 or diastolic blood pressure ≥ 90 mm Hg) at the time of kidney biopsy. The fractions of patients with heavy proteinuria and hypertension at the time of kidney biopsy were not significantly different among white, Hispanic, and Asian/Pacific Islander groups. Of the 124 native kidney biopsies, the majority of cases (64%) fell into Haas subclasses IV or V, which are known independent predictors of progressive disease and poor renal outcomes \[[@B2],[@B8]\]. Only 22 biopsies (18%) were graded as Haas subclasses I or II, reflecting a relatively high threshold for kidney biopsy in our referral region. The distribution of Haas subclass did not differ significantly among race/ethnicity groups. Table [2](#T2){ref-type="table"} shows the demographic characteristics of the IgAN and non-IgAN groups. Among the random sample of patients (N = 149) with non-IgAN primary glomerulopathies, 67 (45%) patients had focal segmental glomerulosclerosis (FSGS), 58 (39%) membranous nephropathy, and 24 (16%) minimal change disease. Seventy-seven (52%) patients were male, 51 (34%) white, 42 (28%) Asian/Pacific Islander, 25 (17%) Hispanic, and 30 (20%) were African American. In contrast to previous reports, the distribution of gender did not differ significantly between the IgAN and non-IgAN groups (P = 0.16) \[[@B8]\]. Patients in the non-IgAN group were, however, significantly older (mean age 42 ± 21 years vs. 37 ± 14 years, *P*= 0.006) compared to patients with IgAN. In addition, the distribution of race/ethnicity differed significantly between the two groups (*P*\< 0.001). This association of IgAN and distribution of race/ethnicity persisted even when stratified by kidney transplant (*P*\< 0.001 for native kidney comparison, *P*= 0.006 for kidney transplant recipient comparison). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Patient demographics: All glomerular lesions ::: Characteristic^a^ IgA nephropathy (N = 149) Non-IgAN glomerular lesions^b^(N = 149) *P*-value^c^ ----------------------- --------------------------- ----------------------------------------- -------------- Mean age (years) 37 ± 14 42 ± 21 0.006 Male, N (%) 89 (60) 77 (52) 0.16 Transplant, N (%) 25 (17) 25 (17) 1.00 Race/ethnicity, N (%)  White 57 (38) 51 (34) \<0.001  African American 4 (3) 30 (20)  Asian/PI^d^ 53 (36) 42 (28)  Hispanic 29 (19) 25 (17)  Other/Unknown 6 (4) 1 (1) ^a^Values represent mean ± standard deviation. ^b^Focal segmental glomerulosclerosis, membranous nephropathy, and minimal change disease. ^c^*P*-value refers to overall ANOVA, Kruskal-Wallis test, or χ^2^. ^d^Asian/Pacific Islander ::: Discussion ========== In a biopsy series of 244 patients with IgAN, Haas found fewer African Americans (in a major urban setting), similar to that noted in other U.S.-based studies of IgAN \[[@B9]-[@B11]\]. While limited by the size of certain ethnic groups in the study population, Haas found no significant difference in renal survival associated with \"white race, black race or Hispanic origin\" \[[@B8]\]. The reason for a lower prevalence of IgAN in African Americans relative to other kidney diseases remains unclear. The frequency of IgAN in African Americans does not appear to be influenced by the higher prevalence of the IgA2 allotype among this group \[[@B12]\]. In our study, the fraction of biopsies in subclasses I and II (18%) was similar to that observed by Haas (23%). However, we observed a higher proportion of biopsies in subclasses IV and V (64% vs. 31%), and a lower proportion of biopsies in subclass III (19% vs. 45%) compared with Haas, possibly reflecting a temporal trend towards a higher biopsy threshold along with intergrader measurement bias. In a study reviewing the pattern of glomerulonephritis in Singapore over the past two decades, Woo and colleagues reported that IgAN was the most common primary GN occurring in Singapore (42% of all primary GNs in the first decade and 45% in the second decade) \[[@B13]\]. In our biopsy population during the same period that we studied, IgAN represented 12.8% of all biopsies with primary glomerular diseases and 8.4% of all biopsies (excluding transplant biopsies for non-glomerular diseases). In China, Li reported that IgAN was the leading cause of ESRD, accounting for approximately 18% of patients \[[@B6]\]. In a national survey of Japanese patients with ESRD, Koyama et al. reported that 28% of new dialysis patients had IgAN listed as their primary cause of ESRD. Moreover, due to the number of additional biopsies showing chronic glomerulonephritis without immunofluorescent microscopic descriptions in the survey, the authors estimated that possibly 40% of newly registered dialysis patients in Japan might have had CKD from IgAN. In contrast, only 0.8% of incident ESRD patients in the U.S. have documented or suspected IgAN \[[@B14]\]. Katznelson and Cecka, using data from the United Network for Organ Sharing (UNOS), have also reported a higher incidence of IgAN and chronic glomerulonephritis causing ESRD in Asian/Pacific Islander American recipients of renal allografts between 1991 and 1996 \[[@B15]\]. In contrast, based on smaller biopsy series, a striking variation in prevalence rates of IgAN has been reported from Europe and South America. In the UK, for example, Ballardie and colleagues noted comparatively low prevalence rates of IgAN in a predominantly white population (Manchester, England) in the early 1970\'s. In the subsequent 15-year period, however, these investigators reported a phenomenal rise in the observed incidence of IgAN (accounting for 31% of all glomerulopathies in 1986), which the investigators felt more likely reflected a higher frequency of detection rather than true rise in disease incidence. Similar prevalence rates have also been documented from isolated white populations in Finland and southern Italy \[[@B16],[@B17]\]. In contrast, few studies have addressed the epidemiology of IgAN in Latin America. In a small Brazilian single-center cohort (N = 205) of primary glomerular diseases, Mazzarolo et al. reported relatively modest prevalence rates (10.2%) of IgAN \[[@B18]\]. A larger series (N = 1,263) of renal biopsies from Peru noted much lower prevalence rates of IgAN, which accounted for only 0.9% of all glomerular lesions over a 10-year period at a central reference renal pathology laboratory in Lima \[[@B19]\]. These differences may be partially attributed to increased screening and disparities in the indication for kidney biopsy. In Japan and South Korea, for example, school-aged children undergo annual screening for urinary abnormalities; kidney biopsy is subsequently recommended for children with evidence of proteinuria or hematuria \[[@B20]\]. More comprehensive yearly health exams are further performed on full-time salaried employees throughout Japan, Singapore, and Hong Kong, making detection more likely in these ethnic groups. Furthermore, a significant reporting bias may also contribute to the higher reported prevalence rates of IgAN in Asian/Pacific Islanders, e.g., in the study by Koyama et al., only 502 (7%) of the approximate 6800 patients diagnosed with IgAN had undergone a confirmatory kidney biopsy \[[@B7]\]. Although the etiology of IgAN remains unknown, there exists a strong suspicion for an environmental antigen trigger combined with a genetic susceptibility factor. Along these lines, several hypotheses have been proposed to account for the reportedly higher prevalence of IgAN in Asian/Pacific Islanders. With respect to potential dietary antigens, Wakai et al. found that high intake of rice and n-6 polyunsaturated fatty acids (PUFA) were associated with an increased risk of IgAN \[[@B21]\]. Recent reports from Japan have also suggested a potential role of *H. parainfluenzae*as a causative agent of IgAN in Japanese children and adults. Such claims are supported by studies showing the glomerular deposition of outer membrane *H. parainfluenzae*antigens and greater levels of plasma IgA1 antibody against OMHP in Japanese patients with IgAN (compared to Japanese patients with other renal diseases) \[[@B22],[@B23]\]. Whether Japanese, or Asian/Pacific Islanders in general, have higher rates of *H. parainfluenzae*colonization and/or infection has yet to be established. The presence of either hypertension or proteinuria ≥ 3.0 g/24 hrs at the time of diagnosis significantly correlated with worsened renal survival in IgAN, even when controlling for serum creatinine at the time of kidney biopsy \[[@B2]\]. We found no difference in the distribution of Haas subclass, hypertension and nephrotic proteinuria among Caucasians, Asian/Pacific Islanders, and Hispanics. Despite ongoing investigative efforts, scant data are available regarding genetic markers that may predispose individuals to progressive disease from IgAN. Recent immunogenetic studies have suggested a potential role for the T-cell receptor (TCR) in the development of immune-mediated diseases. Deenitchina and colleagues found that genetic polymorphism of the TCR constant alpha chain was associated with progression of CKD in a cohort of Japanese patients with IgAN. Although promising, such polymorphisms of the TCR gene have yet to be evaluated in large, prospective studies or by genetic analysis of familial IgAN \[[@B24]\]. Our results contest the assertion that IgAN follows a more severe course in individuals of Asian/Pacific Islander descent. One reason for the similar disease severity of IgAN in our study population may stem in part from the large subpopulation of Filipino patients comprising our Asian/Pacific Islander cohort. It is unclear whether certain subpopulations of Asian/Pacific Islanders, including Filipinos, exhibit IgAN prevalence rates similar to those documented by Koyama and Woo. Anecdotal reports from Thailand and India documenting prevalence rates of 4--9% suggest that IgAN may not have the same epidemiology among all southeast Asians \[[@B1]\]. Despite having higher incidence rates of ESRD than the U.S. white population, the Asian/Pacific Islanders remain a largely unstudied group, for whom more comprehensive data collection is warranted. There are several important limitations to this report. As with any single-center biopsy series, we may have been underpowered to detect a clinically significant difference due to the limited sample size (type II error). In addition, racial admixture may have also confounded the results, as we were unable to subclassify patients in the Asian/Pacific Islander group or account for the growing population of bi- or multi-ethnic individuals in our population. Furthermore, due to the study\'s case control design, and breadth of our referral base (northern California and Hawaii), we were unable to control for the criteria for kidney biopsy. As a result, a biopsy bias may have confounded our results. In other words, Asian/Pacific Islander patients in our referral base with mild to moderate proteinuria and/or hematuria might have been given a presumptive diagnosis of IgAN without nephrology referral or confirmatory kidney biopsy. With regard to disease prevalence, these potential referral and biopsy biases based on race/ethnicity are largely conservative in nature, and would have biased our results towards the null. Finally, we have included data from a modest-sized IgAN transplant population (N = 25), the donor demographics of which were unavailable at the time of the study. However, the association of race/ethnicity and distribution of glomerular lesion persisted, even when stratified by kidney transplant, and thus our overall conclusions remained the same. In addition, a small European study of donor-recipient pairs (average follow-up 7 years) has shown that when a donor kidney with asymptomatic IgA deposits is transplanted into a recipient with ESRD secondary to a disease other than IgAN, the IgA immune deposits in the donor kidney are rapidly removed \[[@B25]\]. Conclusions =========== In conclusion, with the caveats of referral bias and biopsy bias, the race/ethnicity distribution of IgAN differs significantly from that of other major glomerulonephridities. However, among individuals undergoing native kidney biopsy, we see no evidence of a race/ethnicity association with severity of disease in IgAN by clinical and IgAN-specific histopathologic criteria. Further studies are needed to identify populations at higher risk for progressive disease in IgA nephropathy. Competing interests =================== None declared. Authors\' contribution ====================== YH designed the study, collected and analyzed the data, and drafted the manuscript. GC supervised the study design, analyzed the data, and edited the manuscript. EF graded the IgAN histopathologic slides. JO collected, reviewed and graded all histopathologic data, and edited the manuscript. All authors approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2369/5/10/prepub> Acknowledgments =============== Dr. Hall is supported by the American Kidney Fund, Clinical Scientist in Nephrology Award. Dr. Chertow is supported by NIH NIDDK RO1 DK58411-01.

PubMed Central

2024-06-05T03:55:47.658266

2004-9-2

PMC517501

Background ========== Neuroblastoma (NB) is the most frequent extra-cranial solid tumour in children, originating from immature neural crest cells of the sympathetic nervous system \[[@B1]\]. The tumours show remarkable differences in clinical presentation ranging from localized to highly metastatic. Although age and clinical stage are strong prognostic indicators, particular genetic aberrations, i.e. *MYCN*amplification and 17q gain, also have a profound predictive power \[[@B2],[@B3]\]. Presently, three major clinico-genetic NB patient subgroups have been recognized (subgroup 1, 2A and 2B) \[[@B4]\]. Subgroup 1 consists of NB patients with favourable disease stage (stage 1, 2 and 4S), most often infants younger than one year of age presenting with tumours with a near triploid DNA content and a characteristic pattern of chromosomal instability including the consistent presence of an extra chromosome 17. The two other NB patient groups represent mainly older children with high-stage disease (stage 3 and 4) and poor prognosis. Both NB subgroups present with 17q-gain, but are distinguished by presence of *MYCN*amplification and 1p-deletion in subgroup 2B and 11q-deletion often in combination with 3p-deletion in subgroup 2A \[[@B3],[@B5]-[@B9]\]. The first evidence for the occurrence of 11q-deletions in NB was obtained in 1991 \[[@B10]\]. However, it was not until recently that a specific patient subgroup with this particular genetic defect was recognized, representing approximately 20% of cases \[[@B5]-[@B9],[@B11]-[@B13]\]. The recurrent finding of 11q-deletions in NB suggests the presence of a tumour suppressor gene residing on the long arm of chromosome 11. Additional functional evidence for this hypothesis came from the observation that differentiation of NB cells can be induced by transfer of an intact chromosome 11 into a NB cell line \[[@B14]\]. Although both comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) studies indicate that the majority of the 11q-deletions are distal losses encompassing a large portion of the long arm \[[@B5]-[@B9],[@B12],[@B13],[@B15],[@B16]\], detection of rare small or interstitial deletions allowed the provisional localization of an SRO (shortest region of overlap) at 11q23.3 between markers D11S1340 and D11S1299, encompassing a distance of approximately 3 Mb \[[@B16]\]. When a single tumour with two small interstitial deletions is not taken into consideration, the SRO is defined by a small subset of tumours and spans 18 Mb between markers D11S898 and D11S1299 (according to UCSC Genome Browser, freeze version July 2003). This region harbours *SDHD*, which encodes the small subunit D (cybS, cytochrome b558) of the mitochondrial respiratory chain complex II (succinate-ubiquinone oxidoreductase) \[[@B17],[@B18]\] and was recently recognized as a prototype tumour suppressor gene \[[@B19]\]. The first evidence for a role of *SDHD*in tumour development was obtained by the discovery of germline mutations in this gene as the cause for familial paraganglioma (PGL) \[[@B19]\]. Somatic and occult germline *SDHD*mutations were also detected in patients with apparently sporadic pheochromocytoma (PC) \[[@B20],[@B21]\]. It seems that most of the individuals with PC possess *SDHD*mutations in the 5\' portion of the gene causing complete disassembly of complex II, whereas PGL are associated with mutations in the 3\' region of the gene causing partial inactivation of its catalytic activity \[[@B19]-[@B28]\]. PGL and PC are histologically related to NB as they are all neural crest derived. NBs consist of immature neuroblasts, whereas PGL and PC contain mature chromaffin cells. Of further interest is the fact that, in addition to the well established role of *SDHD*in oxidative phosphorylation, *SDHD*has also been presumed to contribute to the function of the mitochondria as oxygen sensors. It was shown that *SDHD*inactivation leads to a pseudo-hypoxic state and upregulation of hypoxia responsive genes, possibly through increased production of reactive oxygen species (ROS) \[[@B23]\]. A hypoxia-induced shift toward a neural crest-like phenotype has been shown to result in more aggressive NB cells with increased potential to metastasize \[[@B29]\]. Consequently, inactivating *SDHD*mutations or reduced activity of *SDHD*might lead to impaired oxidative phosphorylation and hypoxia and thus contribute to NB oncogenesis. In view of the above, we considered *SDHD*as a positional and functional candidate for the presumed NB tumour suppressor gene on 11q23. In order to search for evidence for involvement of *SDHD*in NB development, an extensive series of investigations was performed on the DNA, RNA and protein level. Methods ======= NB patient and cell line samples -------------------------------- Neuroblastoma (NB) tumour samples (at least 70% tumour cells) were collected at the Ghent University Hospital (Ghent, Belgium) (n = 32) and in the Molecular Oncology Unit (Lyon, France) (n = 35). Ethical approval was obtained for the collection of the tumour samples. The latter group includes selected patients with stage 3 or 4 NB without *MYCN*amplification. For all NB patients constitutional leukocyte DNA was available. In addition, 31 NB cell lines were included in the analysis of which karyotypes were available. For 20 of these cell lines, comparative genomic hybridization (CGH) data and/or M-FISH (multicolour fluorescence in situ hybridization) results have been published \[[@B30]-[@B32]\]. For screening of sequence variants in a normal population, leukocyte DNA from 135 unrelated healthy individuals was used. DNA was extracted as previously described \[[@B33]\]. Cultures of NB cell lines N206, SK-N-AS, SK-N-SH, NMB, SK-N-FI, CLB-GA, LA-N-2 and NGP were treated with puromycine (100 μg/ml) during 6 hours in order to prevent possible nonsense mediated RNA decay of variant *SDHD*transcripts. RNA of the cell line pellets (treated and untreated) was extracted with the RNeasy Mini kit (Qiagen) according to the manufacturer, followed by RNase free DNase treatment on column (Qiagen). A fraction of the untreated NB cell line cultures was also used for functional enzyme assays. 11q23 status of samples ----------------------- ### Determination of the 11q23 status in NB cell lines and tumours with LOH or FISH The 11q status of the cell lines was evaluated using FISH. FISH was performed using the LSI *MLL*(11q23.3) SpectrumOrange probe (Vysis) and BAC clone RP11-93E4 for the *CRTAM*gene (11q24.1) in combination with a centromeric probe for chromosome 11. Labelling and FISH was performed as described \[[@B34]\]. For each case at least twenty metaphase chromosomes and 100 interphase nuclei were screened. All NB patients were analyzed with 4 microsatellite markers on 11q23: D11S1986 (11q23.1), D11S1998 (11q23.3), D11S1356 (11q23.3) and D11S1299 (11q23.3), of which D11S1986 and D11S1998 are immediately flanking the *SDHD*gene. In order to discriminate between whole chromosome loss, and unbalanced 11q loss (= partial 11q loss), two microsatellite markers on 11p (D11S922 on 11p15.5 and D11S1324 on 11p14.1) were analyzed in patients that showed allelic imbalance for all 11q markers (positions of the markers are according to the UCSC Genome Browser, freeze version July 2003). Scoring of loss of heterozygosity (LOH) was performed by calculation of the allelic imbalance factor (AIF) \[[@B35]\], whereby AIF \> 2 denotes allelic imbalance, and AIF \> 5 denotes LOH. Experimental conditions for the fluorescent based LOH screening can be obtained from the authors upon request. ### Homozygous deletion screening in NB cell lines Real-time quantitative PCR primers were designed in the four exons of *SDHD*using Primer Express v2.0 (Applied Biosystems) (Table [1](#T1){ref-type="table"}). Exon 1 was too small for primer design in the exonic region; therefore primers flanking the exonic region were designed. Real-time quantitative PCR and quantification was performed as described \[[@B33]\]. MSP --- On the 31 NB cell lines and on another series of 50 NB tumours of which 15 were included in the mutation analysis, methylation-specific PCR (MSP) was performed as described, with minor modifications \[[@B36]\]. MSP primers were designed using the web-based MSP design software MethPrimer <http://www.urogene.org/methprimer/>\[[@B37]\] and checked for specificity using the methBLAST software <http://medgen.ugent.be/methblast/> (Pattyn et al., in preparation). Primers were designed in a CpG island close to the start of the gene (putative *SDHD*promotor) (chr11: 111495002--111495330: UCSC Genome Browser freeze version July 2003) (methylated forward 5\'GTAGTCGGGATCGAGTATTAGTGAGTC3\', methylated reverse 5\'AATAAACCGAAAATCGAAAAACGAT3\', unmethylated forward 5\'AGTTGGGATTGAGTATTAGTGAGTTGT3\', unmethylated reverse 5\'ACTAAATAAACCAAAAATCAAAAAACAAT3\'). Amplification mixtures (50 μl) for the PCR reaction contained 50 ng template DNA, 1× Platinum Taq PCR reaction buffer (Invitrogen), 6 mM MgCl~2~, 200 μM of each dNTP, 1.25 U Platinum Taq polymerase (Invitrogen), 3% DMSO and 300 nM of each primer. The cycling conditions comprised 4 min polymerase activation at 93°C, 40 cycles with denaturation at 93°C for 30 sec, annealing at 64°C (methylated primers) or 65°C (unmethylated primers) for 30 sec and extension at 72°C for 30 sec, and a final extension for 5 min at 72°C. SssI methylase (New England Biolabs) treated DNA, following the manufacturer\'s instructions and normal human genomic DNA were used as a positive and negative control respectively after bisulfite modification. Mutation analysis ----------------- ### DHPLC analysis Intronic primers flanking the *SDHD*exons were designed using Primer Express v2.0 (Applied Biosystems), based on the publicly available *SDHD*genomic sequence (accession number AB026906) (Table [2](#T2){ref-type="table"}). PCR reactions were performed on a PTC-200 DNA engine (MJ Research). Amplification mixtures (25 μl) contained 10 ng template DNA, 1× Platinum Taq PCR reaction buffer (Invitrogen), 2.5 mM MgCl~2~, 200 μM of each dNTP, 1 U Platinum Taq polymerase (Invitrogen) and 500 nM of each primer. The cycling conditions comprised 3 min polymerase activation at 94°C, 35 cycles with denaturation at 92°C for 20 sec, annealing at 60°C for 20 sec and extension at 72°C for 2 min, a final extension for 5 min at 72°C and a slow decrease in temperature to 25°C over 30 minutes. One μl of the PCR products was analyzed on a Ready-To-Run Agarose Gel (1.2%) (Amersham Biosciences). Denaturing high-pressure liquid chromatography (DHPLC) was performed using the Wave system (Transgenomic). The melting profile of each fragment was determined using the Wavemaker software v4.1 (Transgenomic). Crude PCR product was injected into a preheated, fully equilibrated chromatographic column for the DHPLC analysis. Exon 1 fragments were eluted at a temperature of T~m~(= 62.1°C)+0.7°C and T~m~+1.5°C. Exon 2 fragments were eluted at a temperature of T~m~(= 55.7°C)+4.8°C. Exon 3 fragments were eluted at a temperature of T~m~(= 57.4°C)-0.4°C, T~m~+1.1°C and T~m~+3.8°C. Exon 4 fragments were eluted at a temperature of T~m~(= 56.4°C)-0.4°C, T~m~+1.6°C and T~m~+3.2°C. Elution of the fragments was performed using standard conditions according to the manufacturer. Elution profiles were analyzed using the Wavemaker software. ### Sequencing Sequencing was performed on all cell lines and on tumour samples with aberrant DHPLC elution peaks (except from the noncoding region of exon 4 that was sequenced in all NB cell lines without preceding DHPLC mutation screening). Amplified fragments were purified using the Montage PCR96 filter plates (Millipore) or by excision of the fragment of interest from a 1.5% TBE-agarose gel and purification on a GenElute Minus EtBr Spin Column (Sigma-Aldrich). Cycle sequencing was performed using purified amplicons (3--10 ng), the above-mentioned primers (Table [2](#T2){ref-type="table"}) at a concentration of 80 nM and the ABI PRISM BigDye Terminators v3.0 Cycle Sequencing Kit (Applied Biosystems), with the following thermocycling conditions: 25 cycles at 92°C for 10 sec, 55°C for 5 sec and 60°C for 3.5 min. The products were run on an automated sequencer ABI3100 (Applied Biosystems) after isopropanol precipitation. Sequence analysis was performed with the SeqScape v1.1 software (Applied Biosystems). ### Allelic discrimination screening for 2 sequence variants using MGB probes PCR primers and minor groove binder (MGB) probes for sequence variant IVS4-32T\>C were designed using Primer Express v2.0 following the user bulletin guidelines for the design of MGB probes (Applied Biosystems): forward primer 5\'TTTTTTGCAGCCAAGTTATCTGTATAG3\', reverse primer 5\'TGTCCAAGGCCCCTAAAGAA3\', MGB probe allele 1 5\'TGTGGTTTTTtATTGATG3\' labelled with 6-FAM and MGB probe allele 2 5\'TGTGGTTTTTcATTGAT3\' labelled with VIC. To address the frequency of the sequence variant g.7876A\>G (Y93C) in a normal population, the following primers and probe were designed: forward primer 5\'GGCTGCTTATTTGAATCCTTGCT3\', reverse primer 5\'ACTTGCCAGTGACCATGAAGAGT3\' and MGB probe variant allele 5\'ATGGACTgTTCCCTG3\' labelled with VIC. The reaction mixture contained 10 ng of DNA, 100 nM of each MGB probe, 300 nM of each primer and 1× qPCR Mastermix (Eurogentec). For the screening of the g.7876A\>G variant, multiplex PCR was performed using primers and probe of the normal allele of the above-mentioned SNP (IVS4-32T\>C) and primers and probe for the variant allele g.7876A\>G. Reactions were performed on the iCycler Thermal Cycler (Bio-Rad) with the following thermocycling conditions: an initial activation step at 95°C for 10 min, 50 cycles of 95°C for 15 sec and 60°C for 1 min. Allelic discrimination data analysis was performed on the iCycler IQ Optical System Software v3.0a (Bio-Rad). The SNP info for the IVS4-32T\>C variant was submitted to NCBI\'s SNP database (sn\#5606973, *SDHD*\_IVS4-32). Full-length SDHD mRNA amplification ----------------------------------- In order to investigate predicted or putative splice variants caused by the 4 bp-deletion in NB cell line N206 and *SDHD*sequence variants present in other cell lines, the coding region of the full-length *SDHD*mRNA was amplified for cell lines N206, SK-N-AS, SK-N-SH, NMB, SK-N-FI, CLB-GA, LA-N-2 and NGP, before and after puromycin treatment. RNA extraction, DNase treatment and cDNA synthesis were performed as described \[[@B38]\]. Subsequent PCR was performed with forward primer 5\'AGGAACGAGATGGCGGTTCTC3\' (exon 1) and reverse primer 5\'GCTTCCACAGCATGGCAACA3\' (exon 4). PCR products were run on an agarose gel, purified and sequenced using the above-mentioned protocol. The sequence information also provided evidence on the allelic mRNA expression status of *SDHD*. Quantification of SDHD expression using real-time quantitative RT-PCR --------------------------------------------------------------------- Relative *SDHD*expression levels were determined using an optimized two-step SYBR Green I RT-PCR assay \[[@B38]\] with minor modifications in 31 cell lines, 7 normal control samples (human brain, trachea, lung, heart, breast, kidney, liver) and in laser capture microdissected foetal neuroblast cells \[[@B39]\]. The comparative C~T~method was used for quantification. PCR reagents were obtained from Eurogentec as SYBR Green I mastermixes and used according to the manufacturer\'s instructions. Primers in exon 3 were designed using Primer Express (see Primers Exon 3 in Table [1](#T1){ref-type="table"}). Reactions were run on an ABI5700 (Applied Biosystems). Gene expression levels were normalized using the geometric mean of the 4 most stable internal control genes in NB (i.e. *UBC*, *HPRT1*, *SDHA*and *GAPD*) as reported previously \[[@B40]\]. Complex II activity and protein assays -------------------------------------- Enzyme activities were determined spectrophotometrically as previously described \[[@B41]\]. Protein amount was determined with immunoblot analysis of complex II Fp fragment as previously described \[[@B42]\]. Relative protein amounts of complex II compared to complex IV were measured using the TotalLab software (Amersham Biosciences). Ultrastructural analysis of mitochondria in NB cell lines --------------------------------------------------------- Mitochondria of NB cell lines LA-N-2, SK-N-AS, CLB-GA, NGP, CHP-901, SK-N-SH, SK-N-FI, N206, SJNB-12, SJNB-8, NMB, SJNB-10 and IMR-32, breast carcinoma cell line MCF-7 and, Ewing sarcoma cell line SK-N-MC were analysed by electron microscopy. Monolayers from these cell lines were briefly rinsed twice in PBS and then immersed at room temperature in 3 % glutaraldehyde buffered with Na-cacodylate at pH 7.3 for 1 h. After rinses in this buffer with 1 % bovine serum albumin, cells were scraped off with a rubber policeman, and centrifuged with 3 % glutaraldehyde. After washing, the pellets were postfixed in 2 % buffered OsO~4~for 1 h at 4°C. Block staining in uranylacetate (UAc) in 70 % ethanol was followed by dehydration in ethanol and propylene oxide, and embedding in Epon. Ultrathin sections were counterstained with UAc and lead. At least 2 cells in each culture were photographed at a magnification of 20,000 in a Zeiss electron microscope operating at 50 KV. Per culture, 25--63 mitochondria were examined. Their projected length (largest straight distance) was measured and matrix electron density was compared to the surrounding cytosol. Electron microscopic files were made on 11 archived neuroblastic tumours. In addition, previously published cases were examined for mitochondria morphology. Results ======= SDHD deletion, mutation and methylation analysis ------------------------------------------------ ### 11q23-deletion screening Previous karyotyping, comparative genomic hybridization (CGH) and/or M-FISH revealed 11q-deletions in 9 out of 31 neuroblastoma (NB) cell lines (CLB-GA, GI-ME-N, IMR-32, LA-N-6, NBL-S, NGP, SK-N-AS, NMB, N206) \[[@B30]-[@B32]\]. In this study, the presence of 11q23-deletions was confirmed by FISH in all these cell lines except N206 for which the deletion was located distal to the *MLL*locus (11q23.23) (not shown) (Table [3](#T3){ref-type="table"}). Sequencing analysis of *SDHD*on 11q23 demonstrated that the allelic imbalance in NMB does not cause loss of heterozygosity (see later). No previously unnoticed submicroscopic 11q23-deletions were detected. Screening for homozygous deletions in all *SDHD*exons was negative for the 31 NB cell lines. In 20 of the 67 NB tumour samples, loss of heterozygosity (LOH) or allelic imbalance (AI) (AIF \> 2) in the 11q23 region was found (Table [3](#T3){ref-type="table"}): unbalanced 11q LOH (i.e. partial allelic loss of the long arm of chromosome 11) in 2/32 patients of the Ghent University Hospital (Ghent, Belgium) and in 7/35 patients of the Molecular Oncology Unit (Lyon, France) and loss of markers on both chromosome arms (indicating whole chromosome 11 loss, or co-occurrence of 11q and 11p allelic loss) in 3/32 patients of the Ghent University Hospital and 8/35 patients of the Molecular Oncology Unit (Table [3](#T3){ref-type="table"}). The higher frequency of chromosome 11 LOH in the patient subgroup of the Molecular Oncology Unit can be explained by the selection for patient samples of high stage without *MYCN*amplification, in contrast to the other patient subgroup for which samples were unselected. ### Mutation analysis Denaturing high performance liquid chromatography (DHPLC) analysis and subsequent sequencing of the *SDHD*gene in 31 NB cell lines and 67 NB tumour samples revealed the presence of sequence variants in 5 NB cell lines and 4 NB tumour samples (Table [4](#T4){ref-type="table"}). Two variants were considered as bona fide mutations (Figure [1](#F1){ref-type="fig"}). The first, a Y93C missense mutation in cell line NMB, was not detected in 135 unrelated healthy individuals. The second variant detected in NB cell line N206, represented a 4 bp deletion on the exon-intron boundary causing an exon 3 skip leading to a premature stop codon. Interestingly, both effects are located within regions that are frequently affected in paraganglioma (PGL). Unfortunately no normal or primary tumour material of the patients from which the N206 and NMB cell lines were derived was available to test whether these are germline or somatic mutations. In one patient without 11q allelic loss (F11) we observed in both tumour and constitutional DNA a TCTA insertion at position IVS2+37. However, no additional tumour material nor parental material was available for further analysis. So, it remains unclear whether this is a true mutation or a rare polymorphism. In addition, 1 new and 4 known polymorphisms were observed. The H50R variant found in cell line LA-N-2 was described as a polymorphism in several studies \[[@B43]-[@B45]\]. This is also true for the G12S change found in tumour and constitutional DNA of patient F18 \[[@B21]\]. The previously reported polymorphisms IVS3-29A\>G \[[@B25]\] and S68S \[[@B25],[@B27],[@B28],[@B44],[@B46],[@B47]\] were detected in cell lines NGP, NMB and SK-N-FI, in both tumour and constitutional DNA of patients F18 and F35, and in constitutional DNA of patient F22. In all cases, these last two polymorphism (IVS3-29A\>G and S68S) were present together with the IVS4-32T\>C variant, previously described by Taschner and colleagues \[[@B28]\]. Allelic discrimination screening in 135 unrelated individuals revealed an incidence of the IVS4-32T\>C polymorphism of 4.4% (= 6/135; allele frequency 2.2%). This is similar to the incidence found in NB cell lines (3/31 = 9.7%) and NB patient constitutional DNA (3/67 = 4.5%, allele frequency = 2.2%). The presence of the IVS3-29A\>G, S68S and IVS4-32T\>C variants in a cell line (NGP) and two tumours (F18 and F35), in which one of both *SDHD*alleles has been deleted, indicates that all three variants are located on the same allele, representing a low frequent haplotype. ### MSP analysis *SDHD*promotor hypermethylation was tested for 31 NB cell lines and 50 NB patients using methylation-specific PCR (MSP). No evidence for methylation was obtained in any of the analyzed NB cases. ### Analysis of the 4 bp deletion in the cell line N-206 Amplification of the full-length *SDHD*cDNA showed that a 4 bp deletion in the intron-exon boundary in cell line N206 caused skipping of exon 3 leading to a premature stop codon. No alternative transcripts could be detected in cell lines NMB, SK-N-FI, NGP and LA-N-2 carrying basepair variants (and 3 control cell lines without sequence variants SK-N-SH, SK-N-AS and CLB-GA) when grown with or without puromycin (Figure [1](#F1){ref-type="fig"}). The above-mentioned cDNA transcript sequencing revealed that *SDHD*is bi-allelically expressed, thus supporting recent observations in lymphoblastoid cell lines, adult kidney and adult and fetal brain \[[@B19],[@B22]\], but in contrast with the initially reported paternal mono-allelic expression in PGL tissue \[[@B22]\]. SDHD mRNA expression analysis ----------------------------- *SDHD*expression levels were measured using real-time quantitative PCR in 31 NB cell lines, normal foetal neuroblast cells (16, 18 and 19 weeks gestational time) and 7 normal adult tissues (brain, heart, kidney, liver, lung, trachea and breast) (Figure [2](#F2){ref-type="fig"}). The *SDHD*mRNA level was significantly lower in NB cell lines compared to both normal neuroblast cells (Mann-Whitney test: P = 5.31E-06) and normal adult tissue mRNA samples (Mann-Whitney test: P = 1.49E-05). *SDHD*mRNA levels were significantly reduced in cell lines with 11q allelic loss and *SDHD*mutated cell lines (i.e. NMB and N206) (N = 9) compared to cell lines without 11q allelic loss (N = 22) (Mann-Whitney test: P = 1.49E-03). SDHD functional analysis ------------------------ As the *SDHD*gene encodes the small subunit D of the mitochondrial respiratory chain complex II we decided to assess the effect of the basepair variants on the activity of complex II of the respiratory chain by spectrophotometrical measurements in 5 NB cell lines (N206, NMB, SK-N-FI, NGP and LA-N-2) and 3 control NB cell lines without sequence variants (SK-N-SH, SK-N-AS and CLB-GA). No significant differences in complex II enzyme activity could be demonstrated. Although, in LA-N-2 a slight decrease of complex II activity was observed (data not shown). On above-mentioned cell lines and NB cell lines CHP-901, SJNB-12, SJNB-8, SJNB-10 and IMR-32, breast cancer cell line MCF7 and Ewing sarcoma cell line SK-N-MC, immunoblotting of the Fp fragment of complex II showed no significant variation in abundance among the tumour cell lines (data not shown). Ultrastructural morphology of mitochondria in NB cell lines ----------------------------------------------------------- Electron microscopic analysis of NB cell lines revealed that the morphology of the mitochondria is heterogeneous between the different cell lines, with respect to length, dilated intracrista spaces and condensation of the matrix (Table [5](#T5){ref-type="table"} and Figure [3](#F3){ref-type="fig"}). In most of the cell lines the electron dense matrix granules are absent. A striking observation are dilations of the mitochondrial intracrista spaces in most of the NB cell lines including N206 (Figure [3A](#F3){ref-type="fig"}), but not NMB (Figure [3B](#F3){ref-type="fig"}). Cell line LA-N-2 shows very large mitochondria (Figure [3D](#F3){ref-type="fig"}). However, these observations are not the same as described for PGL, where swollen mitochondria are seen with an empty matrix and short or absent cristae \[[@B48]\]. In order to examine whether dilated mitochondrial cristae are a feature of many, or all NBs, we studied the mitochondria in electron micrographs from 11 archived and 22 previously published neuroblastic tumours \[[@B49]-[@B53]\]. Dilated cristae were seen in 11 tumours but they were limited to a minority of the mitochondria (2--23%), in contrast to several of the cell lines of which most mitochondria are altered. In several of the analyzed NB tumours, partially vacuolated matrices were occasionally observed. Discussion ========== In this study, we investigated the possible involvement of *SDHD*in neuroblastoma (NB) tumourigenesis. In a first step, mutation and methylation analyses were performed on a large panel of NB cell lines and tumours. A total of seven sequence variants (in nine different samples) were detected of which two could represent bona fide mutations, i.e. missense mutation Y93C in cell line NMB and a 4 bp deletion in cell line N206. The Y93C sequence variant has not been reported previously and screening of 135 unrelated healthy individuals for this variant was negative. The substituted amino-acid is located within a region of the *SDHD*protein frequently altered due to germline mutations in paraganglioma (PGL) families (loss of Y93 \[[@B22]\] and two missense mutations, i.e. D92Y \[[@B19],[@B28],[@B46]\] and L95P \[[@B28]\]). These residues are part of the third transmembrane helix of the SDHD protein \[[@B54]\]. The second mutation has not been reported either. This mutation results from a 4 bp deletion in the 3\' exon-intron boundary of exon 3 resulting in skipping of exon 3 leading to a transcript with a premature stop codon. The predicted truncated protein has another carboxyterminal amino-acid sequence from H56 on and its normal function is assumed to be impaired as carboxyterminal amino-acids involved in ubiquinone and heme b binding are missing (H71, D82 and Y83) and consequently the structure of the transmembrane subunit and/or association of the catalytic domain subunits SDHA and SDHB to the membrane would be disrupted \[[@B54]\]. The functional consequence of one sequence variant located within an intronic sequence (IVS2+37ins(TCTA)) is more difficult to evaluate due to lack of fresh tumour material, and parental DNA. Further analysis is needed in order to reveal a possible effect on splicing or RNA stability. Finally, one new and 4 known polymorphisms were detected in 5 NB cell lines and 3 tumour samples. Additional screening for homozygous deletions in all cell lines and methylation in cell lines and tumours were negative. Based upon these results, we can exclude a role for *SDHD*as a classical tumour suppressor gene in NB. However, the finding of two apparently bona fide *SDHD*mutations in NB without allelic loss of distal 11q leaves the possibility open that the gene contributes to NB oncogenesis due to haplo-insufficiency, rather than functional inactivation of both alleles. In order to investigate this possibility, we decided to perform further studies at transcript and protein level. Interestingly, *SDHD*expression was shown to be consistently lower in cell lines with 11q allelic loss versus NB cell lines without loss and also significantly decreased in NB cell lines as compared to normal foetal adrenal neuroblast cells of 16, 18 and 19 weeks gestational time with a mean fold difference of 3.61 between neuroblast cells and NB cell lines. A similar correlation between 11q LOH and reduced *SDHD*expression was recently described in colorectal and gastric cancer \[[@B55]\]. Our findings at mRNA transcript level, however, did not match with results obtained from further analysis at protein level. Complex II activity and quantitative protein analysis revealed no significant difference between cell lines with or without 11q allelic loss or *SDHD*mutation. However, measurement of complex II activity might only reflect part of the functional properties of SDHD. Also, measurement of differences in protein quantity is far less sensitive than Q-PCR at transcript levels. Therefore, these observations at present do not fully exclude *SDHD*involvement in NB. Finally, we also looked at the morphologic characteristics of the mitochondria as a possible clue to SDHD dysfunction. In keeping with, at best, partial loss of function of *SDHD*, we did not observe similar gross morphologic changes as reported for PGL with *SDHD*mutations (swelling with loss of matrix density and generalized rarefaction of cristae), the latter being characterized by destabilization of complex II with loss of enzymatic activity \[[@B48]\]. However, most of the cell lines showed dilated mitochondrial cristae. It has been demonstrated that this is a reversible phenomenon, and parallels arise in intracellular ADP/ATP ratio or low energy state \[[@B56]\]. Subsequent combined ultrastructural and biochemical studies from several authors indicated that dilation of cristae follows a decrease in mitochondrial membrane potential that can be provoked by various experimental procedures \[[@B57],[@B58]\]. This configuration was detected in only a small percentage of mitochondria in archived sections of NB tumours and in sections published earlier. Morphologic analysis of mitochondria in NB thus far received little attention. The true significance of the observed mitochondrial morphological changes in NB is intriguing, but does not appear to be related to the mutations we have found. Conclusions =========== In contrast to previous findings in PGL and PC, this study excludes a classical two hit Knudson model for *SDHD*involvement in NB. However, the finding of, albeit rare, bona fide mutations and reduced expression of *SDHD*in NB with 11q allelic loss hints at a possible haplo-insufficient contribution to tumour development. A better understanding of the different functions of SDHD, in particular its possible contribution to energy independent apoptosis involving the release of cytochrome c and procaspases, will allow further functional assays to asses how this gene contributes to tumour development in general, and the high stage NB phenotype in particular \[[@B59],[@B60]\]. Evidence for contribution to a cancer phenotype through haplo-insufficiency has recently been obtained for a number of loci, including *CDKN1B*(p27^Kip1^) \[[@B61],[@B62]\], *TP53*(p53) \[[@B63]\], *DMP1*\[[@B64]\], *PTEN*\[[@B65]\], *APC*\[[@B66]\] and *NKX3.1*\[[@B67]\]. In mouse models for some of these genes, loss or mutation of one allele increased tumour susceptibility despite expression of the remaining wild-type allele \[[@B68]\]. Although the present data on protein and functional level do not provide consistent evidence for the haplo-insufficient involvement of *SDHD*in NB, a bipartite mechanism as tumour suppressor gene for the *SDHD*gene, as described for the *APC*gene can at present not be fully excluded. Following this hypothesis, germline mutations in *SDHD*would predispose to PGL or PC development. Rare somatic mutations and more typically loss of one allele could contribute to the metastasizing NB tumour phenotype (and possible also other tumour types), not as an initiating step but rather as later event in tumour development. However, further evidence is needed to support the haplo-insufficient involvement of *SDHD*in cancer. Ultimately, knockout mice for the *SDHD*gene leading to haplo-insufficiency for *SDHD*in neuroblast progenitor cells, would be the appropriate test to evaluate this hypothesis. List of abbreviations ===================== AIF = allelic imbalance factor CGH = comparative genomic hybridization DHPLC = denaturing high performance liquid chromatography LOH = loss of heterozygosity MGB = minor groove binder MSP = methylation specific PCR NB = neuroblastoma PC = pheochromocytoma PGL = paraganglioma ROS = reactive oxygen species *SDHD*= succinate dehydrogenase, subunit D SNP = single nucleotide polymorphism SRO = shortest region of overlap Competing interests =================== The authors declare that they have no competing interests. Author\'s contributions ======================= KDP carried out the genomic and transcriptomic studies, and drafted the manuscript. JH performed the methylation studies. JS carried out the immunoblottings and spectrophotometric analysis that was evaluated by RVC. AN performed the ultrastructural analysis that was screened and discussed by CV, FR and MP. GL and NVR collected the tumour material. JV and FS participated in the study\'s design and coordination. All authors have reviewed the manuscript and FS and ADP were the final editors of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/4/55/prepub> Acknowledgements ================ We would like to thank E. George for the spectrophotometrical assays, G. De Vos and P. Degraeve for the cell cultures and Petra Van Acker and Inge Vereecke for their help with the DHPLC analyses. This text presents research results of the Belgian program of Interuniversity Poles of attraction initiated by the Belgian State, Prime Minister\'s Office, Science Policy Programming. The scientific responsibility is assumed by the authors. This work was supported by BOF-grant 011F1200 and 011B4300, GOA-grant 12051203 and FWO-grant G.0028.00. Katleen De Preter is an aspirant with the Fund for Scientific Research, Flanders (FWO-Vlaanderen). JV is supported by a post-doctoral grant from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT). Nadine Van Roy is a postdoctoral researcher with the FWO. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Details of sequencing profiles: (A) Deletion of GGCA in cell line N206 causing skip of exon 3 and (B) Y93C mutation in cell line NMB; (C) RT-PCR (reverse transcriptase PCR) on cell lines grown with or without puromycine (T = treated, U = untreated) revealed a transcript variant in cell line N206, caused by the GGCA deletion (lane 1 and 2). :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *SDHD*mRNA levels in NB cell lines (light gray), neuroblast cells (gray) and human normal control samples (white). Significantly reduced *SDHD*mRNA expression levels in NB cell lines with 11q23 allelic loss compared to 11q23 intact NB cell lines (P = 5.31E-06) and normal tissue samples (P = 1.49E-05). :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Mitochondrial ultrastructure shows heterogeneity between cell lines (same final magnification for the 4 images, marker = 0.5 μm): (A) NB cell line N206: dilated crista spaces in small mitochondria with a dense matrix; (B) NB cell line NMB: small mitochondria with narrow cristae and light matrix, so-called orthodox configuration, (C) NB cell line SJNB-8: unusually large mitochondria in orthodox configuration (narrow cristae), some areas in the matrix are cleared and lack cristae; (D) NB cell line LA-N-2: very large mitochondria with dilated cristae and dense matrix. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Primer sequences used for homozygous exon deletion screening with real-time quantitative PCR ::: ***amplicon*** ***forward*** ***reverse*** ------------------ -------------------------------- ---------------------------- ***SDHD*exon 1** 5\'AGGAACGAGATGGCGGTTCT3\' 5\'TCCTAGGGCACCGCAAAC3\' ***SDHD*exon 2** 5\'CCAGTGGTCAGACCTGCTCAT3\' 5\'TGCTGCACTCCACACCATTC3\' ***SDHD*exon 3** 5\'GACTAGCGAGAGGGTTGTCAGTGT3\' 5\'CATCGCAGAGCAAGGATTCA3\' ***SDHD*exon 4** 5\'TGGCACTTTCAGCTTTAACCTTT3\' 5\'CACAGCATGGCAACAGCTTT3\' ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Primer sequences used for denaturing high performance liquid chromatography (DHPLC) and sequencing ::: ***amplicon*** ***forward*** ***reverse*** ---------------------------------------------------------- ----------------------------------- ------------------------------------ ***SDHD*exon 1** 5\'GCACCGCCTCTCGACTTC3\' 5\'TGCTGTGATTTCGGTATTTTCTTC3\' ***SDHD*exon 2** 5\'AACCCCAGTGAAATAGATGCTATCTTC3\' 5\'AGTCCTGCTAAAGGCATGACCATTA3\' ***SDHD*exon 3** 5\'CACTGCCTGTCAGTTTGGGTTAC3\' 5\'GGGCATTTCAATCAACTTCTCCC3\' ***SDHD*exon 4** 5\'TCCCCTAAAGAAGCAAACAGTGAC3\' 5\'GAGCTTAATGGCATGACAAAGCAG3\' ***SDHD exon 4 Noncoding region (only for sequencing)*** 5\'GTGGTTTTTTATTGATGTTATGATTTT3\' 5\'AATCTCAATTTACAGTTGGTAGTATTTT3\' ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### \(A) 11q23 LOH data of NB patients (35 patients form the Molecular Oncology Unit (Lyon, France) (F-samples) and 32 patients from the Ghent University Hospital (Ghent, Belgium) (G-samples)). Based on the allelic imbalance factor (AIF) of 6 markers (4 on 11q23 and 2 on 11p) normal 11q23 status was distinguished from unbalanced 11q LOH and loss of both 11q and 11p (here indicated as whole chromosome loss) (- = no data available) and (B) 11q23 status in NB cell lines based on FISH, LOH, karyotypes, CGH and/or M-FISH ::: **A** ------------------ ---------------------- ---------------- ------------ --- --------- ---------------------- ----- ----- ---- **F1** **normal** no no 3 **G1** **normal** no no 4 **F2** **normal** no no 3 **G2** **whole chr11 loss** no no 4 **F3** **normal** no no 3 **G3** **normal** no no 1 **F4** **normal** no no 4 **G4** **normal** no no 1 **F5** **whole chr11 loss** no no 4 **G5** **normal** no no 2 **F6** **unb \[11q\]LOH** no no 4 **G6** **normal** \- no 3 **F7** **normal** no no 4 **G7** **normal** \- \- 4 **F8** **normal** no no 3 **G8** **normal** no no 1 **F9** **normal** no no 4 **G9** **whole chr11 loss** no no 4 **F10** **whole chr11 loss** no no 3 **G10** **normal** no no 4 **F11** **normal** no no 4 **G11** **normal** no no 1 **F12** **normal** no no 4 **G12** **normal** no no 4 **F13** **normal** no no 3 **G13** **normal** no yes 1 **F14** **whole chr11 loss** no no 3 **G14** **whole chr11 loss** no no 3 **F15** **whole chr11 loss** no yes 4 **G15** **normal** no no 3 **F16** **normal** no no 3 **G16** **normal** \- no 4 **F17** **unb \[11q\]LOH** no no 4 **G17** **normal** no no 3 **F18** **unb \[11q\]LOH** no yes 4 **G18** **normal** no yes 1 **F19** **normal** no no 4 **G19** **normal** no no 4S **F20** **normal** no no 3 **G20** **normal** yes yes 3 **F21** **unb \[11q\]LOH** no no 4 **G21** **unb \[11q\]LOH** no no 3 **F22** **whole chr11 loss** no no 3 **G22** **unb \[11q\]LOH** yes yes 4 **F23** **normal** no no 3 **G23** **normal** no no 4 **F24** **normal** no no 3 **G24** **normal** yes yes 4 **F25** **whole chr11 loss** no no 3 **G25** **normal** no no 4 **F26** **unb \[11q\]LOH** no no 4 **G26** **normal** no yes 4 **F27** **whole chr11 loss** no no 3 **G27** **normal** no no 3 **F28** **normal** no no 3 **G28** **normal** \- no 2 **F29** **unb \[11q\]LOH** no no 4 **G29** **normal** no no 3 **F30** **normal** no yes 4 **G30** **normal** no no 1 **F31** **normal** no no 4 **G31** **normal** \- no 3 **F32** **normal** no no 4 **G32** **normal** no no 4S **F33** **whole chr11 loss** no no 3 **F34** **normal** no yes 4 **F35** **unb \[11q\]LOH** no no 4 **B** **NB cell line** **11q23 status** ***MYCN*ampl** **1p del** **CHP-134** **normal** yes yes **CHP-901** **normal** yes yes **CHP-902R** **normal** yes yes **CLB-GA** **deletion** no no **GI-M-EN** **deletion** no yes **IMR-32** **deletion** yes yes **LA-N-1** **normal** yes yes **LA-N-2** **normal** yes no **LA-N-5** **normal** yes yes **LA-N-6** **deletion** no yes **N206** **normal** yes yes **NBL-S** **deletion** no no **NGP** **deletion** yes yes **NLF** **normal** yes yes **NMB** **normal** yes yes **SJNB-12** **normal** no yes **SJNB-1** **normal** no yes **SJNB-10** **normal** yes yes **SJNB-6** **normal** yes yes **SJNB-8** **normal** yes yes **SK-N-AS** **deletion** no yes **SK-N-BE** **normal** yes yes **SK-N-FI** **normal** no no **SK-N-SH** **normal** no no **SMS-KAN** **normal** yes yes **SMS-KCNR** **normal** yes yes **STA-NB-10** **normal** yes yes **STA-NB-3** **normal** yes yes **STA-NB-8** **normal** yes yes **TR-14** **normal** yes yes **UHG-NP** **normal** yes yes ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### *SDHD*base pair variants found in NB tumour samples and cell lines, the position of the variant, the change in the protein caused by the variant and the conclusion (mutation or polymorphism); also listed for each tumour and cell line are the genomic status for chromosome arm 11q (n.d. = not done) (for cell lines: chromosome 11 centromere copy number versus 11q23 copy number according to FISH, for tumours: normal, unbalanced LOH (unb \[11q\]LOH) or whole chromosome 11 loss according to microsatellite marker analysis), *MYCN*status (normal or amplified), 1p status and tumour stage when available (- = not available). ::: **NB tumour case number / NB cell line** **11q23 status by microsatellite marker analysis** 11q23 status by FISH ***SDHD*base pair variant** **exon** **change in protein** **mutation (M) or polymorphism (P)** **variant present in constitutional (C)/ tumour (T)** ***MYCN*** **1p del** **stage** ------------------------------------------ ---------------------------------------------------- ------------------------------------------------------------- ----------------------------- ------------------ ----------------------------------- -------------------------------------- ------------------------------------------------------- ------------ ------------ ----------- **F11** **normal** n.d. g.6911ins(TCTA) IVS2+37ins(TCTA) ? (see text) ? (see text) C+T normal no 4 **F18** **unb \[11q\] LOH** n.d. g.5842 G\>A 1 G12S P C+T normal yes 4 g.7750 A\>G IVS3-29 A\>G P C+T g.7802 C\>T 3 S68S P C+T g.13678 T\>C IVS4-32 T\>C P C+T **F22** **whole chr11 loss** n.d. g.7750 A\>G IVS3-29 A\>G P C normal no 3 g.7802 C\>T 3 S68S P C g.13678 T\>C IVS4-32 T\>C P C **F35** **unb \[11q\] LOH** n.d. g.7750 A\>G IVS3-29 A\>G P C+T g.7802 C\>T 3 S68S P C+T g.13678 T\>C IVS4-32 T\>C P C+T **LA-N-2** n.d. **2/2heterozygous** g.6854 A\>G 2 H50R P \- amplified no 4 **N206** n.d. **2/2 heterozygous** g.79124del(GGCA) 3 exon 3 skip, premature stop codon M \- amplified yes 4 **NGP** n.d. **2/1 hemizygous** g.7750 A\>G IVS3-29 A\>G P \- amplified yes \- g.7802 C\>T 3 S68S P g.13678 T\>C IVS4-32 T\>C P **NMB** n.d. **4/3 allelic imbalance/ heterozygous (based on sequence)** g.7750 A\>G IVS3-29 A\>G P \- amplified yes 4 g.7802 C\>T 3 S68S P g.7876 A\>G 3 Y93C M g.13678 T\>C IVS4-32 T\>C P **SK-N-FI** n.d. **2/2 heterozygous** g.7750 A\>G IVS3-29 A\>G P \- no no \- g.7802 C\>T 3 S68S P g.13678 T\>C IVS4-32 T\>C P ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Ultrastructural analysis of mitochondria ::: **dilation of cristae\*** **density of matrix°** **configuration\#** **matrix granules°°** **mean projected length (μm)** **max projected length (μm)** --------- --------------------------- ------------------------ --------------------- ----------------------- -------------------------------- ------------------------------- CHP-901 \+ -+ \+ \+ 0,65 1,31 CLB-GA ++ \+ \+ 0,69 1,92 IMR-32 \+ \+ \+ \- 0,69 1,27 LA-N-2 ++ \+ \- 0,82 2,18 MCF-7 \- \+ \+ \- 1,06 3,11 N-206 +++ \+ \- 0,84 1,65 NGP +++ \+ \- 0,84 1,74 NMB \- \- \+ \- 0,68 1,26 SJNB-10 +++ \+ \- 0,63 1,69 SJNB-12 \+ \+ \- 0,47 1,23 SJNB-8 \- -+ \+ \- 1,02 3,40 SK-N-AS ++ \+ \- 0,66 1,47 SK-N-FI \+ \- \+ \- 0,65 1,32 SK-N-MC \- \- \+ \- 0,90 2,03 SK-N-SH \+ -+ \+ \+ 0,56 1,31 \*: +cristae of some mitochondria dilated; ++cristae of many mitochondria dilated; +++all mitochondria have dilated cristae; °: +matrix of most mitochondria is more electron dense than the cytosol; -+part of the mitochondria are dense, but others have a light matrix; -mitochondrial matrix is lighter than or equal to cytosol; \#: +mitochondria with an orthodox configuration are present; in the same cell or culture other mitochondria may have dilated cristae and a dense matrix; °°: -few or no matrix granules; +matrix granules visible in many or most mitochondria, normal image; :::

PubMed Central

2024-06-05T03:55:47.661557

2004-8-24

PMC517502

Background ========== Osteoporosis is a systemic skeletal disorder, characterized by reduction of bone mass, deterioration of bone structure, increasing bone fragility, and increasing fracture risk \[[@B1]-[@B4]\]. It is more frequent among women than among men \[[@B5],[@B6]\]. The development of low bone mass is typically asymptomatic, with many women reporting clinical manifestations including acute back pain, limited back mobility, fragility fractures (hip, vertebrae, proximal femur, distal radius, humerus, tibia), compression of midthoracic vertebrae and upper lumbar vertebrae, progressive deformation of the spinal column (cyphosis), reduced height, and radiculopathies \[[@B2],[@B4]-[@B6],[@B8],[@B9]\]. Several risk factors for osteoporosis have been identified, these include female sex; Caucasian or Asiatic race; advancing age; family history of osteoporosis or fragility fractures; a low body mass index; menopause before age 45 years; prolonged amenorrhea unrelated to menopause; nulliparity; prolonged lactation; diet low in calcium and vitamin D; poor intestinal absorption of calcium; lactose intolerance; excessive caffeine or alcohol consumption; smoking; sedentary lifestyle; and prolonged treatment with thyroid hormones, glucocorticoids (e.g. cortisone), anticonvulsants, aluminum antiacids, and use of anticoagulants \[[@B1],[@B3]-[@B6],[@B8]\]. Approximately 20% of bone mass is genetically determined; however, the risk of osteoporosis can be reduced by optimizing bone mass increasing during youth, conserving bone mass during adulthood, and minimizing bone mass loss during advancing age \[[@B5]-[@B7]\]. Among most important preventive habits are a) weight-bearing exercise (e.g. going up and down stairs, jogging, aerobics, swimming, and isometrics; at least 30 minutes daily), b) diet or supplements containing adequate levels of calcium and vitamin D, and c) absence or cessation of smoking and no greater than moderate alcohol and/or caffeine consumption \[[@B5]-[@B10]\]. A study on US women aged over 25 years found that knowledge about osteoporosis was limited, irrespective of age \[[@B11]\]. Calcium intake was sufficient in most cases, but the amount and type of physical activity was inadequate to achieve enhanced bone mass; most women in this study performed some physical activity in the course of paid work or housework, but did not admit to systematic daily exercise. Other study on Caucasian and African-American women found that most of them had heard about osteoporosis, but few women got both adequate exercise and the recommended intake of calcium per day \[[@B12]\]. Asian women in Australia also had lower calcium intake (\< 800 mg/day) and their knowledge about osteoporosis was limited \[[@B13]\]. There have been three related studies reported in Hispanic women: a study performed in Mexico \[[@B14]\], and two studies on women of Hispanic origin in the US \[[@B15],[@B16]\]. The Mexican study considered women aged 50 -- 59 years, and focused on knowledge about menopause and risk associated with premature menopause. About 90% of subjects were aware of the relationship between menopause and osteoporosis, but most subjects had little knowledge of other risk factors, and incorporated life habits that clearly increased osteoporosis risk. The studies on women of Hispanic origin in the US have yielded somewhat contradictory results. The first of these found that more than 37% of women had adequate preventive habits including the taking of calcium supplements and the performance of regular exercise; much of this was attributed to prior health education, knowledge about osteoporosis, bone-mass evaluation offered by healthcare services, and medical advice \[[@B15]\]. The second study considered both Hispanic and African-American women, and found that most women in both groups had a poor knowledge of behaviors that promote and maintain bone mass \[[@B16]\]. Notably, less than 50% of women performed regular physical exercise, and less than 10% had adequate calcium intake. At present, the Salvadorean Public Health and Social Assistance Ministry does not maintain a specific record of this disease in adults, and most cases are likely classified as dorsalgy, that includes several musculoskeletal diseases such as radiculopathies, cervicalgy, lumbalgy, sciatica, spinal derived pain, and unspecified back pain \[[@B17]\]. This reflects the fact that osteoporosis may be reported as dorsalgy. According to this report from El Salvador, in 2001 dorsalgy was the seventh most frequent cause of morbidity in women aged 50 -- 59 years attending outpatient clinics, with an incidence of 3,983 new cases per 100,000 inhabitants and a total of 8,989 consultations (first-time or subsequent); in women aged 60 years or more, there were 9,754 consultations for dorsalgy in 2001. Bearing in mind the lack of reliable epidemiological data, the present study wished to investigate osteoporosis-related life habits (including exercise and calcium intake) and knowledge about osteoporosis among Salvadorean women aged ≥ 25 years. We investigated possible relationships of these variables with age, educational level, household income, family history of osteoporosis, menopause before age 45 years, fecundity (children per woman), lactose intolerance, caffeine consumption, low in calcium diet, and use of aluminum antiacids. Methods ======= Study design and sampling ------------------------- This was an exploratory study with a cross-sectional design performed between May and September 2003, and it was used to survey a convenience sample comprised of 197 women (73 aged 25 -- 35 years, 74 aged 36 -- 49 years, and 50 aged over 49 years) from urban areas within 6 main municipalities in El Salvador. All subjects were randomly sampled and recruited by personal contact through to visit homes, churches, schools, primary healthcare centers, hospitals, supermarkets, shopping centers, and parks. It was not necessary to obtain any proper informed consent from interviewed women. The participation rate of women was 87.5% (197 out of 225). We selected three age groups of women attending following criterions: women aged 25 -- 35 years are at the period when higher peak of bone mass is reached \[[@B3]\]; women aged 36 -- 49 years are at a period around menopause when bones undergo a slow mineral density loss \[[@B3]\]; finally, most women aged over 49 years have undergone menopause, when osteoporosis clinical manifestations may begin to show \[[@B3]\]. Data collection and instruments ------------------------------- Data were obtained at the time of interview, which were performed by a member of the research team. Prior training in interview techniques was obtained for study. Each staff interviewer subsequently participated in several practice sessions, with an evaluation component to confirm accurate transcription of responses, and comparison and adjustment to ensure good inter-interviewer concordance. The data collection instrument incorporated a personal interview guide comprising five sections, validated through a prior pilot study performed in 12 women (4 in each age group). Among the demographic information was household income. It was classified by the criteria of the Salvadorean Economy Ministry as \"below poverty line\" if monthly income was below the cost of two basic shopping baskets (i.e. \< \$254), and otherwise as \"above poverty line\" \[[@B18]\]. The second survey section generated information on family history of osteoporosis, and asked about the use of aluminum antiacids. Section 3 addressed weight-bearing physical activity (e.g. walking, standing, climbing and descending stairs) both at place of employment and at home, and included questions on weight-bearing exercises including jogging, swimming, aerobics, and isometrics. This survey section was adopted from a previous study \[[@B11]\]. A test/ re-test procedure was administered a week apart on pilot study subjects to calculate reliability of next questionnaire sections. Test/re-test correlation was 0.67 for physical activity section. Section 4 produced information on diet: for each of a list of 31 dietary and non-dietary items (including dairy products, baked products, meat, vegetables, fruits, and calcium supplements), the subjects were asked to estimate frequency of consumption (daily, weekly, fortnightly, or monthly). Similar checklists have been used in related previous studies \[[@B19]\]. Given the high coffee consumption in Central America, the subjects were asked about their degree of coffee consumption. Test/re-test correlation was 0.72 for dietary and non dietary calcium intake section. Final section (5) comprised eight open questions designed to assess nine knowledge dimensions about osteoporosis in subjects. The nine knowledge dimensions regarding osteoporosis, their specific questions and corresponding scores (from 0 to 42) are shown in Table [1](#T1){ref-type="table"}. These questions served as a backbone for the interview and they were similar to other set used in a previous study \[[@B11]\]. Test/re-test correlation was 0.59 for osteoporosis knowledge questionnaire section. Data analysis ------------- Each subject\'s responses to the questions in the fifth section were analyzed by the principal researcher, who compared the answers with a semantic map (Fig. [1](#F1){ref-type="fig"}) developed by the authors on the basis of findings from previous studies on osteoporosis \[[@B1],[@B2],[@B5]-[@B7]\]. This approach allowed for quantification on knowledge about osteoporosis so: 5 points assigned for responses denoting knowledge of three or more concepts (each node in semantic map represents a concept), 3 points for responses denoting knowledge of 2 concepts, and 2 points for responses denoting knowledge about one out of osteoporosis-related concepts as reported previously \[[@B11]\]; except for osteoporosis information source dimension since its sub-score depended on number of information sources that women were able to mention, so that they only got one point per each source up to a maximum of 5 points. The total score achievable was 42 points. The total number of hours of weight-bearing physical activity was estimated by adding the subtotals for activities at home, activities at worksite, and that of weight-bearing exercises. Total calcium intake was estimated by adding the estimated subtotals for consumed dairy products, other dietary intake, and calcium supplements, including calcium content per portion × the number of portions per day. Obtained values were compared with reference values \[[@B20]\]. Data are cited in the text as means ± standard errors. For variables with homogeneous variance, means were compared by two-way analysis of variance and by Tukey tests for pairwise comparisons. For variables with non-homogenous variance, data were compared by the Kruskal-Wallis test (ANOVA on ranks) followed by Dunn\'s test for pairwise comparisons. These analyses were performed using SigmaStat version 2.03. Correlation analysis was used to determine relationships between knowledge scores and WBPA including exercise and calcium intake. To develop predictive models, we used a multivariate analysis based on multiple linear regression as contained in the program AMOS version 5 (Small Waters Corp.). Results ======= Characteristics of the interviewed women ---------------------------------------- The participant characteristics of each age group are shown in Table [2](#T2){ref-type="table"}. Frequency of women with none or only a primary education increased with age; conversely, frequency of women with secondary or higher education decreased with age. Household income reported that most women (\> 61%) were below the poverty line; although the number of women living on poverty conditions fluctuated with age. The frequency of nulliparity among interviewed women declined with age, whereas both the frequency of parity among interviewed women and the fecundity rate (children per woman) increased with age as anticipated. In all age groups, more than 44% of interviewed women reported a family history of osteoporosis, which included extended family: grandmothers, mothers, aunts, elder sisters, and cousins. Median, mean (SEM), and range of age of menopause in interviewed women were: 44, 42.3 (± 0.8), and 27--53 years, respectively. About 20% of interviewed women had lactose intolerance or ingested aluminum antiacids, both of these characteristics were unrelated of age. In addition, most women (\> 69%) were coffee-consumers and the consumption of this drink increased with age. Knowledge about osteoporosis ---------------------------- The sub-scores per knowledge dimensions about osteoporosis and age groups are shown in Table [3](#T3){ref-type="table"}. The total scores of knowledge regarding osteoporosis were similar in the three age groups of women (median 14 in 25- to- 35-years; 17 in 36- to 49-years; 15 in over 49 years). Most of interviewed women (75%) had enough knowledge about osteoporosis regarding the concept of disorder and its risk factors, sex-related factor, and prevention behaviours, irrespective of age (Table [3](#T3){ref-type="table"}). Conversely, these women got less knowledge scores for diagnosis and treatment of osteoporosis than other examined dimensions. In all three age groups, women with secondary or higher education obtained significantly higher total knowledge scores (*F*= 22.46, *p*\< 0.001) than women with lower educational level (Fig. [2](#F2){ref-type="fig"}). There was not a significant relationship between age and educational level (*F*= 1.38, *p*= 0.223). Only 5% of the women with higher education obtained total scores of less than 12 points, and 75% of them obtained total scores of more than 25 (table [3](#T3){ref-type="table"}). Multivariate analysis (factors age, educational level, family history of osteoporosis, household income, early menopause, fecundity) explained only about 37% of variance in total score (R^2^= 0.367, p \< 0.001) (Fig. [3](#F3){ref-type="fig"}). The most meaningful predictors of total score were educational level (R = 0.47, p \< 0.001), household income (R = 0.18, p = 0.015), and early menopause (R = 0.18, p = 0.003). Physical activities ------------------- The amount of weight-bearing physical activity was similar in the three age groups (mean 8.7 ± 0.5 hours/day in women 25- to 35 years; 8.9 ± 0.5 hours/day in those 36- to 49 years; 8.3 ± 0.6 hours/day in those over 49 years). Analysis of variance with factors of age and family history of osteoporosis indicated that neither factor produced a significant effect (F = 0.23, p = 0.794; F = 0.04, p = 0.852), nor the interaction between them was significant. The amount of weight-bearing physical activity reported at the worksite was markedly and significantly lower in over 49 years than in the other two age groups (H = 33.15, *p*\< 0.001; Fig. [4](#F4){ref-type="fig"}). Conversely, physical activity at home was higher in women over-49 years than reported in the other two age groups (H = 19.34, p = 0.002; Fig. [5](#F5){ref-type="fig"}). The amount of weight-bearing exercise was low in all three groups, with means about 2 hours per week in the 25- to 35-years, and less than 1 hour per week in the older age groups (Fig. [6](#F6){ref-type="fig"}). About 75% of 25- to 35 years appear to do less than 2.7 hours of weight-bearing exercises per week; while about 75% of women in the older age groups reported doing no exercises (Fig. [6](#F6){ref-type="fig"}). However, the total duration of exercise did not vary significantly among the three age groups, or between women with or without a family history of osteoporosis (*H*= 10.25, *p*= 0.069). Multivariate analysis employing dependent variable of amount of total physical activity and candidate predictors age, educational level, household income, family history of osteoporosis, early menopause and fecundity, appeared to explain only 5% of total variance (R^2^= 0.048, p = 0.038). Use of dependent variable of physical activity at worksite explained about 26% of variance (R^2^= 0.264, p \< 0.001; Fig. [7](#F7){ref-type="fig"}); the most effective predictors were educational level (R = 0.361, p \< 0.01) and age (R = -0.234, p = 0.002). Similarly, use of dependent variable of physical activity at home explained about 27% of variance (R^2^= 0.273, p \< 0.001; Fig. [8](#F8){ref-type="fig"}); again the most effective predictor was age (R = -0.471, p \< 0.001). Use of dependent variable of exercise activity explained only 13% of variance (R^2^= 0.128, p \< 0.001); the most effective predictor was family history of osteoporosis (R = 0.169, p = 0.011). Total osteoporosis knowledge score was not predicted on the amount of weight-bearing exercise (R = 0.081, p = 0.218). Although, total osteoporosis knowledge scores were significantly associated with WBPA at worksite (R^2^= 0.07, p \< 0.001), at home (R^2^= 0.05, p = 0.002) or during exercise (R^2^= 0.04, p = 0.004), squared correlations were rather low in all of cases. Dietary calcium intake ---------------------- Figure [9](#F9){ref-type="fig"} summarizes the data on total calcium intake (mg/day) in the three age groups, subdivided into women above and below the poverty line. Independently of income, about 75% of women aged less than 49 years ingest less than 600 mg/day of calcium (i.e. only about 60% of the recommended daily intake, 1000 mg/day). In the over-49 age group (recommended daily intake 1200 mg/day), there is a marked difference in calcium intake between women above and below the poverty line; with women below the poverty line typically showing very low calcium intake (over 75% ingest less than 600 mg/day), whereas calcium intake in women above the poverty line is higher (though nevertheless lower than the recommended daily intake in over 75% of subjects). Calcium intake was estimated through the use of dairy products, with 75% of women ingesting less than 410 mg/day, in all three age groups below the poverty line, and in the two younger age groups above the poverty line. In the over-49 above-poverty-line group, 75% of women ingest less than 775 mg/day. Dairy products contributed on average 57% of total calcium intake in women below the poverty line, versus 68% of total calcium intake in women above the poverty line. Women in the over-49 above-poverty-line group ingested significantly more total calcium (H = 18.36, p = 0.003) and significantly more dairy calcium (H = 18.97, p = 0.002) than women in all other age/income groups (Figs. [10](#F10){ref-type="fig"}, [11](#F11){ref-type="fig"}). Supplementary calcium intake did not vary significantly among the age/income groups (H = 9.04, p = 0.108), neither was there any significant interaction between these factors. Multivariate analysis with dependent variable of total calcium intake and candidate predictors age, educational level, household income, lactose intolerance, coffee consumption and use of aluminum antiacids, provided a model that explained only 19% of total variance (R^2^= 0.191, p \< 0.001) (Fig. [12](#F12){ref-type="fig"}). With the addition of dairy calcium intake, the model explained 20% of total variance (R^2^= 0.204, p \< 0.001) (Fig. [13](#F13){ref-type="fig"}). In both cases, the most effective predictors were lactose intolerance (total calcium intake R = -0.239, p \< 0.001; dairy calcium intake R = -0.272, p \< 0.001), household income (R = 0.229, p = 0.003; R = 0.232, p = 0.002), age (R = 0.191, p = 0.005; R = 0.198, p = 0.004), and coffee consumption (R = -0.146, p = 0.025; R = -0.141, p = 0.029). Women who had moderate osteoporosis knowledge had an increase in their intake of calcium that was significant (R = 0.142, p = 0.045). Although total osteoporosis knowledge scores were significantly associated with total calcium intake (R^2^= 0.06, p \< 0.001) or dairy calcium intake (R^2^= 0.04, p = 0.008) or non-dairy calcium intake (R^2^= 0.03, p = 0.016) or calcium supplements (R^2^= 0.07, p \< 0.001), squared correlations were rather low in all cases. With the addition of calcium supplement intake only 12% of total variance was accounted for (R^2^= 0.119, p = 0.003). The greatest predictors were a woman\'s use of aluminum antiacids (R = 0.232, p \< 0.001) and her educational level (R = 0.207, p = 0.004). Discussion ========== The total scores regarding nine knowledge osteoporosis dimensions were similar in all age groups of interviewed women (range of median 14 -- 17, average 12.1 -- 14.8) out of a possible 42 points. A similar study found Taiwanese women got a mean score of 15 out of 44 points related to six osteoporosis knowledge dimensions \[[@B21]\], whereas surveyed American women of three age groups got averaged knowledge scores from 32 to 44 points out of 183 \[[@B11]\]. In all cited cases, the obtained scores indicate that knowledge about osteoporosis is poor or limited among surveyed subjects so health educational programs and health services regarding osteoporosis are necessary for Salvadorean women, as it is also suggested for Taiwanese \[[@B21]\] and American women \[[@B11]\] of all ages. The present results also indicate that Salvadorean women with secondary or higher education have significantly better knowledge of osteoporosis than women with a low educational level, regardless of age. Similarly, other study found that better educated Chinese women in Singapore seem to know more about osteoporosis than those ones worst educated \[[@B22]\]. A previous study has likewise found that osteoporosis-related knowledge is independent of age \[[@B11]\]. Our multivariate analyses indicated that the most effective predictors of osteoporosis-related knowledge were educational level, household income, and early menopause. This latter factor perhaps affects osteoporosis-related knowledge through to give brochures and magazines to women at shopping centers, supermarkets, physician\'s clinics, schools, and colleges by non governmental organizations such as Salvadorean Demographical Association, dairy good producers such as New Zealand Dairy Board and Australian Milk Products, and some pharmaceutical laboratories, especially those produce calcium supplements. As well as information presented on television and in the press. Besides, short counselling sessions about preventive aspects of osteoporosis are given by some physician\'s private clinics, because there is not a settled public health education program about osteoporosis in El Salvador. This country is not only case in Latin America, since Mexican women got more osteoporosis information from mass communication media than health education activities of public institutions \[[@B14]\]. However, total knowledge about osteoporosis may not lead to an improvement in health lifestyle; it is necessary to know more about some specific aspects as osteoporosis risk factors and to acquire healthy habits to reduce the risk for low bone mass. In multivariate analyses, knowledge about osteoporosis was not a significant predictor of either amount of physical activity or total calcium intake. Previous studies likewise found that knowledge of osteoporosis does not correlate with risk-reducing life habits \[[@B11],[@B12]\]. These authors suggested that osteoporosis-related knowledge among women of their sample was limited, the information was not fully understood or poorly internalized. They also suggested that it is unlikely that this type of knowledge will provide a basis for decisions about life style or unhealthy habits \[[@B11]\]. These comments are perhaps similarly applicable to the subjects in our study. Other previous studies have obtained results consistent with this view that osteoporosis-related knowledge is often poorly integrated and internalized, and does not lead to improved health behaviours \[[@B12],[@B16],[@B23],[@B24]\]. Our data on physical activity in the three age groups indicate that the amount of physical activity engaged in paid work currently declines with increasing age, whereas the amount of physical activity during housework appears to increase with increasing age. This trend was confirmed by multivariate analysis, which indicates that the most effective predictors of amount of physical activity at worksite or at home were age and educational level. Educational level attained likely increases the likelihood of obtaining paid work. However, recreational activities and other health-preventive behaviours such as to do isometric exercises may also determine total physical activity. Considering all subjects together, most reported physical activity (96%) was performed in the course of daily housework, walking to work or shops, or standing at work or at home; only 4% was directly applicable to weight-bearing exercise. About 50% of women in the youngest age group did more than 2 hours of physical activity per week, but the amount of physical activity in the older women was markedly lower, and even in the youngest age group very few women performed the recommended daily minimum of 30 minutes of exercise per day \[[@B5],[@B6]\]. Similar results were obtained in a previous study, which found that fewer than 50% of African-American and Hispanic women interviewed in the US do 60 minutes of physical exercise per week \[[@B16]\]. Very few of the women interviewed for the present study employ any of the weight-bearing physical activities that are known to be especially effective for increasing bone mass and thus perhaps reducing the risk of osteoporosis, such as isometrics \[[@B25]\]. Similar findings have been obtained in studies of Caucasian women in the US \[[@B11],[@B12]\] and of African-American and Hispanic women residing in the US \[[@B12],[@B16]\]. Independent of age and income, most women interviewed (50 -- 75%) have a daily calcium intake of less than 60% of the recommended level, thus increasing the risk of osteoporosis. Similarly, in another studies of Caucasian and African-American and Hispanic women in the US \[[@B12],[@B16]\], and Asian and Caucasian women in Australia \[[@B13]\], most of these women did not fulfil the suggested calcium intake. By contrast, in other recent study of Caucasian women in the US \[[@B11]\], only about 20% of women ingested less than 60% of the recommended daily calcium. The difference between our results in El Salvador and those obtained in the study of Caucasian women in the US \[[@B11]\] may, at least in part, reflect differences in standard of living between these two countries. Specifically, the generally higher incomes in the US may be associated with healthier diet, higher educational level, better access to healthcare, and better public education about general health and the prevention of diseases like osteoporosis. Additionally, calcium fortified foods are more readily available in the US. The average proportion of calcium intake as dairy products was 57% in women below the poverty line and 68% in women above the poverty line; both values are close to those reported for Caucasian women in the US \[[@B11]\]. These authors did not detect any significant variation with income, but note that their sample was smaller (n = 75) and more homogeneous (in terms of educational level and household income) than the sample of our study. The relationships between household income and calcium intake observed in our multivariate analyses likewise indicate that calcium intake varies with income. The other important predictor of calcium intake in this analysis was lactose intolerance, reported by 19% of the women interviewed; dairy calcium intake was likely lower in these women. As lightly lower prevalence of lactose intolerance (16%) was reported in the study of Caucasian women in the US \[[@B11]\]. The consumption of caffeine-containing drinks, especially coffee, has been shown to influence calcium intake, since these beverages often replace milk and milk-based beverages due to dairy products are relatively expensive in El Salvador and many Salvadorean families (42.9%) have a low purchasing power, and live below the poverty line \[[@B26]\]. Excessive caffeine ingestion is also reported to accelerate bone resorption \[[@B5],[@B6]\], and prevents intestinal calcium absorption \[[@B9]\]. Calcium intake also varied with age, being significantly less among younger women. Similarly, most of interviewed Caucasian and African-American young adult women in the US did not get recommended calcium intake per day \[[@B12]\]. This may be because younger women tend to perceive dairy products as having a high content in animal fats, and thus tend to reject them, as it has been suggested by previous authors \[[@B19]\]. Auld *et al*. also suggested that Hispanic women (particularly adolescents and young women) in general tend to reject dairy products more than Anglosaxon and Asiatic women. The key practical implications of this study relate to suggest set up of primary healthcare programs regarding osteoporosis for Salvadorean women as it has also suggested for Taiwanese women \[[@B21]\], and Hispanic and African-American women \[[@B16]\], as well as training of healthcare professionals, which in light of the present study need to pay special attention to the following aspects of osteoporosis prevention: a\) The type and frequency of physical exercise. b\) Diet-related risk factors, including inadequate intake of calcium, vitamin D, and phosphorus; also the adverse effects of drinking coffee or other low-calcium drinks instead of milk or milk-based drinks. c\) The importance of monitoring menstruation frequency, since normal circulating estrogen levels and normal menstrual cycles are important for maintaining normal bone metabolism. In addition, it is clearly important to set up health education programs to focus on integration and internalization of knowledge about osteoporosis. Although our results indicate that many women have modest knowledge about osteoporosis concerning risk factors and preventive behaviours, this knowledge often does not translate to appropriate changes in healthy life habits as it is shown through a weak association between total osteoporosis scores and exercise or calcium intake, so that osteoporosis knowledge is not well internalized among interviewed Salvadorean women. The lack of time to practice most adequate osteoporosis preventive exercise may accounts for the weak association between foregoing variables since majority of interviewed women perform WBPA (\> 8 hours/day), but as labour activities or housework. The low calcium intake among subjects may be explained through they have a low purchasing power and do not have the habit to ingest enough dairy products, the main diet calcium source \[[@B9]\]. Non-dairy calcium intake including supplements was also low (\< 370 mg/day), so that they did not fulfil the recommended daily calcium intake from this source either. Finally, we would suggest that osteoporosis education campaigns should be directed at families as well as individuals, with the aim of fomenting within-family education of children about the importance of calcium in the diet, whether as dairy products or in other calcium-rich products such soya milk and oat-based drinks and desserts. Conclusions =========== It showed that Salvadorean women with secondary or higher education have significantly better knowledge about osteoporosis than women with lower educational level, regardless of age. However, this knowledge does not appear to lead to improved life style or preventive habits of osteoporosis. The amount of weight bearing physical activity (mainly walking or standing) during paid work or housework was high in the three age groups of interviewed women (over 8 hours/day). However, very few of the women interviewed for this study did not perform any of the weight-bearing physical activities that are known to be especially effective for increasing bone mass and thus reducing the risk of osteoporosis, such as going up and down stairs, jogging, aerobics, swimming, and isometrics. Most Salvadorean interviewed women have a daily calcium intake of 60% or less than the recommended level depending of lactose intolerance, household income, age, and coffee consumption. The former variables directly affect both calcium source diversity and intake frequency. Age would influence dairy calcium intake, since younger women tend to perceive dairy products as having a high animal fat content, and thus reject them. The coffee consumption would affect calcium intake, since this beverage often replace milk. Therefore, all these factors together may be increasing the risk to develop osteoporosis among Salvadorean women. List of abbreviations ===================== WBPA: Weight-bearing physical activity ANOVA: Analysis of Variance SEM: Standard error of mean Competing interests =================== None declared. Authors\' contributions ======================= RHR performed the design of the study, coordinated the study, performed data analysis, and drafted the paper. SMG participed in the design of the study and provided input into the paper. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2474/5/29/prepub> Acknowledgements ================ This research was supported by Universidad Andres Bello. The authors are indebted to Dr. Manuel Aldegunde and Ms. Veronica Sosa for help with the manuscript and they wish to thanks the health education worker, Mrs. Blanca Ruiz, and Nursing graduates: Mairena Escalante, Jacqueline Hernandez, Minerva Hernandez, Veronica Lozano, Alcira Portillo, Natalia Quijada, Damian Rodriguez, and Isabel Velasco for their skillful assistance with the survey administration. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Semantic map showing an osteoporosis knowledge structure.**Semantic map showing an osteoporosis knowledge structure related to several aspect concerning to concept, risk-factors, prevention, and diagnosis of this skeletal disorder. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Total osteoporosis knowledge scores.**Total osteoporosis knowledge scores obtained by Salvadorean women in each age group (25 -- 35 years, n = 73; 36 -- 49 years, n = 74; over 49 years, n = 50), subclassified by educational level (none, primary, secondary, higher). Data are means ± SEM. Bars with distinct letters differ significantly within each age group (p \< 0.05; two-way ANOVA followed by Tukey test). :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Predictive model of influences of surveyed variables on total knowledge scores.**Predictive model of the surveyed variables: age (Age), education (Educatn), family history of osteoporosis (Fam\_Hist), household income (Income), menopause (Menopaus), and fecundity (Fecundity) influencing on total osteoporosis knowledge score (Tot\_scor; R^2^= 0.37, p \< 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Weight-bearing physical activities performed at worksite.**Weight-bearing physical activity (hours/day) at worksite performed by Salvadorean women in each age group (25 -- 35 years, n = 73; 36 -- 49 years, n = 74; over 49 years, n = 50), subclassified by family history of osteoporosis (osteoporosis in family or no osteoporosis in family). Data are means ± SEM. Bars with distinct letters differ significantly (p \< 0.05; Kruskal-Wallis\' test followed by Dunn\'s test). :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Weight-bearing physical activities performed at home.**Weight-bearing physical activity (hours/day) at home performed by Salvadorean women in each age group (25 -- 35 years, n = 73; 36 -- 49 years, n = 74; over 49 years, n = 50), subclassified by family history of osteoporosis (osteoporosis in family or no osteoporosis in family). Data are means ± SEM. Bars with distinct letters differ significantly (p \< 0.05; Kruskal-Wallis\' test followed by Dunn\'s test). :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Weight-bearing physical activity during exercise.**Weight-bearing physical activity during exercise (hours/week) performed by Salvadorean women in each age group (Age 1: 25 -- 35 years, n = 73; Age 2: 36 -- 49 years, n = 74; Age 3: over 49 years, n = 50), subclassified by family history of osteoporosis (osteoporosis in family or no osteoporosis in family). Data are means ± SEM. Bars with distinct letters differ significantly (p \< 0.05; Kruskal-Wallis\' test followed by Dunn\'s test). :::  ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Predictive model of influences of surveyed variables on WBPA performed at worksite.**Predictive model of the surveyed variables: age (Age), education (Educatn), family history of osteoporosis (Fam\_Hist), household income (Income), menopause (Menopaus), and fecundity (Fecundity) influencing on amount of weight-bearing physical activities performed at worksite (WBPAwork; I; R^2^= 0.26, p \< 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. :::  ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Predictive model of influences of surveyed variables on WBPA performed at home.**Predictive model of the surveyed variables: age (Age), education (Educatn), family history of osteoporosis (Fam\_Hist), household income (Income), menopause (Menopaus), and fecundity (Fecundity) influencing on amount of weight-bearing physical activities performed at home (WBPAhome; II; R^2^= 0.27, p \< 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. :::  ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Total calcium intakes among Salvadorean women classified by age and household income.**Total calcium intakes among Salvadorean women in each age group (Age 1: 25 -- 35 years, n = 73; Age 2: 36 -- 49 years, n = 74; Age 3: over 49 years, n = 50), subclassified by household income (above or below the poverty line). Shaded boxes extend from the 25^th^to the 75^th^percentile; whiskers indicate the 5^th^and 95^th^percentiles; dots indicate individuals lying outside these percentiles; the mean is indicated by a solid line, the median by a dashed line. :::  ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **Total calcium intake among Salvadorean women classified by age and household income.**Total calcium intake among Salvadorean women in each age group (25 -- 35 years, n = 73; 36 -- 49 years, n = 74; over 49 years, n = 50), subclassified by household income (above or below the poverty line). Data are means ± SEM. Bars with distinct letters differ significantly (p \< 0.05; Kruskal-Wallis\' test followed by Dunn\'s test). :::  ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### **Dairy calcium intake among Salvadorean women classified by age and household income.**Dairy calcium intake among Salvadorean women in each age group (25 -- 35 years, n = 73; 36 -- 49 years, n = 74; over 49 years, n = 50), subclassified by household income (above or below the poverty line). Data are means ± SEM. Bars with distinct letters differ significantly (p \< 0.05; Kruskal-Wallis\' test followed by Dunn\'s test). :::  ::: ::: {#F12 .fig} Figure 12 ::: {.caption} ###### **Predictive model of influences of surveyed variables on total calcium intake.**Predictive model of the surveyed variables: age (Age), lactose intolerance (Lactose), coffee consumption (Coffee), use of aluminum antiacids (Antiacid), household income (Income), and education (Educatn) influencing on total calcium intake (Total\_Ca; I; R^2^= 0.19, p \< 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. :::  ::: ::: {#F13 .fig} Figure 13 ::: {.caption} ###### **Predictive model of influences of surveyed variables on dairy calcium intake.**Predictive model of the surveyed variables: age (Age), lactose intolerance (Lactose), coffee consumption (Coffee), use of aluminum antiacids (Antiacid), household income (Income), and education (Educatn) influencing on dairy calcium intake (Dairy\_Ca; II; R^2^= 0.20, p \< 0.001, n = 197). The numbers on single- or double-headed arrows are regression weights or correlation coefficients, respectively. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Knowledge dimensions about osteoporosis, specific questions, and corresponding scores get through concept integration. ::: Knowledge dimensions about osteoporosis and corresponding questions None related concept, sub-scores One related concept, sub-scores Up to two related concepts, sub-scores ≥ Three related concepts, sub-scores ---------------------------------------------------------------------------------------------------------------------- ---------------------------------- --------------------------------- ---------------------------------------- -------------------------------------- **Concept related***What you can tell me about osteoporosis?* 0 2 3 5 **Symptom related***What changes do you note in your body if you have osteoporosis?* 0 2 3 5 **Affected bones**If one bone or more was mentioned when previous question was done. 0 2 3 5 **Sex-related factor***Are women and men equally prone to osteoporosis, or is it more frequent in one of the sexes?* 0 2 \-\-- \-\-- **Risk-factor related***What factors increase the risk of suffering osteoporosis?* 0 2 3 5 **Prevention behavior related***How can you reduce the risk of suffering osteoporosis?* 0 2 3 5 **Diagnosis related***How can osteoporosis be detected?* 0 2 3 5 **Actual treatment related***How can osteoporosis be treated?* 0 2 3 5 **Osteoporosis information source***Where did you get the information about osteoporosis?* 0 2 3 5 Maximum score (Σ) 42 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Descriptive data from a convenience sample, according to women\'s age-group. ::: Characteristics 25--35 years (n = 73) 36--49 years (n = 74) over 49 years (n = 50) ----------------------------------------------------- ----------------------- ----------------------- ------------------------ *Age*-Mean (SEM) 29.8 (0.4) 42.4 (0.5) 64.5 (1.5) *Educational level*(%) None 3 (4.1) 15 (20.3) 14 (28.0) Primary 23 (31.5) 27 (36.5) 25 (50.0) Secondary 17 (23.3) 9 (12.1) 4 (8.0) Higher 30 (41.1) 23 (31.1) 7 (14.0) *Household income*(%) Above the poverty line 28 (38.4) 28 (37.8) 13 (26.0) Below the poverty line 45 (61.6) 46 (62.2) 37 (74.0) *Fecundity*(%) Children per woman^†^-Mean (SEM) 2.4 (0.2) 2.7 (0.1) 6.0 (0.5) Nulliparity 16 (21.9) 5 (6.8) 5 (10.0) Parity 57 (78.1) 69 (93.2) 45 (90.0) *Family history of osteoporosis*(%) Yes 40 (54.8) 33 (44.6) 32 (64.0) No 33 (45.2) 41 (55.4) 18 (36.0) *Menopause among women*(Irrespective of age group). Mean age (years) 42.3 ± 0.8 Median age (years) 44.0 Range of age (years) 27.0 -- 53.0 *Menopause before age 45 years*(%) 25--35 years (n = 73) 36--49 years (n = 74) Over 49 years (n = 50) Yes 4 (5.5) 18 (24.3) 18 (36.0) No 69 (94.5) 56 (75.7) 30 (64.0) *Use of aluminum antiacids*(%) Users 16 (21.9) 17 (23.0) 13 (26.0) Non-users 57 (78.1) 57 (77.0) 37 (74.0) *Lactose intolerance*(%) Yes 15 (20.5) 14 (18.9) 9 (18.0) No 58 (79.5) 60 (81.1) 41 (82.0) *Coffee consumption*(%) Yes 51 (69.9) 62 (83.8) 39 (78.0) No 22 (30.1) 12 (16.2) 11 (22.0) Cups a day^‡^-Mean (SEM) 1.8 (0.1) 2.1 (0.1) 1.9 (0.1) ^†^Fecundity rate is only based on data of parity per each age group. ^‡^Consumption rate is only based on coffee-consumer data per each age group. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Osteoporosis knowledge scores according knowledge dimensions and women\'s age-groups. ::: -------------------------------------------------------------------------------------------------------------------- Knowledge dimensions about osteoporosis (expressed as scores).^†^ 25--35 years\ 36--49 years\ Over 49 years\ (n = 73)^a^ (n = 74)^a^ (n = 50)^a^ ------------------------------------------------------------------- --------------- --------------- ---------------- *Concept related* 3 (5) 3 (5) 3 (5) *Symptom related* 2 (3) 3 (3) 3 (3) *Affected bone related* 0 (3) 2 (2) 0 (2) *Sex related* 2 (2) 2 (2) 2 (2) *Risk factor related* 3 (5) 3 (5) 3 (5) *Prevention behavior related* 3 (5) 3 (5) 3 (5) *Diagnosis related* 0 (3) 0 (3) 0 (3) *Actual treatment related* 0 (0) 0 (0) 0 (0) *Osteoporosis information source* 1 (2) 1 (2) 1 (2) Σ 14 (28) 17 (27) 15 (27) -------------------------------------------------------------------------------------------------------------------- ^†^All values are median and 75^th^percentile (in brackets) of reached scores in each knowledge areas, per each age group. ^a^Comparisons among scores of knowledge specific area per each age group were not significantly different (p \> 0.05, Kruskal-Wallis ANOVA on ranks). :::

PubMed Central

2024-06-05T03:55:47.666964

2004-8-26

PMC517503

Background ========== Liver transplantation (LT) remains the only treatment for end-stage liver disease. However the transplant procedure mandates cold perfusion, hypothermic storage, warm ischemia and warm reperfusion of the graft, resulting in Ischemia/Reperfusion (I/R)-induced injury to the transplanted graft. Although the introduction of the University of Wisconsin solution (UW) has improved clinical outcomes, I/R injury remains one of the major clinical problems following LT leading, in cases of marginal donor quality or prolonged cold or warm ischemic time, to the development of graft dysfunction or non function. Recently the cellular events following liver I/R during LT have been brought to sharp focus. Nevertheless, it is still controversial if the major mode of cell death during I/R is apoptosis and/or necrosis \[[@B1]\]. It has been shown in an animal model of viral-induced hemorrhagic liver necrosis, that the liver undergoes several morphologic changes including mineralization \[[@B2]\]. Furthermore, previous clinical reports have documented the development of calcifications in liver upon extensive ischemia \[[@B3],[@B4]\]. The above observations may reflect a high degree of cell damage leading to an enhanced apoptotic cell engulfment by non-professional phagocytes of the liver \[[@B5]\] with subsequent necrosis and biomineralization. In this report, using a combination of cross-sectional imaging, histochemistry, and light (LM) as well as transmission electron microscopy (TEM) we identified liver calcification, in two grafts with severe I/R injury following LT. Case Presentations ================== Case 1 ------ A 65-year-old man of Asian origin underwent LT at our institution on June 2002, for decompensated Hepatitis B related cirrhosis. The patient did not have a history of abnormal calcium metabolism, or hyperparathyroidism. Upon listing his serum ionized calcium was 1.07 mmol/L. The donor was a previously healthy, 42-year-old male, who suffered intracranial bleeding following a motor vehicle crash. He did not suffer any period of hypoxia but he was hypotensive (SBP = 90 mm Hg) prior to procurement. His serum liver function tests were normal prior to harvesting (AST 32 U/L, ALT 40 U/L). The organ was procured by our institution\'s transplant team. During procurement the liver was found to be well perfused with no focal injuries and no macroscopic evidence of steatosis. The recipient underwent an uncomplicated conventional LT without the use of a veno-venous bypass. There were no periods of hypoxia or severe hypotension during transplantation. The cold ischemic time of the graft was 8 hours while the warm ischemic time was 45 minutes. The graft reperfused well and no biopsies were taken. Intravenous methylprednisolone (500 mg) was administered intraoperatively, and postoperatively the patient received induction with Antithymocyte Globulin, which is the protocol followed at our institution. During transplantation the patient received a total of 4 units of packed red blood cells (PRBCs) and 6 units of fresh frozen plasma (FFP). At the time of LT, the international normalization ratio (INR) was 1.53, while serum total bilirubin was 103 μmol/L (Figure [1A,1B](#F1){ref-type="fig"}). On postoperative day 2 the patient had a peak of his serum AST (3469 U/L) and at this point he had further biochemical evidence of primary graft dysfunction, with inability to normalize his INR (Figure [1B](#F1){ref-type="fig"}), and with progressive elevation of his total serum bilirubin (Figure [1A](#F1){ref-type="fig"}). Repeated ultrasonographic examination revealed a patent hepatic artery and portal vein, as well as patent hepatic veins. At this point, a liver biopsy demonstrated severe reperfusion injury with several apoptotic bodies, several dystrophic calcifications (Figure [1E](#F1){ref-type="fig"}), and no evidence of acute cellular rejection. His clinical status deteriorated, he developed multiorgan system failure and died 12 days after his transplantation. No septic focus was identified. Both kidneys harvested from the same donor did not present any signs of delayed graft function after transplantation. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **A:**Serum Bilirubin levels (μmol/L) for patient 1 (solid line) and for patient 2 (dashed line). The arrowheads indicate the time points where calcifications were detected. **B:**International Normalization Ratio (INR) for patient 1 (solid line) and for patient 2 (dashed line). Arrowheads indicate the time points where calcifications were detected. **C:**Computed Tomography of patient 2. The arrow points an area in the right liver with the same density as the spinal column (arrowhead). **D:**Picture of the explant liver during retransplantation of the same patient (case 2). The arrow shows the abnormal area of the right liver correlating with the computed tomography findings. **E:**Light microscopy of epoxy embedded semi-section obtained from the liver biopsy from patient 1. The image shows moderate calcification (microcalcification) throughout the section. The arrow indicates a representative pattern of calcification. **F:**Light microscopy of epoxy embedded semi-section obtained from the tissues of the explant, following liver retransplantation of patient 2. The image shows the interface between calcified region (upper right) and non-calcified adjacent hepatic cells (lower left region). **G:**Light microscopy image showing a higher magnification of calcified region as shown in F. The bright and high contrast regions represent massive mineralization of hepatic cells of the explant, following retransplantation. **H:**Transmission electron microscopy images of ultrathin section obtained from the transitional zone between calcified and non-calcified tissue. Showing the mode of calcification and textural organization of hydroxyapatite crystal aggregates (dark contrast) within cytoplasmic region of the cell. Note alteration of the nucleus in the center. :::  ::: Case 2 ------ A 55-year-old man of Greek origin underwent LT at our institution for ethanol and hepatitis B related cirrhosis, as well as a large hepatocellular carcinoma. The patient did not have a history suggestive of hyperparathyroidism or abnormal calcium metabolism, and upon listing his serum ionized calcium was 0.94 mmol/L. The donor was a previously healthy, 52 year-old male who suffered a closed head injury during a motor vehicle crash. The organ was procured by our transplant team. Prior to procurement the donor did not suffer any periods of hypoxia or hypotension. His liver function tests were normal prior to harvesting (AST 36 U/L, ALT 45 U/L). During procurement the liver was found to be well perfused, with no evidence of aberrant vascular anatomy and no evidence of trauma. Macroscopic examination of the liver did not show any evidence of steatosis. The recipient underwent a conventional LT without venovenous bypass and without intraoperative hypotension. The cold ischemic time was 10 hours while the warm ischemic time was 40 minutes. Following reperfusion, the graft appeared well perfused and again no biopsies were taken. Intraoperatively the patient received 500 mg of methylprednisolone, and postoperatively he was induced with Antithymocyte Globulin. During transplantation the recipient received 3 units of PRBCs and 6 units of FFP. In the early postoperative period the patient had to be explored once for retroperitoneal bleeding associated with hypotension (SBP\< 90 mm Hg). At the time of LT, the patient had an INR of 2.7 and a serum total bilirubin of 104 μmol/L. Following LT, the patient developed severe primary graft dysfunction with rising serum bilirubin and INR (Figure [1A,1B](#F1){ref-type="fig"}), while his serum ALT and AST peaked during the 2^nd^and 3^rd^postoperative day (4700 U/L and 6598 U/L respectively). At that time, ultrasonography demonstrated uniformly patent vessels (hepatic artery, portal and hepatic veins), while Computed Tomography (CT) showed areas in the right lobe of the liver isodense with the spinal column (Figure [1C](#F1){ref-type="fig"}). The patient was listed for retransplantation. During the retransplant procedure the explant liver graft had a \"bony\" consistency in the involved right lobe. Cross-sections of the right lobe showed a \"clay-like\" parenchyma with clear evidence of calcification (Figure [1D](#F1){ref-type="fig"}). Light (LM) and transmission electron microscopy (TEM) investigation of ultrathin sections obtained from specimens selected from biopsies at the interface of calcified and non-calcified tissues showed extensive intracellular calcification within the hepatic cells (Figure [1F](#F1){ref-type="fig"}). High-resolution TEM (HRTEM) images and selected-area electron diffraction (SAED) combined with energy dispersive spectroscopy (EDS) analysis demonstrated the presence of hydroxyapatite (HA) as a solid phase in the calcified region. The adjacent non- or partially calcified hepatic cells displayed extensive nuclear condensation suggestive of significant apoptosis as well as severe vacuolization, suggestive of an extensive apoptotic and necrotic process (Figure [1G,1H](#F1){ref-type="fig"}). The patient had a complicated postoperative course and finally died from ventricular fibrillation unresponsive to electrical cardioversion. Both kidneys harvested from the initial donor were transplanted without any evidence of delayed or primary graft dysfunction/non-function. Conclusions =========== Currently, more than 16,000 candidates are listed with the United Network for Organ Sharing awaiting liver transplantation. Nevertheless, only 4800 cadaveric liver transplants are performed annually in the United States. Due to this discordance between organ demand and supply, it is estimated that approximately 10% of patients in the waiting list will die before obtaining an organ. As a result, novel strategies to expand the donor pool have been explored. With the exception of live donor liver transplantation, the remaining strategies involve the use of older cadaveric grafts, allografts with mild steatosis or even donors with evidence of past hepatitis B or C infection \[[@B6]\]. This is why, one of the major obstacles to be tackled, is the development of clinically significant ischemia and reperfusion injury, which is even more important for \"marginal\" grafts. Every progress towards understanding the molecular events following not only cold storage but also cold and warm reperfusion of the graft could have a significant impact on the current transplantation practices. Indeed, recent data suggest that following I/R there is a balanced apoptosis and occasionally necrosis of hepatocytes translating into cell swelling, distension of various cellular organelles, clumping and random degradation of nuclear DNA, extensive plasma membrane endocytosis and autophagy \[[@B7]\]. Furthermore, when these events become predominant, they can lead, at least in animal models, to the development of calcifications as observed in livers of rabbits infected with rabbit haemorrhagic virus \[[@B2]\]. In the present case report, we have shown that these events can take place in human subjects following liver transplantation. To our knowledge these are the first reported cases in the literature of liver calcification following liver transplantation, presumably secondary to I/R injury. Not only did both patients have biochemical evidence of severe graft I/R injury, they also had biopsy proven I/R induced injury associated with the development of calcifications. Furthermore, both succumbed to the sequelae of this injury. Both recipients received grafts from donors with normal serum biochemistries and no evidence of hepatic trauma or steatosis. Both donors had no evidence of crystal deposition or storage disease, and although we did not perform any donor liver biopsies, the grafts appeared macroscopically normal and perfused well with UW solution. Corroborating to this remains the fact that all four kidneys (from both donors) were transplanted without any problems. Neither recipient had any evidence of calcium metabolism problems, since both had normal serum calcium upon listing. Both recipients had an anticipated intraoperative course without periods of hypotension and without massive transfusion requirements. Finally, both grafts did not demonstrate any vascular problems in the postoperative period by Ultrasonography or CT scan examination or any evidence of intrahepatic thrombosis in postmortem or explant examination. Ischemic stress has been previously reported to induce calcium accumulation at the cell level, either by impaired energy metabolism and/or plasmalemmal alterations. This elevated intracellular calcium concentration is responsible for cytoskeletal modifications, which alter cell shape, and for the activation of phospholipases, which results in perpetuation of membrane damage and finally, mitochondrial calcification \[[@B8]\]. Although, the crystal shape, composition and organization of HA in our samples are similar to those observed in bone and cartilage \[[@B9]\], as well as synthetic HA formed in serum \[10\], the intracellular precipitation of HA within hepatic cells is unique and has not been reported from other physiological and pathological tissues. The observation of calcified, vacuole-like structures in hepatic cells from these two livers could be suggestive of mitochondrial calcification. In addition, the extensive presence of phagocytic structures in the pre-calcified regions of these livers suggests an intense apoptotic/necrotic process undergone after I/R injury in these regions. Further investigation, however, is required to understand the mechanism(s) and the mode of calcification in the liver. In conclusion, we believe that the described phenomenon is underreported at least in the liver transplant literature. Furthermore, it appears that there is a correlation between the development of severe I/R injury leading to apoptosis and/or necrosis and calcifications detectable even by light microscopy. We think that the development of microcalcifications should be studied more extensively in the context of I/R injury following liver transplantation. Although, such a phenomenon appears to correlate with significant I/R injury, evident by biochemical data, it has the potential to be provide further information on the pathways of severe I/R injury post transplant. Competing Interests =================== None declared. Authors Contributions ===================== GNT: conceived the study and wrote the manuscript MA: carried out the electron microscopy studies EC: participated in the design and analyzed the light microscopy results, critical review of the manuscript AE: analyzed light microscopy results AV: carried out electron microscopy studies PM: participated in the design of the study All authors read and approved the final manuscript. Abbreviations ============= **I/R:**Ischemia and Reperfusion **LM:**Light Microscopy **TEM:**Transmission Electron Microscopy **HRTEM:**High-resolution Transmission Electron Microscopy **EDS:**Energy Dispersive Spectroscopy **HA:**Hydroxyapatite Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2482/4/9/prepub> Acknowledgements ================ We thank Mrs. J. Mui (Senior Technician, Facility for Electron Microscopy Research, McGill University) for her technical assistance. GNT was supported by the Gertrude Nemiroff Eliesen Memorial Award, and EC by the Simone and Morris Fast foundation, McGill University Health Center Research Institute and a Junior scholarship from the FRSQ. This work was supported in part by a grant from the Canadian Institutes for Health Research (CIHR) to EC and National Science and Engineering Research Council of Canada (NSERC) to HV. Consent was obtained from patient relative for publication.

PubMed Central

2024-06-05T03:55:47.670019

2004-9-3

PMC517504

Background ========== RNA interference (RNAi) is a process in which the introduction of dsRNAs into cells leads to inactivation of expression of genes containing homologous sequences by sequence-specific degradation of mRNA (for reviews see \[[@B1],[@B2]\]). RNAi has been observed in a variety of organisms, including fruit fly, nematodes, zebrafish, mice and humans \[[@B3]-[@B7]\], and is mechanistically similar to post-transcriptional gene silencing (PTGS) in plants \[[@B8]\] and quelling in fungi \[[@B9]\]. Genetic studies have identified a number of the proteins that are involved in these processes in *Neurospora crassa*\[[@B10]-[@B12]\], *Caenorhabditis elegans*\[[@B13]-[@B15]\] and *Arabidopsis thaliana*\[[@B16]-[@B18]\]. dsRNA has been shown to induce RNAi in cultured *Drosophila*cells \[[@B19]-[@B22]\] and biochemical studies of this system have revealed the involvement of two distinct activities; an RNA-induced silencing complex (RISC), containing both nuclease activity and RNA that carries out enzymatic degradation of target RNA \[[@B19]\], and an RNase III-like protein (Dicer) that is involved in production of short (22 nucleotide) guide RNAs from dsRNA as an early step in the RNAi process \[[@B22]\]. PTGS in plants can operate as a defence mechanism against virus infection (for reviews see \[[@B8],[@B23]\]) and numerous plant viruses encode silencing suppressors, which are thought to have developed as a response to the plant PTGS system \[[@B24]\]. Plant virus-encoded silencing suppressors may target different components of the PTGS system. The HC-Pro protein, encoded by potyviruses such as *Tobacco etch virus*(TEV), was initially characterised as a determinant of virus movement in plants \[[@B25]\] and subsequently was shown to inhibit silencing of transgenes in transformed plants \[[@B26]\]. HC-Pro antagonizes silencing in all tissues \[[@B26]\] and appears to target a step involved in the maintenance of silencing \[[@B27]\]. In contrast, the 2b protein encoded by *Cucumber mosaic virus*(CMV), a cucumovirus, interferes with the systemic spread of the silencing signal, preventing initiation of silencing in newly emerging tissues \[[@B28]\]. A survey of a small number of other different plant viruses showed that the comovirus *Cowpea mosaic virus*, the geminivirus *African cassava mosaic virus*, the potexvirus *Narcissus mosaic virus,*the tobamovirus TMV, the sobemovirus *Rice yellow mottle virus*(RYMV), the tombusvirus *Tomato bushy stunt virus*(TBSV) and the tobravirus *Tobacco rattle virus*(TRV) also were able to suppress GFP silencing \[[@B24]\]. Although in this study the potexvirus PVX did not suppress silencing, using a different assay these authors showed that PVX is in fact able to prevent systemic silencing \[[@B29]\]. Thus, it seems probable that many plant viruses encode proteins that allow them to evade or inhibit PTGS in certain plant species, and that different suppressor proteins target different parts of the PTGS pathway. An amenable system for studying these suppressor proteins would be an aid in determining the molecular basis of their action. Recently it has been demonstrated that an insect virus, flock house virus (FHV) encodes a protein, 2b, which acts as a suppressor of PTGS in plants \[[@B30]\]. We have investigated the effects on RNAi in cultured *Drosophila*cells of expression of some silencing suppressors from plant viruses. Results ======= Effects of transient expression of the TEV HC-Pro and CMV 2b proteins on RNAi in *Drosophila*cells -------------------------------------------------------------------------------------------------- The TEV HC-Pro or CMV 2b proteins were transiently expressed in *Drosophila*DS2 cells and their effect on dsRNA-mediated silencing of a *lac*Z gene examined using a previously described system \[[@B20]\]. Expression of the CMV 2b protein could be detected by immunoblotting in lysates of cells transfected with pMT-2b and induced by addition of CuSO4 to the growth medium (Fig [1A](#F1){ref-type="fig"}, lane 4). No cross-reacting protein was detected in similarly induced cells not transfected with pMT-2b (Fig [1A](#F1){ref-type="fig"}, lane 2) or in control cells, or cells transfected with pMT-2b that were not induced (Fig [1A](#F1){ref-type="fig"}, lanes 1 and 3. In the absence of an antibody to TEV HC-Pro we used an expression plasmid (pMT-HC-Pro/K) that expressed a mutated HC-Pro (TEV K) containing an insertion of 3 amino acids in the central region of the protein \[[@B31]\] as a negative control to verify that any suppressor activity was due to an effect of the HC-Pro protein. This mutant version of HC-Pro was shown previously to be defective in suppression of PTGS in plants \[[@B32]\]. Northern blot analysis of RNA from cells transfected with pMT-HC-Pro or pMT-HC-Pro/K showed that transcript from both plasmids accumulated in the cells (Figure [1B](#F1){ref-type="fig"}, lanes 2 and 3). β-galactosidase activity could be detected by staining in \~80% of *Drosophila*cells when they were transfected with a *lacZ*expression plasmid (pMT/V5-His/lacZ) (Table [1](#T1){ref-type="table"}). \~17% of cells stained for β-galactosidase when they were co-transfected with pMT/V5-His/lacZ and dsRNA corresponding to the first \~500nt of the *lac*Z gene demonstrating induction of RNAi. No significant decrease in the number of cells stained was observed when cells were transfected with pMT/V5-His/*lacZ*and dsRNA derived from the green fluorescent protein gene indicating that the silencing was specific. Co-transfection of *Drosophila*cells with pMT/V5-His/lacZ, *lacZ*-specific dsRNA and an HC-Pro expression vector (pMT-HC-Pro) resulted in staining of \~46% of cells, indicating that RNAi was being suppressed. Transfection of cells with the mutant pMT-HC-Pro-K along with pMT/V5-His/lacZ and dsRNA resulted in no suppression of RNAi. This indicates that a mutation affecting the ability of HC-Pro to suppress PTGS in plants also affects suppression of RNAi in *Drosophila*cells. Transfection of cells with pMT-2b along with pMT/V5-His/lacZ and dsRNA resulted in no increase in the percentage of transfected cells staining for β-galactosidase activity compared to cells transfected only with pMT/V5-His/lacZ and dsRNA. RNAi in stable cell lines expressing TEV HC-Pro or CMV 2b --------------------------------------------------------- Stable cell lines expressing HC-Pro (DS2-HC-Pro), or CMV 2b (DS2-2b) were produced in order to improve the efficiency of the RNAi suppression assay by reducing the number of different nucleic acid molecules needed for co-transfection. Immunoblotting of lysates of DS2-2b cells confirmed that the 2b protein accumulated in these cells (Fig [2A](#F2){ref-type="fig"}, lane 8) and this protein was not produced when the cells were not treated with CuSO4 (Fig [1A](#F1){ref-type="fig"}, lane 7). Northern blot analysis of RNA from the DS2-HC-Pro cells showed that an HC-Pro-specific transcript accumulated in these cells (Fig [1C](#F1){ref-type="fig"}, lane 1). An unrelated cell line (DS2-scAb S20) expressing a recombinant antibody \[[@B33]\] (Reavy *et al*., 2000) was used as a control in order to eliminate the possibility that stable transformation of the cells could interfere with the RNAi mechanism. The transfection efficiency of all transformed lines with pMT/V5-His/lacZ was lower than that of the control DS2 cells (Table [1](#T1){ref-type="table"}); possibly the transformed cells are more recalcitrant to transfection than the control cells or the additional copies of the metallothionein promoter are saturating the induction factors. Nonetheless, RNAi was strongly induced in the DS2-scAb S20 cells as co-transfection with pMT/V5-His/lacZ and dsRNA reduced the number of cells staining for β-galactosidase activity to only 6.5% compared to \~41% when transfected with pMT/V5-His/lacZ alone (Table [1](#T1){ref-type="table"}). Transfection of DS2-HC-Pro cells with pMT/V5-His/lacZ resulted in \~42% of cells expressing β-galactosidase. This was reduced only to \~32% when transfection was carried out using pMT/V5-His/lacZ and dsRNA, showing that RNAi was significantly inhibited in this cell line. In contrast, no suppression of RNAi was observed in the DS2-2b line (expressing the CMV 2b protein) when transfected with pMT/V5-His/lacZ and dsRNA, as the percentage of stained cells was similar to that in DS2-scAb S20 cells transfected with pMT/V5-His/lacZ and dsRNA. Similar results were obtained when the DS2-2b cells were grown in the presence of CuSO4 to induce expression of the virus protein before transfection with pMT/V5-His/lacZ and dsRNA, indicating that the CMV 2b protein could suppress neither the initiation nor the maintenance of the dsRNA-induced silencing in the *Drosophila*cells. Identification of silencing suppression in *Drosophila*cells by another plant virus protein ------------------------------------------------------------------------------------------- We were interested to determine if the *Drosophila*cell system could be used as a screen for RNAi suppression effects caused by other virus proteins. We chose to examine the potential RNAi suppression activity of a protein from the tobravirus TRV. This virus is able to suppress transgene silencing in plants \[[@B24]\] but the specific viral protein responsible for this activity has not been identified. TRV, like the other tobraviruses has a bipartite, positive strand RNA genome \[[@B34]\], however, the larger RNA (RNA1) can infect plants systemically in the absence of RNA2 to produce what is known as an NM-infection. This occurs frequently in particular cultivars of potato and is often associated with increased symptom severity. Clearly, therefore, RNA1 encodes all the functions necessary for virus multiplication including, possibly, suppression of PTGS/host defence. The one protein encoded by RNA1 without an assigned function is a 16K cysteine-rich protein which was, thus, investigated as a candidate silencing suppressor protein. One characteristic of plant virus silencing suppressor proteins is often significant enhancement of disease symptoms when they are over-expressed from a viral vector \[[@B26]\]. Similar results were obtained from preliminary experiments showing that expression of the TRV 16K gene from a PVX vector did increase the severity of symptoms in infected plants. Inoculation of *Nicotiana benthamiana*plants with PVX alone initially induced vein chlorosis and systemic leaf curling, although the plants continued to grow. Inoculation with PVX carrying the 16K gene caused similar initial symptoms but led to tip necrosis and death of the plants \[[@B35]\]. The TRV 16K protein was expressed in *Drosophila*cells after transfection of cells with the expression plasmid pMT-16K, and the 16K protein could be detected by western blotting when the cells were induced with CuSO4 (Fig [2](#F2){ref-type="fig"}, lane 4) but not when cells were not induced (Fig [2](#F2){ref-type="fig"}, lane 3). A stably-transformed cell line (DS2-16k) containing pMT-16k behaved in a similar way (Fig [2](#F2){ref-type="fig"}, lanes 7, 8). No cross-reacting protein was detected in non-transfected cells (Fig [2](#F2){ref-type="fig"}, lanes 1,2). Co-transfection of pMT/V5-His/lacZ with dsRNA and pMT-16K resulted in \~47% of transfected cells staining blue (Table [1](#T1){ref-type="table"}) compared to \~18% when transfected with pMT/V5-His/lacZ and dsRNA. The 16K protein therefore partially suppressed RNAi in *Drosophila*cells. Detection of *lacZ*gene transcripts ----------------------------------- Northern blot analysis of the RNA present in transfected cells confirmed that HC-Pro was effective in preventing cytoplasmic degradation of the *lacZ*transcript. RNA with the expected size of the *lacZ*transcript could not be detected in extracts of DS2 cells that were transfected with pMT/V5-His/lacZ and dsRNA (Fig. [3](#F3){ref-type="fig"}, lane 2). However, intact *lac*Z RNA was present in extracts of DS2-HC-Pro cells that were transfected with pMT/V5-His/lacZ regardless of whether the cells were co-transfected with dsRNA (Fig [3](#F3){ref-type="fig"}, lanes 3 & 4). Similarly, the *lacZ*transcript was intact in DS2-16K cells after transfection with pMT/V5-His/lacZ and dsRNA (Fig [3](#F3){ref-type="fig"}, lane 6). The amounts of *lacZ*transcript in the DS2-Hc-Pro and DS2-16K cells were less after transfection with pMT/V5-His/lacZ and dsRNA than after transfection with pMT/V5-His/lacZ alone indicating partial suppression of RNAi. No *lacZ*transcript was detected in DS2-2b cells after transfection with pMT/V5-His/lacZ and dsRNA (Fig [3](#F3){ref-type="fig"}, lane 10) but the *lacZ*transcript could be detected in DS2-2b cells transfected with pMT/V5-His/lacZ alone Fig [3](#F3){ref-type="fig"}, lane 8). Detection of siRNAs ------------------- Suppression of silencing by viral proteins in plants is often associated with an inhibition of the production of small, 21 to 25 nucleotide RNAs that may be analogous to the 22 nucleotide guide RNAs identified as part of the *Drosophila*RISC complex \[[@B22],[@B27],[@B36]\]. We were able to identify short RNAs specific to the region of transfected dsRNA in extracts of *Drosophila*cells transfected with pMT/V5-His/lacZ and dsRNA (Fig [4](#F4){ref-type="fig"}, lane 2). A lot of larger RNA species were also detected presumably as a result of degradation of the input dsRNA. Unfortunately it is not possible to probe for the presence of short RNAs outwith the region of input dsRNA, as RNAi is not transitive in *Drosophila*\[[@B37]\]. Expression of the plant viral HC-Pro suppressor protein in the DS2-HC-Pro cells did not prevent the accumulation of these small RNAs when transfected with pMT/V5-His/lacZ and dsRNA (Fig [4](#F4){ref-type="fig"}, lane 4). Co-transfection of *Drosophila*cells with pMT/V5-His/lacZ, dsRNA and pMT-16K also resulted in production of small RNA species (Fig [4](#F4){ref-type="fig"}, lane 8) even though the 16K protein also partially suppresses silencing in the *Drosophila*cells. Possibly the inefficiency of the transfection procedure and the failure of the suppressors to completely suppress RNAi in these experiments masks any visible effect by the suppressors on the accumulation of these molecules. Similarly, small RNAs were detected in the DS2-2b cells after transfection with pMT/V5-His/lacZ and dsRNA (Fig [4](#F4){ref-type="fig"}, lane 6). The dsRNA preparations used to induce RNAi were examined to determine if small RNA species of a similar size to siRNAs were present and were the species detected in figure [4](#F4){ref-type="fig"}. No short RNA species of 21--25 nucleotides were observed in the dsRNA preparation used to transfect the cells indicating that the species observed in the transfected cells were produced as a result of cellular processing (Fig [5](#F5){ref-type="fig"}, lane 3). Some larger products were observed and these are likely to have arisen as a result of premature terminations during the dsRNA synthesis reaction. Discussion ========== Suppression of RNAi in *Drosophila*cells by some plant virus proteins indicates that at least part of the processes of RNAi and PTGS is conserved between plants and *Drosophila.*TEV HC-Pro is one of a family of proteins that suppress PTGS in plants and the CMV 2b protein has a similar ability \[[@B26]\]. These proteins are thought to target different components of the PTGS system, as the CMV 2b protein interferes with the spread of a silencing signal after initial induction of silencing, thus, preventing silencing from initiating in newly emerging leaves. The potyvirus HC-Pro protein, however, interferes with the maintenance of silencing in all tissues \[[@B26],[@B27]\]. These differences were reflected in the *Drosophila*cell system where HC-Pro could suppress RNAi but the 2b gene apparently was ineffective. As there is no spread of a silencing signal in cultured *Drosophila*cells, the failure of the CMV 2b protein to act in this system is not unexpected. It was also significant that a mutant version of HC-Pro (K) that is defective in suppression of gene silencing \[[@B32]\] but is effective in proteolytic cleavage \[[@B31]\] was also unable to suppress RNAi in *Drosophila*cells. This strongly supports the idea that the TEV HC-Pro protein targets the same component of the plant PTGS system and the *Drosophila*RNAi system. Our demonstration that the TRV 16K protein also is able to interfere with RNAi in Drosophila cells, leads us to suggest that many other plant virus silencing suppressor proteins are likely to be functional in this system. SiRNAs were detected in our cell lines expressing HC-Pro and the TRV 16K protein even though suppression of silencing was observed. This is not totally unexpected for a number of possible reasons. Firstly, suppression of RNAi is partial in our cultures indicating that some RNAi, and presumably siRNA production, does occur in some of the cells. We have observed with cells expressing the *lacZ*gene alone that there is considerable variation in the amount of staining of individual cells indicating differences in the amount of gene expression in individual cells. There is no reason to suppose that this variability in gene expression is limited to the *lacZ*gene and it is possible that low levels of the suppressors may fail to inhibit silencing in some of the cells within a culture. Secondly, the experimental protocol requires a time delay between transfection of the cells with the *lacZ*plasmid and dsRNA before induction of expression of the suppressor proteins in order to allow the cells to recover from the transfection procedure. Some production of siRNAs from the dsRNA may possibly occur in cells during this lag period before the suppressors are induced. For these reasons the usefulness of the *Drosophila*RNAi system for studying the mechanisms of action of plant virus suppressor proteins may be limited. Mutants of *Arabidopsis*that are impaired in PTGS have an increased susceptibility to CMV, showing that PTGS can operate as a defensive system that targets viruses \[[@B18]\]. Plant virus-encoded silencing suppressors are thought to have developed as a response to this defensive aspect of the plant PTGS system, and subsequent studies have shown that, as would be expected, a wide variety of plant viruses encode silencing suppressors \[[@B24]\]. The suppressors encoded by these viruses are unrelated in amino acid sequence, and are likely to act at different points in the silencing process, making them ideal probes to investigate the silencing machinery. We anticipate that using different suppressors from a wide range of viruses may permit a detailed examination of the biochemical process of RNAi in *Drosophila*and possibly other organisms as well as allowing detailed characterisation of the mode of action of the virus suppressor proteins. Furthermore, it is becoming apparent that RNAi or PTGS may play a role in processes other than defence against foreign RNAs. Transformation of plants with TEV HC-Pro or rgs-CaM, a plant-encoded PTGS-suppressor protein related to calmodulin, interrupted normal development and led to the formation of differentiated tumours at the stem/root junction \[[@B38]\]. The *Arabidopsis*gene CARPEL FACTORY is related to *Drosophila*Dicer and is involved in plant development and fertility \[[@B39]\], and the EGO-1 gene of *C. elegans*also appears to have a role in both RNAi and germ-line development 15\]. Components of the RNAi mechanism including a homolog of *Dicer*are also involved in synthesis of short temporal RNAs that regulate developmental timing in *C. elegans*\[[@B40]-[@B42]\]. Intervention with plant virus silencing suppressors may therefore have significant utility in determining the involvement of RNAi in development and differentiation in plants and animals, and possibly in manipulating these processes. Conclusions =========== These results indicate that at least part of the process of RNAi in *Drosophila*and PTGS in plants is conserved, and that plant virus silencing suppressor proteins may be useful tools to investigate the mechanism of RNAi. Methods ======= Plasmid constructions --------------------- A region (nucleotides 1055--2449) of the TEV genome containing the HC-Pro sequence was amplified by reverse transcription -- polymerase chain reaction (RT-PCR) using as a template RNA from an infected plant, and primers HC-Pro-1 (5\'-CCGGTACCATGAGCGACAAATCAATCTCTGAGGC-3\') and HC-Pro-2 (5\'-GGCTCGAGCTACACATCTCGGTTCATCCCTCC-3\'). The primers add an ATG codon to the start of the open reading frame and the HC-Pro gene was cloned as a *Kpn*I-*Xho*I fragment into pMT/V5-HisA (Invitrogen) to produce plasmid pMT-HC-Pro. The same primers were also used to clone a mutant HC-Pro gene (TEV-K; \[[@B31]\]) from transgenic plants to produce plasmid pMT-HC-Pro/K. The 2b gene was amplified from RNA2 of CMV isolate Fny using primers 392 (5\'-GAACCATGGAATTGAACGTAGGTGC) and 393 (5\'-GGGTACCTCAGAAAGCACCTTCCGCC) and cloned into pGemT (Promega) before subcloning into pMT-V5-HisA. The TRV 16K gene was amplified from RNA1 of TRV isolate PpK20 using primers 433 (5\'-TCATCATGACGTGTGTACTCAAGGG-3\') and 434 (5\'-AAGGTACCATCAAAAAGCAAACG-3\') to insert *Bsp*HI and *Kpn*I sites upstream and downstream, respectively, of the gene. The PCR product was cloned into pMT/V5-HisA to produce plasmid pMT-16K. The nucleotide sequences of the cloned PCR inserts were confirmed by sequencing. dsRNA synthesis --------------- cDNA corresponding to \~500 bp of the 5\' end of the *lac*Z gene was amplified using pcDNA3.1/HisB/lacZ (Invitrogen) as a template and primers *lacZ*-1 (5\'- [TAATACGACTCACTATAG]{.underline}GGAGACCCAAGCTGGCTAGC-3\') and *lacZ*-2 (5\'- [TAATACGACTCACTATAG]{.underline}GGCAAACGGCGGATTGACCG-3\'). Both primers contain T7 RNA polymerase promoter sequences (shown underlined). The PCR product was used to direct synthesis of dsRNA using T7 RNA polymerase (Invitrogen) after which the DNA template was removed by DNase digestion. Cell culture and transfection ----------------------------- DS2 cells, DES expression medium and the *lacZ*expression plasmid pMT/V5-His/lacZ were supplied as part of the *Drosophila*Expression System (Invitrogen) and cells were grown according to the manufacturer\'s instructions. Cells were grown in 60 mm dishes and transfected by calcium phosphate co-precipitation with various mixtures of 10 μg each of pMT/V5-His/lacZ and the suppressor expression plasmid DNAs and 5 μg dsRNA. In control transfections, 10 μg of an empty expression plasmid (pMT/V5-HisB) replaced the suppressor expression plasmid. After transfection the cells were washed twice in DES medium and grown for eight hours before expression of proteins was induced by addition of CuSO~4~to a final concentration of 500 μM. Stably transformed lines expressing the HC-Pro, 2b or a6k genes were established by co-transfection of cells with pMT-HC-Pro or pMT-2b and pCo-Hygro (Invitrogen) followed by selection of transformed cells in medium containing hygromycin. Cells were stained 48 hrs after transfection to detect *lacZ*gene expression using a β-Gal Staining Kit (Invitrogen). Four randomly selected fields of view each containing \~100 cells were selected in each of duplicate plates and the number of cells staining blue was counted. Immunoblotting -------------- Cell lysates were analysed by polyacrylamide gel electrophoresis and separated proteins were transferred to nitocellulose using a carbonate buffer \[[@B43]\]. The blots were probed with an anti-CMV 2b \[[@B44]\] or anti 16K antibody \[[@B45]\] followed by a goat anti-rabbit alkaline phosphatase conjugate. Blots were developed using SigmaFast NBT/BCIP substrate (Sigma, Poole, UK). Northern blot analysis of RNA from *Drosophila*cells ---------------------------------------------------- RNA was extracted from *Drosophila*cells using TriPure Isolation Reagent (Boehringer). Samples of RNA were separated by electrophoresis on formaldehyde/agarose gels, transferred to nylon membrane and probed with digoxigenin-labelled DNA probes corresponding to \~500nts at the 5\' end of the *lacZ*gene or to the HC-Pro cDNA described above, as appropriate and essentially as described \[[@B46]\]. The probes were made by PCR using primers lacZ-3 (5\'- GGAGACCCAAGCTGGCTAGC-3\') and lacZ-4 (5\'- GGCAAACGGCGGATTGACCG-3\') for the *lacZ*probe, and HC-Pro-1 and HC-Pro-2 for the HC-Pro probe. Digoxigenin-labeled RNA Molecular Weight Marker II (Roche Molecular Biochemicals) was run as a size marker. For detection of short (\~23nt) RNA species total RNA preparations from *Drosophila*cell cultures were fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad), following the manufacturers instructions. \~20 μg of the short RNA species were separated by electrophoresis in a 15% polyacrylamide gel containing 7 M Urea and electroblotted as described by Llave *et al.*\[[@B27]\]. After electrophoresis the gel was stained with ethidium bromide and photographed under ultra-violet light before the RNA species were transferred to Hybond N+ membrane (Amersham) by electroblotting. For the detection of siRNAs, a digoxigenin-labelled RNA probe complementary to the 5\' region of the *lacZ*gene as described above was synthesised from a PCR fragment containing the gene fragment downstream of a T7 RNA polymerase promoter, and hybridised to the blots according to the manufacturer instructions (Roche Diagnostics). Induction and detection of siRNAs to GFP or to 35S promoter sequences in agroinfiltrated plant tissue was performed as described by Canto *et al*. \[[@B47]\]. Competing interests =================== None declared. Authors\' contributions ======================= BR conceived of the study, and participated in its design, carried out the *Drosophila*expression experiments, northern blots and drafted the manuscript. SD carried out the immunoassays. TC carried out some of the siRNA assays. SM participated in the design of the study, carried out some of the siRNA assays and contributed to the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ We thank Hugh Barker for useful discussions and help with photography of cells. We thank Vicki Vance for providing us with TEV HC-Pro mutant K. SCRI is grant-aided by the Scottish Executive Rural affairs Department. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Expression of plant virus silencing suppressor genes in *Drosophila*cells.**A. Immunoblot of lysates from control cells (lanes 1,2 and 5,6), cells transiently transfected with pMT-2b (lanes 3,4) or cells stably transformed with pMT-2b (lanes 7,8). Cells represented in lanes 2, 4, 6 and 8 were induced by addition of CuSO~4~and cells represented in lanes 1, 3, 5 and 7 were uninduced. Lanes 9 and 10 were lysates from control plant or plant infected with CMV respectively. Lysates were analysed by polyacrylamide gel electrophoresis, proteins transferred to nitrocellulose by electroblotting and probed with an anti-2b antibody. The position of migration of a SeeBlue Plus 2 Pre-stained molecular weight marker (Invitrogen) band is shown. B. Northern blot analysis of RNA from control cells (lane 1) or cells transfected with pMT-HC-Pro (lane 2) or with pMT-HC-Pro/K (lane 3). Total cell RNA was separated on 1% agarose gels, transferred to nylon membrane and probed with an HC-Pro specific probe. The position of migration of a digoxigenin-labeled RNA molecular weight marker (Roche Molecular Biochemicals) band is shown. C. Northern blot analysis of RNA from DS2-HC-Pro cells (lane 1) or control cells (lane 2). RNA was examined as above. The position of migration of a digoxigenin-labeled RNA molecular weight marker band is shown. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Expression of TRV 16k protein in *Drosophila*cells.**Immunoblot of lysates from control cells (lanes 1,2 and 5,6), cells transiently transfected with pMT-16k (lanes 3,4) or cells stably transformed with pMT-16k (lanes 7,8). Cells represented in lanes 2, 4, 6 and 8 were induced by addition of CuSO~4~and cells represented in lanes 1, 3, 5 and 7 were uninduced. Lanes 9 and 10 were lysates from control plant or plant infected with TRV respectively. Lysates were analysed by polyacrylamide gel electrophoresis, proteins transferred to nitrocellulose by electroblotting and probed with an anti-16k antibody. The position of migration of SeeBlue Plus 2 Pre-stained molecular weight markers (Invitrogen) bands is shown. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Northern blot analysis of *lacZ*RNA in DS2 cells.**Upper Panels. Northern blot analysis of total cellular RNA probed with a *lacZ*-specific probe corresponding to \~500 bp of the 5\' end of the *lac*Z gene. RNA was extracted from DS2 cells (lanes 1, 2, 7 and 9), DS2-HC-Pro cells (lanes 3 and 4), DS2-16k cells (lanes 5 and 6) or DS2-2b cells (lanes 8 and 10). All cells were transfected with pMT/V5-His/lacZ and lanes 2, 4, 6, 9 and 10 were co-transfected with pMT/V5-His/lacZ and dsRNA. The *lacZ*specific band migrated between 2661nt and 4742 nt digoxigenin labelled RNA size markers. Lower Panels -- Stained gels showing loadings of *Drosophila*rRNA before blotting. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Detection of small *lacZ*gene-specific RNA.**Total RNA was isolated from *Drosophila*cell cultures and fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad). Fractionated samples were analysed in 15% polyacrylamide-UREA gels and stained with ethidium bromide before the RNA species were transferred to Hybond N+ membrane (Amersham) by electroblotting. The membrane was probed with an antisense digoxigenin-labelled lacZ transcript corresponding to \~500 nt at the 5\' end of the *lacZ*gene. The upper panel shows the small RNA species detected by probing with a lacZ gene-specific probe in DS2 cells (lanes 1, 2), DS2-HC-Pro cells (lanes 3, 4), DS2-2b cells (lanes 5, 6) and DS2-16k cells (lanes 7, 8). Cells represented in lanes 1, 3, 5 and 7 were transfected with pMT/V5-His/lacZ and those represented in lanes 2, 4, 6 and 8 were transfected with pMT/V5-His/lacZ and dsRNA. The line to the right indicates the position of GFP specific siRNAs produced in plants as described by Canto et al. (47). The lower panel shows the RNA species detected by staining of the gel before electroblotting. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Small *lacZ*gene-specific RNA is not present in the dsRNA preparation used to induce RNAi.**RNA preparations from *Drosophila*cell cultures were fractionated by chromatography using sepharose CL-2B agarose (Sigma) in Micro Bio-Spin columns (Bio Rad). Fractionated samples were analysed in 15% polyacrylamide-UREA gels, and electroblotted. A digoxigenin-labelled RNA probe complementary to the 5\' region of the *lacZ*gene was used for the detection of siRNAs in lanes 1--3. Induction and detection of siRNAs to 35S promoter sequences in agroinfiltrated plant tissue was performed as described by Canto et al. (47) and were used as markers in lanes 4 and 5. The upper panel shows the small RNA species detected by probing with a *lacZ*gene-specific probe in DS2 cells transfected with pMT/V5-His/lacZ (lane 1) or non-transfected DS2 cells (lane 2) or in the dsRNA preparation used to transfect DS2 cells to induce RNAi (lane 3). Also shown are the small RNA species present in plant tissue agroinfiltrated with a binary vector (lane 5) or in non-agroinfiltrated tissue (lane 4) detected using a digoxigenin-labelled RNA probe to 35S promoter sequences present in the binary vector as markers. The positions of migrations of the small (\~21nt) RNAs are indicated by arrows. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effects of plant virus gene silencing suppressors on RNAi in *Drosophila*cells. ::: Cell Type Transfection \% cells stained \* -------------- ----------------------------------------- --------------------- DS2 pMT/V5-His/*lacZ* 82.86 ± 9.12 DS2 pMT/V5-His/*lacZ*+ dsRNA 17.70 ± 4.25 DS2 pMT/V5-His/*lacZ*+ dsRNA + pMT-HC-Pro 45.78 ± 4.89 DS2 pMT/V5-His/*lacZ*+ dsRNA + pMT-HC-Pro-K 14.62 ± 2.81 DS2 pMT/V5-His/*lacZ*+ dsRNA + pMT-2b 11.13 ± 1.58 DS2 pMT/V5-His/*lacZ*+ dsRNA + pMT-16K 47.25 ± 3.4 DS2-HC-Pro pMT/V5-His/*lacZ* 42.04 ± 3.07 DS2-HC-Pro pMT/V5-His/*lacZ*+ dsRNA 31.70 ± 2.46 DS2-scAb S20 pMT/V5-His/*lacZ* 40.67 ± 2.36 DS2-scAb S20 pMT/V5-His/*lacZ*+ dsRNA 6.50 ± 1.2 DS2-2b pMT/V5-His/*lacZ* 41.16 ± 2.36 DS2-2b pMT/V5-His/*lacZ*+ dsRNA 9.17 ± 2.56 \* Four randomly selected fields of view each containing \~100 cells were selected in each of duplicate plates and the number of cells staining blue was counted for each experiment. The figures shown are the means from four experiments for the DS2 cells and three experiments for the DS2-HC-Pro and DS2-ScAb S20 cells. :::

PubMed Central

2024-06-05T03:55:47.672071

2004-8-25

PMC517505

Background ========== The development of the Internet has encouraged doctors to use computers and hospitals to use wireless communications \[[@B1]\], since wireless technology offers many benefits over its wired counterpart, including ease of installation and access to network information \[[@B2]-[@B6]\], and higher productivity and convenience \[[@B2]\]. One study using personal digital assistants (PDAs) connected to a network showed that the device was of limited use in transmitting data in prehospital stroke management \[[@B7]\]. Another study showed that cellular phones, pagers, or other radio-based devices will remain an important communication mode in the near future \[[@B8]\]. The advancement of wireless local area network (WLAN) technology provides the potential to allow physicians to obtain a patient\'s information anywhere, even before the patient reaches the emergency room (ER) (Orthner, personal communication). Timely access to a patient\'s information may fundamentally improve patient care \[[@B9]\] in both pre- and in-hospital settings, due to earlier doctor interventions. At present, patient data such as electrocardiograms (EKG) and demographics are seldom sent from the prehospital environment to the ER before the ambulance arrives \[[@B10]\]. As a result, some preventive measures have to be given, regardless of need (e.g., aspirin \[[@B11]\] or thrombolysis \[[@B12]\] for presumed acute myocardial infarction. However, despite the potential benefits of wireless technology in prehospital settings, the application of this technology has been slow and few related studies have been carried out. The objective of this study was to assess the ability of wireless technology to facilitate data communication between a prehospital setting and an ER (Orthner, personal communication). The idea was for all the data collected by paramedical personnel to be transmitted to an ER server from the patient\'s home, on the way to the ER, or upon arrival at the ER. Thus the transmission rate is crucial to the usefulness and applicability of the technology. To test the feasibility of wireless data transmission under the various scenarios, two separate WLANs were created, one around our office and another in a house. In this report, we discuss our testing of the wireless technology, and its potentials and limitations in simulated prehospital settings. Methods ======= The WLAN products used (Aironet 340 and 350 series wireless client adaptors and access points (APs); Cisco) offered 11-megabits-per-second (Mbps) transmission rates, built-in security features (including 40- to 128-bit encryption) and Web-based management. The transmission rates of files of different sizes were measured with different APs, patch antennae, clients, and transfer methods. The security of patient data was ensured using a centrally managed Access Control Server (ACS). Other issues such as standards, roaming, and cost are also discussed here. Within a WLAN, data are transmitted between a server and its wireless clients via an AP antenna. Both workstations and laptops were used here as servers, and file transfer rates were measured for both systems. We used Gateway Select series, Dell Inspiron series, and a Toshiba Satellite laptop computer as clients. The computers had CPU operating at 0.8--1.2 GHz, 256--1024 MB of RAM, and 10--40 GB hard drives, and all ran the Microsoft Windows 2000 Professional operating system. PDAs (Ipaq Pocket PCs, models 3550 and 3570, 200-MHz CPU, 32--64 MB RAM, Compaq) were also tested as wireless clients. The wireless coverage was tested using two APs (Aironet 340 and 350 series, Cisco) and a patch antenna (S2406P, Cushcraft Corporation). The feasibility of using cellular phones (StarTac 7868, Motorola) in data transmission in the area not covered by the WLAN was also tested. The various software used in the study for wireless client management, file transfer, and access control included the Aironet Client Utility (Cisco), Link Status Meter (LSM, Cisco), the ACS (V3.0, Cisco), Phone Tools (BVRP Software) for faxing, and file transfer protocol (FTP) for measuring the file transmission rate. The software LSM classifies the link status as the percentage of maximum signal strength and quality: \"excellent\" (\>75%), \"good\" (40--75%), \"fair\" (20--40%), or \"poor\" (\<20%); where signal strength and quality refer to the client adapter\'s radio signal at the time packets are being received, quantified as bytes transmitted and received and the errors that occur. Detailed descriptions of the mentioned software are available from the manuals provided by the vendors. The WLAN and its configuration ------------------------------ The Aironet 340 and 350 series APs were tested by a two-step approach. In the first step, one AP was connected directly with the server that was not connected to the campus Ethernet backbone. In the second step, the AP was assigned a public Internet Protocol (IP) address and connected to the Ethernet backbone in the Susan Mott Webb Nutrition (Webb) Building at the University of Alabama at Birmingham. The IP address was assigned to an AP through either HyperTerminal or a Web console using a Web browser. An administrator ID and password were then created to enhance the Web console security. The client computer required a type II PCMCIA (Personal Computer Memory Card International Association) card slot. Every client needed a functional IP address to become associated with the AP. The Wired Equivalent Privacy (WEP) keys were enabled for both the AP and the clients to ensure two-way authentication. Comparison of different coverage of APs and patch antennas ---------------------------------------------------------- The coverage of the WLAN was found to highly structure dependent. The floor of the Webb building measures about 60 by 25 meters. A single Aironet 340 AP was unable to cover the entire floor with a \"good\" link status. This was achieved using two (more powerful) Aironet 350 APs. Achieving the \"excellent\" link status on the floor required the use of the S2406P (Cushcraft) patch antenna. The wireless clients associated with the AP had a \"fair\" or \"good\" status one floor up and one floor down from the floor where the AP was located. There was a small area outside the 5-floor building in which the clients could associate with the AP with a \"poor\" status. Results ======= Link statuses around a simulated patient\'s home ------------------------------------------------ In order to test the feasibility of implementing an isolated LAN around a patient\'s home, we chose a two-story house with the layout shown in Figure [1](#F1){ref-type="fig"}. Two simulated scenarios were tested: one with an ambulance parked on the street next to the house (Figure [1A](#F1){ref-type="fig"} and [1C](#F1){ref-type="fig"}), and the other with the ambulance parked next to the house in its parking lot (Figure [1B](#F1){ref-type="fig"} and [1D](#F1){ref-type="fig"}). Two connection modes under each scenario were also tested: one mode used the AP-client connection (Figure [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}) and the other used a peer-to-peer connection without the AP (Figure [1C](#F1){ref-type="fig"} and [1D](#F1){ref-type="fig"}). The link statuses both inside and outside the house were at least \"good\", and some of the area close to the AP in both scenarios had \"excellent\" coverage. This suggested that an ambulance with a patch antenna could communicate at a reasonable rate with handheld devices operating inside the patient\'s home, through either an AP or a direct peer-to-peer connection. File transfer rate with laptops ------------------------------- To quantify the baseline signal strength, one Aironet 350 AP was tested in the open area: \"excellent\", \"good\", \"fair\", and \"poor\" link statuses were obtained within 10, 25, 50, and 100 feet (\~4, 10, 20, and 39 meters), respectively. As shown in Table [1A](#T1){ref-type="table"}, the file transfer rate for a 10-MB file was 224--2,000 kbps, depending on the distance. Interestingly, higher rates were reached for files of size 10--100 MB. The different methods and directions of file transfer might affect the rate. Methods such as FTP transfer, copying and pasting between folders, and Microsoft Briefcase synchronization were tested. Other factors that may affect transmission were also tested, including the initiation direction (pulling or pushing, in terms of the choice of client and server; see below) and relative physical motion. One Gateway workstation and two Inspiron 4000 laptops were used. To simplify the test, a single medium-sized file (50 MB) was chosen for the purpose. We chose a 50-MB file since it corresponds to a typical high-quality EKG image. As indicated in Table [1B](#T1){ref-type="table"}, pushing refers to a transfer from the server to clients if initiated from the server side, and from a client to the server if initiated from the client side; whereas pulling refers to the transfer from the server to clients if initiated from the client side, and from a client to the server if initiated from the server side. Pulling a file led to a higher rate of transfer, in all link statuses (i.e., distances). The speed was lower when two clients transferred the 50-MB file at the same time, and it was generally lower for file transfer between two clients than between a server and a client (Table [1B](#T1){ref-type="table"}, Sections III-IV). As expected, the transfer rate was slower while the client device was moving (as shown in Table [2B](#T2){ref-type="table"}, section V). However, it was still found that in a WLAN environment, paramedics carrying data-collecting devices could move around and transmit data by different methods and directions at a reasonable rate. The simultaneous file transfer that involves multiple clients/users is a more likely scenario in real emergency settings. Under a \"good\" link status, the ability of five laptop computers to pull a single file from the FTP server was tested both individually (Table [2A](#T2){ref-type="table"}) and simultaneously (Table [2B](#T2){ref-type="table"}). The data rate reached 5.9 Mbps when a single client was transferring, and fell to below 2 Mbps when multiple clients were involved simultaneously (based on four independent tests). This lower rate, however, is still within a reasonable range, considering the file size. File transfer rate with Pocket PC and cellular phone ---------------------------------------------------- We chose Ipaq Pocket PCs as PDAs due to their relatively large amount of RAM compared to other PDAs, and a cellular phone (Motorola) as alternative data transmission device when the PDA was outside the WLAN coverage (to simulate the scenario when a long-range antenna, such as a yagi antenna (Cisco), is not available). The transfer rates for single files of different sizes are summarized in Table [3A](#T3){ref-type="table"}. The faxing speed through the phone did not appear to be correlated with the file size, since a 50-fold difference in file size resulted in a 30% difference in the time needed to complete the file transmission. The reason is unknown, and should be further investigated. Enhancing the WLAN security using an ACS ---------------------------------------- A Cisco V3 ACS was used to improve the security of the network \[[@B13]\]. As shown in Figure [2](#F2){ref-type="fig"}, the Active Directory facility of a Windows 2000 Server was used as a network access server to communicate with the ACS for authentication, authorization, and accounting (AAA) \[[@B14]\]. The ACS was able to control client access to the network through the AAA process. A private local area network ---------------------------- When managing a WLAN with many clients, there are often insufficient public IP addresses. The solution is to use either the Dynamic Host Configuration Protocol (DHCP) or a private LAN with a Network Address Translation (NAT) server. DHCP services are not easily managed and sometimes create security challenges to network administrators in determining the user\'s identity. A private LAN seems to be a better solution and more applicable in most ER environments where many wireless clients may transmit at same times, thus maintaining relatively high-speed connections. Figure [3](#F3){ref-type="fig"} shows a private LAN with a NAT server that was configured and tested. All wireless clients were centrally managed through an ACS server. The file transfer rates were similar to those achieved with public IP addresses under similar conditions (data not shown). Cost of a small-scale WLAN -------------------------- Health-care organizations have traditionally been slow in accepting WLAN technology in clinical practice. One of the major concerns has been its cost \[[@B15],[@B16]\], followed by security \[[@B17]-[@B20]\], although the benefits of WLANs have been demonstrated in many fields including telemedicine \[[@B21]\]. The cost of a simple WLAN like the one tested here was calculated (Table [4](#T4){ref-type="table"}). Discussion ========== Synchronization --------------- After collecting all patient data at a patient\'s home, the data must ultimately be transferred to the ER. This involves two critical synchronization steps: (1) from the patient\'s home to a server on the ambulance, and (2) from the ambulance to the ER (while in transit or upon arrival). Here the WLAN was employed for both of these steps, using Microsoft Briefcase and Windows Workgroups. Automatic synchronization with the destination server and batch synchronization were desired. The ultimate objective is, however, to link the two synchronization steps using a long-range antenna that reaches up to 25 miles (e.g., a yagi antenna from Cisco). This would significantly shorten the time needed to transfer data from a patient\'s home to the ER, since the data will reach a ER WLAN earlier. We are currently performing the associated experiments. We also tested the use of a cellular phone and other types of PDA (e.g., a Palm Pilot) with network capabilities in transmitting a small (up to 50 MB) but critical file. The results suggested that cellular phones or PDAs with network cards can be effective alternatives to the use of a long-range antenna to transmit data from a patient\'s home to the ER. Wireless transfer of EKG data ----------------------------- EKG data are considered very valuable in the early detection, early intervention, and possibly better outcome of heart attack patients \[[@B9]\]. The use of a wearable device with sensors to monitor specific physiological signals and communicate with a personal server has been reported \[[@B22]\]. Land-based telephone lines have also been employed to transmit EKG data and for monitoring by clinical personnel \[[@B23]\]. In our study, we showed that files up to 5--10 MB (the size of a typical high-quality digitalized EKG image) could be transferred using FTP or other file transfer methods within minutes. Handheld devices such as a Pocket PC and cellular phone may be useful in transmitting EKG files when the ambulance is still at the patient\'s home, as shown in Table [3](#T3){ref-type="table"}. The time required to transmit a file is proportional to its size in the case of a Pocket-PC-to-laptop transfer, but this was found not to be the case between a cellular phone and a fax machine. The reason for this discrepancy is unknown, and needs to be further investigated. The use of a long-range antenna may ultimately be needed to increase the transmission capacity and speed. Interferences ------------- The WLAN operates at 2.42 GHz with an output power of 100 mW, which may pose a risk of interference with medical devices using similar frequencies. Previous studies have shown that a WLAN may interfere with medical devices in close proximity \[[@B24]\] but is unlikely to be interfered with by such devices \[[@B25]\]. Further studies are needed to clearly address the possibility. In another study, infrared modems exhibited a similar performance to a wired system even in an electrically noisy environment \[[@B26]\], indicating that infrared wireless connectivity can be safely and effectively used in operating rooms. These studies suggest that a WLAN can be acceptable for use in prehospital settings if careful interference testing is conducted. Security and privacy -------------------- The major concern over a WLAN is its security \[[@B17]-[@B20]\], especially when personal information is involved. It has been reported that the open-air clear-text transmission of WEP keys and MAC addresses increases network vulnerability \[[@B13],[@B27]-[@B29]\]. One approach to minimizing the risk is to control the access of remote and/or wireless clients through the Remote Access Dial-in User Service and AP management using the Extensible Authentication Protocol. The regulation by the Healthcare Insurance Portability and Accountability Act may further delay an organization\'s decision to adopt WLAN technology, although both the Institute of Electrical and Electronic Engineering (IEEE) 802.3 and the OpenAir standard specifications offer security protection (these are the two major standards in the unlicensed commercial 2.4-GHz WLAN market). According to our experiences, the following steps are required to implement a secure WLAN. First, anonymous access should be disabled and the Service Set Identifier of an AP and data encryption key (WEP key) should be enabled. Secondly, a Web console should be used to designate an administrator and manage APs and clients. Thirdly, an ACS server such as Cisco Secure ACS should be implemented to work with Active Directory in order to offer both device- and user-dependent AAA services. Digital certificates should be applied whenever possible for mutual authentication to protect sensitive information through secure server access and secure Web access. In addition, the physical security of the APs, client, and server computers can never be overemphasized. Standards and interoperability ------------------------------ The IEEE 802.11 specification addresses both the physical and MAC layers (Orthner, personal communication), and the OpenAir 2.4 interface standard is derived from the Wireless LAN Interoperability Forum \[[@B29]\] and needs to be interoperable with the IEEE 802.11 standards. The 5-GHz band WLAN standard (IEEE 802.11a) will become more popular once its cost decreases and the required components become more widely available. The use of standardized compliant devices facilitates communication and interoperability. Limitations of the study ------------------------ The present study was mainly based on the Windows operating system and Cisco wireless products. IEEE 802.11a products for the next generation of WLANs are emerging quickly from various vendors. Hence the stability, compatibility, and interoperability with other vendors require further evaluation. Although currently it is relatively expensive to implement a WLAN using this new protocol, the prices and capabilities are expected to improve within the near future. Conclusions =========== Application of WLAN technology will help both paramedics and other health-care professionals in their daily acquisition of information in a localized area such as within a patient\'s home, an office, a small clinic, or an ER. Implementing a WLAN in a centrally managed and multiple-layer-controlled ACS is the key to ensuring its security and accessibility. Future studies should focus on product capacity, speed, compatibility, interoperability, and security management. Competing interests =================== None declared. Authors\' contributions ======================= D. Chen, the principal investigator, was most involved in conducting the experiments. H.F. Orthner participated in data collection in the simulated patient\'s home. H.F. Orthner was the sponsor and S.-J. Soong and G.J. Grimes were the advisors of the fellowship awarded to D. Chen from the National Library of Medicine, National Institute of Health, and they contributed significantly to the design, coordination, and performing of experiments. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6947/4/12/prepub> Acknowledgements ================ This project has been funded in part with US federal funds from the National Library of Medicine, National Institute of Health, under Contract No. N01-LM-0-3524 and under Fellowship No. F38LM07185. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **An aerial view of the use of a patch antenna at a simulated patient\'s home.**The AP -- clients connection (A and B) and peer-to-peer connection (C and D) configurations were tested. The arrows indicate the locations and the beaming directions of the APs with a patch antenna. The stars indicate the locations of the wireless devices in the peer-to-peer connections. The link statuses were measured as described in the Methods: the solid and dashed circles indicate \"excellent\" and \"good\" statuses, respectively. Panel A: Simulated ambulance parked on the street. Panel B: Simulated ambulance parked in the parking lot of the house. C and D: Peer-to-peer connection was created between two wireless clients. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **The Cisco Secure ACS and its configuration.**The AP was configured to use ACS AAA services for the Extensible Authentication Protocol (EAP)-enabled wireless devices over the WLAN. A Windows 2000 Server running Active Directory (AD) was used to mimic a network access server (NAS) to negotiate with ACS through the Remote Access Dial-in User Service (RADIUS) protocol. The NAS had an enabled RAS connection for the ACS. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **A private WLAN with an ACS.**A workstation running the Linux operating system functioned as a Network Address Translation (NAT) server. The private LAN was linked with the Internet through a switched hub or router. The AP was configured to use ACS for AAA services for the EAP-enabled wireless devices over the WLAN. A Windows 2000 Server running Active Directory was used to mimic a NAS to negotiate with ACS through the RADIUS protocol. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### File transfer directions and rates (kbps, mean ± SD, *n*= 4) ::: **A. File transfer rate in an open area between two laptops for different file sizes and link statuses** ---------------------------------------------------------------------------------------------------------- -------------- -------------- ------ ----- Excellent 400 2000 1660 980 Good 210 890 1110 830 Fair 90 660 740 660 Poor 60 220 250 340 **B. Transfer rates for a 50-MB file between a server and clients under different link statuses** **I. Server to one client** **Pushing** **Pulling** Excellent 3090 ± 70 5540 ± 180 Good 2870 ± 280 5530 ± 130 Fair 630 ± 500 2070 ± 550 **II. One client to server** **Pushing** **Pulling** Excellent 2600 ± 110 5210 ± 410 Good 2710 ± 330 4350 ± 1030 Fair 340 ± 80 750 ± 440 **III. Two clients to server** **Client 1** **Client 2** Excellent 2160 ± 60 3130 ± 320 Good 2140 ± 370 3230 ± 40 **IV. Client 1 to client 2** Excellent 2400 ± 70 Good 2560 ± 130 **V. Server to one client while moving** Excellent 2500 ± 500 Good 1200 ± 410 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Rate of file transfers involving multiple clients (kbps, mean ± SD, *n*= 4) ::: **A. Transferring files individually** ------------------------------------------ ------------- ------------- ------------- ***Client*** ***50 MB*** Gateway Solo 5830 ± 1340 Toshiba Satellite 350 5250 ± 600 Inspiron 8000 5260 ± 990 Inspiron 4000-1 4890 ± 490 Inspiron 4000-2 5960 ± 910 **B. Transferring files simultaneously** ***Client*** ***50 MB*** ***40 MB*** ***30 MB*** Gateway Solo 1330 ± 180 1430 ± 140 1740 ± 490 Toshiba Satellite 350 1600 ± 340 1580 ± 160 1610 ± 240 Inspiron 8000 1400 ± 190 1470 ± 140 1500 ± 130 Inspiron 4000-1 1490 ± 200 1580 ± 240 1590 ± 260 Inspiron 4000-2 1240 ± 440 1540 ± 190 1560 ± 280 ***20 MB*** ***10 MB*** ***1 MB*** Gateway Solo 1690 ± 200 1900 ± 400 7140 ± 110 Toshiba Satellite 350 1850 ± 60 1860 ± 350 6830 ± 120 Inspiron 8000 1730 ± 170 1820 ± 220 4360 ± 2800 Inspiron 4000-1 1690 ± 190 2000 ± 470 3780 ± 1060 Inspiron 4000-2 1840 ± 310 1770 ± 400 3160 ± 1400 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Pocket PC and cellular phone file transfers (SD: 10\~25%, *n*= 4) ::: **A. Pocket PC to laptop** **B. Cellular phone to a fax machine** ---------------------------- ---------------------------------------- ------ ----- 10 \<1 10 134 100 2 100 155 1000 15 500 166 5000 150 1500 180 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Cost of a WLAN of similar scale to the one implemented here ::: ***Component*** ***No. needed*** ***Unit price (US \$)*** ***Total US\$*** ---------------------------------- ------------------ -------------------------- ------------------ Workstation and accessories 1 2500 2500 Laptop and accessories 2 2500 5000 Pocket PC and accessories 3 600 1800 Palm Pilot and accessories 2 500 1000 APs 2 400 800 Patch antennas 2 200 400 Wireless adaptors 5 300 1500 Cellular phone and network costs 1 250 250 **Total (US \$)** **13250** **Note**: software costs are usually associated with the purchase of hardware and not included. :::

PubMed Central

2024-06-05T03:55:47.674441

2004-8-31

PMC517506

Background ========== Much research has been carried out with the aim of influencing the performance of clinicians. The results have varied \[[@B1],[@B2]\]. As with any human behaviour, clinical practice is difficult to change. Some strategies that have been evaluated, like passive dissemination of clinical practice guidelines, have had little or no effect on practice \[[@B3]\]. Others, like educational outreach visits (\"academic detailing\") and multifaceted interventions, may be more effective than passive interventions \[[@B1]\]. The reasons why clinical practice sometimes is not consistent with current best evidence varies across clinical problems and from one clinician to another. A logical consequence of this is to tailor quality improvement strategies to address specific barriers \[[@B4]\]. Several trials of tailored interventions have been conducted. The methods used for identifying barriers to change have varied and there is limited evidence of the relative usefulness of different approaches. However, the choice of method for identifying barriers has implications, particularly with regards to resources, since some methods are time consuming and demand the involvement of many individuals. This represents a practical and financial constraint. On the other hand, if such approaches lead to the identification of important barriers that otherwise would have been overlooked, they may be worth the effort. In this article we describe a simple approach we have used to identify barriers to changing professional practice. This was done as the first step in a process of developing an intervention to improve the pharmacological management of hypertension and hypercholesterolaemia \[[@B5]\]. The intervention focused on three specific recommendations in clinical practice guidelines for hypertension and hypercholesterolaemia \[[@B6]-[@B8]\] based on evidence of a gap between the recommendations and current practice in Norway: • Contrary to recommendations, physicians seem to rarely estimate the risk of cardiovascular disease before initiating treatment \[[@B9]\] • Sales of thiazides are low, despite these drugs being recommended as first-line medication \[[@B10]\] • Relatively few patients reach recommended treatment goals \[[@B11],[@B12]\] We also report the results of a post hoc exercise and a survey we carried out to evaluate our approach to identifying barriers and interventions. Methods ======= We developed the intervention through a process of identifying barriers to implementation of recommendations and measures specifically addressing these barriers (\"tailoring\"). The methods we used were structured reflection, searching for other relevant trials targeted at improving the management of hypertension or hypercholesterolaemia, conducting a survey among general practitioners and discussion with physicians during pilot testing of the intervention. Structured reflection --------------------- The three authors reflected over possible barriers based in part on our own experience as physicians working in primary care in Norway. We used a worksheet to structure our reflection (see Additional file 1). The worksheet included factors that might act as barriers in the practice environment, the professional environment, and related to physicians\' knowledge, skills and attitudes. One worksheet was completed for each targeted behaviour: increasing the use of cardiovascular risk assessment before initiating treatment for hypertension or hypercholesterolaemia, increasing the prescribing of thiazides for the treatment of uncomplicated hypertension, and increasing the proportion of patients on medication for hypertension and hypercholesterolaemia that reach recommended treatment goals. The worksheet was used to facilitate our group discussion of possible interventions to address the identified barriers. Our research group had recently completed a trial of a strategy for guidelines implementation when we were planning this study \[[@B13]\]. In that study the multifaceted intervention consisted of several passive components. Information and materials were distributed by mail and to a large degree we relied on the physicians themselves to make an effort at changing their practice. The observed changes in practice were small. In another trial we had found that the use of active sick leave for back patients was significantly increased through a proactive intervention compared to a passive one \[[@B14]\]. Based on these experiences our research group decided to test an active strategy in this study. Therefore we decided to use outreach visits (\"academic detailing\") prior to considering specific barriers. We considered systematic reviews of interventions to improve professional practice when we designed our strategy \[[@B1]\]. We searched the Cochrane Group of Effective Care and Organisation of Care <http://www.epoc.uottawa.ca> trial register for trials of interventions targeted specifically at the management of hypertension or elevated cholesterol in general practice. Questionnaire to physicians --------------------------- We surveyed general practitioners about some of the interventions about which we were uncertain after our structured reflection. The details of the survey have been described elsewhere \[[@B9]\]. Briefly, 265 physicians who had participated in an earlier trial conducted by our research group \[[@B13]\] were asked to complete a questionnaire as part of the study-evaluation. We used that opportunity to seek answers to the following questions: 1\. Do physicians assess cardiovascular risk before prescribing antihypertensive or cholesterol-lowering drugs? 2\. If not, would physicians be more likely to do so it they received a fee for this? 3\. Do physicians comply with current regulations limiting the reimbursement of cholesterol-lowering drugs? The last question was asked for two reasons. Firstly, we were considering making risk assessment a condition for reimbursement of the drugs. Secondly, the existing regulations were a possible barrier to adhering to our recommendations because they conflicted with these. Pilot testing ------------- During pilot testing of the intervention at two practices, which were selected for convenience, comments from physicians relevant to possible barriers were noted. We also informally evaluated each component of the intervention. Post hoc focus groups and structured reflection exercise -------------------------------------------------------- After we had finished designing the intervention we had the opportunity of testing our method of structured reflection at a gathering of international researchers in the Research Based Education and Quality Improvement group (ReBEQI) <http://www.rebeqi.org>, December 2003. Each participant was asked to complete a worksheet to identify barriers and possible interventions related to the low use of thiazides among general practitioners. They were randomly allocated to four different groups where they collaborated on completing the worksheet. They were also asked to grade the importance of each barrier or intervention as minor, moderate or major. We disregarded those rated as minor. We compared the results from the four groups with the barriers and interventions we had identified. Post hoc survey of physicians exposed to the intervention --------------------------------------------------------- While conducting the randomised trial to test the effectiveness of our multifaceted intervention we carried out telephone interviews with physicians allocated to the experimental group. They were asked if they adhered to our recommendations and, if not, why. The responses where noted down during the interviews. Results ======= Barriers and interventions -------------------------- Figure [1](#F1){ref-type="fig"} illustrates the timeframe for the methods used to identify barriers and interventions. Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"} give an overview of the barriers and interventions that we identified for each clinical problem. Many of the barriers were related to a lack of knowledge and could be addressed through educational interventions. The use of educational outreach visits was logical since we had planned to use an active intervention, based on our previous experience, and since this type of intervention has consistently lead to improved professional behaviour in randomised trials \[[@B1]\]. Similarly, based on previous experience and the capabilities of the software we hade developed \[[@B13]\], we planned on using an electronic risk calculator, electronic prescriptions, patient information materials, and computerised reminders. The search (July 2001) of the EPOC trial register for randomised trials with the word \"hypertension\" in any field yielded 58 references. Most were excluded after reading the abstracts, leaving eight, for which the full text was reviewed \[[@B15]-[@B22]\]. This did not lead to any changes in our intervention strategy. A search for randomised trials with the word \"cholesterol\" yielded 13 references. The full text was reviewed for only one of these \[[@B23]\]. This also provided little further guidance for designing our intervention. The nine trials that we reviewed are summarised in table [4](#T4){ref-type="table"}. The survey results did not indicate that a fee for estimating cardiovascular risk before initiating drug therapy would affect practice \[[@B9]\]. The survey results also indicated that physicians are largely not affected by conditions for drug reimbursement \[[@B9]\]. Moreover, there were no mechanisms in place to enforce such regulations. We did not identify additional barriers during pilot testing of the intervention with five physicians in two practices, but several of those already identified were confirmed, particularly barriers to prescribing thiazides. Based on our findings and an assessment of the feasibility and evidence of effectiveness for various interventions, we designed a multifaceted intervention. The elements of the intervention are described in table [5](#T5){ref-type="table"}. We also considered a number of interventions that we excluded. For example: • We considered placing computers in waiting rooms so that patients could assess their cardiovascular risk before seeing the physician, but concluded this would be costly and difficult to implement. • We considered providing pre-printed prescriptions, but found this would not to be relevant because most physicians use computerised systems for prescribing. • We considered exposing conflicts of interest among clinical specialists who advocated using other first line drugs than thiazides, but elected not to do so. • We considered exposing techniques used in pharmaceutical advertisements, such as using relative risk reductions rather than absolute risk reductions \[[@B24]\], but concluded this would have at best a limited impact. Post hoc focus groups and structured reflection exercise -------------------------------------------------------- Nineteen researchers were divided into four groups. All groups considered advocacy by drug companies to be a major barrier to change. Routines or habits were also included as an important barrier by all the groups, as well as lack of knowledge concerning the effectiveness of thiazides, their favourable adverse effects profile, and their low cost. All the groups also mentioned competing guidelines or diverging opinions as part of the problem. Three of the groups considered local or national opinion leaders as potential barriers to change. Patients\' expectations or perceived expectations were also mentioned by three of the groups. The interventions recommended by the groups to address the identified barriers are presented in table [6](#T6){ref-type="table"}. All the groups suggested the use of computerised reminders to address physicians\' lack of knowledge or their habits and routines. All the groups also suggested some form of interactive education, mainly as a counter force to promotional activities by the pharmaceutical industry, and patient information was suggested by three of the groups. Two suggested training physicians to address patient expectations. Two groups suggested developing clinical guidelines and two suggested audit and feedback, but one group considered this to be of minor importance. Post hoc survey of physicians exposed to the intervention --------------------------------------------------------- Among the 195 physicians exposed to the intervention, 149 (76%) were contacted during the trial period and agreed to answer our questions. No major additional barriers were identified. However, some physicians questioned whether adhering to the recommendations would represent a good use of resources, specifically the recommended treatment goals. Discussion ========== Addressing barriers to change with tailored interventions makes sense and there is some empirical support for this \[[@B1]\]. It is unclear, however, what methods are the most useful for identifying barriers and interventions. Several qualitative methods can be used to identify barriers, such as interviews, focus groups and observation. These methods may be valuable, but they are relatively labour-intensive. We used a simpler approach to identifying barriers to change. Would the use of other methods have provided us with important additional information? Pilot testing and discussions with five physicians in two practices and interviews with 140 participating physicians did not indicate additional barriers. The post-hoc focus groups with international experts did not add much with regards to barriers and interventions. Several of these groups included \"routines and habits\" as a potential barrier, which was not explicitly mentioned among the barriers identified by the investigators. However, all interventions that were mentioned by more than one of the groups in the post-hoc focus group exercise were included in our multifaceted intervention. Our use of computerised reminders was based on the assumption that this would help to establish new routines, although we did not record routines and habits as a barrier when we developed the intervention. There are inherent weaknesses in our approach. One is that the investigators undertaking the structured reflection were few and we were prejudiced by our own experiences. The lack of patient involvement is another limitation, which possibly lead to an under-emphasis of patient-mediated interventions. A weakness with the group of international researchers who participated in the post-hoc focus groups is their lack of familiarity with the Norwegian context. A number of trials of tailored interventions have been conducted. The methods used to identify barriers to change have varied. Some investigators have simply hosted a meeting \[[@B25],[@B26]\], others have used questionnaires \[[@B23]\], conducted focus-groups \[[@B27]-[@B30]\] or interviews \[[@B31]-[@B33]\], or both \[[@B34]\]. Others have used a combination of several qualitative methods \[[@B35]-[@B37]\]. Some investigators have used identification of barriers as an intervention in itself \[[@B19],[@B38],[@B39]\]. The methods that were used have been poorly described in most of these studies. Conclusions =========== Our simple approach to identifying barriers to improving practice appears to have been effective in identifying all of the important barriers, and it was efficient. However, we do not know for certain what barriers other methods would have identified or whether the intervention could have been more effective, if we had used other methods. Further work to address these questions is planned, including direct comparison of alternative methods and evaluations of theory-based approaches <http://www.rebeqi.org>. The effectiveness of our multifaceted intervention is under evaluation in a randomised controlled trial. Competing interests =================== None declared. Author contributions ==================== All the authors participated in the process of structured reflection and in conducting the survey of physicians. AF was responsible for reviewing the results from previous research and pilot testing of the intervention. AF drafted the article while SF and ADO contributed to critical revisions of the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/4/23/prepub> Acknowledgements ================ We thank Kari Håvelsrud for her contributions in carrying out the surveys among physicians, and the ReBEQI-group (Research Based Education and Quality Improvement) for participating in the post hoc exercise. We also thank Dr. E. Borna in Oslo, Norway, and the physicians at Sentrumsklinikken, Asker, Norway for piloting the intervention, and the 149 physicians who participated in the telephone interviews. This work was carried out as part of a project funded by the Norwegian Ministry of Health and the Norwegian Directorate for Health and Social Affairs. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flow chart indicating time frame for methods used to identify barriers and interventions. \* The trial period ended in December 2003, and data collection and analysis will be completed in September 2004. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Barriers to carrying out cardiovascular risk assessment, and possible interventions to address these ::: -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Possible barriers** **Possible interventions** ------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------------- Time-consuming procedure for the physician \- Easy-to-use tools (e.g. risk calculator for computer)\ - The patients or an assistant could carry out the risk assessment\ - Offer economic compensation Physician has no risk assessment tool at hand \- Provide risk assessment tool The patients are focused on single risk-factors, not the global picture \- Patient-information Physicians are not used to risk estimation, not educated to do this \- Educational outreach visit\ - Training Lack of knowledge among physicians of the relevance of global risk assessment \- Information/education (outreach visit) Physicians have more trust in their own clinical judgement than tables or charts \- Education (e.g. case discussions during outreach visit) Differences in opinion among physicians on the importance of treatment of hypertension and hypercholesterolaemia \- Use opinion-leaders and convince clinicians of the high degree of consensus in clinical guidelines May be uncomfortable for physicians to discuss risk-factors with patients \- Patient-information\ - Offer strategies on how to communicate such information with patients -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Barriers to prescribing thiazides for the treatment of hypertension, and possible interventions to address these ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Possible barriers** **Possible interventions** ----------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------- Physicians are neither familiar with the relevant brand-names nor to the use and follow-up of these drugs \- Pre-printed prescriptions, also in electronic format\ - Patient information\ - Support for the clinical follow-up Few other clinicians use these drugs \- Patient information\ - Active promotion of thiazides (through educational outreach visits)\ - Point out the consensus among guidelines that thiazides are a first-line drug Specialists may be prescribing other drugs \- Identify opinion leaders that advocate the use of thiazides\ - Look into possible conflicts of interest Advocacy by pharmaceutical companies \- Point attention to the importance of clinically relevant endpoints when studies are quoted (during educational outreach visits)\ - Review advertisements to identify the main lines of reasoning that are being used Physicians are worried about possible side-effects and lack of anti-hypertensive effect. \- Educational outreach visits Thiazides considered old-fashioned \- Argue that these drugs have been thoroughly tested over many years (during educational outreach visits) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Barriers to reaching recommended treatment goals and possible interventions to address them ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Possible barriers** **Possible interventions** --------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------- Physicians are not accountable to anyone \- Feed-back on to what extent treatment goals are reached among his/her pool of patients (audit) Physicians are unsure of what treatment goal to use \- Give clear treatment goals (during outreach visit)\ - Point at the relatively high degree of consensus among guidelines (during outreach visit) Reluctance and unclear strategy among physicians on how to deal with insufficient treatment \- Support for decision-making if goal is not reached Physicians may be underestimating the consequence of under-treatment \- Educational outreach visit ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Studies targeting the management of hypertension and/or hypercholesterolaemia ::: **Study** **Objective** **Intervention** **Comment** ----------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Bass 1986 \[15\] Improve the detection and management of hypertension Medical assistant oversaw screening, attended to education, compliance and follow-up We did not consider this to be a feasible intervention in our setting Aucott 1996 \[19\] Implement guidelines for cost-effective management of hypertension on medication use and cost, blood pressure control, and other resource use Intensive guideline-based education and supervision (identification of clinical champion, faculty education and development, assignment of PharmD, clinic-based education and precepting of clinicians, monthly feedback to practice) Most elements of this multifaceted intervention were already included in our own. The trial was conducted in a general internal medicine teaching clinic, which limits the relevance to our primary care setting Rossi 1997 \[16\] Alter prescribing habits for the treatment of hypertension Guideline reminders placed in the charts of patients Computerised reminders were already included as part of our multifaceted intervention Goldberg 1998 \[17\] Increase compliance with national guidelines for the primary care of hypertension (and depression) Academic detailing with or without continuous quality improvement (CQI) teams Academic detailing (outreach visit) was already included as part of our multifaceted intervention. The study-findings did not support the use of CQI teams Maclure 1998 \[18\] Increase understanding of the way in which dissemination of evidence changes medical practice Media stories, national warning letter, teleconference, small group workshops, and newsletters Our outreach visits were planned as interactive sessions, thus serving the same purpose as small group workshops or teleconferences. We did not believe that passive distribution of material would be useful Hetlevik 1998 \[21\] Implement clinical guidelines in the treatment of hypertension Computer based clinical decision support system, mailed feedback of current practice, invitation to seminar at conference Most interventions were already included in our multifaceted intervention. We did not believe that inviting to conference-seminar would be useful van der Weijden 1999 \[23\] Assess the feasibility and evaluation needs of a cholesterol guideline Group education, desktop supportive materials, feedback on performance, and face-to-face instruction on location Most interventions were already included in our multifaceted intervention Montgomery 2000 \[20\] Have an effect on absolute cardiovascular risk, blood pressure, and prescribing of cardiovascular drugs Computer based clinical decision support system plus cardiovascular risk chart; or cardiovascular risk chart alone Both interventions were already included in our multifaceted intervention Demakis 2000 \[22\] Improve resident physician compliance with standards of ambulatory care (including hypertension) Computerised reminder system Intervention was already included in our multifaceted intervention ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### The final multifaceted intervention ::: **Educational outreach visit** ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- \- Presentation focusing on three main messages:  1. Relevance of risk estimation and how to do it, including strategies on how to communicate information about risk to patients.  2. Information on evidence in support of effect and the unjustified fear of adverse effects regarding thiazides, pointing at the consensus that exists among guidelines. Attention also directed to the importance of clinically relevant endpoints when studies are quoted.  3. Clear recommendations justified by referring to high degree of consensus among guidelines. \- Guidelines handed out, directing attention to the authors (opinion leaders) **Audit & feed-back at outreach visit** \- To what extent treatment goals are achieved. \- Drug-choice profile on anti hypertensives \- Level of risk among patients on treatment, compared to a sample (men 40--65 years) not on treatment **Computerised reminders** \- Risk assessment \- First-choice antihypertensive drugs \- Treatment goals **Risk assessment tools as charts and in electronic format** **Patient-information material** \- The relationship between single risk factors and global risk \- Thiazides and beta-blockers. \- Treatment goals ::: ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Interventions to address identified barriers (main results from post-hoc focus group and structured reflection exercise) ::: **Barrier: Marketing activities by pharmaceutical industry** ----------------------------------------------------------------------- ----------------------------- ------------------------------------------------------------------------------------ ----------------------------------- **Importance of barrier\*** **Interventions** **Importance of interventions\*** Group 1 3 \- Competing approaches (educational materials; interactive educational workshops) 2 Group 2 3 \- Outreach visits 3 Group 3 3 \- Small group peer comparison 1--2 Group 4 3 \- Continuing education system Not graded **Barrier: Routines and habits** **Importance of barrier\*** **Interventions** **Importance of interventions\*** Group 1 3 \- Computerised reminders 3 \- Audit and feedback 2 \- Intention plus/trial of behaviour 2 Group 2 2 \- Reminder 3 \- Direct mail 3 Group 3 2/3 \- Audit and feed back with peer comparison 1 Group 4 Not graded \- Computerised reminders Not graded **Barrier: Lack of knowledge** **Importance of barrier\*** **Interventions** **Importance of interventions\*** Group 1 2 \- Educational material/guidelines 2 \- Interactive educational meetings 2 Group 2 2 \- Information to patients 2 \- Local quality circles 2 Group 3 Not graded \- Financial incentives 3 \- Reminders/Computerised Decisions Support Systems 2 Group 4 Not graded \- Continuing medical education Not graded \- Computerised reminders **Barrier: Opinion leaders (or specialists) or competing guidelines** **Importance of barrier\*** **Interventions** **Importance of interventions\*** Group 1 3 \- Develop national guidelines 2 Group 2 3 \- Use opinion leaders 3 Group 3 2 \- Guidelines shared by primary and specialist physicians 1--2 Group 4 Not graded \- Not explicitly addressed \- **Barrier: Patient expectations** **Importance of barrier\*** **Interventions** **Importance of interventions\*** Group 1 2 \- Patient materials 3 \- Educational meetings for general practitioners 2 Group 2 Not mentioned \- None \- Group 3 Not graded \- Skills programme training 2 Group 4 Not graded \- Information leaflet to patients about options Not graded \*1 = minor, 2 = moderate, 3 = major :::

PubMed Central

2024-06-05T03:55:47.677626

2004-9-3

PMC517507

Background ========== Lymphatic filariasis affects approximately 120 million people worldwide. Ten percent of these infections are attributed to *Brugia malayi*and *Brugia timori*\[[@B1]\]. Thick blood smear examination is the routine parasitological method used for diagnosis and prevalence studies in the *Brugia*endemic countries of Malaysia and Indonesia \[[@B2],[@B3]\]. This diagnostic method depends on the detection of microfilariae in the peripheral blood, and due to the nocturnal periodicity of microfilaremia in these areas, requires nighttime collection and survey, which is often unpopular with the local population. Furthermore, this method is relatively insensitive \[[@B4]\] and difficult to perform accurately and with consistency in field situations. Conversely, serological diagnostic methods exhibit better sensitivity than detection of microfilaria by thick blood smear, allow the detection of amicrofilaraemic infections among \"endemic normals\" and afford daytime finger-prick blood sampling (thus overcoming the inconveniences associated with night blood sampling, thereby encouraging greater cooperation with the local population and facilitate field work) \[[@B5]\]. However, reports on antigen detection test for brugian filariasis have not demonstrated high levels of sensitivity \[[@B6],[@B7]\]. Thus in the absence of a good antigen detection test for *Brugia*infection, anti-filarial IgG4 assay may be the next best alternative for detection of brugian filariasis \[[@B8]\]. Anti-filarial IgG4 levels have been demonstrated to be elevated in active filarial infection \[[@B9]-[@B13]\] and decline post-treatment \[[@B14]-[@B17]\]. Detection of anti-filarial IgG4 antibodies has also been used for epidemiological assessment of filariasis \[[@B13],[@B18],[@B19]\]. Studies assessing antibody prevalence of lymphatic filariasis have employed assays that use either soluble worm antigens or recombinant antigens \[[@B12],[@B13],[@B18]-[@B21]\]. These antigens may not bind to the same set of anti-filarial antibodies and probably display different cross-reactivities to antibodies against other infections. Thus differences in the antigens employed may be expected to affect the results of antibody prevalence studies. Therefore, the present study aimed to make direct comparison of two antigens i.e. soluble adult worm antigen (SWA) and a recombinant antigen (BmR1), in IgG4 assays on the same set of serum samples. Previously, an ELISA employing soluble adult worm antigen (SWA-ELISA) had been performed to determine prevalence of anti-filarial IgG4 antibodies on a set of serum samples from Indonesia. These samples were collected from: 1\. A transmigrant population that migrated from a non-filarial endemic region to an area endemic for Brugian filariasis and; 2\. Life-long residents of the Brugian endemic area \[[@B22]\]. In the present study a rapid test based on *B. malayi*recombinant antigen (BmR1 dipstick \[Brugia Rapid™\]) and detection of IgG4 antibodies were evaluated using the same set of serum samples. The BmR1 dipstick test has previously been shown to be highly specific and sensitive for the detection of brugian filariasis. In a study involving four international laboratories, the BmR1 dipstick was found to be 93% sensitive and 100% specific when tested with 535 serum samples from patients with various infections and healthy controls \[[@B8]\]. In another multicenter validation study, 97% sensitivity and 99% specificity were recorded when the BmR1 dipstick was tested with 753 serum samples \[[@B23]\]. The present study demonstrated that interpretations of some aspects of the seroepidemiology of filarial infection are affected by the kind of antigen employed in the assay. Thus this study, which utilized the same set of serum samples on assays using two kinds of antigens, highlights the role of the kind of antigen employed in the comparison of results of prevalence studies. Materials and methods ===================== Sera and study population ------------------------- The details on the sera and study population are as described previously \[[@B22]\]. Briefly, serum samples were collected cross-sectionally from a total of 247 transmigrants and 133 life-long residents (LLR) from Budong-budong, a district of Mamuju Regency in South-Sulawesi, Indonesia, which is endemic for nocturnal-periodic *B. malayi*\[[@B24],[@B25]\]. The samples from the transmigrant population are valuable as they could help determine the pattern of acquisition of infection with increasing length of exposure to Brugian filariasis. The transmigrant population had traveled to their new homesteads in groups; they came from the same village or region in Bali or Lesser Sunda islands as part of the government-sponsored relocation programme. Each year a new settlement was founded close to the former one (between 10 and 20 kilometers) which accommodated groups of transmigrants from 2--3 different regions together with migrants from Polmas, an over-populated area in South Sulawesi, to promote integration of different tribes. Transmigrants were grouped together according to the year of arrival in the new settlement. A total of 6 transmigrant units, settled between several months and 6 years prior to the survey, were included in the study together with 2 villages of indigenous Sulawesians (LLR), which were situated closely to the transmigrant areas. Those aged ≤ 15 years were classified as children, while adults were classified as those aged 16 years and older. The mean age of children in transmigrant population and LLR population was 10.2 years and that of adults was 32.6 years. Soluble worm antigen (SWA) -------------------------- Adult *B. malayi*worms were purchased from TRS labs, Athens, Georgia, USA. Female worms were freeze dried, ground to powder, dissolved in phosphate buffered saline (PBS), homogenized and slowly stirred overnight at 4°C. The protein concentration was determined by 2,2\'-biquinoline-4,4\'-dicarboxylic acid disodium salt hydrate (BCA) method before storage at -20°C. BmR1 dipstick ------------- This BmR1 dipstick (Brugia Rapid™) was performed as described previously and according to the instructions of the manufacturer \[16, Malaysian Bio Diagnostics Research Sdn. Bhd., Bangi, Selangor, Malaysia\]. The BmR1 recombinant antigen was expressed from *B*m*17DIII*DNA sequence, GenBank accession no. [**AF225296.**]{.underline} Southern blot hybridization assays performed on cDNA libraries of L3, L4, mf, adult male and adult female *B. malayi*demonstrated that the DNA sequence is present in all of the five kinds of libraries (Rahmah *et al*., unpublished data). Preliminary immunohistological studies suggest that the expressed antigen is found in the epithelial membranes of the adult female uterus (Rahmah *et al*., unpublished data). Statistical Analysis -------------------- The results obtained with BmR1 dipstick were compared to the results obtained previously with SWA-ELISA. The similarities and differences in the results were analyzed by comparing proportion of related samples using McNemar test; and comparing proportions of unrelated samples by using Pearson Chi-square (if indicated Fischer exact test was used instead). Results and Discussion ====================== Figure [1](#F1){ref-type="fig"} shows the antibody prevalence to *B. malayi*, as determined by BmR1 dipstick assay and SWA-ELISA, in the transmigrant population who had resided for various lengths of time in the endemic area and the antibody prevalence in the LLR population. The first detection of IgG4 antibody by the BmR1 dipstick was recorded at 3 years post-exposure, the antibody prevalence increased from 0% in the new arrivals (≤ 1 month and 2--4 months) to 7.4%, 11.1%, 39.1% and 42 % in populations exposed to the infection for 3, 4, 5 and 6 years respectively. Comparison of IgG4 assays using the two kinds of antigen demonstrated similarities in three areas. First, using both SWA-ELISA and BmR1 dipstick, the total prevalence of specific IgG4 in the transmigrant population was found to increase with increasing length of residence in the endemic area. Thus the BmR1 dipstick test confirmed the previously reported finding which demonstrated that the development of anti-filarial IgG4 correlated with the duration of exposure in previously unexposed population \[[@B22]\]. Second, after a period of 5--6 years of being exposed to the filaria infection, anti-filarial IgG4 prevalence by BmR1 dipstick in the transmigrant population (42%) was comparable (p = 0.763) to the antibody prevalence in the LLR population (39.8%); this finding was also previously reported with SWA-ELISA \[[@B22]\]. Thus the results with BmR1 dipstick is in agreement with the previous finding that approximately 5--6 years of exposure is required for the anti-filarial IgG4 in the transmigrant population to reach levels comparable to life-long residents. This is also reported to be the period needed for the detection of microfilaria in the peripheral blood \[[@B22]\]. Third, the overall prevalence of anti-filarial IgG4 was found to be higher in males than in females, by both BmR1 dipstick (p = 0.019) and by SWA-ELISA (p = 0.001). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### IgG4 antibody prevalence in transmigrant population who resided in *B. malayi*endemic areas for various lengths of time and in life-long residents (LLR), as determined by BmR1 dipstick and SWA-ELISA. :::  ::: Differences between the two kinds of antigen were also observed (Figure [1](#F1){ref-type="fig"}). First, except for year 5 transmigrants (p = 0.146), the percent antibody prevalence recorded in the transmigrant population and in the LLR population by BmR1 dipstick were significantly lower than that detected by the SWA-ELISA i.e. year 3, p = 0.00; year 4, p = 0.00; year 6, p = 0.041; LLR, p = 0.00. Second, using SWA-ELISA, anti-filarial IgG4 was first detected at 2--4 months post-exposure in the population of transmigrants, while using BmR1 dipstick the first detection of IgG4 antibody was recorded after three years of residence in the endemic area. Figure [2a](#F2){ref-type="fig"} and [2b](#F2){ref-type="fig"} shows the IgG4 antibody prevalence among children and adult populations as determined by BmR1 dipstick and SWA-ELISA respectively. There was no significant difference detected in antibody prevalence between adults and children in the transmigrant population and in the LLR population when BmR1 dipstick was used (year 3, p = 0.594; year 4, p = 0.066; year 5, p = 0.907; year 6, p = 0.061; LLR, p = 0.074). Using SWA-ELISA, except for early transmigrant settlers (2--4 months residents), the IgG4 prevalence in the transmigrant population was reported to be significantly higher in adults than in children \[[@B22]\]; however in the LLR population, antibody prevalence in children was not significantly higher than in adults (p = 0.316). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### IgG4 antibody prevalence in transmigrant adult and children populations who resided in *B. malayi*endemic areas for various lengths of time and in life-long residents (LLR). **a**(top) IgG4 antibody prevalence as determined by BmR1 dipstick assay. **b**(bottom) IgG4 antibody prevalence as determined by SWA-ELISA. (Note: Previously published in Parasitology 2001; 122, pg. 636 \[Reproduced with permission\]). :::  ::: Thus the BmR1 dipstick test demonstrated that the establishment of anti-filarial IgG4 in the children and adults of the transmigrant population occur at comparable rates; this is not in agreement with the previous finding, using SWA-ELISA, that demonstrated IgG4 was established more rapidly in the adult population than in children \[[@B22]\]. The above observations may be due to a mixture of filarial antigens in SWA, some of which may recognize antibodies produced by exposed but not infected individuals, and/or by individuals who have cleared the infection either due to treatment or spontaneous death of worms. Although IgG4 detection significantly increases the specificity of antibody assays in brugian filariasis \[[@B12]\] the specificity of parasite extract-based assays and recombinant antigen-based assays may not be the same. In a recent study in a non-filaria endemic area in Brazil, the presence of *Strongyloides*antibody responses was found to be associated with higher antifilarial IgG4 responses in assay that uses crude filaria extract as compared to assay that uses *B. malayi*Bm14 recombinant antigen \[[@B26]\]. The discrepancy between the results of SWA-ELISA and BmR1 dipstick may also be partly due to the greater sensitivity of the former as compared to the latter. Out of 120 (of 381) discrepant results, 111 were positive by SWA-ELISA but negative by BmR1 dipstick. Out of these 111 samples, 38 had low ELISA titers (cut-off value \> 4.02 but \< 4.5). If the stringency of the cut-off value of the SWA-ELISA is increased from 4.02 to 4.5, then the discrepancy can be considerably reduced to 88 (of 381), with 73 positive by SWA-ELISA but negative by BmR1 dipstick. On the other hand, since BmR1 dipstick test has been reported to be highly specific \[[@B8],[@B23]\], it is unclear why there were eight individuals who were positive by BmR1 dipstick but negative by SWA-ELISA. Due to the problem of maintaining the antigenicity of the BmR1 antigen when shipped from Malaysia to Netherlands, the dipstick (immunochromatography) assay, which can be transported at room temperature, was employed to test the BmR1 antigen. However the difference in the assay formats is unlikely to be the reason for the lower overall antibody prevalence levels seen with the latter. This is because we have previously shown that ELISA using BmR1 was less sensitive (*albeit*equally specific) than BmR1 dipstick test \[[@B27]\] in detecting *B. malayi*infection. Thus in this study if the ELISA format had been used to determine prevalence of IgG4 antibodies to BmR1 instead of the dipstick format, the SWA-ELISA would still be detecting significantly more positives than the BmR1-ELISA. Differences between the two antigens were also observed in the pattern of IgG4 antibody prevalence in children (Figure [2a](#F2){ref-type="fig"} &[2b](#F2){ref-type="fig"}). Using BmR1 dipstick, positivity of the test in children was first demonstrated at 3 years (2 of 35, 5.7%), followed by no positive child detected at year 4 (0 of 17), positive antibody prevalence at year 5 (3 of 8; 37.5%) which is significantly greater than at year 3 (p = 0.011). This is followed by a nonsignificant decrease (p = 0.589) in antibody prevalence by year 6 (6 of 22; 27.3%). IgG4 prevalence at year 6 and LLR (30 of 87; 34.5%) was also found to be not significantly different (p = 0.542). After year 4, the IgG4 prevalence in children seemed to achieve a stable level that was similar to the antibody prevalence in the LLR population children. In adults, infection was also initially detected at year 3 and there appeared to be a pattern of increasing antibody prevalence with increasing time of residence (9.1% at 3 years; 16.7% at year 4, 40% at year 5; 53.6 % at year 6). Furthermore, the detection rate at year 6 was not significantly different (p = 0.766) from that seen in the LLR population adults (50%). Thus using the BmR1 dipstick, the increasing total IgG4 prevalence with exposure to brugian filariasis was mostly due to the increasing positive cases in the adult population. It is tempting to speculate that the differences in the pattern observed in children and adults are due to the greater rate of aborted infections and/or spontaneous clearance of the infection in children than in adults. The overall lower worm burden in children, due to physiological differences, may enable higher rate of spontaneous clearance of infections in children than in adults. The difference in physiology between adults and children has been demonstrated by the greater number of natural killer cells, and T-and B-lymphocytes in children as compared to middle aged people \[[@B28]\]. In addition CD4/CD8 T-cells have also been reported to decline from a young age onwards \[[@B29],[@B30]\]. However, since this is a cross-sectional study in which data for each period of residence were obtained from different groups of individuals, this hypothesis could not be confirmed. Conversely, results of the SWA-ELISA demonstrated initial antibody positivity in children at 2--4 months, followed by increasing antibody prevalence with time of residence. IgG4 prevalence at year 6 was found to be significantly lower than the antibody prevalence in children of the LLR population (p = 0.007). In the adult population, except for the earlier initial detection, a similar pattern was observed with the results of the BmR1 dipstick i.e. increasing rate of antibody prevalence in the transmigrants with exposure; and the IgG4 prevalence at year 6 is not significantly different (p = 0.430) than the antibody prevalence in the LLR adult population. Conclusions =========== This study highlights that assays using both BmR1 and SWA antigens demonstrate an increasing prevalence of specific IgG4 antibodies in the transmigrant population with increasing length of residence in an area endemic for brugian filariasis, and, by six years residency that the antibody prevalence was similar to that observed in the LLR population. This study also documented three main differences in results derived from assays using two antigens i.e. 1\. Earlier detection and higher rate of antibody prevalence by SWA-ELISA as compared to the results demonstrated by BmR1 dipstick; 2\. Similar rate of acquisition of antibody prevalence in children and adults by BmR1 dipstick; whereas by SWA-ELISA adults were found to become antibody positive faster than in children; 3\. By BmR1 dipstick the increasing total prevalence of IgG4 with exposure was primarily due to the adult population, whereas by SWA-ELISA this was attributed to both children and adult populations. This study demonstrates that some aspects of seroepidemiology of *Brugia malayi*infection may vary with the kind of antigen used in the assay. Thus comparison of results of different studies must take into account the kind of antigen employed, especially if one study uses native antigen and another uses recombinant antigen. It would be interesting to compare seroepidemiological data of IgG4 assays using two different recombinant antigens on the same population. Competing interests =================== Rahmah Noordin is the inventor of the commercialized BmR1 dipstick test (Brugia Rapid™) Authors\' contributions ======================= RN drafted the paper and supplied the BmR1 dipstick test; SW & AM supplied the sera and edited the paper; LBH performed most of the statistical analysis; ES and MY conceived the study and contributed significantly in editing of the paper: In addition ES performed the BmR1 dipstick test and some statistical analysis. All authors read and approved the final manuscript. Acknowledgements ================ This study was funded by European Commission (EC) grant, No. ICA4-CT-2001-10081 and The Netherlands Foundation for the Advancement of Tropical Research (W93-266).

PubMed Central

2024-06-05T03:55:47.681058

2004-8-12

PMC517508

Background ========== During the past 2 decades, tuberculosis -both pulmonary and extrapulmonary- has re-emerged as a major health problem worldwide. Hematologic abnormalities have been described in association with mycobacterial infections for almost 100 years. Patients with both pulmonary and extrapulmonary tuberculosis (TB) may demonstrate peripheral blood abnormalities and findings may be minimal or profound \[[@B1],[@B2]\]. A comprehensive review of the literature reveals a few case reports documenting tuberculosis as a cause of severe hematologic conditions such as hemophagocytic syndrome, thrombotic thrombocytopenic purpura and immune hemolytic anemia. Immune thrombocytopenic purpura (immune TP) associated with tuberculosis is exceedingly rare event. We report the case of immune TP associated with tuberculosis that was presented with severe hemorrhagic diathesis. Case Presentation ================= A 29-year-old previously healthy immigrant male patient from Kazakhstan was admitted to hospital with new-onset severe hemoptysis, macroscopic hematuria and extensive cutaneous petechiae on lower extremities. He appeared ill and poorly nourished. The patient was oriented and well cooperated, and there was no previous history of hematologic or liver or another disease and recent medication. He presented with unexplained weight loss of 2 months duration along with intermittent fever, night sweats and cough. The physical examination revealed a blood pressure of 100/70 mm/Hg, pulse 100/min, a temperature 37.2°C, extensive cutaneous petechiae on lower extremities, hemorrhagic bulla on tongue and on mucosa of oral cavity, and amphoric soufflé on apex of right chest. No organomegaly or lymphadenomegaly or evidence of another disease such as chronic liver disease was detectable. The initial complete blood count revealed a white blood cell 25.1 × 10^9^/l (58% neutrophils, 29% bants, 9% lymphocytes and 4% monocytes), hemoglobin 11.2 gr/dl, hematocrit 36%, MCV 84 fl, reticulocytes 1% and platelet count 7.6 × 10^9^/l. Erythrocyte sedimentation rate was 110 mm/h. A peripheral smear was remarkable for a paucity of platelets. Coagulation profile \[prothrombin time (PT), activated-partial thromboplastin time (aPTT), fibrin degradation products (FDP)\] were normal. A bone marrow aspiration demonstrated hypercellularity of all cell lines with normal maturation of myeloid and erythroid precursors. Megakaryocytes were increased in number with normal morphology. On bone marrow aspiration hemophagocytosis was not observed. A chest X-ray (Figure [1](#F1){ref-type="fig"}) and computed tomography (CT) (Figure [2](#F2){ref-type="fig"}) demonstrated bilateral patchy infiltrates and walled cavities on left and right upper lobes. Acid-fast bacilli were strongly positive in sputum (Figure [3](#F3){ref-type="fig"}). Bone marrow aspirate and urine for acid-fast bacilli were negative. Sputum culture yielded mycobacterium tuberculosis complex. The following laboratory studies were normal or negative: biochemical tests (glucose, urea, creatinine, uric acid, sodium, potassium, calcium, chloride, phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, bilirubin, total protein and albumin), rheumatoid factor, anti-nuclear antibody, anti-platelet specific antibodies, Coomb\'s tests, HIV, hepatitis B and C virus, blood culture, bone marrow aspirate culture and abdominopelvic CT. No granuloma or hemophagocytosis was detected on bone marrow biopsy. The patient was started on rifampin 10 mg/kg/d, isoniazid 5 mg/kg/d, ethambutol 25 mg/kg/d, pyrazinamide 40 mg/kg/d, pyridoxine 75 mg/d and intravenous immune globulin (IVIg) 1 g/kg/d given for 2 days. On day 2 of hospitalization and treatment of anti-tuberculous therapy prednisolone 1 mg/kg/d was added. On day 8, platelet counts started to increase and on day 12 of the treatment it reached to 187 × 10^9^/l level. Patient improved on day 10 and he did not have any complaints on day 14; at time of discharge. He received a total of 6 red blood cell (RBC) units throughout hospitalization. During his hospitalization findings of hemolysis or gastrointestinal bleeding and massive bleeding in another site except hematuria and hemoptysis were not established. A complete blood count at discharge demonstrated a WBC 17 × 10^9^/l, Hb 9.6 g/dl, and platelet count 310 × 10^9^/l (Table [1](#T1){ref-type="table"}). Corticosteroids were discontinued on day 14 of therapy and the patient was discharged and recurrent thrombocytopenia was not established after withdrawal of corticosteroid therapy. Ninety days after discharge, the patient was well with a platelet count of 300 × 10^9^/l (Table [1](#T1){ref-type="table"}) and he had no side effect thought to be secondary to anti-tuberculous drugs. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Anteroposterior chest radiograph showing cavitary lesions in both lungs :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### CT showing cavitary lesions in both lungs :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Sputum showing positivity of acid-fast bacilli :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of patient ::: Day Hb (g/dl) Hct (%) WBC (×10^9^) Plt (×10^9^) Treatment ----- ----------- --------- -------------- -------------- --------------------------------------- 1 11.2 36 25.1 7.6 Anti-tbc drugs + IVIg 2 8 25 23.1 9.6 Anti-tbc drugs + IVIg+Pred+1 RBC Susp 3 8.1 25 22.7 17.7 Anti-tbc drugs+ Pred+ 2 RBC Susp 4 10.3 33 20.6 15.1 Anti-tbc drugs+ Pred 5 8.6 27 24.5 12.3 Anti-tbc drugs+ Pred+ 1 RBC Susp 6 8.2 24 25.4 4.0 Anti-tbc drugs + Pred + 2 RBC Susp 7 10.1 29 20.6 4.7 Anti-tbc drugs+ Pred 8 10.5 32 17.7 20.2 Anti-tbc drugs+ Pred 9 9.9 31 15.8 36.6 Anti-tbc drugs+ Pred 10 10.3 33 18 59 Anti-tbc drugs+ Pred 11 9.4 28 14 100 Anti-tbc drugs+ Pred 12 9.7 28 18.1 187 Anti-tbc drugs+ Pred 13 9.1 26 15.2 324 Anti-tbc drugs+ Pred 14 9.6 28 17 310 Anti-tbc drugs 45 12.3 37 15.5 304 Anti-tbc drugs 90 14 43 11 300 Anti-tbc drugs Abbreviations: Anti-tbc drugs: anti-tuberculous drugs, Pred: Prednisolone, Susp: Suspension ::: Discussion ========== Tuberculosis differs from many other infectious maladies in having particular social and geographic distributions. The disease was under control in developed nations and getting under control in developing nations, until the emergence of HIV infection and the advent of multidrug resistant strains of mycobacteria \[[@B3]-[@B5]\]. Various hematologic abnormalities such as anemia, leukocytosis, monocytosis, lymphopenia, leucopenia, thrombocytopenia, thrombocytosis, leukemoid reactions and pancytopenia have been seen in tuberculosis \[[@B1],[@B2]\], but severe thrombocytopenia and presenting of tuberculosis as immune thrombocytopenic purpura is extremely rare and there are a few reports about tuberculosis induced immune thrombocytopenic purpura published in the world literature \[[@B6]-[@B10]\]. The case that we report could be confused by coincidental presentation of adult idiopathic thrombocytopenic purpura and tuberculosis, by drug-induced thrombocytopenia, thrombotic thrombocytopenic purpura (TTP)-hemolytic uremic syndrome (HUS), hemophagocytic syndrome and disseminated intravascular coagulation (DIC) associated with TB. Idiopathic thrombocytopenic purpura (ITP, also known as primary immune thrombocytopenic purpura) is an acquired disease of children and adults defined as isolated thrombocytopenia with no clinically apparent associated conditions or other causes of thrombocytopenia. Adult ITP typically has an insidious onset with long-lasting histories of purpura (thrombocytopenia for \>6 months) and spontaneous remission is uncommon and is likely to be incomplete \[[@B11]-[@B13]\]. Steroids are the conventional first line therapy for adult ITP. Platelets counts increase within one week in responding patients and usually reach peak values by two to four weeks. However, in most patients, thrombocytopenia recurs when steroids are tapered or discontinued. Also in adult, IVIg is used when clinical situations require a transient increase of the platelet count and a typical response is an increase in platelet count several days after the infusions are initiated and return to the pretreatment level within several weeks \[[@B11],[@B13]\]. In our case, we excluded the adult ITP not only by basing on standard criteria \[[@B12]\], but with response to steroids and IVIg therapy since thrombocytopenia did not recur after withdrawal of prednisone and IVIg therapy. Also we excluded other causes of thrombocytopenia such as hemophagocytic syndrome, TTP, combined autoimmune cytopenias with history, clinical and laboratory findings, and examination of bone marrow aspiration and biopsy that were described in case presentation. Several factors are known to cause bleeding in association with infections, of which thrombocytopenia is the most common. The etiology of thrombocytopenia in most cases appears to be increased destruction of platelets such as due to DIC or septicemia without evidence of DIC or platelet adherence to damaged vascular surfaces or direct platelet toxicity caused by the microorganism or involvement of bone marrow. Adult acute immune thrombocytopenic purpura is defined as a bleeding disorder in otherwise healthy person caused by transient destruction of platelets. Although the most important therapy for infection-related thrombocytopenia is that directed at the underlying infection, treatment decisions for immune thrombocytopenic purpura remain controversial and may include single or combination therapy with corticosteroids, intravenous immunoglobulin (IVIg) according to degree of thrombocytopenia or hemorrhage \[[@B11],[@B14]\]. The case that we reported was presented with symptoms of phthisis lasting for more than 2 months and severe hemoptysis, macroscopic hematuria and extensive cutaneous bleeding findings lasting for one week. Based on the clinical, radiological (X-ray and CT of chest) findings, demonstration of positivity of acid-fast bacilli in sputum and with exclusion of other causes of thrombocytopenia, immune thrombocytopenic purpura due to pulmonary tuberculosis was diagnosed. We applied anti-tuberculous therapy combined with corticosteroids and IVIg because of severe thrombocytopenia and severe hemorrhagic diathesis. Clinically, steroids are known to ameliorate the purpuric bleeding in patients before the platelet count actually increases. The early effect is due to decrease of vascular permeability. The effect of the steroids in the thrombocytopenia is probably complex and it is late effect. The mechanism of action of IVIg is unclear, but studies suggest blockage of the Fc receptors of the reticuloendothelial cells and suppression of antibody production and binding which may be a result of anti-idiotype antibodies that bind antiplatelet antibodies and modulate the immune response \[[@B14]\]. In our case, corticosteroids were discontinued on day 14 of therapy and the patient was discharged and recurrent thrombocytopenia was not established after withdrawal of corticosteroid therapy. These observations suggest that tuberculosis is the cause of thrombocytopenia in our patient. He received a total of 6 RBC units throughout hospitalization. He did not have findings of Coomb\'s positive or negative hemolytic anemia or microangiopathic hemolytic anemia, hemophagocytic syndrome, gastrointestinal bleeding and massive bleeding to thoracic cavity. But he had severe hemoptysis and macroscopic hematuria. Ninety days after discharge, the patient was in well health with a platelet count of 300 × 10^9^/l and he had no side effect thought to be secondary to anti-tuberculous drugs \[[@B15],[@B16]\]. The patient is still in our follow-up without relapsing of thrombocytopenia. In conclusion, since the incidence of tuberculosis is currently increasing in worldwide countries and it may present with different hematologic manifestations, in case of immune thrombocytopenic purpura tuberculosis should also be recalled. Finally, further studies are needed in order to fully characterize the pathophysiology and immunological abnormalities in tuberculosis-related immune thrombocytopenic purpura.

PubMed Central

2024-06-05T03:55:47.682216

2004-9-6

PMC517509

Background ========== The superfamily of nuclear hormone receptor comprises a group of transcription factors that play significant roles in response to a number of biological regulators. In addition to the pregnane X receptor (PXR, NR1I2), the constitutive androstane receptor \[[@B1]\] (CAR, NR1I3) plays a role in the transcriptional activation of genes that encode xenobiotic/steroid and drug metabolizing enzymes, such as cytochrome P450 (CYP) 2Bs, 2C19, 3As, multidrug resistance-associated protein 2 (MRP2), UDP-glucuronosyltransferase (UGT1A1), and 5-aminolevlinic acid synthase 1 (ALAS1) \[[@B2]-[@B8]\]. In response to xenobiotic PB, and other PB-like ligands such as 1,4-bis \[2-(3,5-dichlorpyridyloxy)\]benzene (TCPOBOP) in rodents \[[@B9]\] and 6-(4-Chlorpphenyl)imidazo \[2,1-b\]\[[@B1],[@B3]\]thiazole-5-carbaldehyde *O*-(3,4-dichlorobenzyl)oxime (CITCO) in humans \[[@B10]\], high doses of acetaminophen \[[@B11]\], and bilirubin \[[@B12]\], CAR forms a heterodimer with retinoid X receptor alpha (RXRα) and subsequently binds to the direct repeat (DR-4) motifs in such as the phenobarbital (PB)-responsive enhancer module (PBREM) in the far upstream promoter regions of mouse, rat and human CYP2B genes. In contrast, androstanol and androstenol were initially identified as inverse agonists \[[@B13]\] that reverse the constitutive transactivating potency of CAR. Recently, the mRNA expression of nuclear receptors, such as peroxisome proliferater-activated receptor alpha (PPARα), retinoic acid receptor (RAR)-related orphan receptor (ROR) and RER-ERBα, have been reported to show circadian rhythms in the liver \[[@B14]-[@B17]\]. Hepatic PPARα mRNA and protein levels follow a diurnal rhythm which parallels that of circulating corticosterone. In addition, REV-ERBα expression is regulated by a circadian positive feedback loop attributable to the function of BMAL1/CLOCK heterodimers, and is negatively controlled by circadian negative lobe PER/CRY heterodimers \[[@B18],[@B19]\]. Circadian variations in the chronopharmacokinetics and chronopharmacology of various drugs such as theophylline and propranolol have been recently reported \[[@B20]\]. Furthermore, daily fluctuations in hepatic P450 monooxygenase activities responsible for the first phase of metabolism of various xenobiotics are well known. For example, Cyp2a4, Cyp2a5, CYP7, and CYP3A are among those that show circadian rhythmicities \[[@B21]-[@B23]\] that result from the preceding rhythmic oscillations of transcription factors including nuclear receptors. We previously determined the transcriptional start site of the rat *Car*gene (Kanno et al., 2003), resulting in the discovery of the putative REV-ERBα/ROR responsive element (RORE) at around -1.2 kb on the basis of published genomic sequence (Mazny et al., accession number AC099236). Thus, expression of the *Car*gene is expected to occur in antiphase to that of the *Per1*gene and in phase with the *Bmal1*gene. In the present study, the expression profile of the *Car*gene in rat liver was studied in comparison with those of the clock gene *Bmal1*, clock-controlled gene *Rev-erbα*and CAR-dependent PB-inducible *CYP2B1/2*gene. Results ======= Rat hepatic expression of nuclear receptor CAR mRNA follows a circadian rhythm ------------------------------------------------------------------------------ Apart from the clock gene BMAL1 and clock-directed gene REV-ERBα, a time-dependent profile of CAR mRNA expression was observed for the rat liver. CAR mRNA levels oscillated during the day in phase with BMAL1 and in antiphase with REV-ERBα mRNAs, with a peak at ZT20 and a trough at ZT8, and a peak/trough ratio of 2.0 (Figs. [1](#F1){ref-type="fig"}, [2A](#F2){ref-type="fig"}). The CYP2B mRNA expression profile was resembled to the circadian oscillation of CAR mRNA but in a much more blunted manner (Fig. [2B](#F2){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Diurnal variations in CAR mRNA in the rat liver.**Animals were sacrificed every 4 hours at Zeitgeber times (ZT) 4, 8, 12, 16, 20 and 24/0. mRNA levels of CAR, CYP2B1/2, BMAL1, REV-ERBα and GAPDH were amplified by semi-quantitative RT-PCR. After oligo(dT)-primed cDNA was synthesized from rat liver total RNA, PCR was conducted with an initial enzyme activation step at 95°C for 5 min followed by divergent cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec and extension at 72°C for 60 sec; CAR (27 cycles), CYP2B1/2, REV-ERBα and BMAL1(30 cycles), and GAPDH (24 cycles). The reaction products were separated by agarose gel electrophoresis and stained with ethidium bromide. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Diurnal difference in CAR and CYP2B mRNA levels in the rat liver.**Animals were sacrificed at ZT8 and ZT20 (n = 3--4), and CAR (**A**) and CYP2B (**B**) mRNA levels were measured by semi-quantitative RT-PCR as described in the legend to Fig. 1. The results were normalized against those for GAPDH. The columns and bars represent the means ± SD with a significant difference at \*: *p*\< 0.01 :::  ::: Diurnal difference in the induction of CYP2B by phenobarbital ------------------------------------------------------------- Since CAR is associated with the induction of metabolic enzymes such as CYP2B, CYP3A, and UGT1A1, the circadian rhythmicity of CAR mRNA expression may be reflected in the diurnal-difference of PB-induction of CYP2B1/2 mRNA. Therefore, we investigated the time-dependent difference of the effect of PB-treatment on the induction of CYP2B1/2 mRNA. CYP2B1/2 mRNA expression was comparatively evaluated at ZT13 and ZT1 after 5-hours of PB treatment during ZT8 to ZT13 (the minimum zone of CAR mRNA expression) and ZT20 to ZT1 (during which the expression of CAR mRNA was maximal), respectively. Hepatic CYP2B1/2 mRNA was induced 2.2-fold over the control level in the rats treated with PB between ZT8 and ZT13 \[daytime treatment\]. In contrast, it was increased by 3.8-fold of the control level when the rats were treated from ZT20 to ZT1 \[nighttime treatment\] (Fig. [3](#F3){ref-type="fig"}). These data suggest that the diurnal-difference in CYP2B1/2-induction might be affected by the circadian rhythm of CAR mRNA expression. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Diurnal difference of CYP2B induction.**Animals were sacrificed at ZT13 and ZT25/1 5-hour after the injection of PB (gray columns) or veihcle (black columns) during ZT8-13 \[Day\] and ZT20-1 \[Night\], respectively. Oligo(dT)-primed cDNA was synthesized from rat liver total RNA from each animal, and CYP2B mRNA levels were measured by STBR Green real-time RT-PCR. The results were normalized against those of GAPDH. The columns and bars represent the means ± SD with significant differences compared to the individual controls at \*, ^\#^: *p*\< 0.05 :::  ::: Discussion ========== We previously reported that the induction of rat CYP2B1/2 by PB is absent in the lung in contrast to the marked response in the liver due to the improper splicing of CAR mRNA during its maturation. \[[@B24],[@B25]\]. The longitudinal expression of CAR mRNA along the gastrointestinal tract increases from the duodenum to the terminal jejunum and then decreases toward the distal ileum while only marginal expression can be observed in the stomach and colon, implying a role for endogenous ligands such as bilirubin glucuronides secreted in the duodenum \[[@B26]\]. A single transcriptional start site was determined by comparison between the full-length mRNA and genomic sequences. In the present study, we investigated whether the expression of hepatic CAR mRNA shows circadian rhythmicity, because clock-controlled regulation is expected due to the presence of putative RORE in the promoter region and electrophoretic gel mobility-shift assay showed a slowly migrating band binding to the RORE probe using nuclear proteins (data not shown). The CAR mRNA level was found oscillation daily with a peak at ZT20 and a nadir at ZT8. In contrast, BMAL1 mRNA peaked at ZT24/0 and hit the bottom at ZT12 with a 4-hours retardation, and REV-ERBα mRNA showed a peak at ZT8 and a trough at ZT20 exactly in antiphase with CAR mRNA (Fig. [1](#F1){ref-type="fig"}). In contrast to the self-sustained central clock present in the brain, peripheral circadian clocks are retrained by humonal factors such as glucocorticoid hormones \[[@B27],[@B28]\], as reflected in the diurnal rhythms observed for PPARα and REV-ERBα. Glucocorticoids are also responsible for the induction of human CAR mRNA and protein via a distal glucocorticoid response element in the 5\'-franking region of the gene \[[@B29]\], and the same might be true for its rat counterpart, which was inducible by dexamethasone (data not shown). PPARα mRNA levels followed a similar diurnal rhythm to that of the plasma level of corticosterone, which is low in the morning (around ZT2), and increases in the afternoon to reach a peak 2--3 hours before the lights out (ZT9.5). Therefore, CAR mRNA oscillation might not be retrained by the physiological diurnal variation of glucocorticoids in rats. Recently, bilirubin was reported to be an endogenous activator of the CAR gene, which is in turn associated with the induction of bilirubin metabolising proteins, such as organic anion transporter SLC21A6, glutathione-S-transferase (GST), UGT1A1 and MRP2. Blood-bilirubin level reaches a minimum at the end of the light period and a maximum at the end of the dark period \[[@B30]\]. It is probable that blood bilirubin may contribute to the retraining of CAR expression to optimise bilirubin clearance. Hepatic CYP2B1/2 mRNA level was found to be synchronized with the CAR mRNA oscillation (Fig. [1](#F1){ref-type="fig"}). In the clock-controlled gene cascade or network, the circadian rhythm of CYP2B1/2 mRNA expression might be partially, if not fully, explained by the hepatic CAR level. Furukawa et al. showed that hepatic P450-dependent monooxygenase activities measured by the *O*-dealkylation of 7-alkoxycoumarin fluctuate daily in F344 rats with high values during the dark period \[[@B31]\]. In addition, these fluctuations are regulated by a central clock present in the suprachiasmatic nucleus \[[@B32]\]. Further, cholesterol 7-α hydroxylase (CYP7), coumarin 7-α hydroxylase (Cyp2a4) and coumarin 15-α hydroxylase (Cyp2a5) exhibit circadian rhythmicities. These enzymes are transcriptionally regulated by albumin D-site-binding protein (DBP), which is another primary clock-controlled gene expressed according to a robust daily rhythm in the SCN and several peripheral tissues. Besides DBP, REV-ERBα is transactivated by the binding of the BMAL1-CLOCK heterodimer to the E-box motif in its enhancer region \[[@B33]\], and is down-regulated by the clock gene PER-CRY heterodimer. Neuronal PAS domain protein 2 (NPAS2) is highly related in primary amino acid sequence to CLOCK, being able to dimerize with BMAL1 as in the case of CLOCK. Furthermore, BMAL1-NPAS2 heterodimer was found to transactivate the same target genes as those of BMAL1-CLOCK such as *Per1*, *Per2, Cry1 and Rev-erb*α. Recently, the transcription of *Alas1*gene encoding for the aminolevulinate synthase 1 (*Alas1*) that is rate-limitting enzyme in a heme biosynthesis was reported to be controlled in the circadian clock mechanism. Although *Alas1*is regulated transcriptionally by CAR-modulators having DR4 motifs in the promoter region as well as *CYP2B1/2*, BMAL1-NPAS2 and BMAL1-CLOCK heterodimers would be responsible for the daily physiological fluctuation in phase with *Rev-erbα*\[[@B34]\]. The circadian transcription of CAR and CYP2B1/2 is likely directly, indirectly or in combination dominated by these peripheral clocks and clock-controlled genes. The direct role of RevErb in the regulation of CAR will have to be established in further studies. For example ChIP analysis would be required to show diurnal occupancy of the putative RORE in the CAR promoter, and it has not yet been shown that Rev Erb α can modulate the transcription of the CAR promoter. We were also interested in whether the PB-dependent induction of CYP2B1/2 mRNA is affected by the diurnal rhythm of CAR. As shown in Fig. [3](#F3){ref-type="fig"}, PB-treatment at night \[ZT20-1\] was 1.7-fold more effective than treatment during the daytime \[ZT8-13\] in terms of the induction of CYP2B1/2 mRNA. Although the timing of the injection of PB and monitoring of CYP2B1/2 mRNA levels adopted in this work might not have been optimal, the results obtained suggested that the diurnal difference in the expression of xenosensor genes may underlie chronopharmacokinetics and chronopharmacology in a clinical setting. Conclusions =========== Nuclear receptor CAR mRNA expression oscillates during the day with a peak at ZT20 and trough at ZT8 in antiphase with REV-ERBα, as expected due to the presence of putative ROREs in the promoter region. Since the magnitude of PB-induction of CYP2B1/2 mRNA showed at least a 1.7-fold difference during the day, the diurnal-difference of CYP2B-induction by PB might be controlled by the circadian rhythm of CAR mRNA expression. Methods ======= Animals and treatments ---------------------- Eight week-old male Wistar rats (Clea) were kept under a 12-hours light-dark (LD12:12) cycle and provided food and water *ad libitum*. After more than 2 weeks of housing, the rats were killed at Zeitgeber times (ZT) 0, 4, 8, 12, 16, 20 and 24: ZT0 was lights-on and ZT12 is lights-out. For the PB-induction of CYP2B, the rats were i.p. injected with PB at ZT8 and ZT20 and sacrificed at ZT13 and ZT1, respectively. The livers were then dissected and used for the isolation of total RNA. RNA analysis by RT-PCR and Real-Time RT-PCR ------------------------------------------- Total RNA was extracted from rat liver homogenate using an RNeasy Kit (QIAGEN, Hilden, Germany). After incubation at 65°C for 10 min, the extracts were quickly placed in an ice-cold water bath. Oligo-dT primed cDNA was synthesized from 1 μg of total RNA using RTG You-Prime First-Strand Beads (Amersham Biosciences, NJ), and left at room temperature for 1 min. Reverse transcription was then performed at 37°C for 1 hour to obtain cDNA. PCR was next performed in a total reaction mixture (25 μl) containing 1 μl each of RT-reaction mixture, Ex Taq DNA polymerase (Takara, Japan) and each of primer pair. cDNA was amplified for 24 (GAPDH), 27 (CAR) or 30 (BMAL1, REV-ERVα, CYP2B) cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 1 min in a thermal cycler. The reaction products were separated by agarose gel electrophoresis and analyzed by a Flour Imager (Amersham Biosciences) after staining with ethidium bromide. Real-time PCR was carried out for the quantitation of each transcript in a reaction mixture consisting of 2 μl of the cDNA, 1 μl each pair of primers, 21 μl of water and 25 μl of iQ SYBER™ Green Supermix (BIO-RAD, CA). PCR was performed with an initial enzyme activation step at 95°C for 5 min followed by 50 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec and extension at 72°C for 45 sec in a real-time DNA thermal cycler (iCycler™, BIO-RAD). The following oligonucleotides were used as forward and reverse primers, respectively: 5\'-ACCAGTTTGTGCAGTTCAGG-3\' and 5\'-CTTGAGAAGGGAGATCTGGT-3\' for CAR, 5\'-GAGTTCTTCTCTGGGTTGCTG-3\' and 5\'-ACTGTGGGTCATGGAGAGCTG-3\' for CYP2B1/2, 5\'-AACATGGCACTGAGCAGGTCTCC-3\' and 5\'-GGCATGTCCTATGAACATGTACC-3\' for REV-ERBα, 5\'-GCAAACTACAAGCCAACATTTCTAT-3\' and 5\'-CTTAACTTTGGCAATATCTTTTGGA-3\' for BMAL1, and 5\'-ACCACAGTCCATGCCATCAC-3\' and 5\'-TCCACCACCCTGTTGCTGTA-3\' for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The amplified cDNA was quantitated by the number of cycles (or cross point) at which the fluorescence signal was greater than a defined threshold during the logarithmic phase of amplification. The results were shown relatively to the control level after normalization to that of GAPDH. Competing interests =================== None declared. Authors\' contributions ======================= K.Y. conceived of the study, carried out all experiments and drafted the manuscript. S.O. and T.H. contributed to the experiment, and N.T. participated in the design of the study and its coordination. Y.I. participated in the design of the study and drafted the manuscript in collaboration with K.Y. All authors read and approved the final manuscript.

PubMed Central

2024-06-05T03:55:47.684275

2004-8-28

PMC517510

Background ========== Fay, Pfeiffer, Cronin, Le, and Feuer \[[@B1]\] showed how to calculate the age-conditional probabilities of developing a disease (ACPDvD) from registry data. Throughout this paper we use \"cancer\" as our disease of interest, but the method applies to specific types of cancer as well as other diseases where information is collected by population based surveillance methods. Fay et al \[[@B1]\] provided a formula (see equation 1 below) to calculate ACPDvD after inputing the rate function by age of (1) first incidence of cancer per person-years alive, (2) death from cancer per person-years alive, and (3) death from other causes per person-years alive. Fay et al \[[@B1]\] used a simple piecewise constant model for the three rate functions, which have constant rates within each age group. Here we detail two more complicated models for the rates. The first model is a segmented regression model or joinpoint model for the rates, where the rate function is a series of linear functions that join at the mid-points of the age groups, and the rate function is constant before the first mid-point and after the last \"mid-point\" (because the last interval goes to infinity, the last \"mid-point\" is not really a mid-point at all, see below). We will call this model the MAJ (mid-age group joinpoint) model for the rates. In Figure [1](#F1){ref-type="fig"} we show how both the piecewise constant model and the mid-age group joinpoint model apply to all invasive cancer incidence from the Surveillance Epidemiology and End Results (SEER) program of the U.S. National Cancer Institute in 1998--2000. Figure [1](#F1){ref-type="fig"} uses the SEER 12 registries which cover about 14 percent of the U.S. population, covering 5 states (Connecticut, Hawaii, Iowa, New Mexico, Utah), 6 metropolitan areas (Atlanta, Detroit, Los Angeles, San Francisco-Oakland, San Jose-Monterey, Seattle-Puget Sound) and the Alaska Native Registry (see \[[@B2]\]). Similar graphs showing the MAJ model can be made for the other rates required in the calculations, death from cancer and death from other causes per person-years alive. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### SEER 12 all invasive cancer incidence rates, 1998--2000, all races, both sexes: Piecewise constant and mid-age joinpoint methods. :::  ::: Notice that the MAJ model gives a more smoothly changing and probably a better modeled rate. The only place where the MAJ model may not perform better than the piecewise constant model is at peaks or valleys, where there may be some bias. In Figure [1](#F1){ref-type="fig"} we see that the smoothness of the MAJ appears to produce more plausible estimates for ages 0 through 85 and from ages 90 and above, and the only age group with a noteworthy bias problem is 85 to 90. Thus, for almost all of the age range the MAJ model is more plausible. A problem with the mid-age group joinpoint model is that it requires numeric integration for its calculation. The second model uses a series of piecewise constant values to approximate the mid-age group joinpoint model. We call this second model the PMAJ (piecewise mid-age group joinpoint) model. The PMAJ does not require numeric integration, so it is much faster than the MAJ model. The PMAJ model is a piecewise constant model that only differs from the piecewise constant model of Fay et al \[[@B1]\] in that the pieces are smaller and the corresponding values of the rates are motivated by the MAJ model. Starting with version 5.0, the freely available DevCan software \[[@B3]\] uses the PMAJ method. (There was a small calculation error in versions 5.0 and 5.1 that has been corrected in version 5.2). DevCan calculates ACPDvD or age conditional probability of dying from a disease for U.S. cancer data or for user supplied data. The outline of this paper is as follows. The review and overview section reviews the issues in estimating the age conditional probability of developing disease from surveillance data. This section includes a motivation for using this type of statistic to describe population data. The review and overview section additionally gives graphical descriptions of the MAJ and PMAJ methods. The paper is structured so that readers not interested in the details may skip the next two sections and the appendix, which give precise and notationally involved definitions of the MAJ estimators. The examples and discussion section gives examples of the estimator of ACPDvD using three different methods for estimating the rates, the simple piecewise constant method proposed in Fay et al \[[@B1]\], the MAJ method, and the PMAJ method. In supplimental material \[see [Additional file 1](#S1){ref-type="supplementary-material"}\] we compare the PMAJ method with the method of Wun, et al \[[@B4]\], since the latter method was the method used in versions of the DevCan software before version 5.0. Review and overview =================== Consider a surveillance program like the SEER program of the U.S. National Cancer Institute. This program attempts to count every incidence of cancer within the catchment area of the program. Because cancer is a disease in which the rates of the disease are highly dependent on age, in order to give interpretability to the counts within the SEER registries, we must somehow account for the age distribution in the popoulation. One simple and popular statistic is the age adjusted rate or directly standardized rate (DSR). In the SEER Cancer Statistics Review \[[@B2]\] DSRs are used to compare different cancer sites, trends on specific cancer sites over time, and rates by sex and race. The DSR is calculated by a simple weighted sum of the age specific rates for each 5 year age group, where the weights are proportional to the U.S. 2000 population. Thus, the DSR may be interpreted as the rates adjusted as if all the populations being compared had age distributions similar to the U.S. 2000 population. The DSRs are useful for gaining an overall picture of how the incidence and mortality of each cancer effects different populations (e.g., different races, SEER population at different times), while controling for the effect of differing age distributions between populations being compared. A disadvantage of the DSR is that it is hard to relate to an individual\'s risk. For example, Table I-4 of the SEER Cancer Statistics Review, 1975--2000 \[[@B2]\] states that the DSR for breast cancer for females for the years 1996--2000 is 135 per 100,000 person-years. The average American woman may wonder, how does that relate to my risk? Will I be likely to get breast cancer in my lifetime? If I am 40 years old now, what is my risk of getting breast cancer in the next 10 years given that I have survived to this old without getting it? These questions are the motivation for using the age conditional probability of developing disease (ACPDvD), and in order to estimate the ACPDvD for female breast cancer, we require information not only about the rate of female breast cancer but also about the rates of dying from female breast cancer and dying from other causes. The ACPDvD uses cross-sectional incidence and mortality rates to estimate the age-conditional probabilities of developing disease in a hypothetical cohort in which we assume the age specific rates do not change over time. This gives a personal interpretation to the cross-sectional data, allowing statements like the following: if the incidence and mortality rates remain at their present values (as observed in SEER 12, 1998--2000), then a female born today would have a 13.5% chance of developing breast cancer over her lifetime (see Table [2](#T2){ref-type="table"}). We can also calculate ACPDvD over intervals. For example, a female who has reached 40 years old without developing breast cancer has a 1.5% chance of developing breast cancer by the time she is 50. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Age Conditional Probability of Developing Different Types of Invasive Cancers (in Percent) from SEER 12, 1998--2000 ::: Start Age End Age Model All Invasive (Both Sexes) Prostat(Male) Breast (Female) Acute Lymphocytic Leukemia (Both Sexes) ----------- --------- --------------------- --------------------------- --------------- ----------------- ----------------------------------------- 0 20 Piecewise const 0.3158 0.0009 0.0015 0.0669 PMAJ, interval = .5 0.3260 0.0011 0.0021 0.0633 MAJ 0.3260 0.0011 0.0021 0.0633 0 50 Piecewise const 4.0690 0.2002 1.9188 0.0837 PMAJ, interval = .5 4.1657 0.2550 1.9492 0.0808 MAJ 4.1657 0.2550 1.9492 0.0808 40 50 Piecewise const 2.5260 0.2032 1.5131 0.0053 PMAJ, interval = .5 2.5976 0.2579 1.5169 0.0055 MAJ 2.5975 0.2579 1.5169 0.0055 0 Inf Piecewise const 42.0876 17.4952 13.6471 0.1154 PMAJ, interval = .5 41.7547 17.3375 13.5477 0.1121 MAJ 41.7574 17.3389 13.5485 0.1121 60 61 Piecewise const 1.2340 0.5989 0.3822 0.0009 PMAJ, interval = .5 1.0852 0.4946 0.3627 0.0009 MAJ 1.0852 0.4946 0.3627 0.0009 64 65 Piecewise const 1.2758 0.6131 0.3872 0.0009 PMAJ, interval = .5 1.4453 0.7440 0.4045 0.0010 MAJ 1.4453 0.7440 0.4045 0.0010 60 65 Piecewise const 6.0331 2.9128 1.8777 0.0042 PMAJ, interval = .5 6.0622 2.9492 1.8758 0.0044 MAJ 6.0622 2.9492 1.8759 0.0044 ::: Calculation of the ACPDvD is somewhat complicated, and we describe the complications in relation to the simple DSRs. Consider first the age specific incidence rates which are used to calculate the DSRs. These rates simply count the number of incident cases of a particular disease (e.g., female breast cancer) within each age group and divide by the total number of person-years estimated by the population. For counts of a single year, the person-years are estimated by the mid-year population of the catchment area (for sex-specific cancers like prostate cancer or female breast cancer, we only use the population of the appropriate sex). Note that the incident cases may include individuals who have previously been diagnosed with the cancer and have developed a new primary cancer. For the ACPDvD for any specific disease we would like the rate of first incidence per person-years alive and disease-free. Thus, there are two difficulties, (1) the usual age specific incidence rates include persons with multiple primary cancers, and (2) the denominators include persons who have previously been diagnosed. Merrill and Feuer \[[@B5]\] discuss both difficulties and adjust for them creating risk-adjusted cancer incidence rates. Merrill and Feuer \[[@B5]\] study the effect of these adjustments for several cancer sites. To handle the first difficulty, (similar to \[[@B5]\]) we can remove cases where we have a record of a previous diagnosis of that particular type of cancer. Because the registries in SEER were not all begun at the same time, to avoid bias the DevCan program only searches the records for previous cancers back until the year when the last registry was added. This year is denoted the follow-back year. (If the disease of interest is any malignant cancer, then the difficulty is handled differently. Although at each cancer record we do not record what specific types of cancers were previously diagnosed for the person, we do know whether any tumors were previously diagnosed. Thus, if the disease of interest is any malignant cancer and if the record states there was a previously diagnosed tumor, then we assume that the previously diagnosed tumor was malignant, and do not count that case as a first incidence.) To handle the second difficulty, the additional person-years in the denominator, Merrill and Feuer \[[@B5]\] adjust the denominator by multiplying the age-specific population by 1 minus an estimate of the prevalence of the disease in the population. Merrill and Feuer \[[@B5]\] also estimate the prevalence of medical procedures which remove individuals from the at-risk population, such as hysterectomy which removes the risk of uterine cancers. In calculating the ACPDvD we use only first incident of the disease of interest as in \[[@B5]\], but we correct for the denominators in a different way using an assumption and some mathematics from the theory of competing risks. This second correction is detailed with precise mathematical notation in Fay et al \[[@B1]\]; here we give more heuristic arguments. In the following let the disease of interest be \"cancer\". The ACPDvD between ages *x*and *y*, given alive and cancer-free at age *x*, may be written as the fraction,  To calculate the numerator, we integrate over the probability that the first cancer occurred at exactly age a. In math notation this probability is  where *f*~*c*~(*a*) is a probability function representing the probability that the first cancer occurred at exactly age a. One key result described in Fay et al \[[@B1]\] is that *f*~*c*~(*a*) can be written as the product of two functions, *λ*~*c*~(*a*) = the probability that the first cancer occurred at exactly age a, given the individual is alive just before age *a*, and *S*~*a*~(*a*-) = the probability that the individual is alive just before age *a*. The function *λ*~*c*~(*a*) is known as a cause-specific hazard function, and it is estimated by some function of the age-specific rates, such as the piecewise constant model of Fay et al \[[@B1]\] or the MAJ model introduced in this paper (see Figure [1](#F1){ref-type="fig"}). Using standard results for continuous survival data, we can write *S*~*a*~(*a*-) as  where *λ*~*a*~(*u*) ( = the probability that the individual died at age *u*, given the individual is alive just before age *u*) is the usual hazard function. We estimate *λ*~*a*~(*u*) using some function of the age-specific rates. Thus, the numerator can be written as  If we use the MAJ for both hazard functions, then there is no closed form solution. To see why this is so, note that within the exponential, the integral of a piecewise linear function is the sum of a series of quadratic functions, and the overall integral has no closed form solution. This problem motivates the piecewise mid-age joinpoint (PMAJ) model, where we use a series of piecewise constant functions to approximate the MAJ model. Figure [2](#F2){ref-type="fig"} gives the PMAJ model together with the piecewise constant model used by Fay et al \[[@B1]\] for 70 to 90 year olds from the SEER 12, 1998--2000 rates for all invasive (first) cancer incidence rates per person-years alive. Remember, although both Figure [1](#F1){ref-type="fig"} and Figure [2](#F2){ref-type="fig"} plot incidence rates, we additionally need similar rate functions for mortality rates to calculate the ACPDvD. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### SEER 12 all invasive cancer incidence rates, 1998--2000, all races, both sexes: Piecewise constant and PMAJ methods. :::  ::: Now consider the denominator of the ACPDvD, the probability of being alive and cancer-free at age *x*, denoted . For reference, in Table [1](#T1){ref-type="table"} we give the notation. The only change from the notation in Fay et al \[[@B1]\] is that we use the subscript *a*to represent all causes of events instead of a blank subscript. For example, we let *S\**(*u*) = . Other notation in this paper is defined as it is introduced. Fay et al \[[@B1]\] assumed that the risk of death from other causes does not change if you have previously been diagnosed with cancer, then used the key result mentioned above together with some algebra and calculus to derive the denominator. Then the ACPDvD between the ages of *x*and *y*given alive and cancer-free just before age *x*is ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Notation ::: Random Variables and Parameters --------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------- *T*= age at death *T*\* = age at first cancer or death before cancer *J*= type of death *J*\* = type of event  (*J*= *d*) = death from cancer  (*J*\* = *c*) = first cancer  (*J*= *o*) = death from other causes  (*J*\* = *o*) = death before first cancer *λ*~*c*~(*t*) = rate at *t*for first cancer given alive = rate at *t*for first cancer given alive and cancer-free *λ*~*o*~(*t*) = rate at *t*for death before cancer given alive = rate at *t*for death before cancer given alive and cancer-free *λ*~*d*~(*t*) = rate at *t*for death from cancer given alive *λ*~*a*~(*t*) = rate at *t*for death given alive = rate at *t*for first cancer or death before first cancer given alive and cancer-free Observations Within the age interval, \[*a*~*i*~, *a*~*i*~+1), and within the calendar interval of interest we observe\... *c*~*i*~= number of first cancer incident cases = estimate of person-years alive associated with *j*= *c*, *d*, *o*(DevCan uses the sum of mid-year populations during the calendar interval of interest) *d*~*i*~= number of cancer deaths *o*~*i*~= number of other deaths :::  The details of the MAJ and the PMAJ models are given in the next two sections. Readers only interested in the practical ramifications of the choice in models may skip to the examples and discussion section. Mid-age group joinpoint estimator ================================= In Fay et al \[[@B1]\], the rates were estimated by a piecewise constant model. Here we use a mid-age group joinpoint (MAJ) model, where we draw lines connecting the midpoints of the intervals except the first and last interval. The first interval is constant until the midpoint, and the last interval is constant after a nominal \"midpoint\". This nominal \"midpoint\" is half the length of the previous age interval from the beginning of the last interval, and would be the midpoint if the last age interval was the same length as the previous interval. We introduce new notation for breaking up the ages. Fay et al \[[@B1]\] used 0 = *a*~0~\<*a*~1~\< ··· \<*a*~*k*~\<*a*~*k*+1~= ∞. Here we use a joinpoint model with joins at the midpoints (and nominal midpoint),  Let  (The indices start at -1 so that the index values for the rate estimators, , match up with the count notation of \[[@B1]\].) The MAJ estimator for the rate of event *j*(for *j*= c, d, or *o*) at *t*~*i*~(for *i*= 0,1,\..., *k)*is  where *j*~*i*~is either *c*~*i*~, *d*~*i*~, or *o*~*i*~as defined in Table [1](#T1){ref-type="table"}. (Note that , where  is the piecewise constant function used in \[[@B1]\]). We define  and . For *j*= *a*, MAJ estimator for the rate at *t*~*i*~is  Then for t ∈ \[*t*~*i*~, *t*~*i*+1~) for *i*= 1,\..., *k*, we define  as the point on the line defined by connecting the points (*t*~*i*~, ) and (*t*~*i*+1~, ). In other words,  Where  and  Thus, *α*~*j*,-1~=  and *β*~*j*,-1~= 0, and similarly by taking limits as *t*~*k*+1~→ ∞ then *α*~*j*,*k*~=  and *β*~*j*,*k*~= 0. Now  for *u*∈ \[*t*~*i*~, *t*~*i*+1~) is  Note that (for ℓ = 0,1,\..., *k*)  so that for *i*= 0,1,\...,k,  Also notice that (when *u*\< ∞)  Therefore when *u*∈ \[*t*~*i*~*, t*~*i*+1~),  Let (*x, y*) be the estimator of *A*(*x, y*) using the MAJ model. The two integrals we need to estimate for (*x, y*) are of the type,  where in the numerator of (*x, y*) we need  (i.e., *j*= *c*and *h*= *a*in equation 7), and in the denominator of (*x, y*) we need . Suppose, without loss of generality, that *t*∈ \[*t*~*i*~*,t*~*i*+1~), then  where *R*~*j*,*h*~(*t*~ℓ~, *v*) (for ℓ = - 1,0,1,2,\..., *i*and *v*≤ *t*~ℓ+1~) is defined implicitly (see the Appendix). Then,  Piecewise mid-age group joinpoint estimator =========================================== In the MAJ model we divided up the age line into *k*+ 2 intervals. Here we define those intervals in both the *t*~*i*~notation and the *a*~*i*~notation.  In the MAJ model the rates for the first and the last intervals are represented by lines with zero slope, and the rates for the *i*th interval (*i*= 1,\...,*k*) for the *j*th rate type (*j*= *a*, *c*, *d*, *o*) is a line defined by connecting the points (*t*~*i*-1~, ) and (*t*~*i*~, ) (see equations 2 and 3 for definition of ). In the PMAJ model we divide the *i*th interval into *m*~*i*~equal sized intervals, and use a piecewise constant estimate on each of those *m*~*i*~intervals. One way to define *m*~*i*~is to chose *m*~*i*~so that each equal sized interval is 1/2 year long. In other words, *m*~*i*~= 2(*t*~*i*~- *t*~*i*-1~). This is the definition of *m*~*i*~that we use for the DevCan software (starting with version 5.0, see \[[@B3]\]), but all the following holds for arbitrary *m*~*i*~. In Figure [2](#F2){ref-type="fig"} we show the PMAJ model with half-year intervals and the piecewise constant model for the US all invasive cancer mortality rates for ages 70 through 90 years. Here are the details. Consider the *h*th (for *h*= 1,\..., *m*~*i*~) of the *m*~*i*~intervals within interval *i*(for *i*= 1,\...,*k*) for rate type *j*(for *j*= *a*, *c*, *d*, *o*). This interval is  For convenience we introduce new notation for the ends of this interval, let  so that *t*~*i*-1,0~= *t*~*i*-1~and  = *t*~*i*~. At the beginning of this interval the value of the rate is  (see equations 4 and 6 for definitions of *α*~*j*,*i*-1~and *β*~*j*,*i*-1~). Similarly at the end of this interval the rate is  For the PMAJ model we simply assume a constant rate equal to the average of the beginning and the end values of the rate over this interval. In other words, under the PMAJ model for any *t*∈ \[*t*~*i*-1,*h*-1~*,t*~*i*-1,*h*~) we estimate the rate with  Since the PMAJ model is a piecewise model, we can use Appendix A of \[[@B1]\] to express the estimator of age conditional probability of developing cancer. The only hard part is correctly defining the starting and ending of each piecewise interval. The ends of these intervals are  For convenience write these interval ends with only a single index as  where  and *m*~0~= 1. In other words, *t*~-1~= *τ*~0~and for *i*= 0,1,\..., *k*, then *t*~*i*~= *τ*~*g*~(*i*) and *t*~*i*,*h*~= *τ*~*g*~(*i*)+*h*, where . Now we can follow very similar notation to Appendix A of \[[@B1]\]. We now repeat that Appendix with the modifications to notation required for the PMAJ model. Let the estimator of *A*(*x,y*) under the PMAJ model be denoted (*x,y*). Let *τ*~*i*~≤ *x*\<*τ*~*i*+1~and *τ*~*j*~\<*y*≤ *τ*~*j*+1~for *x*\<*y,i*≤ *j*, and *j*≤ *M*+ 2. For convenience we regroup the ages after inserting group delimiters at *x*and *y*. Let the new delimiters be 0 = *b*~0~*≤ b*~1~≤ *b*~2~≤ ··· ≤ *b*~*M*+3~= ∞ where *b*~0~= *τ*~0~,\..., *b*~*i*~= *τ*~*i*~, *b*~*i*+1~= *x, b*~*i*+2~= *τ*~*i*+1~,\..., *b*~*j*+1~= *τ*~*j*~, *b*~*j*+2~= *y, b*~*j*+3~= *τ*~*j*+1~,\..., *b*~*M*+3~= *τ*~*M*+1~= ∞. We let  and similarly  and . In this notation, the probability of developing cancer by age *y*given survival until age *x*is *A*(*x, y*) = *A*(*b*~*i*+1~, *b*~*j*+2~), and under the PMAJ model we estimate it with  Because  or  may equal zero and *b*~ℓ+1~may equal infinity, we let . These integrals are  where the case *λ*= 0 and *b*~ℓ+1~= ∞ is one of the \"impossible\" hypothetical cohorts (see Section 3.1 of \[[@B1]\]). Thus, we obtain,  Examples and discussion ======================= In this section we explore several different methods for estimating the rate functions, all using the formula of Fay et al \[[@B1]\] (e.g., all using equation 1). This comparison explores the differences between the piecewise constant method proposed in Fay et al \[[@B1]\], the PMAJ method, and the MAJ method. A different comparison emphasizing differences between versions of the DevCan software is described in the supplemental material \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. For all of the examples we use data from 1998--2000 \[[@B6]\]. The incidence data come from the Surveillance, Epidemiology, and End Results (SEER) program of the (U.S.) National Cancer Institute, and mortality data from the (U.S.) National Center for Health Statistics. We use the SEER 12 registries which cover about 14 percent of the U.S. population. We only use the mortality data covering the same area as the SEER 12 registries cover. Because the SEER 12 registries have complete coverage only back through 1992, we only look back in the database until 1992 to delete any incident case that had previously been diagnosed with the cancer of interest. These incident cases are deleted so that they are not counted when estimating the counts of first cancer incidence (the *c*~*i*~values). The mid-year population estimates (the *n*~*i*~values) come from the sum U.S. Census estimates of mid-year populations from 1998, 1999, and 2000 for the SEER 12 catchment areas for the appropriate sex group (e.g., males for prostate cancer). In Table [2](#T2){ref-type="table"} we show the results for all invasive cancers and acute lymphocytic leukemia for both sexes, prostate cancer for males, and breast cancer for females. We see the PMAJ values approximate the MAJ values very well. In conclusion, we have described several methods for estimating rates for input into a formula to calculate ACPDvD, and we have shown that the PMAJ method provides fast and reasonable estimators for the rates. Appendix: Calculation of *R*function ==================================== Recall that *R*~*j,h*~(*t*~ℓ~, *v*) represents an integral with 4 parameters. We can write it as  To simplify notation substitute let *t*~ℓ~ = *u*and *α*~*j*ℓ~ = *α*~*j*~*,β*~*j*ℓ~= *b*~*j*~,*α*~*h*ℓ~= *a*~*h*~, and *β*~*h*ℓ~= *b*~*h*~. Thus,  Case 1: *b*~*j*~= 0 and *b*~*h*~= 0 ----------------------------------- For our application, whenever *v*→ ∞ then *b*~*j*~= 0 and *b*~*h*~= 0, so this is an important special case. When *b*~*j*~= 0 and *b*~*h*~= 0 and *a*~*h*~= 0 and we obtain  which goes to ∞ when *v*→ ∞. When *b*~*j*~= 0 and *b*~*h*~= 0 and *a*~*h*~≠ 0 and we obtain  which goes to *a*~*j*~/*a*~*h*~when *v*→ ∞. Case 2: General Case with *v*\< ∞ --------------------------------- To calculate the integral, *R*(*u, v, a*~*j*~, *b*~*j*~, *a*~*h*~, *b*~*h*~) for finite *v*, we can use an adaptive use of Romberg\'s algorithm for numeric integration (we follow closely Lange \[[@B7]\], pp. 210--211). Let  Divide the interval \[*u, v*\] into *n*equal subintervals of length (*v*- *u*)/*n*, and let  Then lim~*n*→∞~*T*~*n*~= *R*(*u, v, a*~*j*~*, b*~*j*~, *a*~*h*~, *b*~*h*~). A more accurate approximation uses Romberg\'s algorithm,  Let  be our estimate of *R*. The algorithm we use to calculate  is as follows: 1\. Choose *n*. 2\. Calculate *T*~*n*~. 3\. Calculate *T*~2*n*~. 4\. For i = 1 to *I*~*max*~do: • If  then let  and stop. • Otherwise calculate , and continue. For example, one could use *n*= 100 and *δ*= 10^-5^and *I*~*max*~= 100. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Comparing the method of Wun, Merrill, and Feuer (1998) to the PMAJ method. We calculate lifetime risks of developing certain cancers for different race and sex combinations. For each lifetime risk we give the old method of Wun, Merrill, and Feuer \[4\], the PMAJ method, and the percent difference. In general, the two methods agree to within about 2 percent. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ I would like to thank Kathy Cronin for suggesting the PMAJ method and thank her and Ram Tiwari for reading and commenting on drafts of this article.

PubMed Central

2024-06-05T03:55:47.685629

2004-7-27

PMC517511

Introduction ============ The development of an effective vaccine against HIV has been hindered by a variety of problems. The high mutation rate of the virus itself is such that it represents a moving antigenic target during the course of an infection \[[@B1]-[@B4]\]. Furthermore, HLA-A and -B expression is directly downregulated by HIV (via intracellular blocking of class I MHC-export to the cell surface by HIV-1 Nef and Vpu), so that efficient antigen presentation is compromised \[[@B1],[@B5]\]. Compared to administration of protein antigen or naked DNA, an infectious vector could be more effective at enhancing antibody and cytotoxic responses against a transgene product. Application of such a strategy, however, has been often complicated by the development of neutralizing immune responses, principally antibodies, against vector coat antigens \[[@B6]-[@B10]\]. These neutralizing antibodies arise because the viral vectors enter cells largely through endocytic pathways. Their capsids, like most other particulate antigens, are processed at the time of infection and presented to the immune system. Resulting immune responses neutralize subsequent injections of the vector, and so limit the ability of that vector to be used repeatedly to boost immune responses. This limitation can be circumvented by repeatedly changing the serotype of the antigen-carrying vector, or by using recombinant *Tag*-deleted SV40-derived gene delivery vectors (rSV40s) for immunization. Several studies have shown, both directly and indirectly, that rSV40 vectors do not elicit detectable neutralizing antibodies \[[@B11]-[@B13]\]. Even repeated administration of single \[[@B11],[@B12]\] or different \[[@B13]\] rSV40 vectors in normal, immunocompetent hosts does not generate antibodies against the vector capsid proteins sufficiently to impair the ability of these vectors to deliver their genes efficiently *in vivo*. The explanation for this unusual state of affairs may lie in the fact that SV40 enters cells via caveolae and thence travels directly to the nucleus, bypassing cellular antigen processing \[[@B14]-[@B16]\]. Thus, only proteins expressed by virus can elicit immune responses. Since, for *Tag*-deleted rSV40 vectors (unlike wild type SV40), capsid proteins are not expressed, immune responses can only be generated by transgene products \[[@B11],[@B12]\]. Whether for this or for other reasons, rSV40 vectors can be used multiple times to prime and/or boost immune responses against antigens encoded by the transgenes they carry \[[@B13],[@B14]\]. We have previously shown that powerful transgene-specific cytolytic and serum antibody responses can be detected in mice inoculated with rSV40 carrying the cDNA for SIVmac239 envelope glycoprotein gp130 \[[@B12]\]. Four to five monthly immunizations were adequate to produce \>50% specific lysis of envelope-expressing target cells, even with effector:target ratios of 10:1 \[[@B12]\]. Other investigators have reported that co-administration of vectors carrying immunostimulatory cytokines was useful in augmenting anti-lentiviral immune responses \[[@B17]-[@B19]\]. IL-15 has various immunostimulatory and immunomodulatory effects, among which is the ability to upregulate activated T cell proliferation and induce cytotoxic T cell activity \[[@B20]\]. It also promotes cytotoxic T cell memory \[[@B21],[@B22]\]. Both antibody and cell-mediated immune responses may be useful to protect from HIV infection and progression to AIDS \[[@B23]-[@B26]\]. However, there is a particularly good correlation between long-term non-progression to AIDS and strong CTL responses in HIV-positive individuals \[[@B22],[@B27]-[@B31]\]. Weak CTL responses are generally seen in those who progress rapidly to disease, and in children. Because of the importance of a virus-specific cytotoxic T cell (CTL) response, one of the major aims of any vaccine should be to elicit strong HIV-specific CTL responses \[[@B32],[@B33]\]. We used rSV40s to study the generation and longevity of both humoral and cell-mediated responses in an effort to generate immune responses against the HIV-1 envelope glycoprotein, gp120. We also tested whether co-immunization regimens involving rSV40 delivery of both IL-15 and gp120 augmented and/or accelerated SV40-mediated immune responses further. Methods ======= Cell Lines ---------- The murine mastocytoma cell line P815 (ATCC, Bethesda, MD, USA) was used, and maintained in culture with Dulbecco\'s Modified Eagle\'s medium (DMEM), supplemented with 10% newborn calf serum (NCS) (Gibco BRL/Life Technologies, Grand Island, NY, USA). COS-7 cells (ATCC, Bethesda, MD, USA), were used to expand stocks of recombinant SV(gp120) and SV(mIL-15) viruses. Cytotoxic lymphocytes were obtained from spleens of immunized mice, and cultured in RPMI-1640 (Gibco BRL/Life Technologies, Grand Island, NY, USA) supplemented with 10% NCS (RPMI-10). Rabbit kidney fibroblasts (RK13 cells) and CV-1 cells (African green monkey kidney cells) were obtained from ATCC (Bethesda, MD, USA). RK13 cells were used to propagate stocks of VCB41, a vaccinia virus vector carrying HIV-1NL4-3 envelope gp120 sequence. Mice ---- BALB/cJ mice aged 6--8 weeks were purchased from Jackson Laboratories, Bar Harbor, ME, USA. They were fed and housed in accordance with American Association for Accreditation of Laboratory Animal Care standards. Use of mice in the laboratory protocols described was approved by the Thomas Jefferson University Institutional Animal Care and Use Committee. Generation of SV(gp120) ----------------------- A 1.6 kb DNA fragment encoding gp120 from HIV-1NL4-3 was made by PCR using primers with engineered restriction sites. This PCR product was cloned into pT7A5 (a plasmid containing an SV40 genome, in which large T antigen gene was replaced by cytomegalovirus (CMV) immediate early promoter and downstream polylinker), giving pT7A5-gp120. To make SV(gp120), the SV(gp120) genome was released from the carrier plasmid by restriction digestion, and used to make recombinant virus in COS-7 cells as described previously \[[@B34]\]. Virus stocks were purified and titered, as described elsewhere \[[@B34]\]. SV(HBS), a control virus for these studies, carries hepatitis B surface antigen (HBsAg), and has been reported previously \[[@B11]\]. Generation of SV(mIL-15) ------------------------ To generate a recombinant SV40 virus with the murine IL-15 transgene (SV(mIL-15)), mIL-15 cDNA was cloned into pSL-4p, which contains a *Tag*-deleted SV40 genome \[\[[@B35]\], Vera, *et al*., in preparation\], to yield prSVmIL-15. Virus was made from this plasmid in COS-7 cells as previously reported \[[@B32]\]. Immunization of mice -------------------- Mice were given monthly 1 × 10^9^infectious units (IU) of SV(gp120) ± SV(mIL-15) intraperitoneally (IP). In some studies, final administrations included both IP and subcutaneous (SQ) inoculations. SV(HBS) was used as a control antigen-carrying vector. Specific immunization schedules are described in the **Results**section, below. Stably-transfected P815 target cells for cytotoxicity assays ------------------------------------------------------------ Production of HIV Env-expressing stably transfected targets is similar to the procedure used for generating SIV Env-expressing targets \[[@B12]\]. Briefly, gp120 cDNA was cloned into pCDNA3. The resulting plasmid, pcgp120, was co-transfected into P815 cells together with the neomycin resistance-carrying plasmid, pSV2Neo. Transfected cells were selected in G418-supplemented DMEM-10, then cloned by limiting-dilution. Viable clones were expanded, assayed for gp120 expression by flow cytometry, and maintained thereafter in G418-supplemented medium. Flow cytometric detection of cell surface gp120 expression ---------------------------------------------------------- Flow cytometry was used to verify gp120 expression on the surface of P815 cells. A recombinant vaccinia virus carrying HIV-1NL4-3 gp120 (VCB41, NIH AIDS Reference Reagent Repository Program (NIH-ARRRP)) was used both as a positive control for gp120 expression and also to generate gp120-expressing target cells in some experiments. Cells that had been stably-transfected with plasmid gp120, or infected with VCB41 both expressed gp120, as assayed by flow cytometry (Coulter-Epic, Kimmel Cancer Center, TJU) (data not shown). The gp120-expressing P815 population was then cloned by limiting dilution. Clonal outgrowths were then reanalyzed by cytofluorimetry (FACS, data not shown) and the single clone expressing the highest levels of gp120, clone 24, was used in subsequent studies as a target for cytotoxicity assays. Anti-gp120 binding-antibody detection using a CELISA ---------------------------------------------------- An ELISA method was used to assay the activity of anti-gp120 antibodies elicited by immunization of the mice with SV(gp120) ± SV(mIL-15). The strategy for our appraoch to testing for antibodies vs. HIV Env is similar to one we have used to measure binding activity vs. SIV Env \[[@B12]\]. Briefly, a cell-based assay was developed using VCB41-infected P815 cells (cells were infected with virus for 48 hours prior to being used in assay) as control targets. Sera were taken from mice at 2- and 4-week intervals after immunization(s). Antibody reactivity vs. cell membrane-expressed gp120 was tested by measuring A~405nm~of test sera vs. VCB41-infected P815 cells, subtracting A~405nm~due to binding to wild type (wt) VV-infected P815 cells, and also subtracting A~405nm~of control sera from mice immunized in parallel with a control rSV40, SV(HBS) Measurement of cytotoxic lymphocyte activity by specific lysis of ^51^Cr-labeled target cells --------------------------------------------------------------------------------------------- Wild type (wt) P815 cells, or clone 24 P815 cells expressing gp120 were the target cells for unselected lymphocytes from spleens of mice immunized with SV(gp120) (± SV(mIL-15)), or SV(HBS) as control. Where SV(gp120) was used alone, P815 cells infected with wt vaccinia virus or VCB41 were target cells for spleen cells of immunized mice. Mice were boosted simultaneously with 1 × 10^9^IU intraperitoneally (IP) and 1 × 10^8^IU subcutaneously (hind footpads) usually 4 d before assay, but up to 1 month prior to assay, to test cytolytic lymphocyte memory. Spleen cell concentrations adjusted to 2 × 10^6^/ ml with RPMI-10. In some assays of cytotoxic lymphocyte memory, effector cells were harvested 1 month after the final injection. Effector cells from immunized mice were prepared as described, but in addition, were incubated with 5 μg/ml Concanavalin A (Con A, Sigma Chemical Co., St. Louis, MO) overnight, prior to assay. Con A-stimulated cells were then harvested, washed once in RPMI-10, and then used with target cells in the assay. P815 target cells were washed, then labeled with ^51^Cr (ICN Biomedicals, Inc., Irvine, CA, USA) (100 μCi per 1 × 10^6^cells) at 37°C, 5% CO~2~for 4 h as described previously \[[@B12]\]. Afterwards, target cells were washed, then plated in triplicate with effector cells (splenocytes) at effector:target (E:T) ratios of 20:1 and 10:1, and incubated at 37°C, 5% CO~2~for 4 hours. Supernatant ^51^Cr was counted (1282 Compugamma CS, LKB) \[[@B12]\]. Mean specific lysis was calculated as: Mean c.p.m. for gp120-immunized effector cells mixed with gp120-expressing targets, minus the mean c.p.m. control (SV(HBS))-immunized effector cells vs gp120-expressing targets, and expressed as a percentage of the maximal target cell lysis (target cells incubated with 1% Triton-X). Background release of ^51^Cr from wild type target cells was subtracted. Thus: \% specific ^51^Cr release = {\[c.p.m. ^51^Cr released by SV(gp120)-immune populations from gp120-expressing P815 cells\] minus \[c.p.m. ^51^Cr released by SV(HBS)-immune lymphocytes from gp120-expressing P815 cells\]}, divided by \[c.p.m. ^51^Cr released by Triton X-100 from gp120-expressing P815 cells\]. The same calculations were done for lysis of wild type P815 cells by gp120-immune and control-immune effector populations. These numbers were then subtracted from the calculated ^51^Cr release above to determine the gp120-specific lysis of target cells by SV(gp120)-immunized effector cells. Western analysis of IL-15 expression in SV(mIL-15)-transduced P815 cells ------------------------------------------------------------------------ P815 cells were transduced with SV(mIL-15) ×1 at m.o.i. = 100. Culture supernatants were harvested at several times post-transduction, and stored at -80°C. At day 6 post-transduction, a well of cells was harvested and lysed (2% NP40, 50 mM Tris pH7.4,150 mM NaCl, 1 mM EDTA, 10% Glycerol + protease inhibitor cocktail (25× stock Complete™ EDTA-free protease inhibitor cocktail, Roche Diagnostics GmbH, Mannhein, Germany)). Remaining wells were activated non-specifically with 5 mg/ml Con A, and supernatants harvested at various times thereafter. 3 days after con A stimulation, cells were lysed as described above. 50 μg of each culture supernatant or lysate were loaded on a 4--20% Tris-HCl gradient gels (Ready Gel, Bio-Rad, Hercules, CA, USA). 50 ng recombinant human IL-15 was used as a positive control. Samples were electrophoresed, and blotted to PVDF membranes (Immobilon™-P, Millipore Corporation, Bedford, MA). Blots were blocked overnight at 4°C with 5% milk in PBS + Tween-20 (0.05%). Rabbit anti-mouse IL-15 (Abcam, Cambridge, UK) was used as primary antibody, (diluted 1:500 with PBS-Tween), for 2 h at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) was used at 1:10,000 dilution in PBS-Tween, for 1 h at room temperature. Signal was detected with chemiluminescence reagent (ECL Plus, Amersham Pharmacia Biotech UK Ltd., Little Chalfont, UK,) Assaying for IFNγ production stimulated by IL-15 ------------------------------------------------ COS-1 cells were infected with SV(mIL15) or SVLUC (carrying luciferase) as a negative control. 24 h later, the media were changed and cells incubated 48 h in 500 μl of RPMI 10% serum/well. Fresh mouse spleen cells (5000/well) then cultured 48 h with 100 μl of cell supernatant + 100 μl of RPMI 10% serum. IFNγ ELISA (Pharmingen) was performed on the supernatant from these cultures. Results ======= Stably transfected HIV-1 gp120-expressing P815 cells ---------------------------------------------------- P815 cells stably transfected to express HIVNL4-3 gp120 were selected and cloned by limiting dilution (see **Methods**). We used flow cytometry to identify the clone most strongly positive for cell membrane gp120. Compared to other stably-transfected clones, \"clone 24\" expressed gp120 at the cell membrane best (data not shown). VCB41-infected and SV(gp120)-transduced P815 cells also expressed substantial cell membrane gp120. Control wtP815 cells, or P815 cells infected with wt VV did not (data not shown). Therefore, clone 24 cells were used to assay gp120-specific immune responses. In both antibody and cytotoxicity assays, two different types of background were subtracted from the responses of gp120-immunized mice: serum binding or cellular reactivity from gp120-immunized animals vs. wt P815 cells and reactivity from control (i.e., SV(HBS))-immunized rSV40-immunized mice vs. clone 24 cells. Thus, data presented below reflect gp120-specific responses against clone 24. Immunization with SV(gp120) --------------------------- Normal BALB/c mice were inoculated with SV(gp120), and their sera were assayed for reactivity vs. gp120 by CELISA. The details of this cell-based ELISA, or CELISA, as described in **Methods**. Specific binding antibody activity was first statistically significant, compared to prebleed sera 2 weeks after the second inoculation of SV(gp120) (P = 0.000332, using two-tailed Student\'s t-test) and reached a plateau after the third inoculation (P = 0.000000316 by the same analysis) (Figure [1](#F1){ref-type="fig"}). Additional immunizations beyond the third did not further increase detectable antibody levels (data not shown). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Serum antibody against HIV-1NL4-3 envelope glycoprotein gp120 in mice receiving multiple inoculations of SV(gp120) BALB/cJ mice were immunized at monthly intervals with 1 × 10^9^infectious units (IU) SV(gp120), IP. They were bled biweekly. Gp120-specific antibody reactivity was assayed by CELISA, as described in **Methods**and in reference \#12. Specific binding of HIV-1 Env is shown here as specific A405nm, ± S.E.M. :::  ::: Cytolytic responses against gp120: testing for cytotoxic lymphocyte memory -------------------------------------------------------------------------- An effective anti-lentiviral immunization regimen should generate cytotoxic memory cells. To see if SV(gp120) treatment could do this, mice were immunized once with SV(gp120) IP, then sacrificed 1 month later, without further treatment. In order to lyse target cells, committed cytotoxic cells require activation. However, to avoid antigen-specific selection and specific stimulation only of gp120-reactive cytotoxic cells, splenic lymphocytes were non-specifically stimulated by overnight incubation with Con A. A single immunization with SV(gp120) alone elicited only weak memory lytic responses (≤ 10% specific lysis) against gp120-expressing target cells (Figure [2](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Specific cytolytic activity against HIV-1NL4-3 gp120 in mice immunized with SV(gp120) and assayed one month after final injection. BALB/cJ mice were immunized twice with SV(gp120) IP at monthly intervals. Splenocytes were harvested one month after final inoculation. Unselected effector cells were added to ^51^Cr-labelled target cells and specific lysis of gp120-expressing cells was calculated as described in **Methods**, ± S.E.M. Results shown here represent ≥ 3 independent determinations per data set. :::  ::: A second group of animals received a second inoculation with SV(gp120) one month after the first, then were assayed the same way one month later for anti-gp120 cytolytic activity. These mice made stronger specific memory responses (15--20% specific lysis) than did animals given only a single inoculation (P ≤ 0.04, by Student\'s t-test, comparing 2 injections with just one) (Figure [2](#F2){ref-type="fig"}). To test whether SV(gp120) could elicit very long term cytotoxic lymphocyte memory, mice were immunized monthly ×8 with SV(gp120) IP. A final IP inoculation with SV(gp120) was given *1 year after their eighth immunization*. They were sacrificed 4 days later, and direct gp120-specific splenocyte cytotoxicity was measured. Unselected spleen cells from all animals made very strong (≥ 50% specific lysis) gp120-specific cytolytic responses (mean specific lysis of 61% ± 4.2). IL-15 expression and secretion in SV(mIL-15)-transduced P815 cells ------------------------------------------------------------------ Because higher levels of durable memory cytotoxic responses could be achieved with repeated injections of SV(gp120), and lower levels were seen with 2 injections, we tried to accelerate development of such responses using IL-15, delivered by transduction. To determine if IL-15 could be expressed by transduction, P815 cells were transduced with SV(mIL-15) at m.o.i. = 100. Culture supernatants were harvested 36, 72 and 144 hours later, at which point the cultured cells were activated with Con A. Culture supernatants were collected at 24 and 72 hours post-activation. Supernatants were assayed for IL-15 secretion by Western analysis as described in **Methods**, using rabbit antibody vs. murine IL-15 (Figure [3](#F3){ref-type="fig"}). The positive control, recombinant human IL-15, has approximately 60% sequence homology with murine IL-15). IL-15 secretion was detectable, but just barely so, in unstimulated culture supernatants. It was abundant by 72 hrs post-stimulation. These data were used in planning co-transduction experiments. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Western analysis of IL-15 expression in culture supernatants from SV(mIL-15)-transduced P815 cells P815 cells were transduced with SV(mIL-15) at a m.o.i. = 100. 1.5, 3 and 6 days post-transduction, culture supernatants were collected. Cultures were then stimulated with Con A and additional supernatants harvested 24 and 72 hours later. IL-15 secretion into supernatants was visualized by Western analysis. Recombinant human IL-15 (rhIL-15) was the positive control (non-adjacent lane). Supernatants from unstimulated cultures were tested in parallel (non-adjacent lane). :::  ::: The functionality of the IL-15 produced in this fashion was tested by exploiting the ability of IL-15 to elicit production of IFN-γ by lymphocytes. Thus, CV-1 and COS-7 cells (African green monkey kidney cells) were transduced by SV(mIL-15), then cultured for 72 hrs. Control cultures of the same cells were transduced with SVLUC (carrying luciferase as a transgene). Normal mouse spleen cells were cultured for 42 hrs in 200 μl of the resulting culture supernatants. Production of IFN-γ by the spleen cells was measured by ELISA. Supernatants from COS-7 and CV-1 cells elicited respectively 2056 ± 363 pg/ml and 880 ± 196 pg/ml IFN-γ. Supernatants from SVLUC-transduced cells did not elicit detectable interferon secretion by spleen cells (\<20 pg/ml). Effects of IL-15 in cytotoxic lymphocyte responses against gp120 ---------------------------------------------------------------- To determine whether coordinate administration of SV(mIL-15) plus SV(gp120) improved cytolytic responses against gp120, mice were given two sets of injections IP, one month apart. Normal BALB/c mice received IP with 10^9^IU of rSV40: one group was given SV(mIL-15) alone, and one group SV(gp120) alone. Three other groups received both SV(mIL-15) and SV(gp120): either SV(mIL-15) followed 3 days later by SV(gp120), or SV(gp120) first, followed by SV(mIL-15). The final group was given both SV(mIL-15) and SV(gp120) simultaneously. The 3 day separation between the two vectors was used because of the strength of the signal for secreted IL-15 by Western blotting at 72 hours post-stimulation (see above). One month later, unselected spleen cells were assayed as described above for cytolytic activity against gp120-expressing clone 24 cells. Adding IL-15 to the immunization regimen greatly increased gp120-specific cytolytic responses (Figure [4](#F4){ref-type="fig"}). Also, the timing of cytokine administration relative to SV(gp120) inoculation significantly affected the responses seen. Mice given SV(mIL-15) 3 d after SV(gp120) did not make detectable gp120-specific cytotoxic responses. Simultaneous inoculation with SV(mIL-15) and SV(gp120), however, increased specific cytolysis to ≥ 20%, which was significant at E:T = 20:1 (P ≤ 0.05, using Student\'s t-test) compared to SV(gp120) alone. The most dramatic results were observed when SV(mIL-15) was administered 3 d before SV(gp120). Those mice demonstrated highly significant augmentation by SV(IL-15) of gp120-specific lysis, which was ≥ 60% at both 20:1 and 10:1 E:T ratios (P ≤ 0.02 using Students\' t-test, compared with immunization of SV(gp120) alone). Mice injected ×2 with SV(mIL-15) alone made no significant gp120-specific cytolytic responses at either 20:1 or 10:1 effector:target ratios. Mice given only SV(gp120) demonstrated ≈ 10% specific lysis at E:T = 20:1. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Specific cell-mediated responses against gp120-expressing target cells by splenic effectors from co-immunized mice Mice were given two monthly injections with either SV(mIL-15), SV(gp120) or both cytokine and antigen sequentially or simultaneously IP. One month after the final inoculation(s), unselected spleen cells were assayed for specific cytolytic activity against gp120-expressing clone 24 cells labeled with ^51^Cr, as described in **Methods**. Results shown here represent ≥ 3 independent determinations per data set. :::  ::: Effect of IL-15 co-administration on anti-gp120 antibody responses ------------------------------------------------------------------ Mice receiving SV(mIL-15) and/or SV(gp120) (or the control vector, SV(HBS)) according to the schedules outlined above were tested to determine the effect, if any, of such co-administration in anti-gp120 serum antibody responses. CELISA and calculation of gp120-specific antibody binding were performed as described in **Methods**. Slight binding antibody activity was detected 2 weeks after the first inoculation(s), in all SV(gp120) recipient groups. Levels of SV(gp120)-induced groups made detectable antibody responses were not appreciably affected by coadministration of SV(mIL-15) (data not shown). Discussion ========== In this study, we used rSV40 vectors to elicit HIV-1NL4-3 gp120-specific cytotoxic lymphocyte and antibody responses. We have observed that these vectors may be administered repeatedly to boost those responses. Further studies also suggested that such responses are durable *in vivo*. Our results here demonstrate several important strengths of using rSV40 vectors to immunize against lentiviral antigens: Among these are the ability of the vector to be administered multiple times without eliciting neutralizing responses \[[@B11]-[@B13]\], and the magnitude of the cytotoxic responses to the vector-encoded lentiviral target antigen. When SV(gp120) was given alone, i.e. without added SV(mIL-15), levels of specific cytotoxicity increased with additional SV(gp120) injections: After 2 injections, ≈ 20% specific lysis was seen, which increased to \>70% specific lysis after 7 injections. The potency of rSV40 immunization to elicit cytotoxic immune responses is underscored by the fact that these responses were measured in direct ^51^Cr-release assays: unselected lymphoid organ populations were added directly to labeled target cells at low E:T ratios, and specific ^51^Cr release was measured. Analysis to confirm CD8 expression, or expression of other CTL markers was not performed on the effector cells. However, it is unlikely that these data reflect the cytotoxic activity of NK cells. NK cytolytic activity is non-specific and does not increase with repeated immunization. The patterns of ^51^Cr release observed in the current studies were extensively controlled to ascertain the antigen-specificity of the cytolysis observed: background lysis of wild type P815 cells was subtracted, as was lysis by lymphocytes from mice immunized with an irrelevant rSV40 vector. We also found that cytolysis increased with increasing numbers of immunizations, which is not a characteristic of NK cell-mediated lysis. Since a key goal for a vaccine against HIV is to generate immune responses that are durable *in vivo*, we tested whether cytotoxic lymphocyte activity elicited by SV(gp120) immunization, was detectable one month after inoculation. Thus, cytolytic responses, assayed one month after a second injection, were ≈ 20%, which is comparable to those of splenic cytotoxic cells assayed four days following a third inoculation (data not shown). Further, mice given multiple injections of SV(gp120), then rested *for one year,*gave ≈ 70% specific lysis when challenged with SV(gp120). Therefore, SV(gp120) administration may thus favor development of cytotoxic lymphocyte memory. In an attempt to accelerate and to improve upon these specific cytotoxic and particularly cytotoxic memory responses, we co-immunized mice with SV(gp120) and a rSV40 carrying mouse IL-15. IL-15 promotes cytotoxic lymphocyte responses, and in particular, cytotoxic memory responses \[[@B23],[@B24]\]. The biological effects of IL-15 are less well understood than are those of some of the other immunostimulatory cytokines that have been applied to these types of immunization protocols, such as IL-2, IL-12 and IFN-γ. IL-15 is not a T cell-derived product, but rather appears to be produced by a variety of cells, such as epithelial cells, stromal cells and muscle. It acts on activated T cells, sometimes similarly to IL-2, but it has activities distinct from those of IL-2. IL-15 may play a role in T cell activation in the CNS. It also promotes cytotoxic responses, cytotoxic T cell memory, and natural killer (NK) cell maturation \[[@B33],[@B34]\]. Accordingly, our analysis of the contribution by IL-15 to cytotoxic responses, focused mainly on the ability of SV(mIL-15) to augment specific cytotoxic responses of spleen cells from animals rested 1 month following immunization. Because quiescent cytotoxic T cells are not strong effectors, we non-specifically activated the splenocytes prior to assay with Con A. Non-specific activation was used to avoid specifically enriching effector cell population for gp120-specific cells *in vitro*. Furthermore, low effector:target ratios (20:1 and 10:1) were used in these assays. Our immunization protocols tested both simultaneous and staggered administration of rSV40s carrying HIV-1NL4-3 gp120 and murine IL-15. IL-15 co-immunization dramatically accelerated cytotoxic responses, depending on the immunization regimen used: Animals given SV(mIL-15) alone made no gp120-specific cytolytic responses. Mice receiving 2 treatments with a mixture of SV(gp120) and SV(mIL-15) gave much higher specific lysis, depending on the coadministration regimen, as compared to those receiving SV(gp120) alone (≈ 10% specific lysis). Thus, among mice given staggered injections of SV(mIL-15) and SV(gp120), the order of cytokine administration greatly affected the response: if SV(gp120)was given first, no detectable gp120-specific cytolysis was observed. However, if the cytokine was given first, followed 3 days later by SV(gp120), ≥ 60% specific lysis was seen at both 20:1 and 10:1 effector:target ratios. Why the order of cytokine administration should affect antigen-specific responses so dramatically is not yet clear. Cytokine given after, or together with antigen, may have insufficient time to augment cytotoxic responses. In addition, Western analysis of IL-15 production showed that IL-15 secretion was not detectable in supernatants beyond 36 hours, but could be stimulated subsequently. Thus, a specific, possibly brief, window for IL-15 expression and secretion may need to be attained, in order for its effects on gp120-specific responses to be detectable. We observed very high levels of specific lysis by these unselected effector populations following just two tandem injections of SV(mIL-15) followed by SV(gp120). The strong anti-lentiviral cytolytic responses we report were observed in a strain of mouse, BALB/cJ, that generally mounts relatively weak type 1 T cell responses. The finding of \>60% cytolysis with two administrations of SV(gp120) + SV(mIL-15), suggests that a strategy similar to that described herein may be helpful in individuals who would generate relatively low cytolytic responses. Serum antibody levels assayed by CELISA where SV(gp120) was administered alone, multiple times, were detectable after two immunizations, and continued to increase up to week 4 following the third immunization. These responses were not further enhanced by subsequent boosting immunizations. While specific antibody responses against gp120 were detected in all experimental groups following SV(gp120) and SV(mIL-15) co-immunization, IL-15 co-administration did not augment anti-gp120 antibody levels, compared to gp120 alone. This was to be expected, since IL-15 reportedly acts primarily on T cell and NK cell functions, rather than on humoral immune responses. Our data argue in favor of using IL-15 as an adjuvant for antigen-specific immune responses, particularly cytotoxic lymphocyte responses. We also demonstrate that a single transgene, administered multiple times (\>3), may be very effective at eliciting both humoral and cell-mediated responses. These results thus both corroborate and extend our previous observations \[[@B13],[@B14],[@B32]\], and suggest that combining rSV40s encoding antigens and immunostimulatory cytokines sequentially in multi-administration regimens may provide high levels of long-lasting immunity against the target antigen. Conclusions =========== Recombinant SV40-derived gene-delivery vectors, being transparent to the immune system, can be given multiple times to prime and boost immune responses against the delivered antigens. Anti-vector immunity does not overwhelm responses against the target antigens. As well, these vectors elicit very high levels of antibody, and especially cell-meditated immunity. Finally, combining the delivery of rSV40s bearing antigens with those bearing cytokines such as IL-15 can enhance levels of immunity, particularly long-term immunity. Clearly, much work remains. However, this approach offers promise as a strategy to immunize against pathogens for which classical approaches have not been adequately effective. List of Non-Standard Abbreviations Used ======================================= ::: {#T1 .table-wrap} Table 1 -------------------------------- ------------------------------------------------------------------------ **[Abbreviation]{.underline}** **[Meaning]{.underline}** CELISA cell-based ELISA E:T effector cell:target cell ratio gp120 major HIV envelope glycoprotein, 120 kDa HBS hepatitis B surface antigen IFNγ interferon-gamma mIL-15 mouse interleukin-15 NIH-ARRRP National Institutes of Health, AIDS Research Reference Reagent Program pNPP p-nitrophenyl phosphate VCB41 strain of vaccinia virus carrying gp120 coding sequences wt wild type -------------------------------- ------------------------------------------------------------------------ ::: Competing Interests =================== None declared. Authors\' Contributions ======================= HJM devised all the assay systems for cell- and antibody-mediated immunity against lentiviral antigens, performed all the immunization studies and assays. HM also wrote this manuscript. PYT generated the SV(gp120) construct. MV and PF generated the SV(mIL-15) and SVLUC constructs described here and performed the ELISA for IFNγ stimulated by SV(mIL-15). DSS is the Principal Investigator for this work, oversaw and planned the experimental strategies, worked with HJM in interpreting the experimental data and writing the manuscript. Acknowledgements ================ The authors would like to thank Drs. Jean Boyer, Scott Cairns, Judy Lieberman, David Weiner and John Zaia, and the late Nava Sarver for their advice. This work was supported by grants AI46253 and AI48244.

PubMed Central

2024-06-05T03:55:47.688445

2004-8-23

PMC517512

Background ========== Vigorous exercise is considered one of the key-components of a healthy lifestyle and cardiovascular fitness. Higher levels of exercise are associated with lower risks of hypertension, diabetes, osteoporosis, colon cancer, coronary heart disease, depression, anxiety, and has been shown to enhance weight control \[[@B1]-[@B3]\]. While earlier public health recommendations targeted vigorous physical activity, current recommendations include both vigorous and moderate physical activity \[[@B4]\]. Current vigorous activity recommendations include \"increasing the proportion of adults who engage in vigorous physical activity that promotes the development and maintenance of cardiorespiratory fitness three or more days per week for 20 or more minutes per occasion\" \[[@B5]\]. Although the benefits of exercise are well documented, research shows that only 15--25% of adults are engaging in vigorous exercise three or more times a week for at least 20 minutes \[[@B2],[@B6]\]. More than 60% of American adults are not exercising at a moderate or vigorous level frequently enough to reap the health benefits \[[@B2]\]. Nearly 25% of all U.S. adults report no leisure time exercise at all \[[@B6]\]. Numerous studies have examined predictors of exercise maintenance among participants in organized exercise programs \[[@B7]\]. However, data using longitudinal community based studies are sparse. Two community studies that examined the maintenance and adoption of exercise showed that maintenance of vigorous activity over one year was associated with attitudes toward exercise, exercise knowledge, female gender, and self-efficacy \[[@B8]\]. Over two years, vigorous physical activity maintenance was associated with self-efficacy and younger age for initially active men and with education for initially active women \[[@B9]\]. Adoption of moderate activity over one year was associated with health knowledge \[[@B8]\]. Adoption of vigorous activity over two years was associated with self-efficacy, younger age, and neighborhood environment in men, and education, self-efficacy, and friend and family support in women \[[@B9]\]. Although more research is being conducted on correlates of adoption and maintenance of exercise, more longitudinal research is needed among demographically diverse community samples \[[@B10]\]. Considering only 10--25% of community residents have been successful in adopting even short-term leisure time exercise \[[@B8],[@B11],[@B12]\] there is a need to learn more about the people in the community who adopt and continue exercising. Research on potential correlates of long-term maintenance, initiation, and change in these correlates is needed to better understand how changes in life circumstances might be associated with changes in vigorous exercise. The present study sought to cross-sectionally and prospectively examine the correlates of regular vigorous exercise in a sample of community adults over a four year period. Specifically, this study attempted to expand on the current literature by understanding the relationship between regular vigorous exercise and exercise adoption and maintenance by evaluating three comparisons in a community sample: 1) cross-sectional differences in characteristics of regular vigorous exercisers compared to non-exercisers, 2) predictive factors for maintenance of vigorous exercise over time in people who are already exercising at a high level as compared to those who do not maintain their exercise level, and 3) predictors of adoption of exercise in community adults who were sedentary at baseline as compared to those who do not adopt vigorous exercise. Methods ======= Sample ------ The study sample included 917 women and 229 men who completed baseline surveys as part of a 3-year community-based weight gain prevention trial (Pound of Prevention; POP). Participants were recruited using a variety of methods, including direct mail sent to university employees, and advertisements in community newspapers, health department employee newsletters and radio public service announcements. In addition, recruitment also targeted lower-income women at commercial shopping centers and at community health department clinics. Lower income women were paid \$20 to enroll in the study. All participants were informed that they would be randomly assigned to either a mail-based educational program, or a no contact control group and that they would be measured once per year for a total of 4 years. This study was approved by the University of Minnesota Institutional Review Board. Measures -------- Study participants completed questionnaires and were measured for height and weight at baseline and at three annual data collection visits (Years 2, 3 and 4) following baseline. Measures included in the present study are listed below. ### Vigorous exercise Planned vigorous exercise behavior was assessed using 5 questions from a self-administered version of the Physical Activity History questionnaire (PAH). The PAH is reliable and valid and has been used in several large epidemiologic studies \[[@B13]\]. For this study, exercise activities were limited to those that would deliver significant cardiovascular benefit. Participants rated how frequently in the past year they had engaged in one or more of the activities listed. Response choices on the PAH included; never or less than once per month; 1--3 per month; 1--2 per week; 3--4 per week; or 5 or more per week. Five questions were used in this study to represent high intensity planned exercise; 1) Vigorous jogging, running, backpacking or mountain climbing, 2) Bicycle faster than 10 MPH or exercise hard on an exercise bicycle or rowing machine, 3) Vigorous exercise class or vigorous dance, 4) Brisk walking, hiking, skating or cross-country skiing or 5) other vigorous exercise (including lap distance swimming, vigorous racket sports and other strenuous sports such as competitive basketball, football, volleyball and soccer). ### Hours spent watching television To evaluate competing activities, we evaluated participant\'s report of time spent watching television. Time spent watching television has been positively correlated with body weight, presumably in part due to its displacement of exercise behaviors \[[@B14]\]. Participants reported the number of hours they watch television on an average day. ### Social Support Social support from family and friends has been found to be related to exercise in a number of studies \[[@B9]\]. Participants in this study ranked both their family and friends on the extent to which they were supportive of healthy eating and exercise behaviors on a 5 point scale, with 1 representing \"Not at all helpful\" to 5 representing \"Very helpful\". ### Body Mass Height was measured to the nearest centimeter using a wall-mounted ruler and weight was measured to the nearest half pound using calibrated balance beam scales. Body mass index was calculated using the formula weight (kg)/height (m2). ### Demographic Information All demographic information was self-reported and included age, educational attainment (highest level completed), gender, employment status, income, ethnicity, smoking status and marital status. Statistical Analysis -------------------- Before performing analyses evaluating the predictors of exercise maintenance and initiation, the possibility of an intervention effect was evaluated. Maintenance of high intensity exercise at Year 2 was not significantly associated with treatment, however, maintenance of high intensity exercise at Years 3 and 4 were associated with treatment status. Intervention participants were more likely to be maintaining exercise behavior at Year 3 (X^2^= 14.14, p = .042) and at Year 4 (X^2^= 6.42, p = .011). Initiation of exercise at Year 2 and Year 3 were not significantly associated with treatment status. However, initiation of exercise at Year 4 was marginally positively associated with treatment (X^2^= 3.30, p = .069). Due to these associations, treatment assignment (intervention vs control) was controlled in multiple regression analyses. Participants were classified as regular vigorous exercisers (\> 3 times per week) and non-regular exercisers (\< 3 times per week) based on their responses to the five planned high-intensity exercises on the PAH at each of the 4 assessment points. Using these classifications (yes/no vigorous exercise), the participants were classified as maintainers if they exercised more than three times per week at both baseline and at a subsequent annual evaluation (Year 2, 3, or 4), and non-maintainers if they exercised three times per week at baseline and less than three times per week at a subsequent annual evaluation (Year 2, 3, or 4). Similarly, participants were classified as initiators if they reported less than three exercise sessions per week at baseline and more than three exercise sessions per week at subsequent annual evaluation (Year 2, 3, or 4), and non-initiators if they exercised less than three times per week at both baseline and a subsequent annual evaluation (Year 2, 3, or 4). Descriptive analyses using t-tests and chi-square tests were utilized to assess univariate associations between the baseline predictor variables and exercise status. Maintainers were compared with non-maintainers, and consistently sedentary participants were compared with participants who adopted vigorous activity at Years 2, 3 and 4. Logistic regression was used to further assess the statistical significance of associations between the predictor variables and exercise maintenance and adoption. These univariate logistic regression models predicted exercise maintenance (yes/no) and exercise initiation (yes/no) at Years 2, 3 and 4 using the baseline demographic factors as independent variables. Furthermore, the variables that were significant in the univariate analyses were entered into a multivariate logistic regression model to assess the relative weight and statistical significance of associations between the predictor variables and the variables representing exercise maintenance and initiation. These multivariate logistic regression models predicted exercise maintenance (yes/no) and exercise initiation (yes/no) at Years 2, 3 and 4. In these multivariate regressions, we controlled for exercise level at baseline and treatment status. All statistical analyses were conducted using the SAS Version 6.12 \[[@B15]\]. Since other research studies have indicated gender differences in predictors of exercise maintenance and initiation \[[@B16]\], the gender by exercise interaction was evaluated in a regression model using the frequency of vigorous exercise per week at Year 2 as the dependent variable. The independent variables in this model were the main effects for gender, the main effect for vigorous exercise at baseline, and the gender by vigorous exercise at baseline interaction. The interaction was not significant and thus the data were not stratified for analyses. Results ======= Cross-sectional sample description ---------------------------------- Using the definition of vigorous exercise described above, 564 participants (453 females and 111 males) vigorously exercised more than 3 times per week and 582 participants (464 females and 118 males) did not vigorously exercise more than 3 times per week at baseline. Sociodemographic characteristics of these participants are described in Table [1](#T1){ref-type="table"}. Not surprisingly, participation in vigorous exercise was associated with being employed, non-smoker, lower BMI, fewer hours spent watching television, and higher perceived social support from family and friends. None of the other demographic variables were significantly associated with vigorous exercise at baseline. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Correlates of exercise status at baseline ::: **Exercisers \< 3 X/wk at baseline N = 582** **Exercisers \> 3 X/wk at baseline N = 564** --------------------------- ------------------------ ---------------------------------------------- ---------------------------------------------- **Gender** Female 464 (79.7%) 453 (80.3%) **Age (years)** 37.6 (SD = 6.3) 37.6 (SD = 7.2) **BMI (kg/m2)** 28.1 (SD = 6.3) 26.2 (SD = 5.2)\*\*\* **Employed** Yes 474 (81.4%) 495 (87.8%) **Ethnicity** White 500 (85.9%) 503 (89.1%) **Income group** \< 25 K 218 (37.5%) 190 (33.8%) **Marital Status** Married 288 (49.5%) 266 (47.2%) Sep/Div/Wid 96 (16.5%) 96 (17.0%) Never Married 198 (34.0%) 202 (35.8%) **Education** HS Degree or less 73 (12.54%) 70 (12.4%) Some college 235 (40.4%) 192 (34.0%) College degree or more 274 (47.1%) 302 (53.6%) **Smoking** Yes 131 (22.5%) 80 (14.2%)\*\*\* **TV hours/day** 2.6 (SD = 2.5%) 2.1 (SD = 1.9%) **Social support family** 2.7 (SD = 1.3) 2.9 (SD = 1.3) **Social support friend** 2.6 (SD = 1.2) 3.0 (SD = 1.2)\*\*\* \*\*\* denotes significant difference at the p \< .001 ::: Prospective analyses evaluating predictors of exercise maintenance ------------------------------------------------------------------ ### Prospective univariate evaluations of maintenance of exercise at the three annual evaluations Maintainers and non-maintainers were compared on baseline demographic variables, social support, hours spent watching television and smoking. Similar patterns of associations were found for annual visits at Years 2, 3 and 4. Participants who maintained their exercise level at evaluation Years 2, 3 and 4 weighed less, were employed, Caucasian, of a higher income group, more highly educated, more likely to be non-smokers and watched less television per day at baseline. At evaluation Years 3 and 4, the maintainers were older than the non-maintainers at baseline. The maintainers and non-maintainers did not report differences in marital status or social support. These results are presented in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Associations between exercise maintenance and baseline demographic, smoking, social support and hours watching television. ::: ----------------------------------------------------------------------------------------------------------------------------- **Year 2** **Year 3** **Year 4** --------------------------- ------------------- ------------ ------------- ------------ ------------ ---------- ------------- **Gender** Female 300\ 153\ 214\ 194\*\*\*\ 170\ 209\ 79.4% 82.3% 77.3% 84.4% 78.7% 81.6% **Age (years)** 38.0\ 36.9\ 38.3\ 36.9\*\*\ 38.7\ 37.1\*\*\ SD = 6.6 SD = 8.4 SD = 6.6 SD = 8.0 SD = 6.5 SD = 7.9 **BMI (kg/m2)** 25.5\ 27.8\*\*\*\ 25.7\ 26.7\*\*\ 25.4\ 27.0\*\*\*\ SD = 4.7 SD = 6.0 SD = 4.7 SD = 5.8 SD = 4.6 SD = 5.8 **Employed** Yes 341\ 154\*\*\*\ 250\ 197\ 200\ 217\*\*\*\ 90.2% 82.8% 90.3% 85.7% 92.6% 84.8% **Ethnicity** White 350\ 153\*\*\*\ 259\ 192\*\*\*\ 203\ 222\*\*\*\ 92.6% 82.3% 93.5% 83.5% 94.0% 86.7% **Income Group** \< \$25,000 111\ 79\*\*\*\ 82\ 89\*\*\ 57\ 108\*\*\*\ 29.4% 42.5% 29.6% 38.9% 26.4% 42.2% **Marital Status** Married 181\ 85\ 130\ 108\ 106\ 115\ 47.9% 45.7% 46.9% 47.0% 49.1% 44.9% Sep/Div/ Widowed 59\ 37\ 42\ 39\ 35\ 49\ 15.6% 19.9% 15.2% 17.0% 16.2% 19.1% Never Married 111\ 79\ 105\ 83\ 75\ 92\ 29.4% 42.5% 37.9% 36.1% 34.7% 35.9% **Education** HS or less 35\ 35\ 23\ 40\ 15\ 41\ 9.26% 18.8% 8.3% 17.4% 6.9% 16.0% HS + some college 128\ 64\ 93\ 79\ 73\ 96\ 33.8% 34.4% 33.6% 34.4% 33.8% 37.5% College or more 215\ 87\*\*\*\ 161\ 111\ 128\ 119\*\*\*\ 56.9% 46.8% 58.1% 48.3% 59.3% 46.5% **Smoking** yes 42\ 38\*\*\*\ 29\ 39\*\*\ 24\ 45\*\*\ 11.1% 20.4% 10.5% 16.7% 11.1% 17.6% **TV/day** 1.8\ 2.5\*\*\*\ 1.78\ 2.4\*\*\*\ 1.7\ 2.4\*\*\*\ SD = 1.8 SD = 2.20 SD = 1.6 SD = 2.1 SD = 1.5 SD = 2.1 **Social support family** 3.0\ 2.8\ 3.0\ 2.9\ 3.0\ 2.9\ SD = 1.4 SD = 1.2 SD = 1.4 SD = 1.3 SD = 1.4 SD = 1.3 **Social support friend** 3.0\ 3.1\ 3.0\ 3.0\ 3.0\ 3.0\ SD = 1.2 SD = 1.2 SD = 1.2 SD = 1.2 SD = 1.2 SD = 1.2 ----------------------------------------------------------------------------------------------------------------------------- \*\* denotes significant difference at the p \< .05 \*\*\* denotes significant difference at the p \< .001 ::: ### Prospective multivariate predictors of exercise maintenance Multivariate logistic regression analyses were performed to examine the predictors exercise maintenance at Years 2, 3 and 4. Demographic variables at baseline (BMI, income group, employment status, education, ethnicity, smoking status, hours watched TV/day, age, gender) were entered into logistic regression model along with exercise level at baseline and treatment group. Exercise maintenance at Years 2, 3 and 4 were the dependent variables in the three models. At Year 2, ethnicity (OR = .52, CI = .27--.99), BMI (OR = .93, CI = .89--.96) and exercise at baseline (OR = 1.21, CI = 1.11--1.31) were significant predictors of exercise maintenance. Ethnicity (OR = .463, CI = 0.229--0.933) and exercise at baseline (OR = 1.21, CI = 1.12--1.31) were significant predictors of exercise maintenance at Year 2. Television hours per day (OR = 0.86, CI = .74--.99), BMI (OR = .95, CI = .91--.99) and exercise at baseline (OR = 1.22, CI = 1.13--1.31) were significant predictors of exercise maintenance at Year 4. Prospective analyses evaluating predictors of exercise initiation ----------------------------------------------------------------- ### Univariate evaluations of initiation of exercise at the three annual evaluations Results of univariate analyses relating exercise initiation with baseline variables are shown in Table [3](#T3){ref-type="table"}. Participants who initiated a vigorous exercise program differed from consistently sedentary persons in ways that were similar to those observed for exercise maintenance. These results, however, were weaker in magnitude and not as consistent over the three time periods as the results from the exercise maintenance analyses. At baseline, initiators at Year 2 had a higher income, reported higher social support from family and friends, and had a lower BMI. The only significant difference between the initiators and the sedentary persons was hours watching television per day at Year 3. Finally, initiators at Year 4 were more likely to be married at baseline. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Associations between exercise initiation and baseline demographic, smoking, social support and hours watching television. ::: --------------------------------------------------------------------------------------------------------------------------------- **Year 2** **Year 3** **Year 4** --------------------------- -------------------------- ------------ ------------ ------------ ------------ ----------- ---------- **Gender** Female 108\ 356\ 51\ 305\*\*\*\ 36\ 269\ 80.0% 79.6% 69.9% 81.5% 75.0% 82.5% **Age (years)** 37.9\ 37.5\ 38.2\ 37.4\ 37.4\ 37.4\ SD = 6.3 SD = 6.3 SD = 5.2 SD = 6.5 SD = .6.7 SD = 6.6 **BMI (kg/m2)** 27.34\ 28.4\*\ 27.6\ 28.5\ 28.3\ 28.6\ SD = 6.1 SD = 6.3 SD = 5.5 SD = 6.4 SD = 6.9 SD = 6.4 **Employed** Yes 114\ 360\ 63\ 297\ 38\ 259\ 84.4% 80.5% 86.3% 79.4% 79.5% 79.5% **Ethnicity** White 118\ 382\ 63\ 319\ 42\ 277\ 87.4% 85.5% 86.3% 85.3% 87.5% 84.5% **Income Group** \< \$25,000 41\ 177\*\*\ 26\ 151\ 20\ 131\ 30.4% 39.6% 35.6% 40.4% 41.7% 40.2% **Marital Status** Married 69\ 219\ 33\ 186\ 30\ 156\ 51.1% 49.0% 45.2% 49.7% 62.5% 47.9% Sep/Div/ Widowed 22\ 74\ 13\ 61\ 9\ 52\ 16.3% 16.5% 17.8% 16.3% 18.8% 16.0% Never Married 44\ 154\ 27\ 127\ 9\ 18.8\*\ 32.59% 34.5% 37.0% 34.0% 18.8% 36.2% **Education** HS degree or less 14\ 59\ 7\ 52\ 5\ 47\ 10.4% 13.2% 9.6% 13.9% 10.4% 14.4% HS degree + some college 49\ 186\ 25\ 161\ 19\ 142\ 36.3% 41.6% 34.3% 43.1% 39.6% 43.6% College degree + more 72\ 202\ 41\ 161\ 24\ 137\ 53.3% 45.2% 56.2% 43.1% 50.0% 42.0% **Smoking status** yes 25\ 106\ 15\ 91\ 6\ 85\*\*\ 18.5% 23.7% 20.6% 24.3% 12.5% 26.1% **TV/day** 2.4\ 2.7\ 2.1\ 2.8\*\*\*\ 2.6\ 2.8\ SD = 1.9 SD = 2.6 SD = 1.6 SD = 2.8 SD = 3.2 SD = 2.7 **Social support family** 3.0\ 2.7\*\*\*\ 2.6\ 2.7\ 2.9\ 2.6\ SD = 1.3 SD = 1.3 SD = 1.4 SD = 1.3 SD = 1.2 SD = 1.3 **Social support friend** 2.8\ 2.6\*\*\ 2.5\ 2.6\ 2.5\ 2.6\ SD = 1.2 SD = 1.2 SD = 1.1 SD = 1.2 SD = 1.2 SD = 1.2 --------------------------------------------------------------------------------------------------------------------------------- \*\*\* denotes significant difference at the p \< .001 \*\* denotes significant difference at the p \< .05 ::: ### Prospective multivariate predictors of exercise initiation Logistic regression analyses were also performed to examine the predictors of exercise initiation and Years 2, 3 and 4. Similar to the evaluations for exercise maintenance, demographic variables at baseline which were significant in the univariate analyses (baseline BMI, income group, marital status, gender, smoking, hours spent watching television, family social support and friend social support) were entered into three separate models controlling for exercise level at baseline and treatment group to identify predictors exercise initiation at Years 2, 3 and 4. Four variables were significant predictors of exercise initiation at one or more time points. Exercise level at baseline was positively related to exercise initiation at Year 2 (OR = .23, CI = 1.71--2.90) and at Year 3 (OR = 1.71, CI = 1.23--2.38). Baseline income group (OR = 2.38, CI = 1.08--5.23), marital status (OR = 6.07, CI = 0.39--0.95) and smoking status (OR = 3.53, CI = 0.14--0.91) predicted exercise initiation at Year 4. Discussion ========== This study evaluated the demographic predictors of vigorous exercise initiation and maintenance in a community sample. To our knowledge, this is the first study to look at baseline demographic factors as predictors for exercise adoption and maintenance over a four-year period. This study found that demographic predictors were more consistent for exercise maintenance than exercise initiation over the four years evaluated in this study. Results showed that compared to those who did not maintain vigorous exercise, participants who maintained exercise over a 2, 3 and 4 year period were more likely to be employed, Caucasian, have a higher income, have more education, be a nonsmoker, watch less television and have a lower BMI. One of the interesting findings was that correlates of exercise initiation were less consistent than correlates of exercise maintenance over time. There did not appear to be a consistent pattern that described the associations between the predictors included in this study and vigorous exercise initiation. Although certain predictors were associated with exercise initiation at Years 2, 3 or 4, such as gender or BMI, none of the predictors were consistent over the years measured in the study. These results suggest that people in community populations who initiate vigorous exercise are a heterogeneous group, and there may be greater difficulties predicting who will initiate exercise. It could be much simpler to identify the participants who will be more likely to continue exercise, as compared to the participants who will adopt exercise. It is possible that changes in attitudes, life circumstances (such as sickness), new relationships, or variables that were not measured in this study may be better predictors of exercise adoption or maintenance. However, since we did not measure these variables, we can only speculate whether they may disrupt exercise patterns. The processes of vigorous exercise adoption may be better represented by theoretical understandings of exercise behavior, such as the health belief model \[[@B17]\], transtheoretical model \[[@B18],[@B19]\], social cognitive theory \[[@B20]\], or the theory of planned behavior\[[@B21]\]. These results may have implications for designing and implementing exercise interventions. The results support others studies that suggest targeting exercise maintenance interventions at the lower income participants. However, to achieve the Healthy People 2010 objectives by increasing participation rate in vigorous exercise, interventions need to be designed to promote exercise adoption. Unfortunately, the present study\'s results only suggest that we can not characterize this group well. This lack of characterization of this group may actually be a benefit for interventions. Ethnic group, gender, and socio-economic status did not predict exercise initiation in this study. This suggests that each group was as likely to begin a vigorous exercise program. This is good news as exercise initiation may not be limited to those who can afford health clubs and trainers, or those who are the member of a specific ethnic group or gender. Although previous studies have stratified participants by gender \[[@B16]\], no interaction was found in this study between gender and exercise status. The only associations with gender were seen during Year 3, when fewer women than men maintained exercise and fewer women than men initiated exercise. It is possible that the gender by exercise interaction was not significant due to the limitation of exercise in the vigorous activity range, versus the moderate or mild activity range. One hypothesis is that the predictors of exercise adoption that are gender specific may have to do with moderate or minimal exercise adoption. Strengths and limitations of the present study should be recognized. This is one of a few studies that evaluate predictors of exercise initiation, and the first to evaluate exercise initiation over a four-year period. In addition, the sample is large, diverse, and includes longitudinal measurements. This study did not evaluate the predictors of moderate exercise initiation or maintenance. Thus, these results can not be directly interpreted for public health interventions targeted at increasing moderate activity in the general population. In addition, this study may also be affected by a selection bias. The participants in this study volunteered for a study on weight gain prevention, for which the participants may have expected to include a message on increasing exercise. Of note, 49% of the sample reported vigorous exercise more than 3 times per week at baseline, which is much higher than the 15--25% reported in national surveillance studies \[[@B6],[@B2]\]. Although treatment status was controlled for in the analyses, these participants were recruited for an intervention trial, rather than a cohort trial, which could also contribute to a selection bias. In addition, the measures in this study are self-report and include few theoretically based variables. It is possible that the self-report nature of exercise in this study may have allowed over-reporting of vigorous exercise. Considering these limitations, this study does add to the knowledge base about who initiates and maintains vigorous exercise. The results suggest that vigorous exercise maintenance interventions should continue to target low-income populations and that interventions could incorporate messages regarding smoking, weight control, and television. We found that those who adopt vigorous exercise are a more heterogeneous group, and that no one group is more likely to adopt exercise than the others. This suggests that vigorous exercise intervention messages could be more broadly based. This study also suggests that further research is needed to identify participants and effective interventions for those who begin exercise programs. Competing interests =================== None declared. Authors contributions ===================== KB conceptualized the study, planned and executed the analyses, interpreted the results, and drafted the manuscript. RJ participated in conceptualization of the study, interpretation of the results and assisted in drafting the manuscript. SF participated in conceptualization of the study, interpretation of the results and assisted in drafting the manuscript. All authors read and approved the final manuscript.

PubMed Central

2024-06-05T03:55:47.691324

2004-9-1

PMC517617

Background ========== Ongoing genome sequencing and recent developments in cDNA sequencing projects have led to an exponential rise in the amount of sequence information. This has increased the need for acquiring knowledge from sequences as to their biological function. Annotating a single sequence is the gateway to interpreting its biological relevance. However, the usefulness of these annotations is highly correlated with their quality. *Accurate*annotation has traditionally been maintained manually with the experience of individual experts and the experimental characterisation of sequences. However, the increasing gap between the amount of sequence data available and the time needed for their experimental characterisation demands computational function prediction in complementing manual curation \[[@B1]-[@B4]\]. Commonly, computational functional assignment is based on homologues identified from database searches \[[@B5]\]. Such an automated annotation process provides comparable results due to a uniform analysis of all query sequences across the same databases and the possibility of repeating the annotation to updated sequence data \[[@B6]\]. However, crucial aspects for consideration in automated annotation are i) the problems associated with the databases themselves: sequence errors, erroneous annotation due to spelling ambiguities, incomplete functional annotation, inconsistent functional annotation across databases, consistent but wrong annotation across databases, and ii) the problems associated with the inference, i.e. false positives, where an assignment is made on the basis of a wrongly inferred homology \[[@B3],[@B7],[@B8]\]. A number of excellent annotation systems have been developed to tackle these problems, e.g. RiceGAAS \[[@B9]\], GAIA \[[@B10]\], Genotator \[[@B11]\], Magpie \[[@B12]\], GeneQuiz \[[@B6]\], GeneAtlas \[[@B13]\] and PEDANT \[[@B14]\]. However, little has been done to quantify the annotation accuracy by defined benchmarks *and*establish a method to provide a confidence value for each annotation. The current annotation, written in a rich, non-formalised language also complicates this automated process. We addressed this problem by applying a controlled vocabulary from Gene Ontology (GO) \[[@B15]-[@B17]\]. GO provides consistent descriptions of gene products in a species-independent manner. The GO terms are organised in structured, controlled vocabularies (ontologies) to describe gene products in terms of their associated biological processes, cellular components and molecular functions. An increasing number of GO-mapped sequence databases make it possible to replace traditional database searches with GO-related searches. These include databases such as GenBank \[[@B18]\], SWISS-PROT \[[@B18]\], SwissPROT/TrEMBL \[[@B19]\], the TIGR Gene Index \[[@B20]\] and several other genome databases. Many annotation approaches have now been developed based on Gene Ontology. The uncharacterised sequences are searched across GO-mapped protein databases and assigned with GO terms of the best hits \[[@B21],[@B22]\]. Jensen and co-workers used neural networks to predict specific subsets of GO terms \[[@B23]\]. Furthermore, Schung *et al*predicted GO terms by intersecting domain profiles \[[@B24]\]. The SwissPROT/TrEMBL entries were associated with GO terms by an automated process coupled with manual verification \[[@B19]\]. Text mining and similarity searches were combined to annotate SWISS-PROT and GenBank entries with GO terms \[[@B18]\]. However, these approaches were either applied to specific GO subsets or did not provide defined benchmarks and confidence values for their predictions. We have developed an automated system for large-scale cDNA function assignment, designed and optimised to achieve a high-level of prediction accuracy without any manual refinement. Our system assigns molecular function GO terms to uncharacterised cDNA sequences and defines a confidence value for each prediction. The cDNA sequences were searched against GO-mapped protein databases and the GO terms were extracted from the homologues. In the training phase, these GO terms were compared to the GO annotation of the query sequences and labelled correspondingly. We applied Support Vector Machines (SVMs) as the machine learning method to classify whether the extracted GO terms were appropriate to the cDNA sequence or not. In order to classify the GO terms we used a broad variety of elaborated features (attributes) including sequence similarity measures, GO term frequency, GO term relationships between homologues, annotation quality of the homologues, and the level of annotation within the GO hierarchy. To enhance the reliability of the prediction, we used multiple SVMs for classification and applied a committee approach to combine the results with a voting scheme \[[@B25]\]. The confidence values for the predicted GO terms were assigned based on the number of votes i.e. number of SVMs predicting particular GO term as correct. The performance of the system was benchmarked with 36,771 GO-annotated cDNA sequences derived from 13 organisms. It achieved 80% precision for 74% of the test sequences. We applied our annotation system to predict the function for *Xenopus laevis*, a widely studied model organism in developmental biology. Because many researchers are now focussing on the functional genomics of this organism, a demand exists for a quality annotation \[[@B26]\]. Therefore we applied our system to improve the quality and coverage of the existing annotation. We predicted the function for 17,804 *Xenopus laevis*contig sequences (from TIGR Gene Indices) yielding annotation with good confidence values for more than half of these sequences. Results ======= General workflow of training and classification ----------------------------------------------- The classifier (SVM) needs to specify attribute values (features) for a broad list of samples and a class label for each of these samples. Through the training samples it learns the feature patterns and tries to group them according to their class labels. After training, the algorithm assigns class labels to new samples according to the class that they best match. We selected GO-annotated cDNA sequences for training the SVM classifier. The nucleotide sequences were searched against GO-mapped protein databases and GO-annotations were extracted from the significant hits. Then, each GO term obtained was utilized as a sample for the feature table. The sample GO terms were then labelled as either correct (\"+1\") or false (\"-1\") by comparing them to the original annotation. Note that we applied the relationships of the GO terms based on their graph structure: \"Correct\" was assigned not only if they were exact matches but also if the GO terms were related as either \"parent\" or \"child\" (Figure [1](#F1){ref-type="fig"}). Next, the samples were attached with their features or attributes, calculated from the BLAST \[[@B27]\] results. With this data, the classifier was trained to distinguish between the attribute patterns that contributed to class +1 (correct prediction of a GO term) and class-1 (false prediction). To predict the function of unknown sequences, the same procedure was applied as for the training sequences in order to obtain their GO terms and corresponding attribute values. According to these attribute values, the classifier assigned a class for every GO term of the BLAST hits (Figure [2](#F2){ref-type="fig"}). Datasets for training and testing SVM ------------------------------------- For training and testing the SVM, we selected 39,740 GO-annotated cDNA sequences from the following organisms: *Saccharomyces cerevisiae*(yeast), *Drosophila melanogaster*(fly), *Mus musculus*(mouse), *Arabidopsis thaliana*(*Arabidopsis*), *Caenorhabditis elegans*(worm), *Rattus norvegicus*(rat), *Danio rerio*(fish), *Leishmania major*(*Leishmania*), *Bacillus anthracis Ame*(*Bacillus*), *Coxiella burnetii RSA 493*(*Coxiella*), *Shewanella oneidensis MR-1*(*Shewanella*), *Vibrio cholerae*(*Vibrio*) and *Plasmodium falciparum*(*Plasmodium*) (Table [1](#T1){ref-type="table"}). From these, 55.3% of the cDNA sequences were contributed by *Arabidopsis*, mouse and fly (22.1%, 18%, and 15.2% respectively). Prokaryotic bacteria (*Bacillus*, *Coxiella*, *Shewanella*and *Vibrio*) contributed 20.6% and the remaining 24.1% of the sequences came from rat, fish, worm, *Plasmodium*, *Leishmania*and yeast. Yeast and fly are purely manually annotated datasets. Where as *Bacillus*, *Coxiella*, *Vibrio*, *Shewanella*, *Leishmania*and *Plasmodium*are mostly manually, and the rest mostly automatically annotated datasets. Manual annotation tends to be conservative and sparse, since the GO terms are assigned only if the annotator is highly confident. Therefore, a GO term may be missed due to a poor definition of a false negative. To reduce this critical problems, yeast and fly annotations are accompanied by an \"unknown molecular function\" term for sequences with questionable further functions. To reduce false negatives, we discarded all sequences with these tags for training and testing (yeast: 2999 discarded out of 6355, fly: 8495 out of 14335). The cDNA sequences were searched across the protein databases covering a wide range of organisms from prokaryotes to eukaryotes and SWISSPROT. For 36,771 sequences we got hits with GO terms, contributing to 856,632 sample GO terms and yielding an average of 23.29 GO terms per query sequence (Table [1](#T1){ref-type="table"}). These 856,632 samples were used to train our classifier. Generally, the number of GO terms per sequence was less for prokaryotes than for eukaryotes. Rat had the maximum number of GO terms per sequence (36.9), followed by fish (32.1) and worm (27.13). In contrast, *Shewanella*, *Coxiella*and *Vibrio*sequences had the lowest number of GO terms per sequence (10.78, 12.33 and 12.54, respectively). SVM training and testing ------------------------ ### SVM training We set up multiple classifiers by splitting the whole dataset (856,632 samples) into 99 equal subsets. Note that, amongst these 99 subsets, 96 contained data from a single organism and the remaining 3 from two organisms each. Subsequently, we built 99 classifiers with these subsets. Since the training sets were created organism-wise, the classifiers were trained from different ranges of data, based on purely manual annotation (yeast, fly), mostly automated annotation or a mixture of both. For training each of these classifiers, we performed a model selection (parameter optimisation by cross-validation; see Methods), which yielded varying accuracy values ranging from 78.81% to 96.03%, with an average accuracy of 85.11%. ### SVM testing To test the classifiers performance, we prepared 13 test sets (each set corresponding to a single organism) using the same 856,632 sample GO terms. The prediction quality of all 99 classifiers were assessed by an organism-wise cross-validation approach, i.e. for each organism (test set), we used all the classifiers for prediction except those that corresponded to the same organism. With this approach, we were able to simulate the annotation of a new organism. The number of classifiers used for predictions varied highly across organisms (maximum: *Plasmodium*and *Leishmania*, 98 classifiers; minimum: *Arabidopsis*, 74 classifiers). The quality of the predictions was estimated by comparing the predicted terms with the original annotation and the results were expressed in terms of *precision*and *accuracy*values (see Methods). The average-accuracy refers to the average of the accuracy values attained by all classifiers used for the prediction. The maximum average-accuracy was achieved for fly (81.51%), followed by yeast (80.50%), and the minimum for mouse (76.0%). Additionally, we compared the classification efficiency of the classifier derived from automatic annotation (mouse, worm and *Arabidopsis*) with the manually annotated test sequences (yeast and fly). The prediction of the yeast and fly sequences with the 20 classifiers from the mouse sequences produced an average-accuracy of 79% and 80% respectively. Similar results were acquired with the 25 classifiers from *Arabidopsis*(79% and 80%). Likewise, the worm classifiers (11 classifiers) yielded the average-accuracy of 82% for yeast and 83% for fly. These values were comparable with the average-accuracy of 81% achieved by both, using yeast as test sequences against fly classifiers (16 classifier) and vice-versa (fly test sequences against yeast classifiers). Likewise, we classified the mouse test sequences against yeast classifiers (5 classifier) and fly classifiers yielding 69% and 71% average-accuracy respectively. Combining multiple classification results by the committee approach ------------------------------------------------------------------- Though we already achieved a good accuracy with some of the classifiers, our intention was to improve the precision and, furthermore, to obtain confidence values for the predicted GO terms. To this end, we combined the predictions of multiple classifiers by the committee approach. If a classifier predicted a particular GO term as correct, it contributed a vote. Votes were collected from all classifiers and summed up to yield a final score value. If no vote supported a GO term as correct, it was assigned with the label \"false\". Otherwise, the number of votes provided a measure of the reliability. Figure [3](#F3){ref-type="fig"} shows precision and accuracy versus the number of votes. If we made predictions with a minimum of one vote, we were able to achieve 43% precision and 59% accuracy. When the stringency was raised to 25 votes, a minimum of 25 votes was required to classify a GO term as correct, yielding an accuracy of 84% and precision of 75%. At a cut-off value of 74 votes, we attained 91% precision and 71% accuracy. A cut-off value of 94 votes gave 100% precision and 67% accuracy. Our accuracy reached a plateau at 20 votes. However, it decreased slightly for stringencies of more than 30 votes. Note, that this was due to the increasing number of false negatives. The relation between the precision and the number of votes (Figure [3](#F3){ref-type="fig"}) was used as a means of calibrating to assign the confidence values for new predictions. For each threshold value of the votes, we calculated the sensitivity and the false positive rate to obtain a Receiver Operating Characteristic plot (ROC; Figure [4](#F4){ref-type="fig"}). The graph shows that the classification performance was comparable for different classes of organisms like prokaryotes, single cell eukaryotes and multi-cellular eukaryotes, which reflect the organism-independent performance of our method. Note that for fish, worm, *Plasmodium*and *Leishmania*the classification performance was particularly good due to the low number but well characterised test sequences. We compared the prediction performance for GO terms annotated with the evidence code IEA (automated annotation) and non-IEA (manually verified annotation). All sequences from *Bacillus*, *Coxiella*, *Vibrio*, *Shewanella*, yeast, *Leishmania*, and *Plasmodium*were non-IEA annotated and 99.5% of the fly GO terms were non-IEA annotated. In contrast, all sequences from fish and worm were IEA annotated. The remaining test organisms were mostly IEA annotated (rat: 88%, *Arabidopsis*: 79.4%, and mouse: 69.5%). The classification performances revealed by the ROC plots were comparable between IEA and non-IEA annotated test organisms (Figure [4](#F4){ref-type="fig"}). Therefore, the classifier could reproduce the annotation of other automated systems as well as high-quality manual annotation. We were interested in the coverage of sequences with respect to the average precision of the annotations (shown in Figure [5](#F5){ref-type="fig"}). Considering 1 vote as the cut-off value, we obtained 52% average precision for 98% coverage. We obtained 80% average precision for 74% coverage (cut-off: 34 votes), and 90% average precision for 42% coverage (cut-off: 65 votes). These coverage values varied when regarding the test organisms individually. The coverage for different test organisms at 80% average precision were: fish 97%, *Coxiella*89%, worm 88%, *Vibrio*86%, rat 85%, *Bacillus*83%, *Plasmodium*81%, mouse 78%, *Leishmania*76%, *Shewanella*74%, *Arabidopsis*69%, fly 66% and yeast 57%. *Xenopus*annotation ------------------- We extracted all *Xenopus laevis*contig sequences from the TIGR *Xenopus laevis*Gene Index (XGI) \[[@B28]\] and got a total of 35,251 contig sequences, excluding singletons. We applied our method to predict functional GO terms for these contig sequences. We predicted the function for 17,804 sequences with an average of 12.16 GO terms per sequence. In total, 23.4% of all the GO terms were predicted with less than 50% confidence value, 51.5% of them were between 50% to 80% confidence and the remaining 25% with a predicted confidence value of above 80%. At 80% stringency (predicted if the GO term possessed a confidence value of 80% or more), we made predictions for 9,510 contig sequences including 55,994 GO terms, yielding on average 5.88 GO terms per sequence. To compare the functional abundance of the expressed genome across the organisms, we mapped the predicted GO terms (with at least one vote) to the high-level, i.e. more generalised or high-level terms of the molecular function ontology (\"GO slim\" for molecular function) \[[@B29]\]. These molecular function GO slim nodes were taken from the second level of the molecular function ontology. The distribution of higher-level GO terms were compared between *Xenopus*, fly, yeast and mouse (Figure [6](#F6){ref-type="fig"}). Note that some of the deeper-level terms had multiple paths. They were mapped to two or more higher-level nodes, so that the total sum of the higher-level nodes exceeded 100%. Comparison to the TIGR *Xenopus*annotation ------------------------------------------ TIGR provides a GO mapping for *Xenopus*contigs (TIGR *Xenopus laevis*gene indices). We compared our annotation with the TIGR GO annotation for molecular function. From 35,251 contig sequences, TIGR annotated 5,444 contigs with a total of 16,432 molecular function GO terms. In contrast, our approach was able to predict function terms for 17,804 contigs, i.e. more than three times that of TIGR sequences. Our procedure did not annotate 295 contigs from the TIGR annotated contigs. For the remaining 5,149 contigs, 85% of all TIGR terms were found to be exact with those using our method; 3.2% of the TIGR terms were at a higher-level of the GO tree than our annotation, so in this case we provided annotation at a deeper level; in 0.9% of the cases our annotation was at a higher-level; 8.3% of the cases were completely different; and 0.6% of the TIGR terms were obsolete. We compared the quality of TIGR and that of our annotations by a raising stringency and found that when we applied a confidence threshold of 80% for our annotation, we lost 46.6% of the sequences. This included 1,492 sequences holding equivalent TIGR annotation or 27.4% of the total TIGR annotation. With this stringency, our system annotated 9,510 contig sequences, i.e. twice the TIGR annotation at this quality. We were interested in novel annotated sequences with the highest confidence values and found we could predict GO terms for 557 contigs with a confidence value of 100% (all votes matched). Interestingly, 192 of these lacked any GO annotation by TIGR. Out of these, 184 had got a descriptive TIGR annotation and the rest had not got any. Table [2](#T2){ref-type="table"} shows the novel annotation for these eight sequences. Our novel predictions are as follows: 1) TC212171 and TC196381 are predicted to display *endopeptidase activity*and more specifically *serine-type peptidase activity*(98% and 97% confidence respectively). 2) TC209487 and TC190605 are predicted to be *aminopeptidases,*however for the latter the more specific prediction of *prolyl aminopeptidase activity*is assigned with 86% confidence. 3) TC199713 is predicted as *glutathione peroxidase*at 100% confidence and TC194305 is annotated as *protein kinase*with the same confidence. 4) Both TC187949 and TC210151 are *transmembrane receptors*but the latter one is classified as *frizzled receptor*with 82% confidence. In most of these examples the functional assignment and associated confidence were recorded in multiple levels of granularity. Discussion ========== In this paper, we presented an automatic annotation system that is able to cope with the expanding amount of biological sequence data. Our approach efficiently combines the ongoing efforts of Gene Ontology and the availability of GO-mapped sequences with a profound machine learning system. The GO-mapped databases provide annotation described in a controlled vocabulary and also a measure of reliability, as these GO entries are labelled with their type of origin. Furthermore, GO terms are structured hierarchically, which allow us a twofold use of the information: i) the level within the tree is taken as a classification criterion to distinguish low from high-level annotations during the learning procedure, and, ii) the hierarchical structure allows us to extend hits by slightly moving up and down within a restricted local area of the tree. This may overcome fluctuations of the annotation levels coming from varying annotation experts. Our annotation system exploits the different combinations of attributes and yields functional transitivity: SVM learning and prediction are organism-independent and comparable to manual annotation, which may be supported by the nature of the attributes we utilise. Subsets and overlaps are counted in a balanced fashion to avoid biases due to the complexity of an organism and a potentially correlated complexity of its sequences. The committee approach allows us to improve the prediction quality as well as to assign confidence values for the new predictions in a straightforward manner. Our classifiers performance is hardly limited by the varying quality of the training data, whether manual or automatic annotated. The prediction results of manually annotated test sets with the classifiers based on automated annotation as well as classifiers based on manual annotation were comparable. Regarding the outcome of the overall classifiers, we achieve consistency with existing annotation from automatic annotations. This is the less complex part of our work and shows a comparable efficiency of our system. Additionally, our system reproduces annotation of purely manually annotated datasets (fly, yeast, etc). However, the performance results for these datasets are low in terms of recall, i.e. 47.4% recall with 80% precision compared to 60.6% recall with the same precision of the complete test set. Note that manual annotation tends to be conservative and sparse, yielding stringent true positive definitions, whereas automatically annotated sequences may accumulate information to a greater extent. We were interested in annotating *Xenopus*since it is a familiar model organism. However, the sequences were not very well annotated. Our system was applied to annotate the *Xenopus*contig sequences from TIGR. Through our approach, we annotated 50.5% of all contig sequences available at present, and associated a confidence value for each prediction, yielding roughly three times more sequences as compared to the currently available GO annotation. However, the coverage of annotation to new organism like *Xenopus*is crucial. We were able to attain predictions for 50.5% of all *Xenopus*contig sequences (no singletons). This compares to the applied databases that contained 53% satisfactory annotation for their sequences (not regarding sequences with unknown function terms), and better than the organism specific databases (36%). Obviously, improving the quality and quantity of annotation within the available databases goes along with the coverage exploit of machine learning algorithms for new organisms. In future we want to extend our method with the information from other sources such as domain databases and protein family databases. Conclusions =========== We developed an automated annotation system to assign functional GO terms to an unknown sequence. We used the well-established technique of Support Vector Machines (SVM) for the classification of correct and incorrect GO terms. Our approach benefited from the broad variety of potential attributes used for the functional transitivity and a vast amount of data used for training and validating. The committee scheme exploited in our system provided a means to assign confidence values in a straightforward manner. Our system performance was robust, organism-independent and reproduced the high-quality manual annotation. When applying it to *Xenopus laevis*contig sequences, we obtained a remarkably enhanced annotation coverage compared to the existing annotation. Methods ======= Quality criteria for assessing the performance of the classifier ---------------------------------------------------------------- We used the following statistical terms \[[@B30],[@B31]\]. *Accuracy*was the rate of correct predictions compared to all predictions, Accuracy: = (TP + TN) / (TP + FP + TN + FN),     (1) where TP denotes true positives, FP false positives, TN true negatives and FN false negatives. *Precision*was the portion of true positives with respect to all positives, Precision: = TP / (TP + FP).     (2) Also used were *sensitivity*:= TP / (TP + FN), *specificity*:= TN / (FP + TN), and *false positive rate*:= 1 - specificity. We defined the term *\"coverage-of-sequences\"*as the portion of query sequences for which the classifier delivers a prediction; \"*Precision-per-sequence\"*the (average) portion of correct GO terms for a single query sequence, with respect to all GO terms assigned to it. Note that these terms were defined within our model, i.e. a good \"accuracy\" meant good consistency with respect to our training and test sets. Defining the GO term relationships ---------------------------------- We focused on the molecular function terms from GO, because the information extracted from the gene products is usually more predictive for determining molecular functions than for biological processes or cellular components. The functional terms and their hierarchy were obtained from the web pages of the Gene Ontology Consortium \[[@B29]\] (version of June 2003). In our study, relationships \"is-a\" and \"part-of\" were not distinguished. Note, that the \"part-of\" relationship is rare in the molecular function ontology (26 out of 6521 child-parent relationships). The annotation level varies across databases depending on the curator\'s individual knowledge about the gene product. To consider varying levels of annotation in the databases for similar gene products, we traced the relationships to match GO terms of different granularity for the same function. To find a relationship between two terms, the whole path of a GO term was traced back to the root (the root is the \"molecular function\" node, GO:0003674). We defined the distance between two GO terms as the distance of the shortest path. GO terms are organised in directed acyclic graphs, i.e. a child (more specialised term) may have multiple parents (less specialised terms). Therefore, we defined single path and multiple path relationships. In the case of single path relationships, GO terms had only one possible path to the root. The relationship of the term GO~1~with respect to GO~2~was classified as \"parent\", \"child\", \"sibling\" or \"different\" (Figure [1](#F1){ref-type="fig"}) according to the following rules: GO~2~is a *\"parent\"*of GO~1~if their respective paths P~2~and P~1~intersect in such a manner that P~1~⊂ P~2~,     (3) P~*i*~denotes the set of nodes from GO~*i*~to the root GO~2~is a *\"child\"*of GO~1~if their paths P~2~and P~1~intersect such that P~1~⊃ P~2~,     (4) GO~2~is a \"*sibling\"*of GO~1~if a common parent exists with a distance of one to GO~1~and GO~2~(Figure [1E](#F1){ref-type="fig"}). To avoid ambiguities for less differentiated terms, the sibling relationship was set only, if GO~1~and GO~2~were at least 5 nodes away from the root. The relationship *\"different\"*was set if none of the previously stated criteria was fulfilled. We could apply the single path relationship for most of the GO terms (3665 out of 5391). However, for the remaining 1726 terms more than one path to the root were found. For these cases we defined multiple path relationships and each path was considered individually. The single path relationship was applied to each possible pair of these paths (path for GO~1~and GO~2~, respectively) and is henceforth referred to as \"path-pairs\". This method could yield a list of several relations. To select the appropriate relation from this list, we considered the parent relationship to be most relevant, followed by the child relationship, and the sibling was considered least relevant. We implemented the following order: 1\. The parent relationship was set if at least one of the path-pairs gave a (single path) parent relationship; 2\. The child relationship was set if at least one of the path-pairs gave a child relationship. To avoid a bias due to an overwhelming number of path-pairs that did not match, we set a threshold: we considered this relationship only, if the number of path-pairs with no child relationship was equal or less than four times the number of path-pairs with child relationship; 3\. The sibling relationship was set if at least one of the path-pairs gave a sibling relationship. We again set a threshold: we considered this relationship only, if the number of path-pairs with no sibling relationship was equal or less than twice the number of pairs with sibling relationship; 4\. If none of these criteria could be applied, the relationship \"different\" was set. Note that we also implemented the hierarchy of these relations by tuning the stringencies for the fractions of path-pairs that must match (parent: no threshold, child: 1/4, sibling: 1/2). Data basis used for this study ------------------------------ Since the function transitivity at the protein level is more reliable, we used GO-mapped protein databases for searching homologues. Gene association files were obtained via the Gene Ontology Consortium. By combining the gene association files with corresponding sequence databases we created the unified protein databases. The following organisms were used: yeast, fly, mouse, *Arabidopsis*, worm, rat, fish, *Leishmania*, *Bacillus*, *Coxiella*, *Shewanella*, *Vibrio*, *Plasmodium*, *Oryza sativa*, *Trypanosoma brucei*, and *Homo sapiens*. Apart from this, the SWISS-PROT database was also included \[[@B32]\]. For SVM training and testing we selected 39,740 cDNA sequences from 13 organisms. These cDNA sequences were collected from the following organisms: yeast, fly, mouse, *Arabidopsis*, worm, rat, fish, *Leishmania*, *Bacillus*, *Coxiella*, *Shewanella*, *Vibrio*and *Plasmodium*(see Table [1](#T1){ref-type="table"}). Out of all the known cDNA sequences we extracted 39,740 with GO molecular function terms, discarding incompletely annotated ones, i.e. sequences assigned with the GO term \"molecular function unknown\" (GO:0005554). Computing the attributes ------------------------ Each cDNA sequence was searched across the protein databases, using BLASTX within the HUSAR system \[[@B33]\]. A query sequence was not searched within the database of their own organism. In case of SWISSPROT, hits corresponding to the query (cDNA) organism were filtered out. The BLAST files were parsed using the BLAST parser modules from W3H \[[@B34]\] and a low-stringent e-value cut-off of 0.01 was applied to yield a high number of possible hits. Multiple high scoring segment pairs were combined as described elsewhere \[[@B35]\] and used for computing the alignment features. GO terms for each database hit were extracted by considering only GO terms corresponding to the molecular function and by discarding GO terms that were prefixed with NOT (annotators state that a particular gene product is NOT associated with a particular GO term), or corresponding to \"molecular function unknown\" (GO:0005554). These steps reduced our dataset to 36,771 sequences, contributing to 856,632 samples. Each GO term that occurred in the hits represented a sample entry in the feature table. Below it will be referred to as \"sample GO term\". If a GO term occurred several times in the hits, it was considered only once. We defined 31 attributes for each GO term, representing 5 major classes of information (A)- E)): A\) GO level and path: The GO structure was exploited to derive the first two attributes, A.1.*GO level*: the distance of the sample GO term to the root (molecular function node); A.2. *GO path*: the number of paths from the sample GO term to the root. B\) Alignment quality criteria: These attributes are based on the BLAST alignments. For attributes B.1 - B.4, the best value for the corresponding attribute was taken, if a GO term occurred in more than one hit, B.1. *Expectation value:*the expectation value (\"E-value\") from BLASTX; B.2. *Bit score*: the bit score value provided by BLASTX; We wanted to award alignment length and quality by combining features. This was done with respect to the length of the query *and*the hits to offset biases due to different complexities of the query and subject organisms. Attributes B.3, B.4, C.3 and D.3 were obtained from initial trials with a small dataset (6270 cDNA sequences, data not shown) and applying parameter optimisation to distinguish the samples. B.3. *Query coverage score*(QC~S~): Combined measure of alignment size and quality concerning the query sequence, QC~S~:= (A~L~/ Q~L~) × (I + S),     (5) where A~L~denotes the alignment length, Q~L~the length of the query sequence, I the number of identities in the alignment, and S the number of positively contributing residues in the alignment; B.4. *Subject coverage score*(SC~S~): as in B.3, however only with respect to the corresponding subject sequence (database hit), SC~S~:= (A~L~/ S~L~) × (I + S),     (6) where S~L~denotes the length of the subject sequence; Additionally, we decomposed these attributes into the following further six attributes (B.5 - B.10). For these attributes, we considered the hit with the best coverage score if a GO term occurred in more than one hit (query coverage score for attributes B.5, B.7, B.9, and subject coverage score for B.6, B.8, B.10). B.5. *Query percentage*(QP~C~): Percentage of coverage of the alignment region in the query sequence (with respect to QC~S~), i.e. QP~C~:= (A~L~/ Q~L~) × 100;     (7) B.6. *Subject percentage*(SP~C~) Percentage of coverage of the alignment region in the corresponding subject sequence (with respect to SC~S~), i.e. SP~C~:= (A~L~/ S~L~) × 100;     (8) B.7. *Query identity*(QI): Percentage of identical residues in the BLASTX alignment (with respect to QC~S~); B.8. *Subject identity*(SI): Percentage of identical residues in the BLASTX alignment (with respect to SC~S~); B.9. *Query similarity*(QS): Percentage of similar or positively contributing residues in the alignment (with respect to QC~S~); B.10.*Subject similarity*(SS): Percentage of similar or positively contributing residues in the alignment (with respect to SC~S~). C\) GO frequency related attributes: We extracted information about the frequency of GO terms in the hits by the following attributes: C.1.*GO frequency*(F~G~): the number of hits that contained the sample GO term. C.2.*Number of hits*(T~H~): the total number of hits for the query. C.3. *Frequency score*(F~S~): the number of hits that contained the sample GO term. Unlike C.1, we limited this score to emphasize differences in queries with few hits:  C.4.*Species frequency*: The number of organisms contributing to a sample GO term for a single query sequence; C.5.*Total GO*(T~G~): total number of GO terms from all hits. C.6. *Unique GO*(U~G~): as C.5, except, that GO terms occurring more than once (in the hits) were counted only once. D\) GO frequency by considering relationships: For these attributes we applied the structure of the Gene Ontology graph. Not only perfectly matching terms were considered, but also their defined parents, children or siblings: D.1.*Relative frequency for all (R*^A^): the relationships for the sample GO term with all GO terms that occurred in the hits were calculated. The sum of non-\"different\" relationships i.e. parent, child, or sibling was used for this attribute; D.2.*Relative frequency for unique*(R^U^): similar to attribute D.1, with the exception that GO terms occurring more than once were counted only once. D.3.*Relative frequency for all (limited) (R*^*Alim*^*):*same as attribute D.1, however this score was limited to emphasize differences of queries with few hits:  D.4.*Relative frequency for unique (limited)*(R^*Ulim*^): same as attribute D.2, however this score was limited to emphasize differences of queries with few hits:  E\) Annotation quality related attributes: Quality attributes were selected from the evidence codes provided by the gene association tables of the GO-mapped sequence databases. We selected 9 commonly used evidence codes (TAS, NAS, ISS, IPI, IMP, IGI, IEP, IEA, IDA), resulting in attributes E.1 to E.9. The entries of these attributes for each sample GO term were calculated by summing the occurrences of the corresponding evidence codes of all hits. Training and testing the classifier ----------------------------------- Before training, normalisation was performed. We normalised the attributes by taking the logarithm (log) and log of log if necessary. We used log values for 16 attributes (B.3-B.10, C.3, C.4, D.1, D.2, D.4 and E.1) and log of log for 8 attributes (B.2, C.1, E.2, E.4-E.8). Furthermore, we converted the attribute values into mean 0 and standard deviation 1 by applying the Z-transformation. The feature table contained 856,632 samples and 31 attributes. We split the dataset into 99 training subsets. Each subset comprised of approximately 1% of the samples i.e. 8,566 GO terms. This resulted in 96 organism specific subsets and 3 hybrid subsets. We applied the support vector machines in the implementation of LIBSVM \[[@B36]\], which supports a weighted SVM for unbalanced data. We used a higher penalty (5 instead of 1) for false positives (FP) for the model selection and also the training process to support a high specificity of the resulting classifiers. Also note, that our training set contained a high portion of negative samples (see Table [1](#T1){ref-type="table"}) due to our relaxed E-value threshold. We utilised the radial basis function kernel and set the parameter epsilon (tolerance of termination criterion) to 0.01. The parameter C (regularisation term, cost for false classification) and gamma (kernel width) of the SVM were optimised using a grid search. The grid search determined the combination of C (log2-range: 13 to 15, step 1) and gamma (log2-range: 10 to 15, step 1) with the lowest classification error according to a five-fold cross validation such that each of the 99 data subsets was subdivided into a training set (90%) and a validation set (10%). The validation sets were used to estimate the parameters C and gamma for each of the 99 classifiers individually. Finally, the parameters from the classifier selection were applied to train each of the classifiers with 90% of each data set, respectively. The testing was based on the same 13 organisms and 856,632 GO terms corresponding to 36,771 sequences as described above. We performed the testing by an organism-wise cross-validation so that one organism was used as test set and the remaining ones as the training set. Data availability ----------------- The annotation for *Xenopus laevis*contig sequences is downloadable at <ftp://genome.dkfz-heidelberg.de/pub/agd/gene_association.agd_Xenopus>. We followed the standard GO annotation style (using Gene ontology guideline). The evidence code is always IEA. The confidence value is included for each GO term. Authors\' contributions ======================= The main work was carried out by AV. RK and KG conceived the idea of the study. AV and RK drafted the manuscript. FS developed and JM applied the machine learning strategy. KG implemented the databases in SRS. RE and SS supervised the work. All authors participated in reading, approving and revising the manuscript. Acknowledgments =============== We thank the Gene Ontology Consortium and all groups that established GO association databases for making their data available through the web. This work was funded by the German Cancer Research Center (DKFZ), the Deutsche Krebshilfe and the Nationales Genom-Forschungs-Netz (NGFN). We also thank Suresh Kumar Swaminathan and Coral del Val for their suggestions during the preparation of the manuscript, and Andrea Mclntosh-suhr and Christopher Previti for proofreading. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### A schematic representation of possible GO term relationships: A: GO~1~is a \"parent\" of GO~2~in a single path relationship. B: GO~1~is a \"parent\" of GO~2~in a multiple path relationship. C: GO~1~is a \"child\" of GO~2~in a single path relationship. D: GO~1~is a \"child\" of GO~2~in a multiple path relationship. E: GO~1~and GO~2~are \"siblings\" in a single path relationship. F: GO~1~and GO~2~are \"siblings\" in a multiple path relationship. MF denotes the molecular function node (root). :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### General prediction scheme: The training sequences (S1) with known function (GO~x~, GO~y~, GO~z~) were searched across the protein databases, yielding hits with molecular function GO terms (GO~1~, GO~2~, GO~3~, GO~4~, GO~5~, GO~6~) and their features (see methods), sketched as dots in a two-dimensional feature space. If GO terms of the hits compared to GO terms of the query, they were classified as +1 (correct, green), and -1 otherwise (red). The classifier (SVM) separated the classes by an optimal separating hyperplane (OSH). Unknown sequences (S2) were searched in the same manner and the GO terms (GO~n~, GO~m~, GO~o~) were extracted. Their features were calculated and mapped into the feature space. The corresponding labels were assigned (correct/false). :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Accuracy and precision against the number of votes: The accuracy and precision values of the test data is plotted against the number of votes. An increasing number of votes increased the precision monotonically. Higher stringency yielded a sparse lowering of the accuracy due to the rate of false negatives. The relation between the precision and the number of votes was used for assigning confidence values for new predictions. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### ROC plots for the classifiers performance: ROC plots for the results of all organisms tested and the average of all test sequences. The classification performance for different classes of organisms like multi-cellular eukaryotes, single-cell eukaroyotes and the prokaryotes were compared. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Precision against the sequence coverage: Average precision against sequence coverage for all 13-test organisms (circles). The red line denotes a fitting curve. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Comparison of GO slims between *Xenopus*, fly, yeast and mouse: Distributions of higher-level GO terms (,,GO slim\", see text) for *Xenopus*, fly, yeast and mouse. The sum of all high-level terms may exceed the total number of the annotated terms, since some terms may have more than one high-level \"parent\" terms due to multiple paths. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Training and test dataset: Datasets used for training and testing the support vector machines. The columns are: 1. The number of cDNA sequences for training. 2. The number of cDNA sequences with BLAST hits having GO molecular function terms. 3. The average number of GO molecular function terms per cDNA sequence of the BLAST-hits. 4. The classification of GO terms coming from the hits, positive if the GO terms were similar to original annotation, negative otherwise. ::: **Organisms** **Number of cDNAs** **cDNA with MF GO** **Number of GO/cDNA** **Class distribution** --------------- --------------------- --------------------- ----------------------- ------------------------ ------ Rat 1039 1036 36.90 25.7 74.3 Fish 1061 1044 32.10 39.2 60.8 Fly 5840 5574 25.47 23.4 76.6 Worm 4272 3458 27.13 39.5 60.5 *Plasmodium* 274 271 23.67 28.0 72.0 *Leishmania* 82 82 20.51 35.1 64.9 Yeast 3356 2972 18.60 23.7 76.3 *Bacillus* 2729 2577 13.63 35.4 64.6 *Coxiella* 931 900 12.33 37.0 63.0 *Shewanella* 2413 2303 10.78 33.0 67.0 *Vibrio* 1832 1804 12.54 31.9 68.1 *Arabidopsis* 8807 8120 26.66 30.2 69.8 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The novel predictions with high confidence values (100%). The columns correspond to TC (TIGR-id) number, GO term level in GO-structure, the confidence values and the GO number and GO terms. ::: **TC Numbers** **GO level** **Confidence value** **GO ID** **GO terms** ---------------- -------------- ---------------------- ------------ --------------------------------- TC212171 4 100% GO:0008233 peptidase activity 5 100% GO:0004175 endopeptidase activity 5 98% GO:0008236 serine-type peptidase activity 3 98% GO:0016787 hydrolase activity TC196381 5 100% GO:0004175 endopeptidase activity 4 98% GO:0008233 peptidase activity 3 98% GO:0016787 hydrolase activity 5 97% GO:0008236 serine-type peptidase activity TC209487 2 100% GO:0003824 enzyme activity 3 100% GO:0016787 hydrolase activity 5 90% GO:0004177 aminopeptidase activity 4 85% GO:0017171 serine hydrolase activity TC187949 4 100% GO:0004888 transmembrane receptor activity 3 97% GO:0004872 receptor activity TC194305 2 100% GO:0003824 enzyme activity 3 99% GO:0016740 transferase activity 3 99% GO:0016301 kinase activity 6 97% GO:0004672 protein kinase activity TC210151 3 100% GO:0004872 receptor activity 4 97% GO:0004888 transmembrane receptor activity 6 82% GO:0004928 frizzled receptor activity TC199713 6 100% GO:0004602 glutathione peroxidase activity 3 98% GO:0016491 oxidoreductase activity 5 85% GO:0004601 peroxidase activity TC190605 2 100% GO:0003824 enzyme activity 3 100% GO:0016787 hydrolase activity 4 87% GO:0017171 serine hydrolase activity 6 86% GO:0016804 prolyl aminopeptidase activity :::

PubMed Central

2024-06-05T03:55:47.694712

2004-8-26

PMC517704

Background ========== Pulmonary surfactant is a phospholipid-protein complex that lines the inner surface of the lung and is essential for normal pulmonary function. Surfactant acts to promote lung stability by reducing surface tension within the lung, while also protecting against inhaled pathogens. Surfactant is composed of approximately 90% lipids and 10% proteins by weight. The lipid component is primarily phospholipids with phosphatidylcholine (PC) being the most abundant, and the protein component comprised of four surfactant-associated proteins designated SP-A, SP-B, SP-C and SP-D \[[@B1]\]. The reduction of surface tension within the lung is a result of the interaction between surfactant phospholipids and the two hydrophobic surfactant proteins, SP-B and SP-C \[[@B1]\], while the two hydrophilic proteins, SP-A and SP-D, are members of a family of innate immune molecules called collectins \[[@B2]\]. Collectins opsonize bacteria and viruses and enhance their phagocytosis by macrophages and neutrophils \[[@B2]\]. Alterations of the pulmonary surfactant system, including decreased total surfactant levels, have been implicated in the pathophysiology of acute lung injury. Multiple studies of patients with a variety of lung diseases have shown that surfactant levels are decreased in the inflamed, injured, or infected lung \[[@B3]-[@B5]\]. In agreement, decreases in alveolar surfactant lipid pools have also been observed in several animal models of lung inflammation induced by both direct insults to the lung, such as bacterial infection \[[@B6]\], oxygen toxicity \[[@B7],[@B8]\], endotoxin administration \[[@B9]-[@B11]\], and by indirect insults, such as N-nitroso-N-methylurethane \[[@B12]\] and cecal ligation and perforation \[[@B13]\]. Alveolar metabolism of surfactant is a complex process, primarily involving type II epithelial cells that synthesize, secrete and clear surfactant from the airspaces \[[@B14]\], along with phagocytic cells such as macrophages and neutrophils that participate in surfactant clearance \[[@B15],[@B16]\]. In a situation of pulmonary inflammation, altered type II cell metabolism has been thought to play a role in the alterations of surfactant lipid levels. Viviano et al. observed a decrease in alveolar surfactant levels and a corresponding increase in intracellular surfactant after lipopolysaccharide (LPS) administration \[[@B10]\]. Additionally, exposure of rat lungs ventilated *ex vivo*to LPS resulted in the presence of giant lamellar bodies within the type II cells \[[@B17],[@B18]\]. Collectively these studies suggest that type II cell metabolism is altered after LPS administration and that a possible explanation for decreased surfactant pool size may be increased clearance of surfactant lipids by the type II cells. Additional experimental evidence has also implicated recruited inflammatory cells as having an impact on surfactant pool sizes. Both neutrophils and macrophages recovered from LPS exposed lungs had a greater capacity to internalize surfactant-like lipids compared to control cells *in vitro*\[[@B16]\]. Therefore these recruited inflammatory cells may also have a significant impact on surfactant pool size by increasing the overall surfactant lipid clearance. The objective of the current study was specifically to investigate the mechanisms of *in vivo*surfactant clearance in a rodent model of acute pulmonary inflammation induced by intrapulmonary instillation of LPS. We hypothesized that during pulmonary inflammation there is altered clearance of alveolar phospholipid by both alveolar type II cells and inflammatory cells within the airspaces. We analyzed *in vivo*clearance of surfactant lipids by a variety of pulmonary cells at 18 and 72 hours after LPS instillation. Results indicated that increased clearance of surfactant lipids in our model of acute pulmonary inflammation is primarily due to enhanced lipid uptake by the inflammatory cells recruited to the airspace, whereas uptake by type II cells was unaltered. However the *in vivo*surfactant uptake by inflammatory cells in the current study was significantly less than that predicted by previous *in vitro*studies \[[@B16]\]. Methods ======= Materials --------- Dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), dipalmitoylphosphatidylglycerol (DPPG) and cholesterol were purchased from Avanti Polar Lipids (Birmingham, AL). L-α-dipalmitoyl \[2-palmitoyl-9,10-^3^H(N)\]PC was obtained from DuPont New England Nuclear (Boston, MA). Elastase for type II cell isolations was purchased from Worthington Biochemicals (Freehold, NJ). Dulbecco\'s PBS, DMEM, and fetal bovine serum (FBS) were obtained from Life Technologies (Gaithersburg, MD). Low-endotoxin BSA, O26:B6 *Escherichia coli*LPS, Rat IgG and all other chemicals were purchased from Sigma (St. Louis, MO). Chloroform and methanol were obtained from EM Science (Gibbstown, NJ). Preparation of Liposomes ------------------------ Small unilamellar liposomes were prepared with a lipid composition similar to pulmonary surfactant: 52% DPPC, 26% egg PC, 15% DPPG and 7% cholesterol by weight with trace amounts of ^3^H-DPPC (12 μCi/mg phospholipid). The lipids were dried under nitrogen, reconstituted in 0.15 M saline and extruded from a French Press cell under 900 psi. This resulted in small unilamellar liposomes at a concentration of approximately 1 mg lipid/ml. Animal Model ------------ Male pathogen-free Sprague Dawley rats (150--200 g; Taconic Farms, Germantown, NY) were used for the current study. For LPS animals, endotoxin (0.1 mg/kg of O26:B6 *E. coli*LPS) was suspended in 300 μl of sterile 0.15 M saline. Control animals received an equal volume (300 μl) of sterile 0.15 M saline. For instillation, animals were anesthetized with halothane such that they remained unconscious throughout the entire instillation procedure and had no cough reflex upon intubation. Animals were placed on a board at a 45° angle, intubated with an 18-gauge blunt ended catheter and either sterile saline or LPS suspension was instilled followed by five 1 ml boluses of air to facilitate the distribution of the instilled fluid. Two hours prior to killing, liposomes (100 μg lipid) were intratracheally instilled in all groups following the identical instillation procedure. Separate groups of both control and LPS animals were sacrificed at 18 or 72 hours after saline or LPS instillation. Whole Lung Studies ------------------ One control animal and one LPS animal were investigated simultaneously, thus all procedures were done in parallel. At 18 or 72 hours after saline or LPS instillation, animals were anesthetized with 0.3 mg of sodium pentobarbital and 700 U of heparin. After loss of toe pinch reflex, the trachea was cannulated and the rat was exsanguinated via transection of the descending aorta. The chest cavity was subsequently opened and the lungs perfused through the pulmonary artery with 40--50 ml of a calcium buffer (140 mM NaCl, 5 mM KCl, 2.5 mM Na~2~HPO~4~, 10 mM HEPES, 2.0 mM CaCl~2~, and 1.3 mM MgSO~4~at 37°C). The lung from the first animal was carefully removed from the thoracic cavity and placed between saline soaked gauze pads while the second lung was perfused and removed in identical fashion. The time for lung perfusion and removal was approximately 10 minutes. After lungs were isolated from both a control and LPS animal, they were lavaged simultaneously with eight 10-ml volumes of EGTA buffer (140 mM NaCl, 5 mM KCl, 2.5 mM Na~2~HPO~4~, 10 mM HEPES, and 0.2 mM EGTA at 37°C). For each animal the individual lavages were collected and combined to make up the bronchoalveolar lavage fluid (BALF), which was immediately stored on ice. After the lavage procedure, individual lobes were dissected away from the major airways. The lung tissue was then cut into 5 mm pieces in 5 ml calcium buffer and subsequently homogenized with a Polytron PT-MR-2100. After complete homogenization, total lung tissue volume was diluted to 40 ml with calcium buffer and stored on ice. A small aliquot (2 ml) of the total BALF was removed and the remainder was centrifuged at 250 *g*for 10 minutes at 4°C to generate a pellet that was primarily BALF cells. The cell pellet was subsequently suspended in 10 ml of PBS. Lavage cell numbers were determined by a hemocytometer, viability determined by trypan blue exclusion and cell differential determined by Hemacolor staining of cytospins that were prepared using a Shandon Cytospin 2 centrifuge. Liposome-association in total BALF, cell-free BALF, BALF cells and homogenized lung tissue was determined by scintillation counting. Surfactant Phospholipid Measurement ----------------------------------- Alveolar phospholipid levels were measured in the cell-free BALF by phospholipid-phosphorous measurement. Lipids were extracted using the method of Bligh & Dyer \[[@B19]\] and phospholipid levels were determined using a modification of the Duck-Chong phosphorous assay \[[@B20]\]. Briefly, 100 μl of 10% magnesium nitrate in methanol was added to the extracted lipids. After drying, the samples were ashed in a fume hood on an electric rack for approximately 1 min. After 1 ml of 1 M HCl was added, the samples were reheated on a heating block while covered for 15 min at 95°C. After cooling, a 66 μl aliquot of each sample was added to individual wells of a 96 well plate along with 134 μl of a dye consisting of 4.2% ammonium molybdate in 4.5 M HCl with 0.3% malachite green (1:3 vol/vol). The absorbency of each sample was read at 650 nm using a Biorad 550 microplate reader and compared to reference standards on the same plate. Surfactant Protein A (SP-A) Analysis ------------------------------------ Relative quantities of alveolar SP-A from 18-hour control animals, 18-hour LPS animals and 72-hour LPS animals were determined by loading equal volumes of BALF on 15% SDS-PAGE gels under reducing conditions. Total BALF recovery was not different among the individual animals. Proteins were then transferred to nitrocellulose where SP-A was probed with a well characterized polyclonal rabbit anti-rat SP-A \[[@B21]\]. The nitrocellulose was subsequently developed using the ECL system (Amersham Pharmacia Biotech, Piscataway, NJ). Type II Cell Isolation ---------------------- A separate cohort of animals was required for type II cell isolation studies. As with the whole lung studies, type II cell isolation was completed for one control and one LPS rat in parallel for both 18- and 72-hour time points after instillation of saline or LPS. The type II cell isolation procedure was described previously with minor modifications of the original protocol \[[@B22],[@B23]\]. The modifications were designed for isolation of type II cells from inflamed lungs, and included increasing the elastase to 3,000 orcein units/lung, and increasing the surface area for IgG panning by two-fold. Briefly the isolation procedure involved killing the rats, removing the lungs and lavaging the lungs eight times as stated in *Whole Lung Studies.*Subsequently the lungs were lavaged twice with calcium buffer (37°C) and once with elastase solution (3000 orcein/40 ml calcium buffer; 37°C). The lungs were then filled with the elastase solution and suspended in warmed saline (37°C) for 20 minutes. Lung tissue was removed from the major airways, cut into 5-mm pieces and chopped 200 times with sharp scissors in 5 ml of the calcium solution along with 2 mg of DNase. The tissue suspension was added to a flask with 4 ml of FBS and 35 ml of calcium buffer and shaken vigorously for 2 min in a 37°C water bath. The resultant tissue suspension was strained through gauze, and then decreasing sizes of nylon mesh (150-, 15-, and 8-μm). The cell suspension was centrifuged at 250 *g*for 10 min at 4°C, the resulting cellular pellet was suspended in 20 ml of warm DMEM (37°C) and incubated on two IgG coated Petri dishes (150 × 15 mm) for 30 minutes at 37°C. After the incubation period, nonadherent cells were removed from the plate, washed once with PBS, suspended in calcium buffer and used for determination of radiolabel recovery associated with Type II cells. The remaining adherent cells were gently scraped off the plate in 5 ml of DMEM and transferred into a polypropylene tube; this population of cells is referred to as \"plate cells\" and consisted primarily of macrophages and/or neutrophils as described in detail below. The cells were washed once and suspended in calcium buffer for determination of associated radioactivity. A small aliquot from the isolated type II cells and plate cells was saved for differential cell count and viability. Cell purity was determined by counting a minimum of 250 cells from random fields after staining by the Papanicolaou method \[[@B22]\]. Statistics ---------- All data reported are means ± standard error (SE). Repetitions (n) used to calculate means ± SE were from independent experiments, not from replicates within an experiment. An analysis of variance (ANOVA) was used to determine differences between all experimental groups at a specific time point, followed by a Tukey post-hoc test for multiple comparisons. Significance was accepted when p \< 0.05. Results ======= LPS-induced alterations: BALF cells and alveolar surfactant ----------------------------------------------------------- Table [1](#T1){ref-type="table"} reveals total alveolar cell numbers and differentials from the BALF of control and LPS groups killed at the 18- and 72-hour time points. Eighteen hours after instillation of LPS there were significantly more cells recovered in the BALF from the LPS treated animals compared to the saline control group (p \< 0.01). Cell differentials from the two 18-hour groups revealed that the LPS group had primarily neutrophils in the BALF, whereas the cells recovered from the control group were predominantly macrophages. Seventy-two hours after LPS instillation there were significantly greater numbers of cells in the BALF of the LPS group compared to the 72-hour control group (p \< 0.01), but significantly fewer numbers of cells compared to the LPS 18-hour group (p \< 0.01). Cell differentials from the 72-hour groups revealed that the cells were predominately macrophages in both the LPS and control groups. Of note, there were no differences in cell numbers or differentials between the two control groups at the different time points. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Bronchoalveolar lavage fluid characteristics from animals killed 18 and 72 hours after instillation of saline (Control) or lipopolysaccharide (LPS). ::: Control 18 (n = 6) LPS 18 (n = 6) Control 72 (n = 6) LPS 72 (n = 6) ------------------------------------- -------------------- ---------------- -------------------- ------------------ Cell Numbers (10^6^) 12.3 ± 1.1 102.8 ± 8.1\* 11.3 ± 0.9 57.3 ± 6.6\*^\#^ \% Macrophages 98.2 ± 0.3 7.3 ± 0.8\* 97.7 ± 0.4 90 ± 1.5\* \% Neutrophils 1.8 ± 0.3 92.7 ± 0.8\* 2.3 ± 0.4 10 ± 1.5\* Alveolar Phospholipid (mg PL/kg BW) 14.7 ± 0.7 12.8 ± 0.9 14.0 ± 0.4 12.9 ± 0.5 Abbreviations: PL = phospholipid; BW = body weight. Values are mean ± SEM. \* = p \< 0.01 vs. appropriate control; ^\#^= p \< 0.01 vs. LPS 18. ::: Table [1](#T1){ref-type="table"} also displays the total phospholipid levels measured in the BALF of the four experimental groups. There was an approximate 15% decrease in total phospholipid recovered by lung lavage from both LPS groups compared to their respective saline control groups, however this did not reach statistical significance. Of note, there was no difference in mean body weights among the four experimental groups (Con 18: 177 ± 7 g; LPS 18: 180 ± 10 g; Con 72: 186 ± 9 g; LPS 72: 192 ± 7 g). Figure [1](#F1){ref-type="fig"} shows Western blot analysis of SP-A measured in the BALF recovered from animals killed 18 hours post saline instillation (control), and 18 hours and 72 hours after LPS instillation. There was relatively greater quantity of SP-A in the BALF of animals killed at both 18 and 72 hours after LPS instillation compared to the control animals, consistent with previous reports using similar models of intrapulmonary LPS administration \[[@B9]-[@B11]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Alveolar surfactant protein A (SP-A) levels.**Relative quantities of alveolar SP-A were measured by Western Blot. Equal volumes of bronchoalveolar lavage fluid (BALF) from the individual animals were utilized and total BALF recovery was not different among these animals. Lanes 1 and 2 represent individual animals that were lavaged 18 hours after saline instillation. Lanes 3 and 4 represent individual animals that were lavaged 18 hours after instillation of 100 μg/kg O26:B6 lipopolysaccharide (LPS). Lanes 5 and 6 represent animals lavaged 72 hours after instillation of 100 μg/kg 026:B6 LPS. :::  ::: Whole Lung Studies: Clearance of Radiolabel Liposomes ----------------------------------------------------- Figure [2](#F2){ref-type="fig"} shows distribution of the recovered radioactivity associated with the cell free BALF, the isolated BALF cells, and whole lung tissue for the two groups killed 18 hours after instillation of saline or LPS. There was no difference in total lung radioactivity recovered compared to the total radiolabel instilled between the Control 18 and LPS 18 groups (46.0 ± 4.6% and 48.4 ± 4.0%; respectively). There was significantly less radiolabel recovered in the BALF (p \< 0.05) and significantly more radiolabel associated with the BALF cells (p \< 0.01) in the LPS group compared to the control group. There was no difference in radiolabel association with lung tissue between the two groups. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Distribution of radiolabel liposomes 18 hours after instillation of LPS.**Whole lung distribution of the total recovered ^3^H-liposomes instilled 2 hours prior to killing. Animals were killed 18 hours after instillation of saline (Con 18) or 100 μg/kg O26:B6 lipopolysaccharide (LPS 18) where the lungs were lavaged, lavage cells isolated and whole lung tissue homogenized. Radioactivity was subsequently measured in cell free bronchoalveolar lavage fluid (BALF), isolated BALF cells and whole lung tissue homogenate (Tissue). Data are means ± SEM and expressed as a percentage of total recovered radiolabel; n = 6 animals/group. Statistical significance \* = p \< 0.05 vs. respective control group. :::  ::: Figure [3](#F3){ref-type="fig"} displays total recovered radioactivity associated with the cell free BALF, the isolated BALF cells, and whole lung tissue for the two groups killed 72 hours after instillation of saline or LPS. There was no difference in the total lung radiolabel recovered compared to the total radiolabel instilled between the Control 72 and LPS 72 groups (47.3 ± 4.0% and 44.8 ± 3.8%; respectively). There was significantly less radiolabel recovered in the BALF (p \< 0.05) and significantly more radiolabel liposomes associated with the BALF cells (p \< 0.01) in the LPS group compared to the control group. There was no difference in radiolabel associated with lung tissue between the two groups. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Distribution of radiolabel liposomes 72 hours after instillation of LPS.**Whole lung distribution of the total recovered ^3^H-liposomes instilled 2 hours prior to killing. Animals were killed 72 hours after instillation of saline (Con 72) or 100 μg/kg O26:B6 lipopolysaccharide (LPS 72) where the lungs were lavaged, lavage cells isolated and whole lung tissue homogenized. Radioactivity was subsequently measured in cell free bronchoalveolar lavage fluid (BALF), isolated BALF cells and whole lung tissue homogenate (Tissue). Data are means ± SEM and expressed as a percentage of total recovered radiolabel; n = 6 animals/group. Statistical significance \* = p \< 0.05 vs. respective control group. :::  ::: Additional controls were performed in which animals (n = 2) received radiolabel liposomes followed by whole lung lavage and lung tissue homogenization 5 minutes after the instillation procedure. Mean total lung radiolabel recovery for this control group was 77% suggesting that \~25% of the radiolabel liposomes were lost in the instillation procedure (i.e., syringe, catheter), or possibly were adhered to the airway epithelium. The distribution of the recovered liposomal radioactivity within the 5-minute control lungs was 91% associated with the BALF, 2% associated with BALF cells, and 7% associated with lung tissue. Type II Cell Isolation: Cell Recoveries, Purities and Radioactivity per Cell ---------------------------------------------------------------------------- Table [2](#T2){ref-type="table"} demonstrates the cell recovery and cell differential after type II cell isolation for the two experimental groups at the 18-hour time point. Type II cell recovery for the control group ranged from 10.4 × 10^6^to 21.6 × 10^6^cells with a mean of 17.1 × 10^6^. The purity of this cell fraction averaged greater than 80% type II cells and viability was greater than 95%. Plate cell recovery from the control group ranged from 1.8 × 10^6^to 4.7 × 10^6^cells with a mean value of 3.2 × 10^6^. This cell fraction was predominately macrophages and the viability was greater than 90%. For the LPS group at the 18-hour time point, type II cell recovery ranged from 10.6 × 10^6^to 20.5 × 10^6^cells and a mean of 16.3 × 10^6^cells, with a purity greater than 85% type II cells and viability greater than 95%. Plate cells from the LPS 18 group ranged from 2.7 × 10^6^to 13 × 10^6^cells with a mean of 6.6 × 10^6^cells. This cell population consisted primarily of neutrophils and macrophages and viability was greater than 90%. There were no significant differences in cell numbers obtained from control and LPS animals 18 hours after saline or LPS instillation. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Cell recovery and differential after lung digestion and IgG panning for animals killed 18 hours after instillation of saline (Control) or lipopolysaccharide (LPS). ::: Control 18 (n = 7) LPS 18 (n = 7) ---------------------- -------------------- ---------------- ------------ ----------- Cell Numbers (10^6^) 17.1 ± 1.6 3.2 ± 0.5 16.3 ± 1.8 6.6 ± 1.7 \% Type II 81 ± 2 15 ± 7 86 ± 2 14 ± 2 \% Macrophages 9 ± 1 61 ± 5 3 ± 1\* 33 ± 2\* \% Neutrophils 1 ± 1 17 ± 10 4 ± 1 53 ± 3\* \% Other Cells 9 ± 2 7 ± 2 7 ± 2 \- Values are mean ± SEM. \* = p \< 0.01 vs. Control. ::: Figure [4](#F4){ref-type="fig"} reveals the radioactivity per million tissue cells for the isolated type II cells and the other tissue associated cells (plate cells) for the control and LPS 18-hour groups. There were no significant differences in the radioactivity per type II cell or plate cell between the control group and the LPS group at the 18-hour time point after instillation. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Liposome uptake by lung tissue cells 18 hours after instillation of LPS.**Association of ^3^H-liposomes instilled 2 hours prior to killing in cells isolated from lung tissue. Animals were killed 18 hours after instillation of saline (Con 18) or 100 μg/kg O26:B6 LPS (LPS 18). Lungs were lavaged and type II cells and other tissue-associated cells (plate cells) were isolated. Data are means ± SEM and expressed as DPM/10^6^cells, n = 7 animals/group. :::  ::: Table [3](#T3){ref-type="table"} shows the cell recovery and cell differential after type II cell isolation for the two experimental groups at the 72-hour time point. Type II cell recovery for the control group ranged from 21.2 × 10^6^to 40 × 10^6^cells with a mean of 28.4 × 10^6^. The purity of this cell fraction averaged 75% type II cells and viability was greater than 95%. The number of cells obtained from the plate group ranged from 1.8 × 10^6^to 15.9 × 10^6^cells with a mean value of 6.2 × 10^6^with the primary cell type being macrophages with a small percentage of neutrophils and type II cells. Viability for the plate cells were greater than 90%. For the LPS group at the 72-hour time point, type II cell recovery ranged from 19.2 × 10^6^to 64.6 × 10^6^cells and a mean of 33.2 × 10^6^cells, purity greater than 80% type II cells and viability greater than 95%. Plate cells from the LPS 18-hour group ranged from 3.3 × 10^6^to 16.2 × 10^6^cells with a mean of 7.8 × 10^6^cells. This cell population consisted primarily of macrophages and viability was greater than 90%. There were no significant differences in cell recoveries between the control and LPS groups at the 72-hour time point. Of note, there were a greater number of cells recovered from both 72-hour groups compared to both 18-hour groups. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Cell recovery and differential after lung digestion and IgG panning for animals killed 72 hours after instillation of saline (Control) or lipopolysaccharide (LPS). ::: Control 72 (n = 6) LPS 72 (n = 7) ---------------------- -------------------- ---------------- ------------ ----------- Cell Numbers (10^6^) 28.4 ± 3.9 6.2 ± 2.2 33.2 ± 5.6 7.8 ± 2.2 \% Type II Cells 75 ± 2 22 ± 3 83 ± 1 19 ± 3 \% Macrophages 11 ± 2 56 ± 5 12 ± 2 59 ± 4 \% Neutrophils \- 22 ± 7 2 ± 1 22 ± 3 \% Other Cells 14 ± 1 \- 3 ± 3\* \- Values are mean ± SEM. \* = p \< 0.01 vs. Control. ::: Figure [5](#F5){ref-type="fig"} reveals the radioactivity per million tissue cells for the isolated type II cells and the other tissue-associated cells (plate cells) for the control and LPS 72-hour groups. There were no significant differences in the radioactivity per type II cell or plate cell between the LPS group and the control group 72 hours after the appropriate instillation. Of note there was no significant difference in radioactivity per cell for both the type II cells and plate cells between the two 72-hour groups and the two 18-hour groups. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Liposome uptake by lung tissue cells 72 hours after instillation of LPS.**Association of ^3^H-liposomes instilled 2 hours prior to killing in cells isolated from lung tissue. Animals were killed 72 hours after instillation of saline (Con 72) or 100 μg/kg O26:B6 LPS (LPS 72). Lungs were lavaged and type II cells and other tissue-associated cells (plate cells) were isolated. Data are means ± SEM and expressed as DPM/10^6^cells; Con 72 = 6 animals, LPS 72 = 7 animals. :::  ::: Discussion ========== In the present study, we evaluated the clearance of surfactant-like liposomes at 18 and 72 hours following the intrapulmonary instillation of LPS. At both time points, radiolabel liposomes were instilled two hours prior to killing and the distribution within the lung was determined. Radioactivity was measured in cell-free BALF, isolated BALF cells, whole lung tissue, isolated type II cells and remaining tissue-associated cells. At both time points there was a significant increase in clearance of exogenous liposomes from the airspace and a small decrease in alveolar surfactant phospholipid levels in the LPS groups compared to control groups that received vehicle only. This corresponded with increased radiolabel associated with isolated BALF cells and no difference associated with lung tissue at either time point after LPS instillation compared to controls. There was no difference in liposomal radioactivity associated with isolated type II cells or other tissue cells between both LPS groups and their respective control groups, which was in agreement with whole lung measurements. These data suggest that cells recruited into the airspace as a consequence of the LPS-induced inflammatory response can significantly contribute to increased clearance of surfactant-like liposomes from the airspace (Figs. [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}). Eighteen hours after LPS instillation there was an infiltration of neutrophils into the airspace which resulted in a 10-fold increase in BALF cells. At this time point there was a doubling of total liposomal radioactivity associated with these inflammatory cells compared to alveolar cells recovered from control animals. Neutrophils recruited into alveolar spaces during infection are essential for host defense by phagocytosis and killing of bacteria and other infectious agents. Previous studies have documented that surfactant can in fact modulate neutrophil functions, as surfactant proteins A and D can enhance neutrophil uptake of bacteria \[[@B24]\]. Although these phagocytic cells are extremely important in host defense, they have also been implicated as a contributing factor to the lung injury in a variety of inflammatory disorders. In a recent study by Quintero et al., it was demonstrated by confocal microscopy that neutrophils isolated from inflamed lungs were able to significantly internalize and degrade surfactant lipids *in vitro*\[[@B16]\]. Based on *in vitro*measurements of lipid uptake, they estimated that neutrophils could account for up to 48% of the observed clearance and could significantly impact surfactant homeostasis. Data from the current study supports this idea by demonstrating that *in vivo*, neutrophils can indeed contribute to the clearance of surfactant lipids during pulmonary inflammation. At the 72-hour time point after LPS instillation there was an accumulation of macrophages in the alveolar space that resulted in a 5-fold increase in BALF cells compared to control animals. There was a doubling of radiolabeled phospholipids associated with these alveolar cells and no increase in the amount of radiolabeled phospholipids associated with whole lung tissue. This resulted in a significant increased in clearance of total liposomal radioactivity from the BALF, 72 hours after LPS instillation. Alveolar macrophages have been shown *in vitro*to take up and degrade surfactant lipids and have also been shown *in vivo*to account for approximately 20% of surfactant lipid clearance in normal lungs \[[@B16],[@B25]\]. Macrophages isolated from LPS-exposed lungs were shown to have a greater capacity to take up surfactant lipids *in vitro*than macrophages isolated from normal lungs \[[@B16]\], suggesting that in an inflamed lung, the recruited macrophages may have a significant role in surfactant metabolism. Indeed, the *in vivo*data presented in the current study support the theory that activated alveolar macrophages can impact clearance of surfactant lipids. Of note, previous *in vitro*data had predicted that inflammatory cells (neutrophils and macrophages) could account for a 6 to 13 fold increase in lipid clearance \[[@B16]\]. In the current study there was a 5--10 fold increase in alveolar inflammatory cells but only a doubling of cell associated radioactivity and a change in alveolar phospholipid pool size of approximately 15%. Importantly, the current study instilled a similar dose of LPS, had similar end points and the same number of inflammatory cells recovered from the BALF as the aforementioned *in vitro*study. Although the present *in vivo*data demonstrated that the inflammatory cells can indeed contribute to increased lipid clearance as suggested previously, the absolute value was considerably less than that predicted from the *in vitro*studies. Although we do not have an explanation for this quantitative difference between the *in vivo*and *in vitro*observations, it is possible that the process of cell isolation in the *in vitro*study resulted in their activation for subsequent lipid uptake. Alternatively, *in vivo*, multiple factors can impact total surfactant pool size in addition to clearance by inflammatory cells. Type II cells are the predominant cell type that regulates surfactant metabolism, being involved in synthesis, secretion and clearance. Secretion of surfactant phospholipids is solely a property of alveolar type II cells, whereas clearance in a healthy lung is regulated primarily by both type II cells and alveolar macrophages. In one study utilizing uninjured rabbits, it was determined that type II cells accounted for approximately 65% of the clearance of alveolar phospholipid \[[@B25]\]. In a situation of pulmonary inflammation induced by LPS administration, there have been documented decreases in alveolar surfactant phospholipids \[[@B9]-[@B11]\], increases in intracellular surfactant phospholipids \[[@B10]\] and the appearance of giant lamellar bodies within type II cells \[[@B17],[@B18],[@B26]\]. From these observations, LPS induced inflammation could either inhibit surfactant secretion or enhance surfactant clearance by type II cells, resulting in diminished alveolar surfactant pool and increased intracellular pool. We originally hypothesized that after LPS administration there was increased clearance of surfactant phospholipids by type II cells. However, data from this study demonstrated that *in vivo*, surfactant clearance by type II cells was similar at both 18 and 72 hours after intrapulmonary LPS instillation compared to control groups killed at the same time points (Figs [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}). Data from the current study cannot lead to any conclusions pertaining to alterations in type II cell phospholipid synthesis or secretion. As mentioned, we observed a small but not statistically significant decrease in alveolar phospholipid pools after LPS administration, which is in contrast to previous studies by Viviano et al. and MacIntosh et al. that used 1 mg/kg and 0.5 mg/kg of LPS respectively \[[@B9],[@B10]\]. Nevertheless, we did observe a similar increase in alveolar levels of SP-A after intrapulmonary LPS (Fig [1](#F1){ref-type="fig"}), which is a well documented characteristic of lung inflammation after LPS exposure \[[@B9]-[@B11]\]. This increase has been primarily attributed to increased SP-A synthesis by type II cells \[[@B11]\] suggesting that indeed type II cell metabolism was altered in our model. Additionally, these data provide further experimental evidence that surfactant phospholipid and SP-A pools can be independently regulated in response to inflammatory agents. We also investigated additional tissue-associated cells that were isolated after lung digestion (primarily macrophages and neutrophils). Similar to type II cells, surfactant clearance from these tissue-associated cells isolated from the two LPS groups were similar to their respective control groups. In addition, the clearance attributed to type II cells and tissue-associated cells was similar at both time points for the LPS and control groups. This observation is in agreement with a study by Gurel et al., that documented that alveolar type II cells and tissue macrophages contributed equally to the alveolar clearance of phospholipid in adult mice \[[@B27]\]. Conclusions =========== In conclusion, we demonstrated that after intrapulmonary LPS administration in adult rats, the *in vivo*clearance of surfactant lipids by type II cells is unaltered and that the increased clearance of surfactant lipids is primarily due to either neutrophils or macrophages that are recruited to the alveolar space. Authors\' contributions ======================= JLM conceived, designed and performed all aspects of the study, and was the primary participant in its writing. JRW aided in conception and design of study, and participated in its writing. Acknowledgements ================ JLM is a Parker B Francis Fellow in pulmonary research and was a recipient of a Post-Doctoral Fellowship from the Canadian Lung Association and Canadian Institutes of Health Research. Study was funded by RO1 HL-30923 (JRW).

PubMed Central

2024-06-05T03:55:47.698862

2004-7-23

PMC517705

Background ========== Respiratory diseases, infectious and non-infectious, are a prime cause of morbidity, mortality and health system utilization in many countries. Exposure to cigarette smoke is an important factor in causing as well as increasing complications in several pulmonary disorders. The mucociliary clearance constitutes the first line of defense to maintain the airways as free as possible of foreign bodies \[[@B1]\]. Impairment of mucociliary function may be the result of epithelial airway damage, ciliary dysfunction, inflammation, and change in mucus viscosity and/or elasticity. In laboratory studies we have shown that the physical properties of mucus in nonsmokers are not altered by age or by restrictive pulmonary pathology. However, mucus properties show alterations when exposed to tobacco smoke and these alterations are noticeable in the very early stages of smoke exposure, even at an exposure level in the range of 1 to 5 cigarettes per day \[[@B2]\]. The majority of studies on the outcome of tobacco use have been performed in the late phases of the process \[[@B3]-[@B7]\]; the acute and early effects on cilia, mucus and mucociliary clearance after active smoking or side stream tobacco smoke exposure have not been well studied. We hypothesized that studying changes occurring in the initial stages will lead to a better understanding of the multifaceted problems of tobacco exposure. Therefore our laboratory has been developing and using *in vivo*and *ex vivo*epithelial injury models that replicate the features of airway diseases \[[@B8]-[@B12]\]. The mucus blanket in the frog palate is cleared by coordinated ciliary activity in an almost identical fashion to that observed in human airways. We anticipated that a modified frog palate exposure model could better serve our purposes. We were specifically interested in developing an exposure model that would allow us to study the initial effects and mechanisms occurring in an epithelial tissue after exposure to environmental tobacco smoke. We conceived this model to be free of interferences from other agents or systemic physiological responses resulting from other internal or external influences. We also aim to have a better understanding of the mechanism by which ciliated epithelial cells are exfoliated after being exposed to tobacco smoke, as this may relate directly to impaired mucus clearance in several human airway diseases including chronic bronchitis and chronic obstructive pulmonary disease (COPD). Hence we decided first to test our novel *ex vivo*model, and in later studies use *in vivo*models. Methodology =========== Frog palate preparation ----------------------- From a bullfrog, *Rana catesbiana*, the upper portion of the head is removed following the procedures described in previous work \[[@B10],[@B11]\], by cutting with scissors through from the junction of the posterior pharynx and esophagus out to the skin of the back. This procedure was carried out after lowering the body temperature of the frog for 30 -- 60 minutes inside a refrigerator to abolish pain sensations. The palate was examined for macroscopic lesions, such as ulcers, petechia or redness as evidence of inflammation. Only palates free of inflammatory indicators were included in this study. Any blood remaining in the epithelial surface was carefully washed away, then the excised head was placed palate side facing upwards on a piece of gauze saturated with frog Ringer solution (FRS) in a Petri dish. The experimental procedures involving animals were approved by the Health Sciences Animal Policy and Welfare Committee, University of Alberta. The FRS was prepared by mixing standard Ringer injection with sterile water (2:1). The composition of standard frog Ringer (in mmol/l) is 90 NaCl, 3 KCl, 2 CaCl~2~, and 15 NaHCO~3~ (220 mosm/l). The palate was placed inside the frog chamber, a wooden box with a glass top and fitted glass front, and manipulated trough glove openings. The humidity inside the box is maintained at 100% using an ultrasonic Pari nebulizer and the temperature is kept between 22° to 24°C by a rheostat-controlled, externally mounted light source. Before carrying out any measurement, the palate was allowed to stabilize inside the box for 15 minutes before testing. Exposure chamber ---------------- The exposure chamber (Figure [1](#F1){ref-type="fig"}) with a volume \~10 liters had two inlets: one connected to an ultrasonic Pari jet nebulizer system set at 8 L/min measured by a Puritan flow meter to maintain the chamber near 100 % humidity. The other inlet was linked to a burning chamber. The latter, which contained a burning cigarette, was slightly pressurized with air flowing into the chamber at a rate of 2 L/min to promote cigarette combustion. Positive ventilation inside the burning chamber pushed the side stream smoke into the covered but not sealed exposure chamber, which was exhausted into a fume hood. Temperature inside the chamber was maintained to 22°C. and monitored through a thermocouple and digital-display thermometer. The palate was placed inside with the palate side upwards on a piece of gauze saturated with FRS in a glass dish at about five centimeters above the bottom of the chamber. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Box designed to expose the frog palate to cigarette smoke. :::  ::: The exposure chamber was designed to allow a steady flow of side stream tobacco smoke to reach the epithelial tissue. The concentration of cigarette smoke inside the exposure chamber was maintained constant throughout the experiment by the presence of a mixing baffle on the outflow of the chamber. The concentration of tobacco smoke inside the chamber was not directly measured, but is estimated that the half palate was exposed to the smoke of each cigarette delivered, diluted in 180 liters of fresh air. As much as possible, the conditions (humidity, type of solution, temperature, physical manipulation and airflow in and out) in the frog chamber and in the exposure chamber were maintained similar, with cigarette smoke being the independent variable. Frog palate exposure model preparation -------------------------------------- The palate was divided longitudinally in two halves along the midline as shown in Figure [2](#F2){ref-type="fig"}, cutting the epithelium with a scalpel to minimize damage. After five minutes the mucus transport was measured in both halves, left and right, to confirm that both half palates were functioning normally. One side was used as control and the other half was exposed to tobacco smoke. The control half of the palate was left in the frog chamber while the other half was placed in the exposure chamber maintained at similar conditions of 100 % humidity and room temperature. If any or both halves showed a variation in mucus transport greater than 30 % from the baseline, the experiment was aborted. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The frog palate was further divided longitudinally in two halves following the midline. :::  ::: Mucus transport velocity (MTV) determination -------------------------------------------- The palate was placed under a dissecting stereomicroscope provided with a reticulated eyepiece. Mucociliary clearance was determined by observing the movement of particles of charcoal powder gently deposited on a sample of mucus on the palate surface; its clearance was visually monitored and MTV determined. The displacement of 3 -- 5 μL of endogenous frog mucus sample was calculated by dividing the distance traveled by the transit time across the 0.3 inch (7.62 mm) segment marked between 0.1 and 0.4 inches in the graduated eyepiece. At least five measurements of the time required for the mucus sample to travel the defined distance were made every time to obtain control and smoke exposure mucus transport velocity. We used cigarettes regularly available at commercial outlets of a representative brand in terms of customer preference and toxic emissions: nicotine 0.5 -- 2.1 mg, tar 4 -- 24 mg, carbon monoxide 5 -- 25 mg, hydrogen cyanide 0.04 -- 0.21 mg, benzene 0.025 -- 0.069 mg, formaldehyde 0.018 -- 0.1 mg. One cigarette took 17 minutes on average to burn completely in this preparation. Measurements and mucus samples collection ----------------------------------------- After being exposed to one cigarette, the half palate was brought back to the frog chamber and MTV was measured in both control and exposed halves. A sample of mucus was collected from each half, and placed in separate containers and frozen at -80°C in liquid nitrogen followed by storage in a -80° freezer until analysis. The exposed half, brought back to the exposure chamber was further exposed to 3 more cigarettes (51 additional minutes of exposure). Following tobacco exposure, the half palate was again brought back to the frog chamber to measure mucus transport and collect tissue and mucus sample in both halves, control and exposed. One set of half palates (control and exposed to four cigarettes) was stored at 4°C overnight, and transport of mucus was reassessed the next day. Tissue and mucus samples collection ----------------------------------- Sample of mucus from both halves were immersed in glutaraldehyde 2.5%, and placed in properly identified separate containers for storage at 4°C for later Scanning Electron Microscope (SEM) studies. The epithelial tissue was carefully dissected and separated from the palate musculature. Samples of the palate tissue from each half were sectioned, frozen in liquid nitrogen and stored at -80°C for gelatinase zymography studies. Zymography ---------- Samples of tissue, as well as mucus, were taken out of the freezer and ground to a powder in a mortar and pestal. Liquid nitrogen was added to keep the samples frozen. Homogenizing buffer (KCl 100 mM, ZnCl~2~0.5 mM, EDTA 10 mM, Tris-HCl 1 M, pH 6.8) was added to the ground samples (approximately 500 ul buffer per 10 mg tissue sample, 200 buffer per 10 mg mucus sample) that were sonicated for 30 seconds on ice and then centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant was collected for protein assay (BCA protein assay kit, Pierce). Normalizing the protein content as 5 μg, different amount of samples were loaded into the 7.5% separating zymography gel (30% acrylamide/ 0.8% bisacrylamide 3.75 ml, 4 × Tris-Cl/ SDS, pH 8.8 3.75 ml, H~2~O 6 ml, 2% gelatin A 1.5 ml, 10% ammonium persulfate 0.1 ml, TEMED 0.01 ml, for 4 gels) and were run at 100 volts for 20 minutes, 150 volts for 40 minutes. After electrophoresis, the gel was washed 3 times (20 minutes per a time) in 2.5% Triton X-100 at room temperature followed by incubation for 96 hours in zymography development buffer (0.15 M NaCl, 5 mM CaCl~2~, 0.05% Azide NaN~3~, 50 mM Tris-HCl pH 7.6). The gel was then stained for 2 hours with stain solution (Coomassie brilliant blue R-250 1 g/L, methanol: acetic acid: H~2~O= 2.5: 1: 6.5) followed by de-staining (ethanol: acetic acid: H~2~O= 1: 2: 22) overnight. The expression of the gelatinases was shown on the gel as clear bands against the dark background stained with Coomassie blue. The bands were compared with the gelatinase standards for MMP 2 and 9 running in the first lane of each gel. The density of the gels was measured in the Bio-Rad scanning densitometer. Scanning electron microscopy ---------------------------- Samples of mucus and tissue were placed in 2.5 % glutaraldehyde solution immediately after collection and stored at 4°C until processing. The samples were post-fixed in 1 % osmium tetraoxide in Milonig\'s buffer at room temperature for one hour. They were then washed in a series of ethanol (50 -- 100 %), ten minutes at each step, followed by two additional periods of absolute ethanol (10 minutes each). The samples were further dehydrated by critical point drying at 31°C for 5 -- 10 minutes, then mounted on a specimen holder for SEM and dried overnight in vacuum desiccators. In the final stage of preparation for viewing, the samples were sputter coated with gold (Edwards, model S150B Sputter Coater). Samples were viewed using SEM (Hitachi S-2500). Images were scanned directly to a computer and stored as image files for subsequent viewing and analysis. Statistical analysis -------------------- Data are expressed as mean ± standard deviation unless otherwise stated. A paired Student-T test was used for simple comparison. The level of significance was set at 5 %. Results ======= The modified fresh frog palate exposure model was relatively easy to prepare and practical to handle. On gross examination, the surface of the palate exposed to side stream tobacco smoke did not show any macroscopic change in appearance after cigarette smoke exposure (CSE) compared to the control halves maintained in the normal chamber. One half of the palate was used as a control and the other half was exposed to cigarette smoke. Baseline mucus transport velocity(**MTV**) was measured in both half palates prior to exposure of one half palate to cigarette smoke, and were identical (19.5 ± .03 mm/sec). Two additional MTV determinations were carried out in the control half after one and four cigarettes and after 24 h period of recovery during which time the palate was kept at 4°C. A paired T-test showed no statistical difference in mucus transport velocity among the control measurements during the entire experiment (Figure [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Three MTV controls are shown in the graph, no differences were observed among them. After immediate exposure to four cigarettes, mucus transport on the palates was drastically reduced. One of the four cigarette-exposed palate and its respective half palate control were maintained overnight in a refrigerator, and it was not possible to measure a transport time the next day in the exposed palate. :::  ::: A paired T-test of MTV on the control half compared to the exposed half showed that mucus transport velocity in the exposed half palate was reduced (p \< 0.03) immediately after exposure to the side stream smoke of one cigarette compared with the non-exposed half palate. Further exposure to the side stream smoke of three more cigarettes (4 in total) significantly reduced MTV (p \< 0.001), with no signs of recovery after 24 hours. We obtained a sample of mucus from the half palate exposed to four cigarettes and used it on the control half palate to measure MTV. Clearance was within the normal range. Immediately after, the same sample of mucus was tested on the exposed half palate and mucus clearance was again seen to be very slow. Mucus transport had basically ceased in some areas of the tobacco-smoke exposed palate. Scanning electron microscopy (SEM) studies to assess the integrity of the epithelium after exposure to side stream smoke of one cigarette showed areas where the layer of cilia looked disordered. However, we did not see loss of cilia or exfoliation of ciliated epithelial cells after careful examination under the SEM in lower and high power of the entire surface of the sample after this level of exposure. In Figure [4](#F4){ref-type="fig"}, SEM images from palates exposed to the smoke of four cigarettes showed greater epithelial tissue disruption (panels 2 and 3) compared to a control palate (panel 1). Large areas of deciliated cells were observed, as well as exfoliation of intact ciliated cells. Examples of exfoliated cells found on the surface of the epithelium in the representative SEMs are indicated with black arrows. Morphometric analysis of the area of cilia loss from 3 paired palates exposed to four cigarettes, evaluating 12 different areas randomly selected in each palate, showed cilia loss of 51 ± 14 % compared to \< 2 % on control palates. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Magnification is ×400. On the left, the surface of the normal non-exposed palate is shown with a continuous ciliary layer, punctuated with secretory gland openings. The middle and right micrographs show the surface of palates exposed to the smoke of four cigarettes. :::  ::: Gelatinase zymography showed increased activity of MMP-9 in mucus collected from the palates exposed to tobacco smoke of four cigarettes compared to mucus from control palates as shown in Figure [5](#F5){ref-type="fig"}. MMP2 activity was not different in mucus samples obtained from palates exposed, or not to cigarette smoke, but these results are inconclusive. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### A representative gelatinase zymogram on mucus collected from control and cigarette-exposed palates. In the left lane, standards for MMP 2 and 9 are shown. In the next two lanes MMP activity is shown in control mucus (two samples). The next two lanes show MMP activity in two aliquots of the mucus collected from palates exposed to the smoke of four cigarettes. :::  ::: Discussion ========== Major findings in this study include: a) our amphibian mucociliary clearance model appears to be appropriate to study the early effects of tobacco smoke exposure; b) the clearance of mucus is significantly reduced after exposure to side stream tobacco smoke associated to dose response; c) ciliated cells are anatomically and physiologically acutely affected therefore drastically impairing mucus clearance. Acute irritation and inflammation of cilia due to exposure to tobacco smoke could explain this alteration; d) mucus seems to not be physiologically affected at this stage; and e) tobacco smoke triggers an increased activity of matrix metalloproteinases (in particular MMP9) associated with disruption of the extracellular matrix, most likely affecting cellular attachments to the basal membrane generating a substantial exfoliation of the ciliated cells. These findings might assist us in enhancing the knowledge of the changes in the mucociliary apparatus that occur early after exposure to environmental tobacco products, with the goal to understand how these changes relate to the development of chronic airway pathologies in humans. The frog palate has been used for several decades as a model to assess mucociliary clearance \[[@B13]-[@B17]\]. Different species of frogs have also been used, as well as a variety of study designs. Researchers had to deal with several sources of variability, making it difficult to standardize a widely acceptable model. There are disadvantages in our model such that it pertains to a non-mammalian species, in addition that the epithelial tissue is non-respiratory. However as a model it has advantages over some mammalian models like rodents in that the ciliated epithelium has a well developed mucus blanket that work in coordination with cilia similar to the human situation. This exposure model is an isolated system, theoretically free of interferences from other agents or systemic physiological responses resulting from other internal or external influences. In this injury model we randomly exposed either half palate to side stream tobacco smoke and used the opposite half palate as the control (internal control). Since there was no statistical difference in mucus transport between them, previously established in different sets of pairs of palates, one side was arbitrarily selected for control and the opposite half for exposure to tobacco smoke. This study follows an approach utilized previously by Zayas *et al*\[[@B7]\] that compared samples of mucus obtained from both mainstem bronchi in smokers and in nonsmokers. In assessing the mucus transport rate in both half palates before any experimental procedure and to insure that they functioned identically, we established baseline data for our experiment. This helped to reduce error variability due to sex, weight, age, as well as control for any seasonal effect on the mucus transport rate in the frog palates as demonstrated by Rubin *et al*\[[@B18]\]. Since the palate is excised from the frog we may assume that any observed effect or response is a local and direct effect of exposure to side stream tobacco smoke, which is an advantage of our model. From the data obtained, we would conclude that our frog palate exposure model is suitable to study the acute effects of second hand tobacco smoke. We may also conclude that clearability of mucus seems not to be altered at this stage, at this level of exposure and in this particular exposure preparation or design. Therefore, at this stage mucus transport seems not to be functionally affected for being cleared in a normal fashion by non-exposed cilia. However, after more prolonged tobacco exposure there will be modifications in mucus properties, as seen and measured in smoking dogs where after two months of tobacco exposure, the galactose content and the viscoelastic properties of mucus were observed to present alterations. After further exposure the viscosity and elasticity properties of mucus were reversed to quasi-normal levels, but galactose content did not \[[@B12]\]. Hence the model makes it very useful for differentiation of cilia-related effects versus mucus-related effects on mucociliary clearance after acute tobacco exposure. Mucus clearance is a function of cilia beat frequency and mucus viscoelastic properties. A change in mucus clearance may be due to one or the other component or both. In future studies, incorporating high speed digital imaging of the palate surface to determine cilia beat frequency will allow us to further differentiate between cilia related effects versus mucus related effects. Exposure to tobacco smoke with all its noxious agents and components will possibly allow us to separate and study in detail, the timing and appearance of different phases of the organism response. Our exposure model may allow us to individualize and study the inflammation phase resulting from the tobacco exposure. We can then focus on the injury phase occurring in the mucociliary system. Later we will explore mechanisms involved in the healing process. The remodeling of the ciliated epithelium following acute injury will be an important component of our studies and particularly how the remodeling is mediated. The time frame of the injury and recovery phases needs to be determined. Cilia do not work alone, but in association with other cilia to produce metachronal waves. Several metachronal waves may contribute to the propulsion of the mucus layer to flow over irregularities or non-ciliated areas. Our data indicates that acute tobacco exposure may have an initial and early irritation effect, possibly mediated by an unknown mechanism on the exposed palate that leads to inhibition of ciliary beat frequency or discoordination of metachronal waves. Such factors adversely affect clearance by imposing stasis or local eddies resulting in erratic clearance that may be the reason why scanning electron micrographs of palates after one cigarette showed cilia somewhat disordered, but not visibly disrupted. We have shown that tobacco smoke exposure interferes with mucociliary clearance. Sustained exposure may lead to loss of ciliated epithelium associated with activation of matrix metalloproteinases. Significant loss of cilia or ciliated epithelial cells results in disruption or even cessation of mucociliary clearance. Matrix metalloproteinases may be implicated in this injury through disruption of epithelial cell-to-cell or cell-to-basement membrane connections. Further clarification of the mechanisms involved will be undertaken in subsequent studies. Our exposure model can assess physiological, ultrastructural and molecular parameters in response to the initial deleterious effects of acute exposure to side stream tobacco smoke in an epithelial model homologous to the human airways. In these preliminary studies we did not attempt to mimic \"human smoking conditions\". However, our results show that MTV is affected even after exposure to one cigarette. Although concentration of smoke used in the present study is higher than likely encountered in typical environmental tobacco exposures, they are within an order of magnitude of those computed for exposure in poorly ventilated cars or homes. Future studies will try to replicate real conditions faced by non-smokers exposed to environmental tobacco smoke and to characterize the mediation and effectors of this acute injury. Acknowledgements ================ This study was funded by the Alberta Lung Association and the Canadian Institutes of Health Research. The authors would like to thank Dr. Ming Chen for his expert assistance with the scanning electron microscopy.

PubMed Central

2024-06-05T03:55:47.701491

2004-8-20

PMC517706

Background ========== Multiple alignments of protein sequences are important in many applications, including phylogenetic tree estimation, secondary structure prediction and critical residue identification. Many multiple sequence alignment (MSA) algorithms have been proposed; for a recent review, see \[[@B1]\]. Two attributes of MSA programs are of primary importance to the user: biological accuracy and computational complexity (i.e., time and memory requirements). Complexity is of increasing relevance due to the rapid growth of sequence databases, which now contain enough representatives of larger protein families to exceed the capacity of most current programs. Obtaining biologically accurate alignments is also a challenge, as the best methods sometimes fail to align readily apparent conserved motifs \[[@B2]\]. We recently introduced MUSCLE, a new MSA program that provides significant improvements in both accuracy and speed, giving only a summary of the algorithm \[[@B2]\]. Here, we describe the MUSCLE algorithm more fully and analyze its complexity. We introduce a new option designed for high-throughput applications, MUSCLE-fast. We also describe a new method for evaluating objective functions for profile-profile alignment, the iterated step in the MUSCLE algorithm. Current methods --------------- While multiple alignment and phylogenetic tree reconstruction have traditionally been considered separately, the most natural formulation of the computational problem is to define a model of sequence evolution that assigns probabilities to all possible elementary sequence edits and then to seek an optimal directed graph in which edges represents edits and terminal nodes are the observed sequences. This graph makes the history explicit (it can be interpreted as a phylogenetic tree) and implies an alignment. No tractable method for finding an optimal graph is known for biologically realistic models, and simplification is therefore required. A common heuristic is to seek a multiple alignment that maximizes the SP score (the summed alignment score of each sequence pair), which is NP complete \[[@B3]\]. It can be achieved by dynamic programming with time and space complexity O(*L*^*N*^) in the sequence length *L*and number of sequences *N*\[[@B4]\], and is practical only for very small *N*. Stochastic methods such as Gibbs sampling can be used to search for a maximum objective score \[[@B5]\], but have not been widely adopted. A more popular strategy is the progressive method \[[@B6],[@B7]\] (Figure [1](#F1){ref-type="fig"}), which first estimates a phylogenetic tree. A profile (a multiple alignment treated as a sequence by regarding each column as a symbol) is then constructed for each node in the binary tree. If the node is a leaf, the profile is the corresponding sequence; otherwise its profile is produced by a pair-wise alignment of the profiles of its child nodes (Figure [2](#F2){ref-type="fig"}). Current progressive algorithms are typically practical for up to a few hundred sequences on desktop computers, the best-known of which is CLUSTALW \[[@B8]\]. A variant of the progressive approach is used by T-Coffee \[[@B9]\], which builds a library of both local and global alignments of every pair of sequences and uses a library-based score for aligning two profiles. On the BAliBASE benchmark \[[@B10],[@B11]\], T-Coffee achieves the best results reported prior to MUSCLE, but has a high time and space complexity that limits the number of sequences it can align to typically around one hundred. In our experience, errors in progressive alignments can often be attributed to one of the following issues: sub-optimal branching order in the tree, scoring parameters that are not optimal for a particular set of sequences (especially gap penalties), and inappropriate boundary conditions (e.g., seeking a global alignment of proteins having different domain organizations). Misalignments are sometimes readily apparent, motivating further processing (*refinement*). One approach is to use a progressive alignment as the initial state of a stochastic search for a maximum objective score (*stochastic refinement*). Alternatively, pairs of profiles can be extracted from the progressive alignment and re-aligned, keeping the results only when an objective score is improved (*horizontal refinement*) \[[@B12]\]. Implementation ============== The basic strategy used by MUSCLE is similar to that used by PRRP \[[@B13]\] and MAFFT \[[@B14]\]. A progressive alignment is built, to which horizontal refinement is then applied. Algorithm overview ------------------ MUSCLE has three stages. At the completion of each stage, a multiple alignment is available and the algorithm can be terminated. Stage 1: draft progressive -------------------------- The first stage builds a progressive alignment. ### Similarity measure The similarity of each pair of sequences is computed, either using *k*-mer counting or by constructing a global alignment of the pair and determining the fractional identity. ### Distance estimate A triangular distance matrix is computed from the pair-wise similarities. ### Tree construction A tree is constructed from the distance matrix using UPGMA or neighbor-joining, and a root is identified. ### Progressive alignment A progressive alignment is built by following the branching order of the tree, yielding a multiple alignment of all input sequences at the root. Stage 2: improved progressive ----------------------------- The second stage attempts to improve the tree and builds a new progressive alignment according to this tree. This stage may be iterated. ### Similarity measure The similarity of each pair of sequences is computed using fractional identity computed from their mutual alignment in the current multiple alignment. ### Tree construction A tree is constructed by computing a Kimura distance matrix and applying a clustering method to this matrix. ### Tree comparison The previous and new trees are compared, identifying the set of internal nodes for which the branching order has changed. If Stage 2 has executed more than once, and the number of changed nodes has not decreased, the process of improving the tree is considered to have converged and iteration terminates. ### Progressive alignment A new progressive alignment is built. The existing alignment is retained of each subtree for which the branching order is unchanged; new alignments are created for the (possibly empty) set of changed nodes. When the alignment at the root is completed, the algorithm may terminate, return to step 2.1 or go to Stage 3. Stage 3: refinement ------------------- The third stage performs iterative refinement using a variant of tree-dependent restricted partitioning \[[@B12]\]. ### Choice of bipartition An edge is deleted from the tree, dividing the sequences into two disjoint subsets (a bipartition). Edges are visiting in order of decreasing distance from the root. ### Profile extraction The profile (multiple alignment) of each subset is extracted from the current multiple alignment. Columns containing no residues (i.e., indels only) are discarded. ### Re-alignment The two profiles obtained in step 3.2 are re-aligned to each other using profile-profile alignment. ### Accept/reject The SP score of the multiple alignment implied by the new profile-profile alignment is computed. If the score increases, the new alignment is retained, otherwise it is discarded. If all edges have been visited without a change being retained, or if a user-defined maximum number of iterations has been reached, the algorithm is terminated, otherwise it returns to step 3.1. Visiting edges in order of decreasing distance from the root has the effect of first re-aligning individual sequences, then closely related groups Algorithm elements ------------------ In the following, we describe the elements of the MUSCLE algorithm. In several cases, alternative versions of these elements were implemented in order to investigate their relative performance and to offer different trade-offs between accuracy, speed and memory use. Most of these alternatives are made available to the user via command-line options. Four benchmark datasets have been used to evaluate options and parameters in MUSCLE: BAliBASE \[[@B10],[@B11]\], SABmark \[[@B15]\], SMART \[[@B16]-[@B18]\] and our own benchmark, PREFAB \[[@B2]\]. Objective score --------------- In its refinement stage, MUSCLE seeks to maximize an objective score, i.e. a function that maps a multiple sequence alignment to a real number which is designed to give larger values to better alignments. MUSCLE uses the *sum-of-pairs*(SP) score, defined to be the sum over pairs of sequences of their alignment scores. The alignment score of a pair of sequences is computed as the sum of substitution matrix scores for each aligned pair of residues, plus gap penalties. Gaps require special consideration (Figure [3](#F3){ref-type="fig"}). We use the term *indel*for the symbol that indicates a gap in a column (typically a dash \'-\'), reserving the term *gap*for a maximal contiguous series of indels. The gap penalty contribution to SP for a pair of sequences is computed by discarding all columns in which both sequences have an indel, then applying an affine penalty *g*+ *λe*for each remaining gap where *g*is the per-gap penalty, *λ*is the gap length (number of indels in the gap), and *e*is the gap-length penalty (sometimes called the extension penalty). Progressive alignment --------------------- Progressive alignment requires a rooted binary tree in which each sequence is assigned to a leaf. The tree is created by clustering a triangular matrix containing a distance measure for each pair of sequences. The branching order of the tree is followed in postfix order (i.e., children are visited before their parent). At each internal node, profile-profile alignment is used to align the existing alignments of the two child subtrees, and the new alignment is assigned to that node. A multiple alignment of all input sequences is produced at the root node (Figure [1](#F1){ref-type="fig"}). Similarity measures ------------------- We use the term *similarity*for a measure on a pair of sequences that indicates their degree of evolutionary divergence (the sequences are assumed to be related). MUSCLE uses two types of similarity measure: the fractional identity *D*computed from a global alignment of the two sequences, and measures obtained by *k*-mer counting. A *k*-mer is a contiguous subsequence of length *k*, also known as a word or *k*-tuple. Related sequences tend to have more *k*-mers in common than expected by chance, provided that *k*is not too large and the divergence is not too great. Many sequence comparison methods based on *k*-mer counting have been proposed in the literature; for a review, see \[[@B19]\]. The primary motivation for these measures is improved speed as no alignment is required. MAFFT uses *k*-mer counting in a compressed alphabet (i.e., an alphabet in which symbols denote classes that may contain two or more residue types) to compute its initial distance measure. The alphabet used in MAFFT is taken from \[[@B20]\], and is one of the options implemented in MUSCLE. Trivially, identity is higher or equal in a compressed alphabet; it cannot be reduced. If the alphabet is chosen such that there are high probabilities of intra-class substitution and low probabilities of inter-class substitution, then we might expect that detectable identity (and hence the number of conserved *k*-mers) could be usefully extended to greater evolutionary distances while limiting the increase in matches due to chance. We have previously shown \[[@B21]\] that *k*-mer similarities correlate well with fractional identity, although we failed to find evidence that compressed alphabets have superior performance to the standard alphabet at lower identities. We define the following similarity measure between sequences X and Y: *F*= Σ~*τ*~min \[*n*~X~(*τ*), *n*~Y~(*τ*) \] / \[min (*L*~X~, *L*~Y~) - *k*+ 1 \].     (1) Here *τ*is a *k*-mer, *L*~X~, *L*~Y~are the sequence lengths, and *n*~X~(*τ*) and *n*~Y~(*τ*) are the number of times *τ*occurs in X and Y respectively. This definition can be motivated by considering an alignment of X to Y and defining the similarity to be the fraction of *k*-mers that are conserved between the two sequences. The denominator of *F*is the maximum number of *k*-mers that could be aligned. Note that if a given *k*-mer occurs more often in one sequence than the other, the excess cannot be conserved, hence the minimum in the numerator. The definition of *F*is an approximation in which it is assumed that (after correcting for excesses) common *k*-mers are always alignable to each other. MUSCLE also implements a binary approximation *F*^Binary^, so-called because it reduces the *k*-mer count to a present / absent bit: *F*^Binary^= Σ~*τ*~*δ*~XY~(*τ*) / \[min (*L*~X~, *L*~Y~) - *k*+ 1 \].     (2) Here, *δ*~XY~(*τ*) is 1 if *τ*is present in both sequences, 0 otherwise. As multiple instances of a given *k*-mer in one sequence are relatively rare, this is often a good approximation to *F*. The binary approximation enables a significant speed improvement as the size of the count vector for a given sequence can be reduced by an order of magnitude. This allows the count vector for every sequence to be retained in memory, and pairs of vectors to be compared efficiently using bit-wise instructions. When using an integer count, there may be insufficient memory to store all count vectors, making it necessary to re-compute counts several times for a given sequence. Distance measures ----------------- Given a similarity value, we wish to estimate an additive distance measure. An additive measure distance measure d(A, B) between two sequences A and B satisfies d(A, B) = d(A, C) + d(C, B) for any third sequence C, assuming that A, B and C are all related. Ideal but generally unknowable is the *mutation distance*, i.e. the number of mutations that occurred on the historical path between the sequences. The historical path through the phylogenetic tree extends from one sequence to the other via their most recent common ancestor. The mutation distance is trivially additive. The fractional identity *D*is often used as a similarity measure; for closely related sequences 1 - *D*is a good approximation to a mutation distance (it is exact assuming substitution at a single site to be the only allowed type of mutation and that no position mutates more than once). As sequences diverge, there is an increasing probability of multiple mutations at a single site. To correct for this, we use the following distance estimate \[[@B22]\]: *d*~Kimura~= -log~e~(1 - *D*- *D*^2^/5)     (3) For *D*≤ 0.25 we use a lookup table taken from the CLUSTALW source code. For *k*-mer measures, we use: *d*~kmer~= 1 - *F*.     (4) Tree construction ----------------- Given a distance matrix, a binary tree is constructed by clustering. Two methods are implemented: neighbor-joining \[[@B23]\], and UPGMA \[[@B24]\]. MUSCLE implements three variants of UPGMA that differ in their assignment of distances to a new cluster. Consider two clusters (subtrees) *L*and *R*to be merged into a new cluster *P*, which becomes the parent of *L*and *R*in the binary tree. Average linkage assigns this distance to a third cluster *C*: *d*^Avg^~*PC*~= (*d*~*LC*~+ *d*~*RC*~)/2.     (5) We can take the minimum rather than the average: *d*^Min^~*PC*~= min \[*d*~*LC*~, *d*~*RC*~\].     (6) Following MAFFT, we also implemented a weighted mixture of minimum and average linkage: *d*^Mix^~*PC*~= (1 - *s*) *d*^Min^~*PC*~+ *s d*^Avg^~*PC*~,     (7) where *s*is a parameter set to 0.1 by default. Clustering produces a pseudo-root (the last node created). We implemented two other methods for determining a root: minimizing the average branch weight \[[@B25]\], as used by CLUSTALW, and locating the root at the center of the longest span. Sequence weighting ------------------ Conventional wisdom holds that sequences should be weighted to correct for the effects of biased sampling from a family of related proteins; however, there is no consensus on how such weights should be computed. MUSCLE implements the following sequence weighting schemes: none (all sequences have equal weight), Henikoff \[[@B26]\], PSI-BLAST \[[@B27]\] (a variant of Henikoff), CLUSTALW\'s, GSC \[[@B28]\], and the three-way method \[[@B29]\]. We found the use of weighting to give a small improvement in benchmark accuracy results, e.g. approximately 1% on BAliBASE, but saw little difference between the alternative schemes. The CLUSTALW method enables a significant reduction in complexity (described later), and is therefore the default choice. Profile functions ----------------- In order to apply pair-wise alignment methods to profiles, a scoring function must be defined for a pair of profile positions, i.e. a pair of multiple alignment columns. This function is the profile analog of a substitution matrix; see for example \[[@B30]\]. We use the following notation. Let *i*and *j*be amino acid types, *p*~*i*~the background probability of *i*, *p*~*ij*~the joint probability of *i*and *j*being aligned to each other, *S*~*ij*~the substitution matrix score, *f*^*x*^~*i*~the observed frequency of *i*in column *x*of the first profile, *f*^*x*^~G~the observed frequency of gaps in that column, and *α*^*x*^~*i*~the estimated probability of observing *i*in position *x*in the family. (Similarly for position *y*in the second profile). Estimated probabilities *α*are derived from the observed frequencies *f*, typically by adding heuristic pseudo-counts or by using Bayesian methods such as Dirichlet mixture priors \[[@B31]\]. A commonly used profile function is the sequence-weighted sum of substitution matrix scores for each pair of letters, selecting one from each column (PSP, for profile SP): PSP^*xy*^= Σ~*i*~Σ~*j*~*f*^*x*^~*i*~*f*^*y*^~*j*~*S*~*ij*~.     (8) Note that *S*~*ij*~= log (*p*~*ij*~/ *p*~*i*~*p*~*j*~) \[[@B32]\], so PSP^*xy*^= Σ~*i*~Σ~*j*~*f*^*x*^~*i*~*f*^*y*^~*j*~log (*p*~*ij*~/ *p*~*i*~*p*~*j*~).     (9) PSP is the function used by CLUSTALW and MAFFT. It is a natural choice when attempting to maximize the SP objective score: if gap penalties are neglected, maximizing PSP maximizes SP under the constraint that columns in each profile are preserved. (This follows from the observation that the contribution to SP from a pair of sequences in the same profile is the same for all alignments allowed under the constraint). MUSCLE implements PSP functions based on the 200 PAM matrix of \[[@B33]\] and the 240 PAM VTML matrix \[[@B34]\]. In addition to PSP, MUSCLE implements a function we call the *log-expectation*(LE) score. LE is a modified version of the log-average (LA) profile function that was proposed on theoretical grounds \[[@B35]\]: LA^*xy*^= log Σ~*i*~Σ~*j*~*α*^*x*^~*i*~*α*^*y*^~*j*~*p*~*ij*~/ *p*~*i*~*p*~*j*~.     (10) LE is defined as follows: LE^*xy*^= (1 - *f*^*x*^~*G*~) (1 - *f*^*y*^~G~) log Σ~*i*~Σ ~*j*~*f*^*x*^~*i*~*f*^*y*^~*j*~*p*~*ij*~/ *p*~*i*~*p*~*j*~.     (11) The MUSCLE LE function uses probabilities computed from VTML 240. Note that estimated probabilities *α*in LA are replaced by observed frequencies *f*in LE. The factor (1 - *f*~G~) is the *occupancy*of a column. Frequencies *f*~*i*~must be normalized to sum to one if indels are present (otherwise the logarithm becomes increasingly negative with increasing numbers of gaps even when aligning conserved or similar residues). The occupancy factors are introduced to encourage more highly occupied columns (i.e., those with fewer gaps) to align, and are found to significantly improve accuracy. We avoid these complications in the PSP score by computing frequencies in a 21-letter alphabet (amino acids + indel), and by defining the substitution score of an amino acid to an indel to be zero. This has the desired effect of down-weighting column pairs with low occupancies, and can also be motivated by consideration of the SP function. If gap penalties are ignored, then this definition of PSP preserves the optimization of SP under the fixed-column constraint by correctly accounting for the reduced number of residue pairs in columns containing gaps. Gap penalties ------------- We call the first indel in a gap its *gap-open*; the last its *gap-close*. Consider an alignment of two profiles X and Y, and a gap of length *λ*in X in which the gap-open is aligned to position *y*~*o*~in Y and the gap-close to *y*~*c*~. The penalty for this gap is *b*(*y*~*o*~) + *t*(*y*~*c*~) + *λe*, where *b*and *t*are costs for opening and closing a gap that vary according to the position in Y, and *e*is a length cost (sometimes called a gap extension penalty) that does not vary by position. A fixed length cost allows a minor optimization of the scoring scheme \[[@B14]\]. Consider a global alignment of sequences X and Y having lengths *L*~X~and *L*~Y~. If a constant *δ*(the *center*) is added to each substitution matrix score and *δ*/2 is added for each gapped position, this adds the constant value *δ*(*L*~X~+ *L*~Y~)/2 to the score of any possible alignment, and the set of optimal alignments is therefore unchanged. Given a scoring scheme with substitution matrix *S*~*ij*~and extension penalty *e*, we can thus choose *δ*/2 = *e*and instead use *S\'*~*ij*~= *S*~*ij*~+ 2*e*and *e\'*= 0 to obtain the same alignment. The constant 2*e*can be added to the substitution matrix at compile time, and no explicit extension penalty is then needed in the recursion relations. MUSCLE uses this optimization for the PSP function, but not for LE (where the center must be added at execution time after taking the logarithm). Let *f*^*y*^~*o*~be the number of gap-opens in column *y*in Y and *f*^*y*^~*c*~be the number of gap-closes in column *y*. MUSCLE computes *b*and *t*as follows (Figure [4](#F4){ref-type="fig"}): *b*(*y*) = *g*/2 (1 - *f*^*y*^~*o*~) (1 + *h*~*w*~(*y*)*H*),     (12) *t*(*y*) = *g*/2 (1 - *f*^*y*^~*c*~) (1 + *h*~*w*~(*y*)*H*).     (13) Here, *g*is a parameter that can be considered a default per-gap penalty, *h*~*w*~(*y*) is 1 if *y*falls within a window of *w*consecutive hydrophobic residues or zero otherwise, and *H*is a tunable parameter. By default, *w*= 5, *H*= 1.2. The factor *g*/2 (1 - *f*^*y*^~*o*~) is motivated by considering the SP score of the alignment. The gap penalty contribution to SP for a pair of sequences (*A*∈ Y, *B*∈ X) is computed by discarding all columns in which both sequences have an indel, then applying an affine penalty *g*+ *λe*for each remaining gap. It is convenient here to consider that half of the per-gap penalty *g*is applied to the open position and half to the close position. Suppose a gap *G*is inserted into X such that the gap-open is aligned to position *y*in Y. If a sequence *s*∈ Y has a gap-open at *y*, then the SP score includes no open penalty for *G*induced by any pair (*s*, *t*) : *t*∈ X. The multiplier (1 - *f*^*y*^~*o*~) therefore corrects the gap-open contribution to the SP score due to pre-existing gaps in Y. (It should be noted that even with this correction, there are other issues related to gaps and PSP still does not exactly optimize SP under the fixed-column constraint). The increased penalty in hydrophobic windows is designed to discourage gaps in buried core regions where insertions and deletions are less frequent. Note that MUSCLE treats open and close positions symmetrically, in contrast to CLUSTALW, which treats the open position specially and may therefore tend to produce, in word processing terms, left-aligned gaps with a ragged right margin. Terminal gaps ------------- A *terminal gap*is one that opens at the N-terminal position of the sequence to which it is aligned or closes at the C-terminal; as opposed to an *internal gap*. It has been suggested \[[@B9],[@B36]\] that global methods have intrinsic difficulties with long deletions or insertions. We believe that these difficulties are often due to the choice of penalties for terminal gaps. CLUSTALW, which charges no penalty for terminal gaps, tends to fail to open a needed internal gap and thus fail to align terminal motifs; MAFFT, which charges the same penalty for terminal and internal gaps, sometimes aligns small numbers of residues to a terminal by inserting an unnatural internal gap. By default, MUSCLE penalizes terminal gaps with half the penalty of an internal gap. This is done by setting *b*(1), the open penalty at the C-terminal, and *t*(*L*), the close penalty at the N-terminal, to zero (Figure [4](#F4){ref-type="fig"}). The option of always applying full penalties, as in MAFFT, is also provided. We found that the compromise of a half penalty for terminal gaps gave good results for a wide range of input data, but that further improvements could sometimes by achieved by the following technique. If the length ratio of the two profiles to be aligned exceeds a threshold (by default, 20%), then MUSCLE constructs four different alignments in which gaps at both, one or neither terminals are fully penalized. A *conservation score*is defined by subtracting all gap penalties (both internal and terminal) from the alignment score, leaving a sum over profile functions only. The alignment with the highest conservation score is used. Tree comparison --------------- In progressive alignment, two subtrees will produce identical alignments if they have the same set of sequences at their leaves and the same branching orders (topologies). We exploit this observation to optimize the progressive alignment in Stage 2 of MUSCLE, which begins by constructing a new tree. Unchanged subtrees are identified, and their alignments are retained (Figure [5](#F5){ref-type="fig"}). A progressive alignment of the changed subtrees is constructed, producing the same alignment at the root that would be obtained starting from the leaves. Tree comparison is performed by the following algorithm. Consider two trees *A*and *B*with identical sets of *N*leaves. Leaves are identified by consecutive integers (*ids*) 1 through *N*. Call a pair of nodes, one from each tree, equivalent if they are the same leaf or they are internal nodes and their children are equivalent. The left/right position of a child is not considered; in other words, subtree rotations are allowed (because they do not change the results of a progressive alignment). Traverse *A*in prefix order (children before their parent), assigning internal nodes ids *N*+ 1 through 2*N*in the order visited. When visiting an internal node *P*^*A*^, take the ids of its two child nodes *L*^*A*^and *R*^*A*^and use them as indexes into a lookup table pointing to nodes in *B*. If (a) *L*^*A*^is equivalent to a node *L*^*B*^in *B*and *R*^*A*^is equivalent to a node *R*^*B*^, and (b) *L*^*B*^and *R*^*B*^have the same parent *P*^*B*^, then assign *P*^*B*^the same id as *P*^*A*^, to which it is equivalent. When the traversal is complete, a node *b*in *B*is equivalent to some node in *A*if and only if *b*has an id. This procedure is O(*N*) time and space. Defaults, optimizations and complexity analysis ----------------------------------------------- We now discuss the default choices of algorithm elements in the MUSCLE program and analyze their complexity. Complexity of CLUSTALW ---------------------- It is instructive to consider the complexity of CLUSTALW. This is of intrinsic interest as CLUSTALW is currently the most widely used MSA program and, to the best of our knowledge, its complexity has not previously been stated correctly in the literature. It is also useful as a baseline for motivating some of the optimizations used in MUSCLE. The CLUSTALW algorithm can be described by the same steps as Stage 1 above. The similarity measure is the fractional identity computed from a global alignment, clustering is done by neighbor-joining. Global alignment of a pair of sequences or profiles is computed using the Myers-Miller linear space algorithm \[[@B37]\] which is O(*L*) space and O(*L*^2^) time in the typical sequence length *L*. Given *N*sequences and thus *N*(*N*- 1)/2 = O(*N*^2^) pairs, it is therefore O(*N*^2^*L*^2^) time and O(*N*^2^+ *L*) space to construct the distance matrix. The neighbor-joining implementation is O(*N*^2^) space and O(*N*^4^) time, at least up to CLUSTALW 1.82, although O(*N*^3^) time is possible; see e.g. \[[@B38]\]. A single iteration of progressive alignment computes a profile of each subtree from its multiple alignment, which is O(*N*~P~*L*~P~) time and space in the number of sequences in the profile *N*~P~and the profile length *L*~P~, then uses Myers-Miller to align the profiles in O(*L*~P~) space and O(*L*~P~^2^) time. There are *N*- 1 internal nodes in a rooted binary tree and hence O(*N*) iterations. It is often assumed that *L*~P~is O(*L*), i.e. that O(0) gaps are introduced in each iteration. However, we often observe the alignment length to grow approximately linearly, i.e. that O(1) gaps are added per iteration. For example, taking the average over all iterations in all alignments in version 3 of the PREFAB benchmark, Stage 1 of MUSCLE adds 2.8 gaps per iteration to the longer profile. It is therefore more realistic to assume that *L*~P~is O(*L*+ *N*), making one iteration of progressive alignment O(*NL*+ *L*^2^) in both space and time. This analysis is summarized in Table [1](#T1){ref-type="table"}. Initial distance measure ------------------------ One might expect (a) that a more accurate distance measure would lead to a more accurate final alignment due to an improved tree, and (b) that errors due to a less accurate distance measure might be eliminated by allowing Stage 2 to iterate more times. Neither of these expectations is supported by our test results (unpublished). Allowing Stage 2 to iterate more than once with the goal of further improving the tree gave no significant improvement with any distance measure. Possibly, the tree is biased towards the MSA that was used to estimate it, and the MSA is biased by the tree used to create it, making it hard to achieve improvements. The most accurate measure on a pair of sequences is presumably the fractional identity *D*computed from a global alignment, but use of *D*in step 1.1 does not improve average accuracy on benchmark tests. The 6-class Dayhoff alphabet used by MAFFT proved to give slightly higher benchmark accuracy scores, despite the fact that other alphabets were found to correlate better with *D*\[[@B21]\]. We also found that the use of the binary approximation *F*^Binary^gave slightly reduced accuracy scores even when Stage 2 was allowed to iterate. The default choice in MUSCLE is therefore to use the Dayhoff alphabet in step 1.1 and to execute Stage 2 once only. While the impact on the average accuracy of the final alignment due to the different options is not understood, we observe that a better alignment of a pair of sequences is often obtained from a multiple alignment than from a pair-wise alignment, due to the presence of intermediate sequences having higher identities. It is therefore plausible that *D*obtained from the multiple alignment in step 2.1 may be more accurate than *D*obtained from a pair-wise alignment in step 1.1, and this may be relatively insensitive to the method used to create the tree for Stage 1. But this leaves unexplained why *k*-mer counting appears to be as good as or better than *D*in Stage 1. Computing *F*from a pair of sequences is O(*L*) time and O(1) space, so for all pairs the similarity calculation is O(*N*^2^*L*), compared with O(*N*^2^*L*^2^) in CLUSTALW. For a typical *L*around 250, combined with an order of magnitude improvement due to the simplicity of *k*-mer counting compared with dynamic programming, this typically gives a three orders of magnitude speed improvement for computing the distance matrix in MUSCLE compared with CLUSTALW. The default strategy is therefore well justified as a speed optimization, and has the added bonus of providing a small improvement in accuracy. Clustering ---------- MUSCLE implements both UPGMA and neighbor-joining. We found UPGMA to give slightly better benchmark scores than neighbor-joining; UPGMA is therefore the default option. We expect neighbor-joining to give a better estimate of the correct evolutionary tree (see e.g. \[[@B38]\]). However, it is well-known that alignment accuracy decreases with lower sequence identity (see e.g. \[[@B39]\]). It follows that given a set of profiles, the two that can be aligned most accurately will tend to be the pair with the highest identity, i.e. at the shortest evolutionary distance. This is exactly the pair selected by the nearest-neighbor criterion in UPGMA. By contrast, neighbor-joining selects a pair of evolutionary neighbors, i.e. a pair having a common ancestor. When mutation rates are variable, the evolutionary neighbor may not be the nearest neighbor (Figure [6](#F6){ref-type="fig"}). This explains why a nearest-neighbor tree may be superior to the true evolutionary tree for guiding a progressive alignment. Neighbor-joining is naively O(*N*^4^) time, although this can be reduced to O(*N*^3^). UPGMA is naively O(*N*^3^) time as the minimum of an *N*^2^matrix must be found in each of *N*- 1 iterations. However, this can be reduced to O(*N*^2^) time by maintaining a vector of pointers to the minimum value in each row of the matrix. We are again fortunate to find that the most accurate method is also the fastest. Dynamic programming ------------------- The textbook algorithm for pair-wise alignment with affine penalties employs three dynamic programming matrices; see e.g. \[[@B40],[@B41]\]. A more time-and space-efficient implementation can be achieved using linear space for the recursion relations and a single matrix for trace-back (Kazutaka Katoh, personal communication). Consider sequences X and Y length *L*~X~, *L*~Y~. We use the following notation: X~*x*~is the *x*th letter in X, X^*x*^the first *x*letters in X, *S*~*xy*~the substitution score (or profile function) for aligning X~*x*~to Y~*y*~, *b*^X^~*x*~the score for a gap-open in Y that is aligned to X~*x*~, *t*^X^~*x*~the score for a gap-close aligned to X~*x*~, *U*~*xy*~the set of all alignments of X^*x*^to Y^*y*^, M~*xy*~the score of the best alignment in *U*~*xy*~ending in a match (i.e., X~*x*~and Y~*y*~are aligned), D~*xy*~the score of the best alignment ending in a delete relative to X (X~*x*~is aligned to an indel) and I~*xy*~the score of the best alignment ending in an insert (Y~*y*~is aligned to an indel). A match is preceded by either a match, delete or insert, so: M~*xy*~= *S*~*xy*~+ max { M~*x*-1*y*-1~, D~*x*-1*y*-1~+ *t*^X^~*x*-1~, I~*x*-1*y*-1~+ *t*^Y^~*y*-1~}     (14) We assume that a center parameter has been added to *S*~*xy*~such that the gap extension penalty is zero. By considering all possible lengths for the final gap, D~*xy*~= max(*k*\<*x*) \[M~*ky*~+ *b*^X^~*k*+1~\].     (15) Here, *k*is the last position in X that is aligned to a letter in Y. Extract the special case of a gap of length 1: D~*xy*~= max { max(*k*\<*x*-1) \[M~*ky*~+ *b*^X^~*k*+1~\], M~*x*-1*y*~+ *b*^X^~*x*~}.     (16) Hence, D~*xy*~= max { D~*x*-1*y*~, M~*x*-1*y*~+ *b*^X^~*x*~}.     (17) Similarly, I~*xy*~= max { I~*xy*-1~, M~*xy*-1~+ *b*^Y^~*y*~}.     (18) Let the outer loop iterate over increasing *x*and the inner loop over increasing *y*. For fixed *x*, define vectors M^*curr*^~*y*~= M~*xy*~, M^*prev*^~*y*~= M~*x*-1*y*~, D^*curr*^~*x*~= D~*xy*~, D^*prev*^~*x*~= D~*x*-1*y*~; for fixed *x*, *y*define scalars I^*curr*^= I~*xy*~, I^*prev*^= I~*xy*-1~. Now we can re-write (14), (17) and (18) to obtain the following recursion relations: M^*curr*^~*y*~= S~*xy*~+ max { M^*prev*^~*y*-1~, D^*prev*^~*y*-1~+ *t*^X^~*x*-1~, I^*prev*^~*y*-1~+ *t*^Y^~*y*-1~}     (19) D^*curr*^~*y*~= max { D^*prev*^~*y*~, M^*prev*^~*y*~+ *b*^X^~*x*~}     (20) I^*curr*^= max { I^*prev*^, M^*prev*^~*y*~+ *b*^Y^~*y*~}.     (21) An *L*~X~× *L*~Y~matrix is needed for the trace-back that produces the final alignment. ### Inner loop The inner-most dynamic programming loop, which computes the profile function, deserves careful optimization. We will consider the case of PSP; similar optimizations are possible for LE. PSP = Σ~*i*~Σ~*j*~*f*^*x*^~*i*~*f*^*y*^~*j*~*S*~*ij*~= Σ~*i*~*f*^*x*^~*i*~*W*^*y*^~*i*~, where *W*^*y*^~*i*~= Σ~*j*~*f*^*y*^~*j*~*S*~*ij*~. The vector *W*^*y*^~*i*~is used *L*~X~times, and it therefore pays to compute it once and cache it. Observe that a typical profile column contains \<\< 20 different amino acids. We sort the frequencies in decreasing order; the summation Σ~*i*~*f*^*x*^~*i*~*W*^*y*^~*i*~is terminated if a frequency *f*^*x*^~*i*~= 0 is encountered. This typically reduces the time spent in the summation, especially when sequences are closely related. As with *W*^*y*^~*i*~, the sort order is computed once and cached. Observe that the roles of the two profiles are not symmetrical. It is most efficient to choose X, for which frequency sort orders are computed, to be the profile with the lowest amino acid diversity when averaged over columns. With this choice, the summation terminates earlier on average then if the other profile is identified as X. Note that out of *N*- 1 iterations of progressive alignment, a minimum of  and maximum of *N*- 1 profile-profile alignments will include at least one profile containing one sequence only, and in the refinement phase exactly *N*of the 2*N*- 1 edges in the tree terminate on a leaf. At least half of all profile-profile alignments created in the MUSCLE algorithm therefore include a profile of one sequence only. Special cases where one or both profiles is a single sequence can be handled in separate subroutines, saving overhead due to unneeded loops that are guaranteed to execute once only. This optimization is especially useful for the LE function as it enables the logarithm to be incorporated into the *W*vector. Diagonal finding ---------------- Many alignment algorithms are optimized for speed, typically at some expense in average accuracy, by using fast methods to identify regions of high similarity between two sequences, which appear as diagonals in the similarity matrix. The alignment path is then constrained to include these diagonals, reducing the area of the dynamic programming matrix that must be computed. MAFFT uses the fast Fourier transform to find diagonals. MUSCLE uses a different technique which we have previously shown \[[@B21]\] have comparable sensitivity and to be significantly faster. We use a compressed alphabet to find *k*-mers in common between two sequences, then attempt to extend the match. In the case of diagonal identification we found compressed alphabets to significantly out-perform the standard amino acid alphabet \[[@B21]\]. Currently, MUSCLE uses 6-mers in the Dayhoff alphabet for diagonal finding, as for the initial distance measure, though other alphabets are known to give slightly better performance \[[@B21]\]. A candidate diagonal is rejected if there is any overlap (i.e., if a single position in one of the sequences appears in two or more diagonals) or if it is less than a minimum length (default 24). The ends of the diagonal are deleted (by default, the first and last five positions) as they are less reliable. Despite these heuristics, we find the use of diagonal-finding to reduce average accuracy and to give only modest improvements in speed for typical input data; this option is therefore disabled by default. Similar results are seen in MAFFT; the most accurate MAFFT script is NWNSI \[[@B14]\], in which diagonal-finding is also disabled. Additive profiles ----------------- Both the PSP and LE profile functions are defined in terms of amino acid frequencies and position-specific gap penalties. The data structure representing a profile is a vector of length *L*~P~in which each element contains frequencies for each amino acid type and a few additional values related to gaps. We call this data structure a *profile vector*, as distinct from a *profile matrix*, an explicit *N*× *L*~P~multiple alignment containing letters and indels. For *N*\> 20, using profile vectors reduces the cost of computing the profile function compared with profile matrices, and is therefore preferred for use in dynamic programming. In CLUSTALW and MAFFT, the implementation of progressive alignment builds a profile matrix at each internal node of the tree, which is used to compute a profile vector. This procedure is O(*NL*~P~) = O(*N*^2^+ *NL*) in time and space, becoming expensive for large *N*. Observe that the count of a given amino acid in a column in the parent matrix is the sum of the counts in the two child columns that are aligned at that position (Figure [7](#F7){ref-type="fig"}). With a suitable sequence weighting scheme, it is therefore possible to compute the amino acid frequencies of the parent profile vector from the frequencies in the two child profile vectors and the alignment path. This is an O(*L*~P~) procedure in both time and space, giving a significant advantage for *N*\>\> 20. Three issues must be addressed to fully implement this idea: the sequence weighting scheme, inclusion of occupancy factors and position-specific gap penalties, and construction of a profile matrix (i.e., the final multiple alignment) at the root node. ### Sequence weighting For the frequencies in the parent profile vector to be a linear combination of the child frequencies, the weight assigned to a sequence must be the same in the child and parent profiles. This requirement is not satisfied, for example, by the Henikoff or PSI-BLAST schemes, which compute weights based on a multiple alignment. We therefore choose the CLUSTALW scheme, which computes a fixed weight for each sequence from edge lengths in the tree. ### Gap representation To compute gap penalties, we need the frequencies *f*~*o*~of gap opens and *f*~*c*~of gap closes in each position. In the case of the LE profile function, we additionally require the gap frequency *f*~G~. These can be accommodated by storing *f*~*o*~, *f*~*c*~and *f*~*e*~in the profile vector, where *f*~*e*~is the frequency of gap-extensions in the column (meaning that indels are found in a given sequence in the column, the preceding column and in the following column; i.e., a gap-close is not counted as an extension). These three *occupancy frequencies*are sufficient for computing the profile function and the position-specific gap penalties *b*and *t*. Note that we can compute the frequency *f*~G~of indels, as needed for the occupancy factor in the profile function, as follows: *f*~G~= *f*~*o*~+ *f*~*c*~+ *f*~*e*~.     (22) Now consider the problem of computing the occupancy frequencies in the parent profile vector, given only the child occupancy frequencies and the trace-back path for the alignment. Consider first a diagonal edge in the path, i.e. an edge that does not open or extend a gap, following another diagonal edge. In this case, the occupancy frequencies are computed similarly to amino acid frequencies (as a sum in which a child frequency is weighted according to the total weight of the sequences in its profile). For horizontal or vertical edges, i.e. edges that open or extend gaps, the parent occupancy frequencies can be computed by considering the effect of the new column of indels (Figure [8](#F8){ref-type="fig"}). It is straightforward to work through all cases and show that the three frequencies *f*~*o*~, *f*~*c*~and *f*~*e*~are sufficient for their values in the parent profile vector to be computed in O(*L*~P~) time from the child profile vectors and alignment path. ### Construction of the root alignment By avoiding the use of profile matrices, the complexity of a single progressive alignment iteration is reduced from O(*L*~P~^2^+ *NL*~p~) space and O(*L*~P~^2^+ *NL*~P~) time to O(*L*~P~^2^) = O(*L*^2^+ *NL*) space and time. The *NL*term in the time complexity is now due only to the increase in profile length, and is therefore typically much smaller than before. The root alignment is constructed by storing the alignment path produced at each internal node. For each input sequence, the path to the root is followed, inserting the gaps induced by each alignment path at each internal node. This procedure is O(*NL*~P~log *N*) = O(*N*^2^log *N*+ *NL*log *N*) time, and requires O(*NL*~P~) = O(*NL*+ *N*^2^) space for storage of the paths. This is expensive for large *N*, and we therefore optimize this step by using a device we call an *e-string*, a type of edit string. ### E-strings An alignment path can be considered as an operator on a pair of sequences that inserts indels into those sequences such that their lengths become equal. Conventionally, an alignment path is represented as a vector of three symbols representing edges in the graph, say M, D and I (for match, delete and insert, i.e. a diagonal, horizontal or vertical edge). Note that indels in one sequence are inserted only by Ds, indels in the other are inserted only by Is. Define an e-string *e*to be a vector of \|*e*\| integers interpreted as an operator that inserts indels into a string by scanning it from left to right (Figure [9](#F9){ref-type="fig"}). A positive integer *n*means skip *n*letters of the string; a negative integer -*n*means insert *n*indels at the current position. We require the vector to be in its shortest form, so signs always alternate. We represent an alignment path as a pair of e-strings, one for each sequence, assigned to the appropriate edges in the tree. We will typically find that \|*e*\|, the length of the e-string, is much less than *L*~P~, the length of the alignment path. Now consider the effect of applying two consecutive e-strings (\"multiplying\" them). This can be expressed as a third e-string, which can be efficiently computed in O(\|*e*\|) time from the multiplicands. For each leaf (input sequence), the product is computed of e-strings on the path to the root (Figure [10](#F10){ref-type="fig"}). The final e-string obtained at the root is then applied to the sequence. This method does not reduce the big-O time or space complexity, but is much faster than a naive implementation. ### Brenner\'s method Steven Brenner (personal communication) observed that a multiple alignment can be alternatively be obtained by aligning each sequence to the root profile. This requires O(*NL*~P~^2^) time, giving a lower asymptotic complexity in *N*at the expense of an additional factor of *L*~P~. This method gives opportunities for errors relative to the \"exact\" e-string solution (when a sequence misaligns to its copy in the profile), but can also lead to improvements by allowing the sequence to correctly align to conserved motifs that were not apparent when the sequence was added. (Note the resemblance to the refinement stage, which begins by re-aligning individual sequences to the rest). The chances for error are reduced by constraining the alignment to forbid gaps in the root profile. Our tests show that this method gives comparable average accuracy to the e-string solution but to be slower for up to at least a few thousand sequences, and e-strings are therefore used by default. Refinement complexity --------------------- In the following, we assume that an explicit multiple alignment (profile matrix) of all sequences is maintained, and determine the complexity of each step in Stage 3. Step 3.1 determines the bipartition induced by deleting an edge from the tree. This is O(*N*) time, and sufficiently fast that there is little motivation for further optimization. Step 3.2 extracts profiles for the two partitions from the current multiple alignment and computes their profile vectors, which is O(*NL*~P~) time and space. Step 3.3 performs profile-profile alignment, which is O(*L*~P~^2^) time and space. Step 3.4 computes the SP score, which is O(*N*^2^*L*~P~) time and O(*NL*~P~) space (discussed in more detail shortly). A single iteration of Stage 3 is thus O(*N*^2^*L*~P~+ *L*~P~^2^) time and O(*NL*~P~+ *L*~P~^2^) space. There are O(*N*) edges in the tree, so executing this process for all edges is O(*N*^3^*L*~P~+ *NL*~P~^2^) time and O(*NL*~P~+ *L*~P~^2^) space, which is O(*N*^4^+ *N*^3^*L*+ *NL*^2^) time and O(*N*^2^+ *NL*+ *L*^2^) space. Assuming that a fixed maximum number of iterations of Stage 3 is imposed, this is also the total complexity of refinement. We now consider optimizations of the refinement stage. Anchor columns -------------- A multiple alignment can be divided vertically at high-confidence (*anchor*) columns. Each vertical block is then refined separately, improving speed and reducing space due to the O(*L*^2^) factor in dynamic programming. This strategy has been used by several previous algorithms, including PRRP \[[@B13]\], RASCAL \[[@B42]\] and MAFFT. In MUSCLE, the following criteria are used to identify anchor columns. The profile function (LE or PSP) must exceed a threshold, the averaged profile function over a window around the position must exceed a (lower) threshold, and the column may not contain a gap. In addition, the contribution to the averaged score from a single column has a ceiling, reducing skew in the averaged score due to exceptionally high-scoring columns. These heuristics are designed to avoid anchor columns that have high scores but are either artifacts (similar residues found by chance in unrelated regions) or are too close to variable regions. When performing a profile-profile alignment, each anchor column and its two immediate neighbors (which form the boundaries of vertical blocks) are required to remain aligned; i.e., terminal gaps are forbidden except at the true terminals. Introducing this constraint overcomes a small degradation in average alignment quality that is otherwise observed. This implies that the degradation is sometimes due to cases where a well-conserved region is divided into two parts by an anchor column, one of which becomes short enough that it misaligns to a similar short motif elsewhere. SP score -------- Notice that computation of the SP score dominates the time complexity of refinement and of MUSCLE overall, introducing O(*N*^4^) and O(*N*^3^*L*) terms. We are therefore motivated to consider optimizations of this step. We first consider the contribution SP^a^to the SP score from amino acids; gap penalties require special treatment. Let *s*and *t*be sequences, *x*be a column, *s*\[*x*\] be the amino acid of sequence *s*in column *x*, and *S*(*i*, *j*) be the substitution score of amino acids *i*and *j*. It is convenient to impose an (arbitrary) ordering on the sequences and amino acid types. Then, SP^a^= Σ~*x*~Σ~*s*~Σ~*t*\>*s*~*S*(*s*\[*x*\], *t*\[*x*\]).     (23) Define *δ*(*s*, *i*, *x*) = 1 if *s*\[*x*\] = *i*, 0 otherwise, and *n*~*i*~\[*x*\] = Σ~*s*~*δ*(*s*, *i*, *x*). We say *n*~*i*~\[*x*\] is the *count*of amino acid type *i*in column *x*. We can now transform the sum over pairs of sequences into a sum over pairs of amino acid types: SP^a^= Σ~*x*~Σ~*i*~*n*~*i*~Σ~*j*~*n*~*j*\>*i*~*S*(*i*, *j*) + 1/2 Σ~*x*~Σ~*i*~(*n*~*i*~^2^- *n*~*i*~) *S*(*i*, *i*).     (24) Frequencies are computed as: *f*^*x*^~*i*~= *n*~*i*~\[*x*\]/ *N*.     (25) Using frequencies,  For simplicity, we have neglected sequence weighting; it is straightforward to show that (26) applies unchanged if weighting is used. Note that (23) is O(*N*^2^*L*~P~) but (25) and (26) are O(*NL*~P~). For *N*\>\> 20, this is a substantial improvement. Let SP^g^be the contribution of gap penalties to SP, so SP = SP^a^+ SP^g^. It is natural to seek an O(*NL*~P~) expression for SP^g^analogous to (26), but to the best of our knowledge no solution is known. Note that in MUSCLE refinement, the absolute value of the SP score is not needed; rather, it suffices to determine the difference in the SP scores before and after re-aligning a pair of profiles. Let SP(*s*, *t*) be the contribution to the SP score from a pair of sequences *s*and *t*, so SP = Σ~*s*~Σ~*t*\>*s*~SP(*s*,*t*), and denote the two profiles by X and Y. Then we can decompose SP into intra-and inter-profile terms as follows: SP = Σ~*s*∈X~Σ~*t*∈X:*t*\>*s*~SP(*s*, *t*) + Σ~*s*∈Y~Σ~*t*∈Y:*t*\>*s*~SP(*s*, *t*) + Σ~*s*∈X~Σ~*t*∈Y~SP(*s*, *t*)     (27) Note that the intra-profile terms are unchanged in any alignment that preserves the columns of the profile intact, which is true by definition in profile-profile alignment. This follows by noting that any indels added to align the profiles are guaranteed to be external gaps with respect to any pair of sequences in the same profile. It therefore suffices to compute the change in the inter-profile term: SP~XY~= Σ~*s*∈X~Σ~*t*∈Y~SP(*s*, *t*).     (28) This reduces the average time by a factor of about two. We can further improve on this by noting that in the typical case, there are few or no changes to the alignment. This suggests computing the change in SP score by looking only at differences between the two alignments. Let *π*~-~be the alignment path before re-alignment and *π*~+~the path after re-alignment. The change in alignment can be specified as the set of edges in *π*~-~or *π*~+~, but not both; i.e., by considering a path to be a set of edges and taking the set symmetric difference Δ*π*= (*π*~-~∪ *π*~+~) - (*π*~-~∩ *π*~+~). The path *π*~+~after re-alignment is available from the dynamic programming traceback. The path *π*~-~before re-alignment can be efficiently computed in O(*L*~P~) time. Note that in order to construct the profile of a subset of sequences extracted from a multiple alignment, those columns that contain only indels in that subset must be deleted. The set of such columns in both profiles is therefore available as a side effect of profile construction, and this set immediately implies *π*~-~. It is a simple O(*L*~P~) procedure to compute Δ*π*from *π*~-~and *π*~+~. Note that SP^a^is a sum over columns, and there is a one-to-one correspondence between columns and edges in *π*. The change in SP^a^can therefore be computed as a sum over columns in Δ*π*, with a negative sign for edges from *π*~-~, reducing the time complexity from O(*NL*~P~) to O(*N*\|Δ*π*\|). We now turn our attention to SP^g^. We say that a gap G *intersects*Δ*π*if and only if any indel in G is in a column in Δ*π*, and denote by Γ the set of gaps that intersect Δ*π*. If a gap does not intersect Δ*π*, i.e. does not have an indel in a changed column, its contribution to SP^g^is unchanged. It therefore suffices to consider penalties for gaps in Γ, again with negative signs for edges from *π*~-~. The construction of Γ is straightforward in O(*NL*~P~) time. Finally, a sum over pairs in Γ is needed, reducing the O(*N*^2^) component to the smallest possible set of terms. ### Dimer approximation We next describe an approximation to SP that can be computed in O(*NL*~P~) time. Define a two-symbol alphabet {X, -} in which X represents any amino acid and - is the indel symbol. There are four dimers in this alphabet: XX, X-, -X and \--, which denote by no-gap, gap-open, gap-close and gap-extend respectively. Re-write a multiple alignment in terms of these dimers, adopting the convention that dimer *ab*composed of symbol *a*in column *x*-1 and symbol *b*in column *x*is written in column *x*. Now consider the contribution to SP^g^of an aligned pair of dimers, written as *ab*↔*cd*. Clearly XX↔X- adds a gap-open penalty; XX↔-X adds a gap-close (Figure [11](#F11){ref-type="fig"}). To avoid double-counting, we will include only the penalty contribution of indels in the second column. Then XX↔X- adds a per-gap penalty, but XX↔-X adds zero because the second column does not contain a gap. External indels must be discarded; so, for example, \--↔\-- adds zero. In fact, aligning two identical dimers always contributes zero because any indel in the second column is found in both sequences and is therefore external. The contribution of all possible pairs of dimers is unambiguous, with the exception of -X↔\--, which can add a per-gap or extend penalty (Figure [12](#F12){ref-type="fig"}). We approximate this case by assigning it a penalty of *tg*, where *g*is the default per-gap penalty and *t*is a tunable parameter, set to 0.2 by default. With this approximation, dimers can be treated like amino acids: the scores for each aligned pair of dimers forms a substitution matrix (Figure [13](#F13){ref-type="fig"}), and SP^g^can be computed by summing substitution scores over all pairs of sequences. We can now apply Equation 26, re-interpreting the frequency vectors *f*as having 24 components (20 amino acids and four dimers), and compute the change in SP by considering only those columns in Δ*π*. We find use of the dimer approximation to marginally reduce benchmark scores. By default, MUSCLE therefore uses the exact SP score for *N*≤ 100 and the dimer approximation for *N*\> 100, where the higher time complexity of the exact score becomes more noticeable. Evaluation of profile functions ------------------------------- We have previously attempted a systematic comparison of profile functions \[[@B30]\]. The methodology used in that work demanded careful optimization of affine gap parameters for each function. This proved to be time-consuming and tedious, and we therefore tried the following alternative approach, inspired by the notion that a good profile function should be good at discriminating correctly aligned pairs of profile positions from incorrectly aligned pairs. The protocol begins with a set of pair-wise structural alignments. With the sequence of each structure as a query, we used PSI-BLAST to search the NCBI non-redundant protein sequence database \[[@B43]\], giving a multiple sequence alignment (profile) for each structure. Note that we use the term profile in this context to mean the sequence alignment produced by PSI-BLAST, not the scoring matrix. Using the structural alignments as a guide, we then created a database in which columns from the PSI-BLAST profiles were aligned to each other, giving a large set of pairs of alignment columns that we consider to be correctly aligned (the \"true\" database, although there are undoubtedly misaligned sequences and hence some incorrect pairs). By selecting the same number of pairs of columns at random from structures in different FSSP families, we created a similar (\"false\") database of unrelated pairs. A profile function was evaluated by computing the score of each pair of columns in both the true and false databases, and then sorting the results in order of increasing score. The results can be displayed by reviewing the sorted list and, for each score *S*in the list, plotting the number of true pairs with score ≤ *S*(*x*axis) against the number of false pairs with score ≤ *S*(*y*axis); we call the resulting graph a *discrimination plot*. Ideally, all true pairs would score higher than all false pairs, in which case the profile function would be a perfect discriminator and would always produce perfect alignments. A function that perfectly discriminates will appear as a Γ-shaped plot; a function that has no ability to discriminate will appear as a diagonal plot along the line *x*= *y*. If a function F has a discrimination plot that is always above another function G (i.e., D~F~(*x*) \> D~G~(*x*) ∀ *x*, where D~F~is the discriminator plot for F as a function of *x*), then F has a superior ability to discriminate true from false pairs compared with G. If the plots intersect, the situation is ambiguous and neither function is clearly superior. We used sets of structural alignments from \[[@B30]\] (PP) and \[[@B44]\] (PP2). PP contains 588 structure pairs with sequence identity ≤ 30%, z-score ≥ 15, RMSD ≤ 2.5Å and an alignment length of ≥ 50 positions. These criteria were designed to select pairs of structures with low sequence identity and high structural similarity. PP2 contains 500 pairs selected from the FSSP database \[[@B45]\] with ≤ 30% sequence identity, z-score ≥ 8 and ≤ 12, RMSD ≤ 3.5Å and alignment length ≥ 50. The criteria for PP2 were designed to select challenging alignments with low sequence identity and relatively high structural divergence, leading to a high frequency of gaps and therefore, presumably, a stronger dependence on accurate identification of sequence similarity. Results on PP2 show the LE function to have higher discrimination than all other tested functions (historically, the LE function was designed by systematic trial and error using a wide range of different profile functions with feedback from discrimination plots). This is illustrated in Figure [14](#F14){ref-type="fig"}, in which the discrimination plot for LE on PP2 is compared with several other functions: PSP, LA, Yona and Levitt\'s \[[@B46]\], LAMA \[[@B47]\]. Using PP, we again find that LE is superior to LA (not shown), but the comparison with PSP is ambiguous as the discrimination plots intersect (Figure [15](#F15){ref-type="fig"}). A major advantage of this approach is that no gap penalties are required, with the result that once the databases have been constructed, a new function can be tested in seconds rather than the days or weeks that were needed with the earlier methodology. However, some caveats are in order. We are using PSI-BLAST as a gold standard for creating profiles, but PSI-BLAST may introduce biases both due to its selection of sequences for inclusion in the profile and due to errors in alignments of those sequences to the query. If the profile function will be used to align PSI-BLAST profiles, then this is an appropriate experimental design. But in the case of multiple sequence alignment, where profiles are produced iteratively by the profile function itself, the results may not be directly applicable. We also note that any monotonic transformation of the profile function leaves the discriminator plot unchanged as it does not change the sort order of the scores. (A monotonic transformation is F\' = *m*(F) where *m*(*x*) is a monotonically increasing function). However, a monotonic transformation may change the alignments produced by a profile function, so we can regard high discrimination as a necessary but not sufficient condition for a good profile function. One can turn this into a virtue by noting that the discrimination plot allows the relative probability of true versus false to be determined from a score. It is therefore possible to numerically determine a log-odds function from the discrimination plot, which can be evaluated by table look-up. Using discrimination plots for PP2, we found the optimal transformation for LE to be close to linear, in contrast to other functions we tried, including PSP (results not shown). This observation further encouraged us to explore the performance of LE in an MSA algorithm. Testing on multiple alignment benchmarks we find LE to give superior results on BAliBASE, but statistically indistinguishable results on other databases (results not shown). MUSCLE therefore uses LE as the default choice as it sometimes gives better results but has not been observed to give lower average accuracy on any of our tests. It is also useful to introduce a method with a distinctively different scoring scheme as an alternative that may give better results on some input data and may provide unique features for incorporation into jury or consensus systems. One drawback of LE is its relatively slow performance due to the need to compute a logarithm for each cell of the dynamic programming matrix. Complexity of MUSCLE -------------------- The complexity of MUSCLE is summarized in Table [2](#T2){ref-type="table"}. We assume *L*~P~= O(*L*+ *N*), the e-string construction for the root alignment, and a fixed number of refinement iterations. Results ======= MUSCLE offers a variety of options that offer different trade-offs between speed and accuracy. In the following, we report speed and accuracy results for three sets of options: (1) the full MUSCLE algorithm including Stages 1, 2 and 3 with default options; (2) Stages 1 and 2 only, using default options (MUSCLE-prog); and (3) Stage 1 only using the fastest possible options (MUSCLE-fast), which are as follows: *F*^Binary^is used as a distance measure (Equation 2), the PSP profile function is used, and diagonal finding is enabled. Alignment accuracy ------------------ In Tables [3](#T3){ref-type="table"} and [4](#T4){ref-type="table"} we report the speed and accuracy of MUSCLE v3.3, CLUSTALW v1.82, Progressive POA, a recently published method that is claimed to be 10 to 30 times faster than CLUSTALW for large alignments \[[@B48]\], and the MAFFT script FFTNS1 v3.82, the fastest previously published method known to us. On the advice of one of the authors of Progressive POA, we used command-line options selecting global alignment with truncated gap scoring (Catherine Grasso, personal communication). We report results both using distance matrices computed by BLAST (POA-blast) and using the distance method built into the program (POA). We use four sets of reference alignments: BAliBASE v2, PREFAB v3, SABmark v1.61, and a version of SMART from July 2000. The accuracy score is *Q*, the number of residue pairs correctly aligned divided by the length of the reference alignment. For more discussion of the reference data, assessment methodology and a comparison of MUSCLE with T-Coffee and NWNSI, the most accurate MAFFT script, see \[[@B2]\]. Execution speed --------------- To compare speeds for a larger number of sequences, we created a test set by using PSI-BLAST to search the NCBI non-redundant protein sequence database for hits to dienoyl-coa isomerase (1dci in the Protein Data Bank \[[@B49]\]), selecting the highest-scoring 1,000 sequences. This set of sequences had average length 282, maximum length 454 and average pair-wise identity 20%. We aligned randomly chosen subsets of from 200 to 1,000 sequences with each program and noted the total execution time. In the case of 1,000 sequences, the resulting alignments had from 1,100 from 1,400 columns, confirming that it is unrealistic to assume that *L*~P~is O(*L*). Results are shown in Figure [16](#F16){ref-type="fig"}. We have previously shown that MUSCLE-prog is faster than FFTNS1 on a set of 5,000 sequences, for which we estimated that CLUSTALW would require approximately one year \[[@B2]\]. In this test, MUSCLE-fast is approximately 3× faster than FFTNS1 for 200 sequences, and 5× faster for 1,000 sequences. This trend continues for larger numbers of sequences (complete results not shown), showing that MUSCLE-fast has lower asymptotic complexity, due largely to the use of additive profiles for progressive alignment compared with the profile matrices constructed by FFTNS1. Conclusions =========== MUSCLE demonstrates improvements in accuracy and reductions in computational complexity by exploiting a range of existing and new algorithmic techniques. While the design--typically for practical multiple sequence alignment tools--arguably lacks elegance and theoretical coherence, useful improvements were achieved through a number of factors. Most important of these were selection of heuristics, close attention to details of the implementation, and careful evaluation of the impact of different elements of the algorithm on speed and accuracy. MUSCLE enables high-throughput applications to achieve average accuracy comparable to the most accurate tools previously available, which we expect to be increasingly important in view of the continuing rapid growth in sequence data. Availability and requirements ============================= MUSCLE is a command-line program written in a conservative subset of C++. At the time of writing, MUSCLE has been successfully ported to 32-bit Windows, 32-bit Intel architecture Linux, Solaris, Macintosh OSX and the 64-bit HP Alpha Tru64 platform. MUSCLE is donated to the public domain. Source code and executable files are freely available at <http://www.drive5.com/muscle>. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Progressive alignment.**Sequences are assigned to the leaves of a binary tree. At each internal (i.e., non-leaf) node, the two child profiles are aligned using profile-profile alignment (see Figure 2). Indels introduced at each node are indicated by shaded background. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Profile-profile alignment.**Two profiles (multiple sequence alignments) X and Y are aligned to each other such that columns from X and Y are preserved in the result. Columns of indels (gray background) are inserted as needed in order to align the columns to each other. The score for aligning a pair of columns is determined by the profile function, which should assign a high score to pairs of columns containing similar amino acids. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Gap penalties in the SP score**This figure shows a multiple alignment of three sequences *s*, *t*and *u*. The SP score is the sum over all pairs of sequences of their pairwise alignment score. The contribution to the SP score from the pair (*s*, *t*) is computed by discarding columns in which both sequences have indels (arrows). Such indels are said to be external with respect to the pair. Gaps in the remaining columns (gray background) are assessed affine penalties *g*+ *λe*where *g*is the per-gap penalty, *λ*is the gap length, and *e*is the gap extension penalty. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Position-specific gap penalties.**An alignment of two profiles X and Y. Gaps in sequences *t*and *u*are embedded in X. Y contains a single sequence *w*. The gap in *w*(gray background) is inserted to align the profiles and is not part of Y. Consider the SP score for this alignment. We need not consider pairs of sequences in X as their scores are unchanged under all possible alignments of X to Y, leaving the inter-profile pairs (*s*, *w*), (*t*, *w*), (*u*, *w*) and (*v*, *w*). Note that there is no gap penalty for the pairs (*u*, *w*) and (*v*, *w*) as these pairs do not have gaps relative to each other. The remaining pairs (*t*, *w*) and (*u*, *w*) are assessed a penalty *g*+ 3*e*for the gap in Y. The total over all pairs of open or close penalties due to a gap in Y is thus reduced in proportion to the fraction of sequences in X having a gap with the same open or close position. We incorporate this into the PSP score by using position-specific gap penalties *b*(*x*) and *t*(*x*). For example, *b*(*x*) in column 4 of X is half the default value because half of the sequences in X open a gap in that column. Note that there is no open penalty at the N-terminal and no close penalty at the C-terminal. This causes terminal gaps to receive half the penalty of internal gaps. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Tree comparison.**Two trees are compared in order to identify those nodes that have the same branching orders within subtree rotation (white). If a progressive alignment has been created using to the old tree, then alignments at these nodes can be retained as the same result would be produced at those nodes by the new tree. New alignments are needed at the changed (black) nodes only. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Neighbor-joining and UPGMA trees for progressive alignment.**Here we show the same set of four sequences and the order in which they will be aligned according to a neighbor-joining tree (above) and a UPGMA tree (below). Notice that *t*and *u*are the most closely related pair, but (*s*, *t*) and (*u*, *v*) are evolutionary neighbors. With neighbor joining, *t*and *u*are not aligned to each other until the root, in contrast to UPGMA, which aligns *s*and *t*as the first pair. :::  ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Additive profiles.**The profile functions in MUSCLE require amino acid frequencies for each column. Here we show the alignment of two profiles X and Y, giving a new profile Z. Note that the count *n*^Z^~*i*~for amino acid *i*in a given column of Z is the sum of the counts in the child profiles, i.e. *n*^Z^~*i*~= *n*^X^~*i*~+ *n*^Y^~*i*~. In terms of frequencies, this becomes *f*^Z^~*i*~= *N*^X^*f*^X^~*i*~/*N*^Z^+ *N*^Y^*f*^Y^~*i*~/*N*^Z^, where *N*^X^, *N*^Y^, *N*^Z^are the number of sequences in X, Y and Z respectively. Therefore, given a suitable sequence weighting scheme, it is possible to compute frequencies in Z from the frequencies in X and Y. This avoids the step of building an explicit multiple alignment for Z in order to compute frequencies, as done in CLUSTALW and MAFFT. :::  ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Occupancy frequencies in additive profiles.**Here we show an alignment of profiles X and Y giving Z. A column C of indels (shaded background) has been inserted at position *x*in order to align X to Y. To compute the number of gap-extensions in column *x*of Z, three cases must be considered: (1) a gap-extension in the corresponding column of Y, (2) a gap-open in the preceding column of X, and (3) a gap-extension in the preceding column of X. By enumerating all such cases, it is straightforward to compute the occupancy frequencies in Z from the occupancy frequencies of X and Y, plus the alignment path. :::  ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **E-strings.**(1) The effect of the e-string operator \<3,-1,2\> on the sequence MQTIF. A positive number *n*skips *n*letters, a negative number *-n*insert *n*indels. (2) The effect of applying two successive e-strings. In the last line, the result is expressed as a new e-string applied to the original string. (3) We define multiplication on two e-strings as yielding the e-string that is equivalent to applying the two e-strings in order. (4) An alignment path is conventionally represented as a vector of edge types (*M*, *D*and *I*). In this example, *MDMIMM*, shown above a pairwise alignment, is the path that generates that alignment. The alignment can also be generated by a pair of e-strings (shown to the right). An alignment path is therefore equivalent to a pair of e-strings. :::  ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **Root alignment construction.**Here we show the same progressive alignment as Figure 1. Each edge in the tree is labeled with the e-string for its side of the alignment at the parent node. The e-string needed to insert indels into a sequence in the root alignment can be determined by multiplying e-strings along the path to the root. For example, for sequence LSF, the root e-string is \<3,-1,1\>\*\<1,-1,2\> = \<1,-1,1,-1,1\>. :::  ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### **Dimers in the {X,-} alphabet.**Gap penalties for the sequence pair (*s*, *u*) can be computed be considering all aligned pairs of dimers in the alphabet {X,-}, where X is any amino acid and - is the usual indel symbol. Four cases are highlighted. Note that an aligned pair of identical dimers never contribute a gap penalty as any indels in the dimers are necessarily external, as in the left-most example. :::  ::: ::: {#F12 .fig} Figure 12 ::: {.caption} ###### **Problem dimer pair.**The aligned dimer pair -X ↔ \-- causes a problem because its gap penalty contribution cannot be computed without additional information. Note that the first column of indels is external; after this column is discarded, different penalties may be needed, as these two examples show. :::  ::: ::: {#F13 .fig} Figure 13 ::: {.caption} ###### **Dimer substitution matrix.**This matrix specifies the contribution to the total gap penalty for a pair of sequences for each possible pair of aligned dimers. Here, *g*is the per-gap penalty, *e*is the gap-extension penalty. The problem case -X ↔ \-- is approximated as *tg*, where *t*is a tunable parameter. :::  ::: ::: {#F14 .fig} Figure 14 ::: {.caption} ###### **Discrimination plot for PP2.**The *x*axis is the number of true column pairs with scores ≤ *S*for some value *S*, as a fraction of the total number of true pairs; the *y*axis is the number of false column pairs with scores ≤ *S*, as a fraction of the total number of false pairs. The databases were constructed from the PP2 test set. Shown are discrimination plots for the log-expectation (LE), log-average (LA), Yona-Levitt (YL), LAMA, and profile sum of pairs (PSP) functions. The LE function shows higher discrimination over the entire range of scores than any other function we tested (complete results not shown). The poor performance of the \"standard\" PSP function is striking. PSP displays negative discrimination over some of its range where it falls below the diagonal (dashed line). :::  ::: ::: {#F15 .fig} Figure 15 ::: {.caption} ###### **Discrimination plot for PP.**This is similar to Figure 13, except that the database was generated from the PP test set. Here we see an ambiguous result as the discrimination plots for LE and PSP intersect. :::  ::: ::: {#F16 .fig} Figure 16 ::: {.caption} ###### **Execution time as a function of *N*.**This plot shows the execution time as a function of *N*(number of sequences) for the tested alignment methods. Input data is from 200 to 1,000 sequences in increments of 200. Average sequence length is 282, maximum length 454. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Complexity of CLUSTALW. Here we show the big-O asymptotic complexity of the elements of CLUSTALW as a function of *L*, the typical sequence length, and *N*, the number of sequences, retaining the highest-order terms in *N*with *L*fixed and vice versa. ::: **Step** **O(Space)** **O(Time)** ----------------------------- ------------------------------- ------------------------------------ Distance matrix *N*^2^+ *L* *N*^2^*L*^2^ Neighbor joining *N*^2^ *N*^4^ Progressive (one iteration) *NL*~P~+ *L*~P~= *NL*+ *L*^2^ *NL*~P~+ *L*~P~^2^= *N*^2^+ *L*^2^ Progressive (total) *NL*+ *L*^2^ *N*^3^+ *NL*^2^ TOTAL *N*^2^+ *L*^2^ *N*^4^+ *L*^2^ ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Complexity of MUSCLE. Here we show the big-O asymptotic complexity of the elements of MUSCLE as a function of *L*, the typical sequence length, and *N*, the number of sequences, retaining the highest-order terms in *N*with *L*fixed and vice versa. ::: **Step** **O(Space)** **O(Time)** ------------------------------------ ------------------------------------ -------------------------------------------- *K*-mer distance matrix *N*^2^+ *L* *N*^2^*L* UPGMA *N*^2^ *N*^2^ Progressive (one iteration) *L*~P~^2^= *NL*+ *L*^2^ *L*~P~^2^= *N*^2^+ *L*^2^ Progressive (root alignment) *NL*~P~= *N*^2^+ *NL* *NL*~P~log *N*= *N*^2^log *N*+ *NL*log *N* Progressive (*N*iterations + root) *N*^2^+ *NL*+ *L*^2^ *N*^3^+ *NL*^2^ Refinement (one edge) *NL*~P~+ *L*~P~^2^= *N*^2^+ *L*^2^ *N*^2^*L*~P~+ *L*~P~^2^= *N*^3^*+ L*^2^ Refinement (*N*edges) *N*^2^+ *L*^2^ *N*^4^*+ NL*^2^ TOTAL *N*^2^+ *L*^2^ *N*^4^+ *NL*^2^ ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Accuracy scores. The average accuracy, measured by the *Q*score, is reported for each method on each set of reference alignments. ::: **Method** **PREFAB** **BAliBASE** **SABmark** **SMART** ------------- ------------ -------------- ------------- ----------- MUSCLE 0.648 0.896 0.430 0.856 MUSCLE-prog 0.634 0.883 0.427 0.837 FFTNS1 0.619 0.844 0.376 0.815 MUSCLE-fast 0.616 0.849 0.396 0.813 CLUSTALW 0.588 0.860 0.404 0.823 POA-blast 0.577 0.839 0.352 0.788 POA 0.576 0.834 0.280 0.797 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### CPU times. The total CPU time required to create all alignments in each test set, measured in seconds on a 2.5 GHz Pentium 4 desktop computer. ::: **Method** **PREFAB** **BAliBASE** **SABmark** **SMART** ------------- ------------ -------------- ------------- ----------- MUSCLE-fast 540 11 45 30 FFTNS1 720 16 70 46 MUSCLE-prog 3,000 52 429 180 MUSCLE 11,000 81 1,500 560 POA-blast 11,000 90 290 670 CLUSTALW 15,000 160 210 480 POA 24,000 130 380 880 :::

PubMed Central

2024-06-05T03:55:47.704156

2004-8-19

PMC517707

Background ========== An important challenge in Serial Analysis of Gene Expression (SAGE) \[[@B1]\] analysis is the decision whether a gene is differentially expressed between two classes, for example tumoral vs. normal classes. In statistical terms, this essential step is to test the null hypothesis H~0~: \"gene has no differential expression between the two probed classes\". A much more usual approach is to assign an index (*P*-value or Bayes factor, for example) that measures the confidence/significance of the hypothesis and let the biologists themselves to establish a cutoff of what they call significant. This necessity arises because counting sequenced SAGE tags is a process prone to random and systematic errors that affect gene expression abundance estimates. Systematic errors may come from various sources such as GC content bias \[[@B2]\], sequencing errors \[[@B3],[@B4]\] as well as the possibility of non unique tags. This kind of error can be detected/corrected using some bioinformatics procedures such as quality control of automatic sequencing pipe-line \[[@B5]\], or statistical estimation procedures such as \"denoising\" \[[@B6],[@B7]\]. Random errors are due to the inherent stochastic characteristic of SAGE data acquisition: sampling from automatic sequencing. Like colored balls in an urn, sampling and counting SAGE tags from a library is commonly modeled by a Bernoulli Process relying on an infinite population sampling approximation. If an Expressed Sequence Tag (EST) library is non-normalized, its counting data, also known as \"Digital-Northern\", reflects the abundance of genes. Likewise, the Massively Parallel Signature Sequencing (MPSS) \[[@B8]\] technique counts tags to infer the transcriptome, but using a completely different strategy from traditional DNA sequencing methods, that allows augmented high-throughput capability. Therefore, all the results discussed here are readily applicable to \"Digital-Northern\" or MPSS context since, from a mathematical viewpoint, all represent the same bioinformatics problem: counting transcripts (as balls in urns). Nowadays, the variability in SAGE abundance data is modeled only as due to sampling from sequencing, since almost all statistical procedures are performed after aggregation of observations from various libraries of the same class, creating a \"pseudo-library\". See \[[@B9]-[@B11]\] for good reviews on statistical techniques used in SAGE analysis. This extensively used trick tacitly ignores the within-class variability, i.e., the biological variability among individuals within a class (different patients having the same cancer diagnosis, for example), and could lead to overconfident conclusions. Results ======= Here we propose a Bayesian model of mixtures to account for within-class variability as a generalization of the Beta-Binomial model \[[@B12]\]. We also show that the usual \"pseudo-library\" construction is a particular case of our mixture model. Finally, we propose the use of the Bayes Error Rate to intuitively rank the differential expression hypothesis under a Bayesian framework, avoiding several technicalities and difficulties such as: typeI and typeII error analysis, Bonferroni-like multiple testing correction, asymptotic results evocation, imposition of a test statistic and null probability density function (pdf), and so on. Statistical model ----------------- The counting process from automatic sequencing of one single *i*-th library is often modeled as a Bernoulli Process and a fixed unknown tag abundance *π*~*i*~is implicitly assumed. The pdf of the random variable of interest, \"expression abundance\" *π*∈ \[0;1\] among all *n*libraries is unknown, thus each library could be regarded as being created by a realization of *π*. These features lead naturally to mixture models \[[@B13],[@B14]\]:  where: *f*(·) is the unknown pdf of the abundance among same-class libraries parameterized by a vector ***θ***, **X**= (*x*~1~,\..., *x*~*n*~) is the vector of counts in all *n*libraries of same class, **M**= (*m*~1~,\..., *m*~*n*~) is the vector of library sizes and *L*is the likelihood of each *i*-th observation. The common procedure of merging all observations from libraries of the same class, constructing a \"pseudo-library\" before statistical inference, is recognized as a particular case of this mixture model: just assume that all libraries have strictly the same abundance, with no biological variability. Mathematically, this is a function with infinite probability density over one single abundance value *π*= *θ*and zero over every other *π*≠ *θ*, or a Dirac\'s Delta function. Using *f*(·) as a Dirac\'s Delta function constrained to \[0;1\], turn Eq.1 into the familiar and commonly used binomial distribution (see derivation in the Methods section). We believe that Dirac\'s Delta is a naive description of real-life SAGE libraries. The Beta distribution is an alternative with non-zero within-class variance to account for intuitively expected biological differences among them. Using *f*(·) as a Beta in Eq.1, yields the so-called Beta-Binomial model (see derivation in the Methods section). Given the parameter vector ***θ***that describes the random variable *π*of some fixed gene *G*, we must decide if there is a difference between A and B classes (e.g, tumor vs. normal classes). We propose to consider genes as being differentially expressed based on non-superposition of the predictive Beta pdfs of both A and B classes. By \"predictive\" we mean that we use the *a posteriori*mode in the Beta pdfs. The \"non-superposition\" intuitive feature is mathematically written as the Bayes Error Rate *E*\[[@B15]\]:  where *f*(·) is the Beta pdf and \"hat\" over the parameters indicate the values that lead to an *a posteriori*pdf maximum. The *a posteriori*distribution is obtained as usual from Bayesian Statistical Theory (*a priori*pdf choice and detailed derivation are in the Methods section). Intuitively, if the pdfs are \"far apart\", the gene probably has reproducible differential expression between classes. In this case, rarely could one misclassify class A as B and vice-versa. Figure [1](#F1){ref-type="fig"} gives some insight about this fact. Using our proposed approach, the \"far apart\" notion means a small Bayes Error Rate *E*. For adepts of the Frequentist Statistics, this evidence measure could resemble a typeI and typeII errors sum, however it is just an illustrative analogy. As in any significance test method, the experimenter must define what is a high significance *E*value. This cutoff should be guided by external and independent confirmatory assays. To avoid crude decision boundaries, one could rank their significance results but there is no way to avoid some arbitrariness in any kind of statistical test. In the classical Frequentist Statistics framework, it is common to call a result as significant if it presents a *P*-value ≤ 0.01 in a *t*-like-test, hoping that this could control the error at this level. However, due to technical difficulties such as lack of sensitivity of posterior confirmatory methods or high absolute expression (not differential expression) necessity, this apparent statistically sound results could be not useful in a pragmatic sense. That is why we prefer to rank the differential expression results and allow researchers to establish a cutoff compatible with their subsequent application for the selected genes, rather than split them based in assumption-derived error-rate cutoffs. People familiar with the Frequentist Statistics framework could miss multiple testing considerations, typeI/typeII error studies, and so on. However, in the Bayesian framework, several of these concerns are meaningless since we work with parameters space and not with sample space. The bayesians avoid statements about \"data that could be observed but was not\" and work only with available information (prior and experimental), extracting all possible information from data effectively observed. For those genes classified as differentially expressed, one should aggregate intuitive information adding \"error-bars\" to expression ratios. Recently we have developed a method to add credibility intervals to gene expression ratio \[[@B16]\], which could improve posterior analyses such as clustering \[[@B17]\] or comparison with microarray data. Comparison with available methods using publically available data ----------------------------------------------------------------- To show the model is usefulness, we applied it to a tumor vs. normal two-classes comparison problem. We chose a subset of brain tumor SAGE data from The Cancer Genome Anatomy Project\'s SAGE Genie public database web-site \[[@B18]\]. The SAGE Genie performs several bioinformatics protocols to assure the quality of its data with systematic errors cleaning/correction \[[@B19]\]. We used all 4 available libraries in SAGE Genie until Jan/2004 from astrocytoma grade III tumors and almost all (except the fetal library) normal brain libraries (see Methods section for details about libraries). We want to stress 3 typical and important cases: (i) when our measure agreed with other evidence measures accepting null hypothesis H~0~, i.e., there is no evidence of differential behavior between tumor and normal classes; (ii) when our method agreed with others rejecting H~0~, i.e., there is evidence of differential expression; and (iii) when our method showed evidence in favor but other evidence measures showed evidence against the H~0~. Case (iii) is the main motivation of our method since it reveals situations that researchers may call a gene differentially expressed and, in fact, it could be not so significant if biological replicates are taken into account. The other evidence measures used were: the Audic-Claverie bayesian evidence \[[@B20]\], the classical Fisher Exact Test *P*-value, and the classical *χ*^2^*P*-value, all obtained using the IDEG6 web-interface \[[@B21],[@B22]\] (see Methods section). A case (i) prototype is the TTTCAATAGA tag with **X**~T~= (0, 2, 5, 8) and **X**~N~= (1, 1, 0, 0, 0, 7, 2). The Audic-Claverie, Fisher and *χ*^2^methods yield *P*-values of 0.06, 0.44, 0.41, respectively, indicating low evidence against H~0~for all mystical significance level cutoffs ≤ 0.01, ≤ 0.05 or ≤ 0.1. The Bayes Error Rate evidence is *E*= 0.61, an intuitively unacceptable superposition level between the normal and tumoral predictive Beta pdfs, showing that there is no separable behavior between classes. Figure [2a](#F2){ref-type="fig"} shows an obvious superposition between pdf and observations of this two classes. A case (ii) prototype is the AAAAGAAACT tag with **X**~T~= (7, 11, 18, 10) and **X**~N~= (7, 1, 2, 1, 2, 0, 3). All *P*-values are 0.00 (zero), significant at any cutoff level. Our evidence is *E*= 0.03, showing safely that this gene behaves differentially between normal brain and astrocytoma grade III patients. Figure [2b](#F2){ref-type="fig"} shows that two Betas are apart from each other and, even observing clear within-class variability, the expression is different. A case (iii) prototype is the TTGGAGATCT tag with **X**~T~= (7, 239, 244, 123) and **X**~N~= (54, 27, 33, 21, 40, 196, 28). All *P*-values are 0.00 (zero), indicating significant difference between classes. On the other hand, our evidence *E*= 0.73 indicates high superposition between tumor and normal classes. Figure [2c](#F2){ref-type="fig"} shows that within-class variability for tumor class is not negligible. It is obvious that individual libraries confound their results with normal brain libraries, and the Betas have a relatively high intersection. Using a common \"pseudo-library\" approach, one is lead to call this gene as a strong discriminator between classes. We believe that this is a suspect conclusion. There are several other obvious case (iii) examples, such as tag TACAGTATGT in Figure [2d](#F2){ref-type="fig"}, that received *P*-values \< 0.01 from all other methods, and they are the main concern of our method since they may lead to waste of resources in clinical validation efforts of genes that, by SAGE itself, could be left behind in favor of other promising genes. All tag results are available as additional file and graphics for all tags are at the supplemental web-site \[[@B23]\]. One could think about a case (iv) when considering within-class variability leads one to H~0~rejection, but considering \"pseudo-libraries\" leads to H~0~acceptance. This seems to be inconsistent since one expects that, once H~0~is accepted in a simplified model, it should also be accepted in the complete model. In fact, we do not observe such a situation, except by tags with *P*-values or Bayes Error Rate very close to arbitrarily defined cutoff values. We believe that these occurrences are just \"edge effect\" manifestations. Discussion ========== In order to assure that we are dealing with a fundamental question in SAGE analysis, we show more insights analyzing the method\'s robustness using the same data but excluding \"small\" libraries. Also, we draw some parallels between our proposed method and the only available published solution for dealing with within-class variability, a *t*-test approximation \[[@B12]\]. We used our method with all available libraries but some of them are smaller than 50,000 tags (see Table [1](#T1){ref-type="table"}). In the SAGE community, libraries smaller than this arbitrary limit are considered \"small\". Several researchers claim that these are non-representative and should be excluded from analysis. We observed several case (iii) tag examples which remain as case (iii) if we use libraries with size \> 40,000 and \> 50,000 (shown at the supplemental web-site only). Figure [3](#F3){ref-type="fig"} shows a tag example analyzed in these tree setups and it is clear that inclusion of \"small\" libraries gave pretty much the same result, indicating robustness of our method against small class size variations and against \"small\" sized libraries. Moreover, these libraries are not always outliers from biological sampling but seem to be samples like any other. These results suggest that one can use the \"small\" libraries, jointly with non-\"small\" ones, because biological variability seems to be greater than binomial sampling variability. Obviously, we are not recommending to use only \"small\" libraries in SAGE analysis, but suggesting that our method is relatively robust. For low expression genes, the binomial sampling variability should become more relevant as the library size decreases. Also, the results obtained using two/three libraries could be very different from using just one. These proprieties could be tag dependent since some tags could be much noisier than others for biological reasons. Some \"denoising\" procedure could be used before application of our method \[[@B7]\]. Therefore, our findings should be carefully interpreted. To prove that the incoherence of using \"pseudo-libraries\" methods is not a prerogative of tags showing small fold-changes, we analyzed another three very illustrative examples: ATGGCAACAG, GGATGTGAAA, and GTATGGGCCC; which are case (iii) tags. These tags present high fold changes: 7.59, 8.15 and 25.80 fold-change respectively, augmented in pooled tumor libraries. Using the well-known Fisher Exact test, *χ*^2^classical test and the Audic-Claverie\'s method, we get 0.00 (zero!) for all *P*-values of the no differential expression null hypothesis. Using the conceptually different Bayesian *P*-value implemented at SAGE Genie \[[@B24],[@B25]\] we obtain 0.01, 0.00 and 0.00 respectively for posterior probabilities of fold-changes greater than 4-fold. Finally, using our own proposed measure, applied to the pool, we get *E*= 0.00 meaning no superposition between the two classes pdfs. All these results indicate strong significance in differential expression of these tags. However, if we consider within-class variability, the test proposed by Baggerly *et al.*\[[@B12]\] yields 0.08, 0.07 and 0.15 respectively for *t*-test *P*-values, and our method yields Bayes Error Rates of 0.38, 0.37, 0.43 respectively; indicating not so significant evidence in favor of the differentially expressed hypothesis. A closer look at the graphics of these tags induces one to believe that there is no reproducible differential expression because several observations of tumor and normal are superimposed (all graphics available at supplemental web-site \[[@B23]\]). Since we show clearly that methods that use \"pseudo-library\" aggregation could be incoherent in some cases, a natural question is how our proposed method performs compared to the only published solution that accounts for within-class variability, the Baggerly *et al.*\[[@B12]\]*t*-test approximation. Without knowing the true state of all tags, it is impossible to carry out a serious benchmark. Since the interpretation of evidence measures is very different, the performance could be subjected to an arbitrary cutoff selection for each method. Figure [4](#F4){ref-type="fig"} shows a scatter-plot of evidence measures obtained for each of the two methods. It is clear from this graphic that there are many more tags considered as differentially expressed using our method than the *t*-test approximation, considering *E*≤ 0.1 and *P*-value ≤ 0.01. There are also some tags selected by *t*-test and ignored by ours. It is impossible to know which method perform better without the true unknown status of those tags. Looking at individual libraries results, constructed as depicted in Figure [2](#F2){ref-type="fig"} for example, could help in this analysis but this is a subjective procedure. It is important to bear in mind that a difficulty is hidden in the Beta modeling imposed in the very first beginning. If Beta is not a good model for an unknown biological behavior, then some apparent inconsistency could appear in both Baggerly *et al.*\[[@B12]\] and our approaches. However, our general mixture model allows another propositions. Other simplex constrained pdfs, different from Beta, exist but the tractability is much more difficult \[[@B26]\]. We believe that to build a fully non-parametric approach to this problem is a very hard issue, but should be considered as a future challenge. Conclusion ========== Until now, almost all statistical methods for SAGE data analysis tacitly ignore the within-class variability. To our knowledge, the firsts to formally address this issue was Baggerly *et al.*\[[@B12]\] who introduced the Beta-Binomial model as the correct way to model the probability of counting tags instead of Binomial models. They also proposed a *t*-like statistics, outlined a possible hypothesis test using the classical Frequentist Statistics framework and evoked some asymptotic results for *t*pdf justification. In this work we presented the Bayesian alternative for this problem and defined a theoretical model that views Baggerly\'s Beta-Binomial approach or even the common Binomial approach as particular cases of mixture models. Other models are possible modifying the mixing distribution, such as Beta-Poisson \[[@B14]\], or using other simplex constrained pdf \[[@B26]\] to model expression abundance. At last, but not at least, we proposed a method for ranking differentially expressed genes between two classes using the Bayes Error Rate as an intuitive measure of separation between the classes pdfs, avoiding statistical test formalism and its conceptual/practical difficulties. We show that there are cases in which approaches that ignore within-class variability will lead to high significance in differences between tumor and normal classes, but looking carefully at individual observations jointly, one should not attribute such high significance to them since abundance probability density functions have considerable superposition. In conclusion, we recommend that within-class variability must be taken into account in any statistical analysis of SAGE data if replicates are available. We suggest that biological replication should be considered in planning new SAGE experiments. Methods ======= General bayesian model ---------------------- To start generically, suppose that the probability density function (pdf) for the random variable of interest \"expression abundance\" *π*∈ \[0;1\] of some gene *G*is indexed in a model family by means of a parameter vector ***θ***. Therefore, following the usual Bayesian framework, the *a posteriori*pdf that describes the class is:  where: **X**= (*x*~1~,\..., *x*~*n*~) is the vector of counts in all *n*libraries of same class, **M**= (*m*~1~,\..., *m*~*n*~) is the vector of total observations in all *n*libraries of same class, *g*(·) is the *a priori*pdf, and *L*is the likelihood of each *i*-th observation. Note that the product of all likelihood functions over all observations is the so-called Likelihood Function. The counting process from automatic sequencing is often modeled as a Binomial. Since the sample size and the stopping rule are not known in advance the model is not strictly Binomial. We do not need the combinatorial constant in the model, but we write it just because it is commonly used and will vanish in *posteriori*expression anyway. \"Pseudo-Library\" method as particular case -------------------------------------------- Merging all observations from the same class libraries and constructing \"pseudo-libraries\", with the sum of their components, is the standard procedure to use replicates. Our general model is reduced to this (unrealistic) one if one uses *f*(·) as a Dirac\'s Delta in Eq.1:  where: **1**{·} is the indicator function. Using Eq.4 in Eq.3 yield:  where: g(*θ*) = 1, the non-informative uniform *a priori*distribution. The expert recognizes that *θ*\~ Beta(1 + **Σ***x*~*i*~, 1 + **Σ***m*~*i*~- **Σ***x*~*i*~), and the sum of observations is the mathematical translation of \"pseudo-libraries\" construction. Beta-Binomial method as particular case --------------------------------------- The only published solution that allows non-zero within-class variance in SAGE analysis is the Beta-Binomial model \[[@B12]\]. Using *f*(·) as a Beta in Eq.1 we get the Beta-Binomial model as a particular case of general model:  where: *B*(·) is the beta special function, and:  Again, an expert recognizes ***θ*** = (*θ*~1~, *θ*~2~) as the mean and standard deviation (stdv) of a Beta random variable. We prefer this parameterization of Beta distributions instead of the common (*α*, *β*) one because: (i) it is much more intuitive to biologists to deal with mean and stdv than with abstract *α*and *β*, and (ii) as *α*, *β*\> 0, the domain Θ = {(*θ*~1~, *θ*~2~): 0 ≤ *θ*~1~≤ 1, 0 ≤ *θ*~2~^2^\<*θ*~1~(1- *θ*~1~) ≤ 1/4} is bounded and much more amenable to perform the necessary numerical computations. Using Eq.6 in Eq.3 yield:  where: *g*(*θ*~1~, *θ*~2~) is the *priori*pdf. *A Priori*distribution definition --------------------------------- To complete a Bayesian model, it is necessary to choose the *a priori*pdf. We use an uniform distribution over Θ. On the other hand, we know in advance that variance of this model cannot be smaller than the variance eventually obtained if we do not consider within-class variability. Even if the within-class variability is very small, it cannot be estimated as being smaller than the simple sampling error because they are inseparable, and sampling error is the lower limit \[[@B12]\]. As an illustration, the same situation could occur if one takes several diameter measurements of a folded paper ball and a perfect sphere using a common ruler. In the first case, the intrinsic nature of the measured object dominates the measurement variability but, in the second case, we cannot know the diameter of the perfect sphere with better precision than our ruler can measure. This kind of knowledge is naturally incorporated in Bayesian statistics by means of *a priori*distributions. To match our desired features, it is sufficient to define an uniform over the Θ parameter space but constrained at a minimum stdv σ, obtained from the result of no within-class variance model:  over domain Θ = {(*θ*~1~, *θ*~2~): 0 ≤ *θ*~1~≤ 1, 0 ≤ *θ*~2~^2^\<*θ*~1~(1 - *θ*~1~) ≤ 1/4}. Since we showed (Eq.5) that the no within-class variance model is *θ*\~ Beta(1 + Σ*x*~*i*~, 1 + Σ*m*~*i*~- Σ*x*~*i*~), it is easy to obtain our lower stdv boundary from Beta variance:  Therefore, using Eq.9 and Eq.10 in Eq.8, our *posteriori*is completely defined. Differential expression detection --------------------------------- We detect a tag as differentially expressed using the Bayes Error Rate *E*\[[@B15]\] in both predictive Beta pdfs:  where:  Note that *f*(·) is the Beta pdf, as in Eq.6 development. The \"hat\" over ***θ*** = (*θ*~1~, *θ*~2~) indicates values that leads Eq.8 to maximum. As usual, the maximization, subject to constrain Θ defined previously, is made upon logarithm of *posteriori\'s*core since it gives the same estimates as the *posteriori*itself:  Figure [5](#F5){ref-type="fig"} shows an example of this process. See Results section to get an intuitive notion of this evidence measure. Implementation -- numerical analysis ------------------------------------ The method was implemented as R language \[[@B27],[@B28]\] scripts which are freely available under GPL/GNU copyleft license at supplemental web site \[[@B23]\]. At this web page there are details on how to run it locally. Our method is computer-intensive mainly because some numeric maximization and integration are needed. We used efficient R built-in routines to perform such numerical tasks. Remember that maximization needed in Eq.12 is constrained, thus we used simply auxiliary re-parameterization to obtain linear constrains and used the constrOptim R routine. For numerical integration we used the 1-dimensional gaussian quadrature integrate R built-in function. Although numerical integration of Eq.9 should be performed in all \[0;1\] support, the relevant contribution for this integral is concentrated in a much smaller region. Integrating over the formal limits will cause serious numerical errors, and to avoid this problem we approximate our integration region to an interval delimited by 0.005 and 0.995 quantile of each Beta pdf since the relevant density lie in there. The credibility intervals (\"error-bars\") for the expression ratio of interesting tags were obtained as described in our recent work \[[@B16]\]. We chose arbitrarily 68% credibility intervals. Implementation -- Web based interface ------------------------------------- We have also developed an easy-to-use web-based service that performs all calculations at our server and provides password-protected results. Although desirable, for the sake of automatic web hyperlink with SAGE Genie database, it is not necessary to explicitly identify the tags analyzed but rather any (custom) i.d. string. This could increase privacy or make our web-interface useful for \"Digital-Northern\", MPSS or any mathematically related problem of mixtures from binomial sampling. Figure [6](#F6){ref-type="fig"} shows snapshots of the interface. Publically available data ------------------------- The Table [1](#T1){ref-type="table"} list the SAGE Genie\'s library name, Gene Expression Omnibus (GEO) \[[@B29]\] accession code and size of all used libraries. For our aims, it is sufficient to focus the analysis at the tag level. Thus, we process the tag counts and let the identification of tag\'s best gene match as a posterior question that could be carefully done only to really interesting tags. We choose not to process tags whose counts appear only in libraries of one class. It is important to note that all libraries are from bulk material, without cell-lines, and came from patients with similar disease description. The normal libraries came from different normal regions of the brain. We think that this data set is very illustrative since there are biological replicates in the tumor class allowing clear verification of within-class biological variability. On the other hand, taking only one kind of disease, astrocytoma grade III, instead of all brain tumors in the database, leads one to believe that the within-class variability is in fact due to biological diversity of the patients and not due to very distinct molecular profile of distinct brain tumors stored in SAGE Genie\'s database. Therefore, we believe that this *in silico*comparison is well-suited to demonstrate the necessity of dealing with within-class effect, although it is not our aim here to make a detailed or biological analysis of brain tumor data. Comparison with other methods ----------------------------- To bring some intuition about our differential expression evidence measure, we tabulated evidence measure obtained from the famous Fisher Exact Test, the classical Pearson\'s *χ*^2^proportion test and the bayesian Audic-Claverie\'s method. All these tests were performed using the easy-to-use web-interface IDEG6 \[[@B21],[@B22]\]. The \"*P*-values\" are conceptually very different from our evidence measure but are the most used evidence measures. Although numbers cannot be compared, the conclusions obtained from these methods should be since graphical representation of each library observation gives clear indication of incoherence of \"pseudo-library\" methods. The results of the significance measures for all tags are available as additional Excel^©^or OpenOffice^©^interactive files in which the user can set cutoffs for the significance measures, and explore the differences in conclusions. We carry out a qualitative comparison of our method with Baggerly *et al.*\[[@B12]\]*t*-test approximation in a graphical way since it is impossible to judge them without the unknown true status of analyzed tags, given the too different interpretation of numeric values returned. In their Frequentist framework, the estimator *p*~*i*~= *x*~*i*~/*m*~*i*~is used for *π*~*i*~and a linear combination of these abundances is proposed as the correct way to combine results from different libraries:  where *w*~*i*~are the weights that yield an unbiased minimum variance estimator *V*~*u*~for weighted proportion\'s variance and ***θ*** = (*α*, *β*) are the Beta pdf parameters. However, this unbiased variance could be unrealistically small when it becomes smaller than the sampling variability. We know that the variance of this model cannot be smaller than the variance eventually obtained if we do not consider within-class variability. Therefore, they propose the final *ad hoc*estimator: *V*= max \[*V*~*u*~; *V*~*pseudo-lib*~\]     (14) where:  The max(·) function assure that *V*is not unrealistic small when *V*~*u*~is unrealistic small. To *fit*all these parameters, they used the computationally practical Method of Moments. Once *p*~A~, *p*~B~, *V*~A~and *V*~B~are found for classes A and B, these authors test if the proportions are significantly different proposing the use of a *t*~*w*~statistics as following a Student\'s *t*~*df*~pdf:  List of Abbreviations ===================== SAGE: Serial Analysis of Gene Expression MPSS: Massively Parallel Signature Sequencing EST: Expressed Sequence Tag pdf: probability density function GEO: Gene Expression Omnibus Authors\' Contributions ======================= RV conceived and executed this work. HB helped with all biological issues. DFCP helped in differential expression detection methods and implemented the on-line web-based tool. CABP helped with Bayesian statistics and proposed the mixture ideas. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 **Results for all evidence measures**. This file allows the user to interactively define significance cutoffs for ranked tags. The ranks are based on evidence measures against \"no differential expression\" hypothesis, i.e., evidences closer to 0 (zero) denote higher confidence in differential expression and closer to 1 (one) denote no evidence of differential expression. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ RV is supported by FAPESP 02/04698-8 fellowship. We thank Tie Koide for critical reading of the manuscript and BIOINFO-USP/Rede-Vision for computational support. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **The Bayes Error Rate illustration**. This figure shows two illustrations of the proposed use of Bayes Error Rate *E*to define differentially expressing genes based on pdf of expression abundance *π*. The left example shows an obvious superposition of classes\' pdf, thus a gene having this profile does not present evidence of differential expression between classes. The right example shows two pdfs \"far apart\" and genes with this kind of behaviour should be safely considered differentially expressing between two classes. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Maximum *a posteriori*Beta pdfs of classes in main prototype cases**. The predictive pdf and the Bayes Error Rate *E*of both tumoral (T) and normal (N) classes, for examples tags contained in the three important prototype cases described in text, are shown in the figure. The \'x\' and \'o\' marks represent observed abundances in each tumoral and normal. Frame a) shows case (i) example when methods agree with \"no differential expression\" conclusion. Frame b) shows case (ii) example when methods agree with \"differential expression\" conclusion. Frame c) and d) show case (iii) examples when classical *P*-value method leads to significant differential expression between classes and our method indicates pdf superposition if one take within-class variability into account. Individual observations indicate that the classes are not clearly divided, casting doubt on \"differential expression\" conclusion. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Effect of \"small\" size libraries on the final result.**The predictive pdf and the Bayes Error Rate *E*of both tumoral (T) and normal (N) classes for examples tags contained in the three important prototype cases described in text are shown. The \'x\' and \'o\' marks represent observed abundances in each tumoral and normal libraries. Frame a) shows the result using all libraries. The \"small\" libraries (size \< 50,000) are highlighted with their counts over library size. Frame b) shows results excluding those \"small\" libraries. It is clear that results are pretty much the same and that \"small\" libraries are not (necessarily) outliers of the sampling. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Qualitative comparison of Bayes Error Rate and *t*-test approximation.**It is shown the Bayes Error Rate (*E*) versus the *t*-test approximation *P*-value (Baggerly) \[12\] for each tag. The red lines are arbitrary cutoffs that define significance regions E ≤ 0.1 and Baggerly ≤ 0.01. The green line is a LOWESS trend fit. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Illustration of Maximum *a Posteriori*parameter estimation.**The maximum a posteriori parameters of Eq.12 in an artificial example it is shown. The bi-dimensional pdf from which \"hat\" (pointed by the arrow) parameters are extracted is proportional to that described in Eq.8. :::  ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Snapshot of the web-interface for our SAGEbetaBin method.**An illustration of on-line tool implemented to make our method easily available it is shown. Researchers submit their data (A) and receive, by e-mail, an alert when we finished the job along with instructions to get results in a password-protected web-page (B). If the ID supplied is a human tag (C), then results are linked with SAGE genies\' tag-to-gene map. The individual observations graphics, as utilized in this work, is available on-demand (D). :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Brain tumor and normal libraries from SAGE Genie used as real data application. ::: **\#** **Library Name** **GEO accession** **Total Tags** -------- ------------------------------------------------ ------------------- ---------------- 1 SAGE\_Brain\_astrocytoma\_grade\_III\_B\_H1020 GSM697 51573 2 SAGE\_Brain\_astrocytoma\_grade\_III\_B\_H970 GSM14763 106982 3 SAGE\_Brain\_astrocytoma\_grade\_III\_B\_R140 GSM14773 118733 4 SAGE\_Brain\_astrocytoma\_grade\_III\_B\_R927 GSM14766 107344 5 SAGE\_Brain\_normal\_cerebellum\_B\_1 GSM761 50385 6 SAGE\_Brain\_normal\_cerebellum\_B\_BB542 GSM695 40500 7 SAGE\_Brain\_normal\_cortex\_B\_BB542 GSM676 94233 8 SAGE\_Brain\_normal\_cortex\_B\_pool6 GSM763 62451 9 SAGE\_Brain\_normal\_peds\_cortex\_B\_H1571 GSM786 77554 10 SAGE\_Brain\_normal\_substantia\_nigra\_B\_1 GSM14796 42498 11 SAGE\_Brain\_normal\_thalamus\_B\_1 GSM713 24015 :::

PubMed Central

2024-06-05T03:55:47.711674

2004-8-31

PMC517708

Background ========== DNA microarray technology is now playing an increasingly important role in biomedical research. Microarray technology gives one the opportunity to measure gene expression levels of thousands to tens of thousands of genes simultaneously, in order to study the differential gene expression pattern between different developmental stages, diseases states and samples treated with drugs or other compounds. Before comparing data from different arrays to address these biological questions, however, a \"much more mundane but indispensable \" normalization step \[[@B1]\] is currently used in most microarray analyses. Because of the slight difference in RNA quantities, imaging settings and other variables, even in very controlled experiments the intensity levels from different arrays are of different scales and need to be normalized before they can be compared with each other. Various normalization methods have been developed and some are widely used. The simplest method is total intensity based normalization \[[@B2]\]; this approach scales intensity levels of every gene by a constant factor so that total intensities of all the arrays are the same. \"Spiked-in\" based normalization methods scale intensity based on spiked-in standards \[[@B3]\]. Nonlinear normalization methods use local regression to scale intensities to compensate for the intensity-dependent differences between arrays \[[@B4]-[@B6]\]. For most current applications, these normalization methods seem to be adequate. However, the residual left by a less than perfect normalization procedure is another source of non-biological variation that is usually non-desirable, especially when the differences in expression levels are expected to be small \[[@B7]\]. In addition, if the goal is meta-analysis of multiple sets of microarray data \[[@B8],[@B9]\] systematic differences between experiments may result in a normalization artifact. We were therefore interested in developing an approach to analyse microarray data without first performing a normalization step. Our approach was partly inspired by non-parametric statistical methods \[[@B10]\]. For example, nonparametric methods that use ranks \[[@B11],[@B12]\] to compare microarray results, in addition to being distribution free, have the additional advantage of being normalization free. DNA microarray technology has been used widely in biomedical studies. One interesting application is in the area of molecular classification; one popular use is in the comparison of tumor samples. Since clinical and histopathological classification is sometimes difficult and labor-intensive, the use of genome wide expression patterns to classify tumor samples has recently become a very active research area \[[@B13]-[@B16]\]. Although some tumors appear to be amenable to classification using microarray data \[[@B17],[@B18]\], general multiple tumor classification using microarray data has proved to be an interesting and challenging task for several reasons: the general difficulties inherent in multi-class classification problems, the small number of samples available, and the inherent biological variation between specimens, *etc*. We decided to use multi-class tumor classification as a test case to illustrate the power of our approach. We compared our results for a multi-class tumor classification problem with more conventional approaches published by Ramaswamy *et al*. \[[@B19]\] and Yeang CH *et al.*\[[@B20]\]. These authors compared the accuracies of using k-Nearest Neighbors (kNN, 60--70%), Weighted Voting (WV, 60--70%) and Support Vector Machine (SVM, 80%) algorithms in a multi-class tumor classification problem and concluded that SVM is a more powerful machine learning algorithm for this application. Results ======= Normalization Free approach to microarray data analysis ------------------------------------------------------- Generally, measurements on single microarrays give a real-valued intensity level x~i~(1\<= i \<= N) for each gene i on the array, where N is the total number of genes on the array. Without first doing some type of normalization, the intensity level of gene i from array A, x~i~^A^, cannot be directly compared with the intensity level of gene i from array B, x~i~^B^. In this study, we sought an alternative quantity or quantities that can be directly compared between different arrays without compromising important biological information. One obvious candidate is r~i~, the rank of intensity level of gene i on the array. However, we felt that rank is not an adequate measure because information about relative expression level is not represented explicitly. Instead, we decided to use the following measures. Let s~ij~= 1 if(x~i~- x~j~\> 0) 0 otherwise     (1) , where 1\<= i, j \<= N. Basically, s~ij~is the sign of the intensity difference of gene i and j on a single microarray and therefore will remain unchanged under any monotonic transformation of x. Therefore, instead of computing with the absolute expression level of a gene, its relative level to all the other genes on the microarray is used. For each gene i, instead of one real valued x~i~, the approach uses s~i~= (s~i1~, \..., s~ij~, \..., s~iN~), a binary vector of size N. For ease of reference, we will simply refer to this value as the SED (Signs of Expression Difference) of gene i; and the entire matrix (s~ij~) the SED of the array. Given (x~i~), s~ij~is simply and uniquely defined but (s~ij~) does not uniquely determine x~i~so some information is lost by only using (s~ij~) instead of (x~i~). Since r~i~= Σ~j\ =\ 1,\...,N~s~ij~is the rank of gene i in terms of intensity levels, rank information is preserved in (s~ij~). What is lost in the transformation from (x~i~) -\> (s~ij~) is just the intensity differences between the closest ranked genes, which in most cases are small, considering that microarray data are generally considered \"very noisy\". It was our major goal to demonstrate that (s~ij~) has indeed captured important components of the information from (x~i~). Instead of directly using the intensity levels x, and its derivatives such as the mean μ, the standard deviation σ and the signal to noise ratio (S2N) between two sample groups A and B, \[(μ^A^~+~μ^B^)/(σ^A^+ σ^B^)\], we will use (s~ij~) to compare gene expression differences between arrays. Since we expect measurement variations within an array will be less than those between arrays and we take the signs of relative expression differences to get SED, we expect the SED will be less \"noisy\". However, the value of any single s~ij~may still vary between technical and biological replicates. One would expect more s~ij~would change values randomly if the technical replicates was done on arrays that were fabricated in a different run than arrays from same run, for example. Biological variations are expected to be even more frequent. However, we hypothesize that we can perform statistical analysis on the SED, which contains tens of thousands of s~ij~for a single gene, and minimize the impact of such noises. We will also consider (sp~ij~), a natural generalization of the SED concept. Here, sp~ij~is the probability of x~i~\> x~j~. In other words, imagining one can get a large number, n, of either technical or biological replicates of the sample of interest, then sp~ij~= m/n as n -\> 8, where m = Σ~K\ =\ 1,\...,n~s~ij~^K^and s^K^is for replicate K. We will call (sp~i~) = (sp~i1~, \..., sp~ij~, \..., sp~iN~) the SED probabilities of gene i. Note that in calculating both SED and SED probabilities, only intensity comparisons within arrays are involved and therefore forego the normalization step. For example, if gene i is more highly expressed in sample A than B we would expect that more s~ij~^A^than s~ij~^B^would be 1 instead of 0 and the overall (very loosely defined) sp~ij~^A^would be larger than sp~ij~^B^. Since rank can be calculated from SED, any rank based method can be expanded to use SED. A gene i\'s SED can be viewed in two different perspectives. On one hand, it provides information about gene i\'s expression level relative to every other gene on the array, and therefore can be used to examine gene i\'s expression patterns between samples. On the other hand, it also provides information about the expression levels of all the other genes on the array, using the gene i as a control, in essence. Therefore, SED can be used to study questions either at the gene level or at the array level. In this paper, we focus on solving a simpler problem at the array level where it is not necessary to decide whether the expression level of an individual gene is increased or decreased between array A and B and by how much. Rather, it is focused on whether the overall expression patterns are different at all between array A and B. Multi-class classification of tumor samples ------------------------------------------- To test whether (s~ij~) and (sp~ij~) extracts most of the information from (x~i~), we used these values in a test case of a multi class classification problem described by Ramaswamy *et al.*and Yeang *et al*\[[@B19],[@B20]\]. Two algorithms were used to classify each of the 144 tumor samples into one of 14 tumor classes. One is the Naive Bayes (NB) classifier \[[@B21]\] using SED probabilities. The other is the Nearest Neighbor (NN) classifier using SED. In the NB method, to classify a sample T, we first calculate (sp~ij~^C^) for each class C (1 \<= C \<= 14) using training samples, i.e. the 144 samples with T taken away (for details see methods). Then sample T is classified according to: score(T, C)~=~Σ~i\ =\ 1,\...N~Σ~j\ =\ 1,\...,N~log(p~ij~),     (2) where p~ij~= sp~ij~^C^if s~ij~^T^= 1 1 - sp~ij~^C^otherwise T is simply classified to the class C that has the maximum score. In the NN method, we compute instead, for each training sample t, matches(T, t) = Σ~i\ =\ 1,\...N~Σ~j\ =\ 1,\...,N~δ(s~ij~^T^, s~ij~^t^),     (3) where δ(x, y) = 1 if x = y. 0 otherwise Then T is classified to class C of the sample t that has the maximum matches. If one is to give a statistical interpretation of these scores, one can simply view (sp~ij~) as defining a multi-binomial probability model. In addition, one could consider each s~ij~as a draw from a binomial distribution with probability p~ij~= sp~ij~. Then, the score(T, C) is simply a logarithm of the probability p to get all the s~ij~exactly the same as s~ij~^T^under the probability model C where p~ij~= sp~ij~^C^. (Since each class defines a different probability model, score(T,C) for difference class C, in theory, should not be directly compared. Instead, a P value should be calculated from the probability model for Pr(p\<exp(Score(T, C))) and used to evaluate the closeness of sample T to each class C. For simplicity, we are not considering such issues here.) When the NB algorithm was applied to the 144 samples, the accuracy obtained was about 63%; the NN algorithm performed slightly better and gave an accuracy of 70%. Feature selection ----------------- Depending on the algorithm, a better classification result can sometimes be obtained by using a subset of genes \[[@B22],[@B23]\]. We were interested to know whether feature selection helps to increase accuracy in our approach. Within our framework, it is easier to treat (i,j) pairs as selection units. We therefore filtered out (i,j) pairs where the variance of sp~ij~across the 14 tumor classes was less than a pre-determined value and left the rest of the algorithm unchanged. We reasoned that the filtered-out part of the matrix has less discriminating power across the tumor spectrum and might add noise due to the small sample sizes used. Using the NB algorithm, the best result achieved was 70% with a cutoff σ^2^= 0.06, while the NN method with σ^2^= 0.05 gave 77%. Table [1](#T1){ref-type="table"} lists the accuracies achieved using different feature cutoffs. \"Single gene\'s SED\" based classifier --------------------------------------- The above procedure utilized the entire SED matrix as a classifier. In other words, all the relations between genes were considered in the classification. To determine whether the inclusion of the whole matrix was actually required to achieve the current accuracy, we investigated the efficacy of using single gene\'s SED as classifiers. In these cases, we define a classifier based on gene i and its relative expression to every other gene. Therefore, the score~i~(T, C) = Σ~j\ =\ 1,\...,N~log(p~ij~), where p~ij~is as same as mentioned previously. Similarly, matches~i~(T, t) = Σ~j\ =\ 1,\...,N~δ(s~ij~^T^, s~ij~^t^) defines a classifier based on gene i\'s SED. Fig. [1](#F1){ref-type="fig"} shows a display of the cumulative frequency of single gene SED based classifiers versus accuracy for all the 16063 classifiers. In general, single gene-based classifiers performed worse than the whole genome-based classifiers, as expected. Nevertheless, most of the classifiers performed reasonably well, compared with just using single gene expression levels. About 80% of the single gene based classifiers resulted in an accuracy between 40% and 60%, while about half of the classifiers had an accuracy greater than 50%. These results suggest that there is a lot of redundant information in the SEDs and SED probabilities and that our method should be reasonably robust. We then investigated the number of genes that are required to achieve the current accuracy. Fig. [2](#F2){ref-type="fig"} shows the combined results for classifiers using only a subset of genes. Our results suggest that a subset of genes (\~200) is sufficient for predictions and that the prediction accuracy is stable after 1000 genes. Different classification accuracy between tumor classes ------------------------------------------------------- From the analyses described above, we noticed that there was a significant difference in accuracy between different tumor classes. For 3 classes (LY, LE, CNS) we obtained either 100% or close to 100% accuracy (see Table [2](#T2){ref-type="table"} for detail). Since these happen to correspond to the 3 classes with more than double the number of training samples than the other classes, we tested whether this high accuracy is due to the larger sample sizes by using only 8 training samples for every tumor classes. The results were essentially the same, indicating that sample size is not the issue. On the other hand, there are classes where we obtained very poor results; these often happen to be the same classes where SVM in \[[@B19],[@B20]\] performed poorly as well (see Table [2](#T2){ref-type="table"}). We were interested in exploring the possible reasons for misclassification. In Fig. [3](#F3){ref-type="fig"}, a scatterplot of the SED Match scores (without feature selection) between 8 OV samples and 8 CNS samples is displayed. The OV and CNS class were selected since one is \"very hard\" and the other is \"very easy\" to classify. Without trying to be statistically correct, the plot does suggest that samples of the OV class are in general \"farther away\" from each other, compared with those from the CNS class. As this may be one of the reasons that the OV class is harder to classify, algorithms that take this kind of information into account may perform better than the simple ones we have presented here. Discussion ========== Although we used the multi-class tumor classification problem as our test case, our major goal was to illustrate the feasibility of the normalization free SED approach, and not in sample classification *per se*. Therefore, we chose the algorithms NB and NN for their simplicity and not for their performance in solving this specific problem. The performance of a classifier depends, in this case, mainly on the power of its algorithm, and the data representation it used. From a machine learning perspective, one can simply view the intensity -\> SED transformation as a change in data representation, a mapping from the gene\'s attribute, intensity x, to some features SED. It was our goal to demonstrate that the new features (SED and SED probability), in addition to being normalization free, still convey the essential information in the original attribute, the intensity x. Since the data representation is quite different between the intensity x (a real valued quantity) and SED (a binary valued vector of rather large size N), it is difficult to directly compare the two. No obvious yet non-trivial algorithms work with both representations; even if there were such an algorithm it is not clear that it would be the right one to use for comparison as it might well be the case that different data representation works best with different algorithms. Here, we have limited the presentation to some empirical results with SED representation, which are comparable with results using several different algorithms that are based directly on raw intensity \[[@B19],[@B20]\]. Our classification results are close to those obtained with WV and kNN methods, which are based on directly focusing on intensity levels. Previous results using SVM were significantly better, but we feel the differences are due more to the power of the algorithm \[[@B24]\] than the way information is coded. In fact, slightly more accurate results are obtained with modification of algorithms that directly manipulate intensity levels \[[@B25],[@B26]\]. We do not imply that the algorithms (NB and NN) we chose are better than other alternatives (and we do not have empirical evidence pointing either way). Instead, we fully expect more sophisticated algorithms would work better with the SED approach as well. Certainly, SED probability is more information rich than SED. We expect that an SED probability based analysis would perform better than the simple binary valued SED. In this paper, we mainly tested the SED. SED probability is only used for a group of samples, not for single samples. If one limits oneself to use only raw data, then for single arrays one can only get SED. However, if some assumptions about the patterns of gene expression levels can be made, one can certainly get an estimation of SED probability even for a single array. For example, as in some nonlinear normalization algorithms, if one assumes that the variation of expression levels are similar for genes with similar expression levels, then one can estimate SED probability from a probability model. Also, the magnitude of the intensity difference can also be used to help such an estimation. Alternatively, as more and more microarray data become available, one can use other similar samples to get an estimation of a prior SED probability, and then use a Bayesian approach to estimate the sample\'s SED probability. The obvious disadvantage of our SED based approach is that for each gene expression level, one is not dealing with a single real number but instead a vector of size N, where N is in the tens of thousands. This could significantly increase both computing time and memory requirement (however, see methods for details) On the other hand, it also has certain advantages: 1) It is free of normalization noise. Since it is generally believed that biological variation is larger than technical variation and normalization noise is just another source of technical variation, the benefit here is only of a limited scope. However, it may be important when the expression level difference one is interested in is small. 2) In addition to being normalization free, SED and SED probability also have the advantage in being distribution free, and therefore could perform better if the intensity levels were non-normal. 3) SED and SED probability are easier to interpret. SED values can easily be checked against raw intensity levels according to Eq. (1). While SED probability is one step further away from intensity levels, one could still have an intuitive sense of it and make comparisons between different experiments. It would be much harder to have a real grasp of the absolute gene expression level, except that it is \"high\" or \"low\" or somewhere \"in between\"; it is certainly harder to compare between experiments intuitively. We have only tested the SED approach on datasets that are from the same chip format. Data from different chip formats or complete different technology platforms, of course, would be harder to compare. But they are also challenge for normalization based method. It would certainly be interesting to compare SED and normalization based method under these more challenging conditions. If this normalization-free approach (SED) proves to retain the essential biological information in general, its application may be extended to meta-analysis where different datasets could be integrated and intervalidated. The method could also be used when the number of arrays is a limiting factor for experiments. For example, one could take advantage of the massive amount of public array data, obtain prior distribution of SED probabilities from datasets with similar conditions, and analyze new data within a Bayesian framework. If the performance of the nearest neighbor method in general is anywhere close to what we demonstrated in the multi tumor classifications here (as is clear from Eq. (3) and Fig. [3](#F3){ref-type="fig"}, the nearest neighbor method, without feature selection at least, allows direct sample vs. sample comparison. Note also that the samples in the multi tumor problems are from different biological specimens, therefore, large between-sample-variation is expected), it might be used as a microarray database query method, *i.e.*, to find similar microarray results in the database that are \"similar\" to one\'s own, independent of array annotations. It might also be worth noting that the SED approach could easily be applied to other kinds of comparative data analysis for samples with very large numbers of \"noisy\" attributes. The SED approach may also perform better when between-sample-variation is large, especially if such variation contains some rather uninteresting technical measurement errors that would not affect within-sample-variations. Conclusions =========== We have proposed a new approach to analyze microarray data and tested the method on a set of publicly available datasets. The results were comparable to those obtained with some widely used normalization based algorithms. We hope that we have demonstrated that this normalization free method is feasible and promising. We think the SED based, normalization free approach could be used to complement the more popular normalization based approaches in microarray data analysis. Methods ======= Microarray data for multiple tumor samples were downloaded from <http://www.broad.mit.edu/cancer/software/genepattern/datasets/>. Naive Bayes and Nearest Neighbour Classifiers were implemented in the Java programming language. *Ad hoc*analysis was done with perl scripts. Graphics were generated using the R computing environment. Naive Bayes method ------------------ Because of the uneven and relatively small sample sizes for each tumor class (mostly 8 but up to 24), extra care was taken in computing sp~ij~. Assuming a prior probability of 0.5, sp~ij~was estimated by Bayesian posterior probability (m+1)/(n+2) where n is the total number of samples in the class and m is the total number of samples where x~i~\> x~j~. For classes that were over-represented (sample size \> 8), the threshold of sp~ij~was set to \[0.125, 0.875\], since the NB method is sensitive to the extreme values of sp, and samples can be over-predicated without thresholds. In addition, several alternatives were tested to demonstrate that our results were reasonably robust and not sensitive to the particular choices we made: 1\) To examine the influence of the sample size, in a separate analysis the sample size of ME, LE, CNS class was artificially reduced to 8, *i.e.*only the first 8 samples were used to calculate sp~ij~with no significant change of results observed; 2\) Since sp~ij~depends on the sample size n for each tumor class, we have applied a \"sample replacement\" strategy in addition to the usual \"take-one-sample-out\" approach for cross-validation, *i.e.*when one sample is taken out as the test sample, another sample from the same class is duplicated to take its place to keep the sample size constant. Essentially the same results were obtained. Results reported are from the sample replacement runs. In Feature Filtering, the variance of sp~ij~between all 14 classes was calculated as: σ^2^= (Σ~C\ =\ 1,\...,14,~sp~ij~^C^\* sp~ij~^C^)/14 - ((Σ~C\ =\ 1,\...,14~sp~ij~^C^)/14)^2^    (4) with the test sample taken out, and used as the criterion for feature exclusion. Nearest neighbor method ----------------------- Feature filter was done as in the Naive Bayes Method. Software implementation and availability ---------------------------------------- The analysis was done on a computer (Pentium M 1.5 GHz) operating under Microsoft XP. Both Naïve Bayes and Nearest Neighbor Classifiers are implemented in Java. Since SED can be easily calculated from the raw intensities only the later are kept in memory and SED are computed from the intensities on an as-needed bases. Memory needed to analysis the 144 samples is less than 64 MB. The most computationally intensive algorithm that we tried is the Nearest Neighbor method without any feature selections and it takes about 10 sec to calculate SED score for one pair of tumor samples with about 16000 genes. A Java program named SED (including source code) to perform nearest neighbor analysis of microarray samples is freely available by contacting author at <hw14@columbia.edu>. Authors\' contributions ======================= H.W. conceived of the SED study and performed implementations. H.H. refined the approach and provided additional statistical insight on SED. Both authors read and approved the final manuscript. Acknowledgements ================ The authors wish to thank Dr. Randall D. Little for his careful reading and extensive editing of the manuscript. This work is supported by grant R44 AG022242-01 from NIH National Institute of Aging of the National Institutes of Health, USA Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Cumulative Frequency of Single Gene SED classifiers versus cross validation accuracy.**The X axis represents the cross validation accuracy and the Y axis represents the cumulative frequency of the single gene based classifiers. For each point (x,y) on the curve, y equals the percentage of classifiers that have an accuracy less than or equal to x. Solid curve -- NN method with cutoff σ^2^= 0.05. Dashed line -- NB method with σ^2^= 0.06. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Evaluation of the number of genes required to achieve the highest accuracy.**The X axis represents the number of genes used in the classifiers. The Y axis represents the cross validation accuracy. Solid line -- NN method with cutoff σ^2^= 0.05. Dashed line -- NB method with σ^2^= 0.06. X is in log scale. As X increases more genes are included in the classifier in the order they are represented in the original microarray (unselected for performance). :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Scatterplot of SED Match scores of OV and CNS samples.**The X axis represents the SED Match scores from eq. (3) / N^2^, where N = 16063, the total number of genes. The Y axis represents the sample id. Samples 1--8 (shown in red) are from class OV while 9--16 (shown in blue) are from class CNS. Only 8 samples from the CNS class were used for ease of comparison. Other samples gave similar results. For each sample, its Match scores / N^2^against all the other 7 samples within the class are shown. The Match scores of OV class are much more dispersed (to the left), compared with that of CNS class. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The relationship between accuracy (%) and the filter threshold The cross validation accuracies with Naive Bayes (NB) and Nearest Neighbor (NN) Methods are displayed with different filter cutoffs. The percentages of the features (gene pairs) used are listed as well. Since the number of features used are slightly different for different test samples, ranges are shown. ::: Filter Threshold No Filter 0.02 0.04 0.05 0.06 0.08 ------------------- ----------- -------- -------- ---------- ---------- ------------ NB 63 66 68 69 70 68 NN 70 73 75 77 75 74 Features used (%) 100 41--43 12--13 4.9--5.9 1.7--2.1 0.12--0.17 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Summary of cross validation results. All 14 tumor types and their abbreviations are listed in the first column. The sample sizes for each tumor type and the number of successfully classified samples by the SVM algorithm (from ref. \[19\]), the NB (Naive Bayes) algorithm with cutoff σ^2^= 0.06 and the NN (Nearest Neighbor) algorithm with σ^2^= 0.05 are listed. ::: Tumor type (Abbreviation) Sample Size SVM \[19\] NB NN ------------------------------ ------------- ------------ ---- ---- Ovary (OV) 8 3 1 4 Lung (LU) 8 4 1 3 Bladder (BL) 8 5 7 5 Melanoma (ML) 8 5 6 6 Renal (RE) 8 5 5 6 Pancreas (PA) 8 5 6 6 Colorectal (CR) 8 6 1 3 Prostate (PR) 8 6 6 6 Breast (BR) 8 7 3 4 Uterus (UT) 8 7 6 6 Mesothelioma (ME) 8 8 6 6 Lymphoma (LY) 16 16 14 16 Central Nervous System (CNS) 16 16 16 16 Leukemia (LE) 24 24 23 24 :::

PubMed Central

2024-06-05T03:55:47.714644

2004-9-2

PMC517709

Background ========== A bioinformatic approach to identifying cis-regulatory elements controlling transcription has become feasible with the availability of complete genome sequences and large scale expression data using high-throughput methods such as microarrays \[[@B1],[@B2]\] and SAGE \[[@B3]\]. The expression data provides a list of genes whose expression is significantly modified under a particular condition. However, this data does not indicate whether these genes are direct targets of a particular transcription factor or if the changes in expression are the result of an indirect effect caused by altering the expression of other transcription factors that work directly at the promoter. Using information about sequence preference for binding of particular transcription factors, one can identify possible regulatory binding sites within a sequenced genome. However, this approach does not indicate if the sites are functional. We have therefore developed an algorithm that combines both of these approaches to distinguish between the direct and indirect targets that are regulated by a particular transcription factor. We have applied this methodology to study the transcriptional regulatory system that specifies cell mating-type in the yeast *Saccharomyces cerevisiae*\[[@B4]\]. Yeast have three cell types, haploid **a**and *α*cells, and the **a**/*α*diploid, that differ in their ability to mate and in the proteins they express. Cell mating-type is determined in part by *α*2 and **a**1, which are cell-type-specific proteins that are members of the homeodomain (HD) DNA-binding family. In an **a**/*α*diploid cell, *α*2 binds with **a**1 to form a heterodimer complex that represses transcription of haploid-specific genes \[[@B5]\]. The crystal structures of the *α*2 HD binding DNA alone and in complex with **a**1 have been solved, providing models for how these complexes bind DNA \[[@B6],[@B7]\]. Biochemical and mutational analysis of each protein and their DNA-binding sites have defined the requirements for DNA recognition by this complex \[[@B8]-[@B10]\]. Genome-wide expression analysis has also been performed on each of the different cell types \[[@B11]\]. The combination of these resources has allowed us to develop and test algorithms to identify target sites for the **a**1-*α*2 complex. Previous work, using a relatively simple binding site search program identified targets for the *α*2-Mcm1 complex, which represses **a**-cell-type specific genes in *α*and **a**/*α*cells \[[@B12]\]. The more advanced methods described in this paper have helped identify several novel targets of the **a**1-*α*2 complex that may be involved in cell-type specific processes. Interestingly, we identified several genes that are repressed in diploid cells but do not appear to be direct targets of the **a**1-*α*2 complex, suggesting that these genes are controlled by another transcriptional regulatory factor that is directly or indirectly regulated by the **a**1-*α*2 complex. We have also identified a number of **a**1-*α*2 target sites that do not repress adjacent genes. The combination of site preference and expression data is therefore a valuable tool to identify direct functional targets of a transcription factor or complex. Results ======= Development of a search algorithm for targets of the a1-*α*2 complex -------------------------------------------------------------------- To generate an algorithm that combines microarray expression data and mutational analysis of binding sites, we first defined a scoring method that ranks gene expression data. We utilized the microarray expression data from Galitski and coworkers for gene expression in the **a**and *α*haploid and **a/***α*diploid cells, as well as various polyploids \[[@B11]\]. Since the **a**1-*α*2 complex should be absent in any of the homozygous **a**or *α*type polyploids (**a, aa**, \..., *α,αα*,\..., etc.) we expect the expression of haploid-specific genes in these cells to be much higher than in cells that are heterozygous for the *MAT*locus (**a***α*, **aa***α*, **a***αα*, **aa***αα*, etc.). Thus, one term in the scoring function rewards lower expression in heterozygous cell types compared to the homozygous cell types (see the Methods section for details). We expect that most of the haploid-specific genes will be expressed equally in both of **a**and *α*cell types. Consequently, we have introduced a second term in the scoring function that penalizes such differences in expression in the two haploid cell types. This scoring function would identify haploid-specific genes that are repressed in diploid cells, but would not indicate if these genes are direct targets of the **a**1-*α*2 repressor complex. To identify genes from this ranking that are directly repressed by the **a**1-*α*2 complex we used the available mutational data on the **a**1-*α*2-binding site \[[@B9]\]. In these experiments, the effects of single base pair mutations of the **a**1-*α*2 consensus binding site were measured by assaying their ability to repress transcription of a heterologous promoter and by electrophoretic mobility shift DNA-binding assays (EMSA). Under the assumption that the level of expression is proportional to how often that site is unoccupied, we used the effects of the single base mutations to estimate the parameters for the binding energy of sites with different bases at each position. We then used this information to search for potentially strong binding sites in the promoter regions (in practice, 800 bp upstream of the translation start site) of every gene in the genome. This search provides us with a list of genes with putative **a**1-*α*2 binding sites in the promoter, irrespective of functionality. Even under ideal conditions, either of the above lists of genes would not directly indicate a haploid-specific gene directly repressed by the **a**1-*α*2 complex. In addition, our accuracy is limited by the noise in the microarray expression data, as well as by simplifying assumptions made to utilize single-base mutational data to score arbitrary sequences. We therefore decided to rank the genes using a scoring system that takes into account expression patterns across different mating types, as well as the likelihood of finding a good **a**1-*α*2-binding site in the promoter region of the gene. To test the significance of these composite scores, we generated permuted data, which combined random promoters with the expression data. This analysis suggested that the top 10--15 predictions were significant. The results of experiments on the 24 genes with the highest combined scores from our analysis are shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Potential **a**1-*α*2 Binding Sites in Haploid-specific Genes ::: ORF Gene Expression p-val^a^ Binding p-val^b^ Combined p-val **a**1-*α*2 ChIP^c^ -------------- --------- --------------------- ------------------ ---------------- --------------------- *YDL227C* *HO* 0.0006 0.0017 1.1e-6 +^f^ *YLR265C* *NEJ1* 0.0003 0.0053 1.7e-6 \+ *YBL016W* *FUS3* 0.0001 0.0991 1.6e-5+ *YOR212W* *STE4* 0.0020 0.0082 1.7e-5 \+ *YJR086W* *STE18* 0.0008 0.0218 1.7e-5 \+ *YHR005C* *GPA1* 0.0005 0.0437 2.1e-5 \+ *YDR103W* *STE5* 0.0017 0.0298 5.2e-5 \+ *YBR073W* *RDH54* 0.0053 0.0116 6.2e-5 \+ *YGR044C* *RME1* 0.0009 0.0720 6.8e-5 +^f^ *YGL248W* *PDE1* 0.0182 0.0040 7.3e-5 \+ *YPL038W* *MET31* 0.0292 0.0027 8.0e-5 \+ *YDR088C* *SLU7* 0.0303 0.0038 1.2e-4 \- *YGL052W* 0.0117 0.0109 1.3e-4 \- *YJL157C* *FAR1* 0.0013 0.1141 1.4e-4 \+ *YPR122W* *AXL1* 0.0091 0.0163 1.5e-4 +^f^ *YIL099W* *SGA1* 0.0063 0.0267 1.7e-4 \- *YLR233C* *EST1* 0.0226 0.0090 2.0e-4 \- *YKL182W* *FAS1* 0.0578 0.0035 2.1e-4 \- *YMR053C* *STB2* 0.0028 0.0884 2.5e-4 \- *YNL319W* 0.0123 0.0222 2.7e-4 \- *YFR012W* 0.0088 0.0125 2.8e-4 \- *YNL188W* *KAR1* 0.0019 0.1890 3.6e-4 \- *YGL193C* 0.0014 0.2654 3.8e-4 \- *YMR157C* *FMP39* 0.1557 0.0026 4.0e-4 \- *YBR158W*^d^ *AMN1* 0.0037 0.0221 8.7e-5 \+ *YCL066W*^e^ *MATα1* 0.1630 0.9510 1.5e-1 +^f^ ^a^The ranking of haploid-specific gene expression determined by analysis of microarray data \[11\]. ^b^The ranking of potential **a**1-*α*2 target of haploid-specific genes. ^c^A + indicates that the **a**1-*α*2 binds to the promoter by ChIP assay. ^d^Identified in a search for sites with 1500 bp from the start of the ORF ^e^Identified if remove the penalty for expression in one haploid cell type but not the other. ^f^Identified as direct targets of the **a**1-*α*2 repressor complex in previous studies. ::: **a**1-*α*2 binding to the promoter regions of genes identified in the search ----------------------------------------------------------------------------- To evaluate the success of our computational algorithm for identifying direct targets of the **a**1-*α*2 repressor complex, we assayed binding by the complex to the identified promoters using chromatin-immunoprecipitation (ChIP) assays with polyclonal antibody directed against the *α*2 protein. In *α*haploid and **a/***α*-diploid cells the *α*2 protein combines with the MADS-box transcription factor Mcm1 to bind to elements in the promoters of **a**-specific genes to repress their transcription \[[@B13],[@B14]\]. We therefore included in each PCR a primer set for the promoter region of the **a**-specific gene *STE6*to serve as a positive control for the ability to ChIP *α*2 in both haploid *α*and diploid **a/***α*cells. This primer set also allowed us to rule out the possibility that the predicted **a**1-*α*2 target genes were immunoprecipitating because of binding by the *α*2-Mcm1 complex. The primer set for the *YDL223C*promoter, a gene not bound or repressed by the **a**1-*α*2 or *α*2-Mcm1 complexes, was included in the reaction as a negative control for non-specific immunoprecipitation of the DNA. A gel displaying the results for a few of the promoters that were assayed is shown in Figure [1](#F1){ref-type="fig"} and the data summarized for all the promoters that were predicted to be directly repressed by the **a**1-*α*2 complex is listed in Table [1](#T1){ref-type="table"}. In general, there is a very good correlation between the experimental data with the predictions based on our computational algorithm. Almost all of the high scoring genes were bound by the **a**1-*α*2 complex in vivo. Genes that had a combined p-value higher than the threshold of 1.5 × 10^-4^(which corresponds to choosing the top 15 of the list in Table [1](#T1){ref-type="table"}) do not appear to be strongly bound by the complex. This p-value corresponds to roughly a probability of one in six thousand, indicating it is possible to get such combination by chance. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### ChIP assays of promoter fragments that are predicted to be targets of the **a**1-*α*2 complex. ChIP assays with antibody to the *α*2 protein were performed on lysates from *MAT**a***/*MATα*(**a**/*α*) and *mat**a***Δ/*MATα*(Δ/*α*) cells. Total chromatin (TC) and immunoprecipitated (IP) samples were subjected to multiplex PCR with primers flanking potential **a**1-*α*2 sites (labeled *hsg*) in the indicated promoters. Primers for the promoter region of *STE6*, an **a**-specific gene that is repressed by the *α*2-Mcm1 complex in both cell types were used as a positive control for the ChIPs. Primers that hybridize in *YDL223C*, a gene that is not regulated by *α*2, was used as the negative control. The presence of a band over background levels from the test promoter from **a**/*α*lysates but not *mat*Δ/*α*indicates that the **a**1-*α*2 complex is specifically binding to the promoter. Assays for the *GPA1, FAR1, NEJ1, RDH54, MET31, PDE1, AMN1/CST13*and *KAR1*are shown. A summary of the all of the ChIPs performed is in Table 1. :::  ::: In comparison to higher eukaryotes, most yeast promoters are relatively small and contain activator or repressor binding sites within several hundred base pairs of the start site of the open reading frame (ORF) of the gene. However, there are a few genes, like *HO*, whose regulation is controlled by a region several Kb long. Consequently, we did a separate search looking for additional **a**1-*α*2 binding sites that are within a region 1.5 Kb upstream of the ORF. Most of the sites identified in this search were well above the threshold value and were not bound by the **a**1-*α*2 complex in ChIP assays (data not shown). However, the search identified one site upstream of the *AMN1/CST13*gene that was a potential target site. The ChIP analysis verified this as a functional target site for the **a**1-*α*2 complex in vivo (Figure [1](#F1){ref-type="fig"}, Table [1](#T1){ref-type="table"}). Analysis of haploid-specific genes predicted not to be bound by **a**1-*α*2 --------------------------------------------------------------------------- Among the top 35 genes in the list ranked by haploid-specific expression score, more than half do not appear to contain an identifiable **a**1-*α*2 site by our analysis (lack of a significant binding site being defined as binding p-value greater than 2 × 10^-4^) (Table [2](#T2){ref-type="table"}). Although there were no apparent strong affinity **a**1-*α*2-binding sites in the promoters of these genes, it is possible that there are several weak affinity sites in the promoter that were not identified in the search. If present, these sites may work cooperatively to increase binding by the **a**1-*α*2 complex to the promoter and therefore repress transcription. In support of this model we have found that under some conditions weak affinity **a**1-*α*2 sites in the *HO*promoter have a role in repression of the promoter (Mathias and Vershon, unpublished). Promoters with a number of weak affinity sites may therefore be directly regulated by the **a**1-*α*2 complex. However, it is also possible that these genes are indirectly repressed by **a**1-*α*2, through its ability to repress expression of another transcription factor that is required for expression of the genes identified in the microarray. To distinguish between these possibilities we assayed for **a**1-*α*2 binding to these promoters by ChIP (Figure [2](#F2){ref-type="fig"} and Table [2](#T2){ref-type="table"}). As predicted from the site identification analysis, the **a**1-*α*2 complex did not appear to bind to most of these promoters. This result suggests that these genes are not directly repressed by the complex. The one exception to our predictions was that the **a**1-*α*2 complex appeared to weakly bind to the *NEM1*promoter in the ChIP assays (Figure [2](#F2){ref-type="fig"}). Interestingly, *NEM1*is downstream of *GPA1*, a gene that is strongly repressed by the **a**1-*α*2 complex (Fig [1](#F1){ref-type="fig"}). Binding and repression by **a**1-*α*2 at *GPA1*may help binding to weak sites in the *NEM1*promoter. Alternatively, although we sheared the DNA used in the ChIP to an average of less than 500 bp, it is possible that there may have been some fragments that spanned the \~2 kb between the genes, thereby giving a positive result in the ChIP assay. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Haploid-specific Genes that Do Not Contain **a**1-*α*2 Target Sites ::: ORF Gene Expression p-val^a^ Binding p-val^b^ Combined p-val **a**1-*α*2 ChIP^c^ ----------- --------- --------------------- ------------------ ---------------- --------------------- *YIL117C* *PRM5* 0.0011 0.3690 0.0004 \- *YLR159W* 0.0015 0.4050 0.0006 \- *YBR051W* 0.0022 0.4390 0.0010 \- *YLR080W* *EMP46* 0.0024 0.6757 0.0016 \- *YIR039C* *YPS6* 0.0025 0.7843 0.0020 \- *YCL014W* *BUD3* 0.0027 0.3475 0.0009 \- *YPL189W* *GUP2* 0.0030 0.3708 0.0011 \- *YJL077C* *ICS3* 0.0032 0.9540 0.0030 \- *YML042W* *CAT2* 0.0033 0.7564 0.0025 \- *YHR004C* *NEM1* 0.0035 0.6396 0.0022 \+ *YFR046C* *CNN1* 0.0036 0.5712 0.0020 \- *YDR220C* 0.0038 0.5919 0.0022 \- *YPL025C* 0.0041 0.6844 0.0028 \- *YBR006W* *UGA2* 0.0043 0.7684 0.0033 \- *YBR108W* 0.0044 0.4390 0.0019 \- *YFL034W* 0.0046 0.3287 0.0015 \- *YCL027W* *FUS1* 0.0047 0.7170 0.0034 \- *YLR308W* *CDA2* 0.0049 0.3424 0.0017 *YGR014W* *MSB2* 0.0050 0.6554 0.0033 *YJL202C* 0.0052 0.6416 0.0033 ^a^The ranking of haploid-specific gene expression determined by analysis of microarray data \[11\]. ^b^The ranking of potential **a**1-*α*2 target of haploid-specific genes. ^c^A + indicates that the **a**1-*α*2 binds to the promoter by ChIP assay. ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### ChIP assays of **a**1-*α*2 binding to promoter fragments that are not predicted to be targets of the complex. ChIP assays were performed as described in Figure 1. ChIP assays for **a**1-*α*2 binding to the *FUS1*, *BUD3*, *NEM1*, *CAT2*, *YFL034W*and *GUP2*promoters are shown. :::  ::: Comparison with binding site identification by the weight matrix method ----------------------------------------------------------------------- We compare the performance of our algorithm with that of the weight matrix method \[[@B15]-[@B18]\]. In our study, we derived our parameters from a set of artificial sequences. Usually, the weight matrix has to be constructed from a set of known sites. We calculate the weight matrix for **a**1-*α*2 from regulatory elements upstream several known target genes: *HO, GPA1, FUS3, AXL1, STE5, RME1*and *MATα1*. As usual, one is faced with a choice of threshold weight matrix score for selecting putative sites in the yeast genome. For a stringent threshold that corresponds to the top 16 targets, we recovered all the genes, other than *RME1*, used in construction of the weight matrix. However, we did not recover most of the other genuine targets identified, and verified, in this study. If we set the threshold to be lax enough to include *RME1*, we obtained 55 candidate genes, including *STE18*and *RDH54,*but still miss targets like *STE4*. It is likely that most of the 55 putative targets are false positives, as evidenced by lack of haploid-specific regulation in the corresponding gene expression data. Overall, we find our method to be more successful than the weight matrix method. The use of mutational data as opposed to literature based data for sequence preference possibly accounts for part of the success (an advantage we may not have for some other transcription factors). However, much of our success has to do with cutting down of false positive rates by using microarray data judiciously. Analysis of all potential **a**1-*α*2 target sites in the genome ---------------------------------------------------------------- Among the genes identified in the computational analysis, there is a good correlation between the presence of strong **a**1-*α*2-binding sites in their promoter region and repression in diploid cells. This raises the question of whether all **a**1-*α*2-binding sites function as repressor sites. To address this question we searched for all potential binding sites in the yeast genome. As expected, many of the best sites are in the promoters of known or previously identified haploid-specific genes (Table [1](#T1){ref-type="table"}). However, we also identified a number of putative **a**1-*α*2-binding sites within ORFs (Table [3](#T3){ref-type="table"}). To test if the **a**1-*α*2 complex is able to bind to these sites we performed electrophoretic mobility shift assays (EMSAs) with purified *α*2 and **a**1 proteins and radiolabeled oligonucleotides containing these sites (Fig [3A](#F3){ref-type="fig"}). The **a**1-*α*2 complex bound to sites from the *YKL162C*, *CDC25*, *PRM8*, *PRM9*, and *URB1*ORFs with weaker affinity than to a strong binding site from the *HO*promoter, *HO(10)*. However, these sites did have slightly better binding affinity than to the *HO(8)*site, which we have shown is unable to repress transcription on its own (Mathias and Vershon, unpublished). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Potential **a**1-*α*2 target sites in ORFs ::: ORF Gene Expression p-val^a^ Binding p-val^b^ Combined p-val **a**1-*α*2 ChIP^c^ EMSA^d^ ----------- ---------- --------------------- ------------------ ---------------- --------------------- --------- *YKL014C* *URB1* 0.621 0.042 0.025 \- 25× *YGL053W* *PRM8* 0.291 0.011 0.002 \- 5× *YAR031W* *PRM9* 0.891 0.013 0.012 \- 10× *YKL162C* 0.782 0.017 0.014 \- 10× *YLR310C* *CDC25* 0.102 0.018 0.002 \- 5× *YPL061W* *ALD6* 0.597 0.018 0.009 \- *YBR028C* 0.793 0.046 0.036 \- *YOL022C* 0.563 0.023 0.013 \- *YJR016C* *ILV3* 0.248 0.025 0.006 \- *YBR218C* *PYC2* 0.324 0.027 0.008 \- *YFR040W* *SAP155* 0.234 0.165 0.038 \- *YMR269W* 0.332 0.049 0.016 \- *YJL129C* *TRK1* 0.297 0.083 0.024 \- *YCR053W* *THR4* 0.607 0.073 0.044 \- ^a^The ranking of haploid-specific gene expression determined by analysis of microarray data \[11\]. ^b^The ranking of potential **a**1-*α*2 target of haploid-specific genes. ^c^A + indicates that the **a**1-*α*2 binds to the promoter by ChIP assay. ^d^Relative binding affinity in fold decrease in affinity compared to the *HO(10)*binding site. ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **a**1-*α*2 binding in vitro and in vivo to sites in the ORF regions of the genome. (A) An EMSA of purified **a**1 and *α*2 proteins binding to a strong **a**1-*α*2 binding site, *HO(10)*(lanes 1--8) and sites from the *YKL162C*(lanes 9--13), *CDC25*(lanes 14--18), a weak **a**1-*α*2 binding site from the *HO*promoter, *HO(8)*(lanes 19--23), *PRM8*(lanes 24--28), *PRM9*(lanes 29--33) and *URB1*(lanes 34--38). The concentration of the **a**1 protein was held constant at 1.4 × 10^-6^M (lanes 2 and 4--38) and mixed with 5-fold dilutions of the *α*2 protein starting at 8.2 × 10^-8^M (lanes 3, 4, 9, 14, 19, 24, 29 and 34)). The EMSAs shown are phosphorimages of the gels. Lane 1 contains *HO(10)*probe alone, and lanes 2 and 3 contain 1.4 × 10^-6^M **a**1 and 8.2 × 10^-8^M *α*2 respectively. The fold repression by each site in the context of a heterologous promoter is shown below each gene/promoter. (B) ChIP assays for the genes *YKL162C*, *CDC25*, *PRM8*, *PRM9*and *URB1*are shown. ChIP assays were performed as described in Figure 1 :::  ::: Since the **a**1-*α*2 complex was able to bind to these sites with weak to moderate affinity in vitro, it is possible these sites may partially repress transcription on their own. To test this model, we cloned these sites into the context of the *CYC1*promoter driving expression of a *lacZ*gene and measured the ability of the sites to repress transcription of the reporter in diploid cells \[[@B9]\]. The sites from the *CDC25*and *URB1*ORFs did not repress transcription of the reporter promoter in diploid cells (Fig [3A](#F3){ref-type="fig"}). However, the site from *PRM8*ORF, which showed the highest binding affinity among the sites found in ORF regions, weakly (2.8-fold) repressed the reporter promoter. This result indicates that this site can function as a repressor site in vivo if placed in the proper context. We next tested whether **a**1-*α*2 bound to these sites in the normal genomic context in vivo by ChIP assays. None of the sites in the ORF regions were bound by the **a**1-*α*2 complex (Fig [3B](#F3){ref-type="fig"} and Table [3](#T3){ref-type="table"}). This result indicates that while they are competent for weak binding and repression in a heterologous promoter, they are unable to repress transcription in their normal genomic context. Our search also identified several potential **a**1-*α*2 binding sites in the promoter regions of genes that do not appear to be repressed in diploid cells (Table [4](#T4){ref-type="table"}). Only the *COX13*site had moderate binding affinity for the **a**1-*α*2 complex in the EMSAs (Fig [4A](#F4){ref-type="fig"}). However, despite the relatively weak binding affinity of these sites, they were able to partially repress transcription of the reporter in diploid cells (Fig [4A](#F4){ref-type="fig"}). In particular, the sites from the *COX13*and *REX2*promoters showed significant levels of repression. Interestingly, although these sites functioned as repressor sites in the context of the heterologous reporter, except for the *COX13*promoter, most of these sites were not bound by the **a**1-*α*2 complex at their genomic locations by ChIP assays (Fig [4B](#F4){ref-type="fig"}). These results suggest that the genomic context of most of these **a**1-*α*2 sites prevents binding by the complex. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Potential **a**1-*α*2 Target Sites in the Promoters of Non-Haploid-specific Genes ::: ORF Gene Expression^a^p-val Binding p-val Combined p-val **a**1-*α*2 ChIP^b^ ----------- --------- -------------------- --------------- ---------------- --------------------- *YGL191W* *COX13* 0.4111 0.0053 0.0021 \+ *YPL099C* *FMP14* 0.1622 0.0075 0.0012 *YLR059C* *REX2* 0.1532 0.0098 0.0015 +/- *YDR212W* *TCP1* 0.5694 0.0017 0.0010 *YJL124C* *LSM1* 0.0830 0.0120 0.0010 +/- *YAR033W* *MST28* 0.7419 0.0133 0.0099 \- *YMR015C* *ERG5* 0.6021 0.0138 0.0083 \- *YMR291W* 0.2659 0.0218 0.0058 \- *YPL188W* *POS5* 0.2648 0.0230 0.0061 +/- *YDR101C* *ARX1* 0.3694 0.0298 0.0110 \+ *YHR058C* *MED6* 0.4507 0.0350 0.0157 *YGL117W* 0.5577 0.0370 0.0206 *YIL027C* *KRE27* 0.5762 0.0381 0.0220 ^a^The ranking of haploid-specific gene expression determined by analysis of microarray data \[11\]. ^b^A + indicates that the **a**1-*α*2 binds to the promoter by ChIP assay. A +/- indicates weak (2-fold) enhancement of the band in **a**/*α*cells by ChIP assay. ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **a**1-*α*2 binding in vitro and in vivo to putative binding sites in promoters associated with genes which are not expressed in a haploid-specific manner. (A) An EMSA of purified **a**1 and *α*2 proteins binding to a strong **a**1-*α*2 binding site, *HO(6)*(lanes 1--7), *COX13*(lanes 8--11), *REX2*(lanes 12--15), *LSM1*(lanes 16--19) and *FMP14*(lanes 20--23). The concentration of the **a**1 protein was held constant at 1.4 × 10^-6^M (lanes 2 and 4--23) and mixed with 5-fold dilutions of the *α*2 protein starting at 8.2 × 10^-8^M (lanes 3, 4, 8, 12, 16 and 20). The EMSAs shown are phosphorimages of the gels. Lane 1 contains *HO(6)*probe alone, and lanes 2 and 3 contain 1.4 × 10^-6^M **a**1 and 8.2 × 10^-8^M *α*2 respectively. The fold repression by each site in the context of a heterologous promoter is shown. (B) ChIP assays for *COX13*, *REX2*, *LSM1*and *FMP14*were performed as described in Figure 1. :::  ::: Discussion ========== Genome-wide gene expression data using SAGE or DNA microarrays has provided a wealth of information on the regulation of genes under certain conditions or by specific transcription factors. The combination of this information with sequence analysis programs has enabled researchers to identify potential regulatory sites. For example, in a pioneering paper, Tavazoie *et al.*clustered expression data and used multiple local sequence alignment algorithms on the promoter regions of the co-clustered genes to discover regulatory motifs \[[@B19]\]. This approach has been further refined by using Bayesian networks to incorporate additional constraints regarding relative positions and the orientations of the motifs \[[@B20]\]. Another approach has been to break the genes into modules and perform module assignments and motif searches at the same time via an expectation maximization algorithm (as opposed to clustering first and finding motifs later) \[[@B21],[@B22]\]. Although these approaches have worked well at identifying potential targets sites one drawback is that the expression patterns have to cluster well for these methods to work. For a small number of microarray experiments, this may always not be the case. A method that does not utilize clustering is a regression model based analysis to locate \"words\" in the promoter that correlates with modulation of expression \[[@B23]\]. However, this approach is restricted to retrieving functional consensus binding sites in the promoter regions and for transcription factors with low sequence specificity, this approach needs to be modified. Most of these approaches attack the difficult problem of what to do when relatively little is known about the regulatory system and sequence recognition by the protein. Consequently they develop pattern recognition algorithms that are essentially unsupervised. Our focus has been to take advantage, as much as possible, of knowledge about the biological system and use that information combined with expression analysis to identify potential target sites. The minor loss of generality of the tools resulting from such an approach is more than offset by its predictive power. To determine if the changes in expression of a specific gene are the result of a transcription factor working at the promoter we developed an algorithm that combines expression data with information on the binding site preference for a transcription factor. As a test for this algorithm we identified genes in yeast that are direct targets for regulation by the **a**1-*α*2 repressor complex. We also used this method to identify genes that are repressed in diploid cells but that are not direct targets of the complex, as well as functional **a**1-*α*2 binding sites that do not appear to repress transcription in their genomic context. The combination of these sets of findings has provided insight into the regulatory network and mechanism of repression by the **a**1-*α*2 complex. The primary goal of this study was to identify genes that are direct targets for repression by the **a**1-*α*2 complex. There are two major functional subsets among the **a**1-*α*2 target genes identified in this analysis (Table [1](#T1){ref-type="table"}). One, not surprisingly, involves genes that are required for various processes in mating of the two haploid cell-types. These include components of the mating pheromone signal transduction pathway, such as *GPA1*, *STE18*, *STE4*, and *STE5,*which are activated in response to the binding of pheromone from the other cell type \[[@B24]\]. This group also includes genes further down that pathway, such as *FAR1*and *FUS3*, which are required for cell-cycle arrest before mating. A number of these genes have previously been shown or suspected to be under the control of **a**1-*α*2 repressor complex \[[@B25],[@B26]\]. Repression of these genes in diploid cells is biologically important because it prevents further mating by diploid cells. If diploid cells mate they would form triploids or higher ordered genomic polyploids, which are genetically unstable during meiosis and therefore detrimental to cell survival. The second subset of genes identified in the analysis is associated with mating type switching and recombination. The *HO*gene is a known target of the **a**1-*α*2 complex and its promoter contains 10 binding sites of varying affinity \[[@B25]\]. Repression of *HO*is essential in diploid cells because it prevents switching of one of the *MAT*loci to form homozygous **a**/**a**or *α*/*α*diploid cells. Although diploid in genomic content, cells homozygous for the *MAT*loci are competent to mate and therefore would form higher order genomic polyploids that are genetically unstable. We have also shown that *NEJ1*, which is involved in non-homologous end-joining (NHEJ), is a direct target for the **a**1-*α*2 complex \[[@B27],[@B28]\]. It has been proposed that that repression of the NHEJ pathway may promote homologous recombination and crossing over in diploid cells. In addition, we found that *RDH54*, a gene involved in double-stranded DNA break repair, is a direct target for the **a**1-*α*2 complex \[[@B29]\]. This result is somewhat unexpected because *RDH54*is required for meiosis and null mutants show significantly reduced spore viability. It is likely that the **a**1-*α*2 complex only partially reduces the level of expression of the gene and that diploid cells require a lower level of activity of the protein. We also identified several genes that fell outside of these two subsets. One is *RME1*, which encodes a transcriptional repressor of *IME1*, the master regulator of meiosis \[[@B30]-[@B32]\]. **a**1-*α*2-mediated repression of *RME1*is required to allow cells to enter the meiotic pathway in diploid cells. Interestingly, we also found that *PDE1*and *MET31*are weakly, but reproducibly, direct targets for repression by the **a**1-*α*2 complex. The Pde1 protein is a low affinity cAMP phosphodiesterase that appears to have a role in response to stress and cell aging \[[@B33]\]. Repression of *PDE1*in diploids may partially account for the difference of starvation response between haploids and diploids. Met31 is a zinc finger DNA-binding protein that activates genes involved in sulfur metabolism \[[@B34]\]. It is unclear why this gene would be a target for the **a**1-*α*2 complex. It is possible that the presence of an **a**1-*α*2 target site upstream of a gene that has lower expression in diploid cells was fortuitous and that these sites were not functional targets. However, if this was the case then there would be little pressure to conserve these binding sites through evolution. Several closely related species of yeast have been sequenced and comparison of the corresponding promoter regions has led to the discovery of conserved regulatory motifs \[[@B35],[@B36]\]. Although lack of conservation does not imply non-functionality, significant conservation strongly argues for functionality of a putative regulatory element. To investigate this possibility, we performed a phylogenetic comparison to infer whether these sites are preserved among six sequenced *Saccharomyces*species using the PhyloGibbs program \[[@B37]\]. The program identified the **a**1-*α*2 binding site among a promoter set including many known haploid-specific genes (*HO, NEJ1, GPA1, STE4,*and *STE18*). This analysis also showed that the **a**1-*α*2 binding sites in the *RDH54, PDE1*and *MET31*promoters are strongly conserved among multiple species, suggesting that these sites play an important functional role. Our analysis identified a number of haploid-specific genes that do not appear to be direct targets of the **a**1-*α*2 repressor complex (Table [2](#T2){ref-type="table"}). Genes in this list do not contain a recognizable **a**1-*α*2-binding site and, with the exception of *NEM1*, are not detectably bound by the **a**1-*α*2 complex in the ChIP assays. It is possible that **a**1-*α*2 indirectly turns off these genes by repressing an activator protein that is required for their expression. However, besides *MET31*, there were no obvious genes coding for activator proteins that were direct targets of the **a**1-*α*2 complex. It is possible that the haploid-specific genes without **a**1-*α*2 sites are indirectly repressed through more complex mechanisms that involve repression of *RME1*. We also identified potential **a**1-*α*2-binding sites in the genome that do not appear to repress expression of nearby genes. Although sites from the *PRM8*, *PRM9*, *CDC25*, and *LSM1*promoters appear to be moderate binding sites for the **a**1-*α*2 complex in vitro, ChIP and heterologous reporter assays showed these sites are neither bound by the proteins nor are functional repressor sites in vivo. Many of these sites lie in open reading frames of actively transcribed genes and so it is possible that transcription through the binding site or the chromatin structure of the region prevents high affinity binding by the complex. The model that the genomic context of these sites is important for their regulatory activity is further supported by our results that show that some of these sites, such as *COX13*and *REX2*, function as strong **a**1-*α*2 dependent repressor sites in the context of the heterologous promoter. Although **a**1-*α*2 complex is bound to the *COX13*site in vivo it does not appear to repress transcription of this gene in diploid cell. Interestingly, this binding site is very close to the end of the coding region of *IME4*, an inducer of meiosis that is expressed in diploid cells \[[@B38]\]. The *IME4*gene is only expressed in diploid cells and it was thought that the **a**1-*α*2 complex may be indirectly activating its expression by repressing a repressor protein, such as *RME1*. However, the fact that **a**1-*α*2 binds to the downstream region of this gene suggests that it may play a direct role in its expression. Our data shows that the algorithm we have developed is useful in sorting between direct and indirect targets of a transcription factor. Although we have used mutational data to define the binding site for the **a**1-*α*2 complex, in principal binding site sequences derived from site selection experiments may also be used. This analysis may also complement genome-wide ChIP studies to identify the target sites of the transcription factor. Conclusions =========== In summary, we show that combining microarray data with motif analysis, lets us distinguish between the genes that are direct targets of a transcription factor and those that are modulated because of secondary effects. We get excellent agreement of the computational predictions with location analysis by ChIP experiments. We find most of the direct targets of **a**1/*α*2 complex to be involved in the mating pathway, mating type switching, recombination and meiosis. We also found a few weak targets that are possibly involved in sensing and control of the metabolic state. We also see that the sites we predict solely based on single species data are often evolutionarily conserved in other species of *Saccharomyces.* Methods ======= Combined scoring of genes from microarray data and mutational analysis ---------------------------------------------------------------------- We define a scoring algorithm that ranks gene expression patterns. For gene *g*, the score is given in terms of the expression in different types of cells (**a**, *α*and **a**/*α*) Score(*g*) = sgn((*X*~**a**~(*g*) + *X*~*α*~(*g*))/2 - *X*~**a**/*α*~(*g*)) \[(*X*~**a**~(*g*)) + *X*~*α*~(*g*))/2 - *X*~**a**/*α*~(*g*)\]^2^- *A*(*X*~**a**~(*g*) - *X*~*α*~(*g*))^2^. We initially used the logarithm of expression level of the gene *g*for the three cell types for the variables *X*~t~(*g*) with t indicating the type. We have since found that using the complete expression data for the polyploids is a better strategy. In the final results, shown in the paper, *X*~**a**~(*g*) is the average of the log expression for **a**, **aa**and **aaa**. Likewise, *X*~*α*~(*g*) is the average from *α, αα*and *ααα*. *X*~**a**/*α*~(*g*) comes from averaging log expression over **aa***α*, **a***αα*and **aa***αα*. The polyploid averaged quantities tend to be less noisy (demonstrated, for example, by the quantities *X*~**a**~and *X*~*α*~being close to each other for the generic gene, which is not regulated by cell type. This, in turn, allows easier detection of genuine haploid-specific targets. An explanation of the purpose served by different terms in the overall score is described below. The first term scores well when expression in diploids is lower than the average expression in haploids. The second term penalizes the gene if the expressions in different types of haploids are very different. *A*is chosen to be large enough so that known **a**-specific genes and *α*-specific genes score worse than known haploid-specific genes, but not large enough to overwhelm the first term. The optimal *A*is about 10. Comparison of the performance of our algorithm for *A*= 1 and *A*= 10, shows that the biologically known sites almost always stay near the top but further down in the list the second choice is better. The exception is a special gene: *MATα*1. Since *MATα*1 is not present in the *MAT****a***type cell, there is a penalty for expression patterns. Cumulative probability for any gene to have higher score than a gene g is *P*~*exprn*~(*g*), namely, fraction of genes with score higher than g. This scoring function would rank haploid-specific genes high but may not select out genes that are directly regulated by the **a**1-*α*2 repressor complex. In order to select for genes with an upstream region with a strong **a**1-*α*2 repressor, we used the binding site mutational data available \[[@B9]\]. In these experiments, repression of a heterologous promoter, incorporating single site mutations of a consensus binding site of the **a**1-*α*2 repressor, was measured. Under the assumption that the degree of repression is inversely proportional to how often that site is occupied, we derived the expression: 1/Repression ∝ \[1 - 1/(1 + e^*βE*(*S*)^/*z*)\] ≈ e^*βE*(*S*)^/*z*assuming near saturation of binding. The symbol *z*represents the fugacity and *β*is inverse of *k*~*B*~*T*, *k*~*B*~being the boltzman constant. The binding (free) energy is given by *E*(*S*) = Σ~*ib*~*ε*~*ib*~*S*~*ib*~, within the single base model \[[@B15],[@B16]\]. The index *i*runs over the positions in the motif and *b*runs over the bases A, C, G, T.*S*~*ib*~is 1 or 0 depending upon whether the *i-*th base is *b*or not. The parameters *ε*~*ib*~represent effects of single base changes on the binding (free) energy. They are related to the weight matrix parameters \[[@B17],[@B18]\] widely used to characterized variable motifs. Note that e^*βE*(*S*)^/*z*would more commonly be represented as (*K*/ \[Protein\])•exp(Δ*G*(*S*)/*RT*) in the biochemistry literature \[[@B18],[@B39]\]. The independent base model is only an approximation and mutations in nearest neighbor sites could produce effects that we cannot estimate from the existing data. There is a better separation between well-known sites and generic sequences if an extra penalty is added to the score for neighboring base pairs which both differ from the consensus. In this way every base different from consensus and neighboring another base different from consensus draws an additional penalty to the binding energy score. This parameter was set to be ln(2), by experience. Although this method prevented many false positives, it also penalized a few genuine candidates, such as the binding sites in the promoters of *FAR1*and *MATα*1. Thus, from the effect of the single base mutations, we estimated the parameters *ε*~*ib*~. Armed with these parameters, we found the probability *P*(*E*\|*ε*, *L*) that a random sequence of a certain length, *L*, would have a subsequence of binding energy greater than *E*. For gene g, the strongest site in the upstream region of length *L*would have binding energy *E*~*g*~. Low values of *P*~*binding*~(*g*) = *P*(*E*~*g*~\|*ε*,*L*), indicated the presence of a good binding site. The genes were ordered according to the lowest value of a combined p-value, *P*~*exprn*~(*g*) *P*~*binding*~(*g*), and then ranked as candidates for haploid-specific genes that are directly repressed by the **a**1-*α*2 complex. One of the issues in such studies is how to decide on how many of the top candidates are significant. This problem occurs even in solely, sequence based analysis as well \[[@B40]\]. In our study, we generated random combinations of expression p-values with scrambled binding p-values, so that we could choose a cutoff threshold by comparing the ordered p-values with the ordered \"scrambled\" p-values. Figure [5](#F5){ref-type="fig"} plots these sorted p-values against average (log) sorted p-values for the random combinations. The comparison suggests that only about 10--15 top candidates in the list are significant. Weight matrix based search -------------------------- A weight matrix \[[@B15]-[@B18]\] search for binding sites was performed using a set of known sites \[[@B9]\] to construct the matrix. Each matrix entry, *w*~*ia*~, was set to log((*f*~*ia*~+ *δ*)/(*P*~*a*~+ *δ*)), where *f*~*ia*~is the frequency with which base a appears in the *i*th position in the known sites, *P*~*a*~is the frequency with which base a appears in the promoters of genes and *δ*is a small number added to ensure that the weight matrix score is finite even when *f*~*ia*~= 0. Each subsequence, *S*~*ia*~, of length 20 in each promoter was assigned a weight matrix score Σ~*ia*~*S*~*ia*~*w*~*ia*~. After a threshold score is chosen, sites scoring above that threshold are declared to be binding sites. Automated primer generation --------------------------- An automated procedure for generating primers flanking a specified site in the genome sequence, *σ*, was implemented. To each pair of numbers, *d*~*u*~, and *d*~*d*~, representing primer distances upstream and downstream of the candidate binding site respectively, and primer lengths *l*~*u*~, and *l*~*d*~, a score is assigned via *S*(*d*~*u*~, *d*~*d*~, *l*~*u*~, *l*~*d*~,***σ***) = - Σ~*a*~*k*~*a*~(*P*~*a*~(*d*~*u*~, *d*~*d*~, *l*~*u*~, *l*~*d*~,***σ***) - )^2^ where the *P*~*a*~are functions including the melting temperatures, distances of upstream and downstream primers from the candidate site, total length of the bound region, and the difference between the primer melting temperatures. The  s are the preferred values of those functions. The *k*~*a*~are adjusted to reflect the relative importance of the parameters; for example it is more important that the difference in melting temperatures be close to zero than it is that the distance to the upstream primer match the distance to the downstream primer. Values of *d*~*u*~, *d*~*d*~, *l*~*u*~and *l*~*d*~are restricted to those whose corresponding primers have GG, GC, CG, or CC at the end nearest the candidate site. Primers are identified by selecting the values of *d*~*u*~, *d*~*d*~, *l*~*u*~and *l*~*d*~which maximize *S*. \[A web-based interface to this algorithm is available at <http://hill-226-174.rutgers.edu/>\] Chromatin immunoprecipitation ----------------------------- Chromatin immunoprecipitation (ChIP) was carried out as described previously \[[@B41]\] with the following modifications. One liter of JRY103 (*MATα/MAT****a****ade2-1/ADE2 HIS3/his3-11,15 leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3-1/ura3-1 ash1Δ::LEU2/ash1Δ::LEU2*) and JRY118 (*MATα/mat****a****Δ::TRP1 ade2-1/ADE2 HIS3/his3-11,15 leu2-3,112/leu2-3,112 trp1-1/trp1-1 ura3-1/ura3-1 ash1Δ::LEU2/ash1Δ::LEU2*) cultures were grown to an A600 of 0.5 and treated with 1% formaldehyde for 20 min at RT on a rotating shaker at low speed. Cells were collected, washed 2X with cold 1XTBS. Equal volumes of cells were aliquoted into ten 1.5 ml microfuge tubes, washed once with 1.5 ml of cold 1X TBS. The pellets in each tube were resuspended with 400 *μ*l of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate) plus 1 mM PMSF, 1 mM benzamidine, and 1X Protease inhibitor cocktail from Roche (Cat No. 1873580) and also manufacturer recommended concentration of protease inhibitor cocktail from SIGMA (Cat No., P 8215). To this 200 *μ*l of glass beads were added to each tube and lysed using a multitube vortexer at full speed for 30 min at 4°C. The lysate was transferred in a new tube and 400 *μ*l of lysis buffer was added and vortexed briefly. The lysates were centrifuged at 12,000 g for 10 min at 4°C and the supernatants were sonicated at 30% output for four 10 sec cycles with intermittent cooling on ice. The lysates were cleared by centrifugation at 12,000 g for 10 min and 1 mM PMSF was added to the samples. A 1/10^th^volume aliquot was removed and frozen to be used as total chromatin control. The remaining sample was precleared by the addition of 25 *μ*l recombinant protein G-agarose beads, incubated while nutating for 30 min and the supernatant was collected after centrifugation at 12,000 for 5 min. 1 *μ*l of rabbit anti-*α*2 antiserum (a gift from A. Johnson, UCSF) was added to each supernatant of the samples and incubated 12 h on a nutator at 4°C. To immunopreciptiate *α*2 50 *μ*l of recombinant protein G agarose beads (Roche) was added to the samples and nutated for 90 minutes at 4°C. The protein G beads were pelleted, washed once in low salt buffer (0.1%SDS, 1% Triton X-100, 20 mM Tris pH8.0, 2 mM EDTA and 150 mM NaCl), once in high salt (composition same as lowsalt + 500 mM NaCl), once in LiCl buffer (0.25 M LiCl, 1% IGEPAL, 1XTE and 1% Na-Deoxycholate) and twice with 1XTE (pH8.0). The immunoprecipitated DNA was eluted twice with 250 *μ*l of elution buffer (1%SDS and 0.1 M NaHCO3) and the eluates were pooled (500 *μ*l final volume). To this 20 *μ*l of 5 M NaCl was added and incubated 12 h at 65°C. To remove the crosslinks, 10 *μ*l of 0.5 M EDTA, 20 *μ*l of 1 M Tris-HCl, pH 7.5 and 2 *μ*l of proteinase K (10 mg/ml) was added and incubated for 45 minutes at 45°C. The DNA samples were extracted once with Phenol:chloroform:Isoamylalcohol and the DNA was ethanol precipitated, washed once with 70% ethanol and resuspended in 50 *μ*l (IP) or 500 *μ*l (TC) TE. Purified DNA from the immunoprecipitated samples was subjected to multiplex PCR amplification with primers specific for the *STE6*promoter as a positive control for the immunoprecipitation of *α*2 and the *YDL223C*ORF as a negative control for nonspecific immunoprecipitation, along with the specific primers for candidate *α*2-**a**1 target sites. PCRs were carried out in 50 *μ*l containing 10 pmols of each primer, 0.2 mM dNTPs, 2 mM MgCl2, 1X Eppendorf Taq buffer, 0.5X Taq Master buffer and 2.5 U of Eppendorf Taq polymerase. The amplifications were carried out at 94°C for 1 min and 30 secs, followed by 25 cycles of 94°C for 30 secs, 52°C for 1 min, and 72°C for 30 secs and a final extension step of 7 min at 72°C. The PCR products were separated on 2.5% agarose gels. Electrophoretic mobility shift assays ------------------------------------- Oligonucleotides containing the predicted **a**1-*α*2 binding sites from within the ORFs of *URB1*, *PRM8*, *PRM9*, *YKL162C*and *CDC25*and the promoters of *COX13, REX2, LSM1*, and *FMP14*were synthesized, one strand was end-labeled with \[*γ*-^32^P\]-ATP, and then annealed with excess cold complementary oligonucleotide. The *HO(10*) and *HO(8*) **a**1-*α*2 sites within Upstream Regulatory Sequence 1 (URS1) of the *HO*promoter were used as strong and weak binding sites respectively. The EMSA was performed as described previously \[[@B42]\], using a constant 1.4 *μ*M **a**1 and five-fold titrations of *α*2 starting at 82 nM in protein dilution buffer (50 mM Tris pH 7.6. 1 mM EDTA, 500 mM NaCl, 10 mM 2-mercaptoethanol, 10 mg/ml bovine serum albumin). *β*-galactosidase assays ------------------------ Oligonucleotides containing **a**1-*α*2 binding sites were synthesized with 5\' overhangs to allow cloning into the *Xho*I site of pTBA23 (2*μURA3*Amp^r^), a reporter plasmid containing a *CYC1-lacZ*fusion \[[@B43]\]. Reporter constructs were transformed into JRY103 and JRY118 and the *β*-galactosidase activity was measured on three independent transformants, as described previously \[[@B14]\]. List of Abbreviations ===================== SAGE -- Serial Analysis of Gene Expression HD -- Homeodomain ORF -- Open Reading Frame ChIP -- Chromatin immunoprecipitation NHEJ -- Non-Homologous End Joining TC -- Total Chromatin IP -- Immunoprecipitated sample EMSA -- Electrophoretic Mobility Shift Assay PCR -- Polymerase Chain Reaction PMSF -- Phenyl Methyl Sulfonyl Fluoride EDTA -- Ethylenediaminetetraacetic Acid Author\'s Contribution ====================== VHN carried out the ChIP experiments, and wrote parts of the first draft of the manuscript. RAO did the bioinformatics analysis, and wrote programs facilitating primer design. ARB did EMSA and beta-gal assays. JRM constructed the strains and contributed to the design of the ChIP experiment. AKV and AMS supervised and coordinated the computational and the experimental research as well as prepared the manuscript. All authors contributed to the manuscript and approved the final version. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Significance of combined p-values. Natural logarithm of combined p-values for twenty permutations/scrambles generated, sorted and plotted (in blue) against average of log p-value. The genuine combined value is plotted in red. :::  ::: Acknowledgements ================ We thank Alexander Johnson for the rabbit *α*2 antibody and Yvette Green for help with some of the initial ChIP assays. We also thank Rahul Siddharthan for running PhyloGibbs on the candidate direct target promoter set. JM was supported by a Charles and Johanna Busch predoctoral fellowship. This work was partially supported by a grant from the National Institutes of Health to AKV (GM49265).

PubMed Central

2024-06-05T03:55:47.717818

2004-8-26

PMC517710

Background ========== The variable domain of the immunoglobulin heavy chain (IGH) is encoded by the IGHV (variable), the IGHD (diversity) and the IGHJ (joining) genes. In developing B cells these genes are brought together via a process of recombination involving the selection of one of each gene type from sets of genes present within the genome \[[@B1]\]. The bringing together of the selected IGHV, IGHD and IGHJ genes generates combinatorial diversity \[[@B1]\]. The first genes to join are the IGHD and IGHJ genes, followed by the bringing together of the IGHV gene with D-J. Further junctional diversity is generated at the points between the joining genes \[[@B2],[@B3]\]. Junctional diversity results from the loss of nucleotides through the action of unknown exonuclease(s) and from the addition of N \[[@B3]\] and P nucleotides \[[@B2]\]. The final IGH V-D-J rearrangement in mature B cells is finally subject to the process of somatic hypermutation in secondary lymphoid organs which involves the targeted introduction and accumulation of point mutations \[[@B4]\]. The addition of N nucleotides is performed by the enzyme terminal dideoxynucleotidyl transferase (TdT), and in the IGH locus this addition can occur at both the D to J and the V to D-J joins \[[@B5]\]. The regions of N addition are denoted as N regions, and nucleotides that fall between the V and D genes are denoted as N1 regions, while those that lie between the D and J genes are denoted as N2 regions. P nucleotides are derived from the asymmetric opening of hairpin loops that form at gene ends as part of the rearrangement process \[[@B6]\]. The opening of the hairpin loops produces short, self-complementary single stranded extensions that can be incorporated into junctions, or may alternatively be removed via exonuclease activity \[[@B6]\]. It is the self-complementarity of P nucleotides that leads to their palindromic appearance and thus to their name. Hairpin opening is said to produce inserts of 0--4 nucleotides \[[@B2]\]. P nucleotides have been associated with the IGHV and IGHJ genes, as well as with each end of the IGHD gene \[[@B7]\] and estimates of the frequency of P nucleotide addition suggest a presence in about ten percent of sequences \[[@B7]-[@B10]\]. The mechanism of immunoglobulin gene rearrangement was first proposed by Tonegawa in the late 1970\'s \[[@B1]\]. Since that time, much has been learnt about the processes involved. Some areas, however, remain relatively uninvestigated, including the nature of exonuclease removal and the contribution of P nucleotide addition to junctional diversity. The lack of research in these fields may reflect the inherent difficulties in studying the relevant gene sequences, because IGH V-D-J junctions are the result of random and imprecise processes. It can therefore be difficult to distinguish between gene ends and N or P additions. The very few reports of exonuclease removal in the literature mainly describe analysis of murine sequences \[[@B11]-[@B14]\]. These investigations revealed nucleotide loss to be significantly different for murine IGHJ and IGHD genes. Differences were seen in the average exonuclease removal from IGHJ and IGHD gene subgroups, with individual gene subgroups possessing significantly different average levels of nucleotide removal. Influences upon gene processing that have been proposed to explain these observations include the presence of TG motifs \[[@B15]\], the relative location of stretches of 3 or more W (A or T) nucleotides and their positional relationship with respect to 2 or more S (G or C) nucleotides \[[@B12]\], and the presence of TAT motifs \[[@B13]\]. Recent advances in data standardisation in immunogenetics has allowed for improved statistical analysis. The standardisation emanates from IMGT-ONTOLOGY \[[@B16],[@B17]\] upon which one of the most widely used immunogenetics tools, IMGT/V-QUEST, is based \[[@B18]\]. IMGT, the IMmunoGeneTics Information System^*R*^, also offers standardised nomenclature \[[@B19]\] and standardised numbering of positions within immunoglobulin sequences \[[@B20]\]. Despite the importance of IMGT within the field, the tools offered still suffer from shortcomings especially in the analysis of IGH V-D-J junctions. Alternative means of analysing junctions are therefore still sought by researchers \[[@B21],[@B22]\]. The development of a statistically based algorithm for the partitioning of immunoglobulin sequences \[[@B21]\] as an alternative to IMGT/Junction Analysis \[[@B18]\], combined with the large amount of sequence data available through public nucleotide databases, has allowed us to investigate the nature of nucleotide removal from human immunoglobulin heavy chain genes in the expressed repertoire. Improved means of identification of gene ends facilitates the development of datasets with more certain partitioning. This study reports the extent of P nucleotide addition and the nature of exonuclease removal in the expressed human repertoire. Analysis of nucleotide loss and addition within the dataset reveals that different gene subgroups undergo distinct processing by exonuclease(s) and shows that there is no significant contribution by P nucleotides to the diversity of the expressed repertoire. Results ======= Dataset creation ---------------- The collection of human IgM sequences from public databases resulted in a dataset of approximately 1500 sequences. The exclusion of fetal, moderately and highly mutated (\>5 mutations) and disease associated sequences reduced the dataset to 306 sequences. Further exclusions were made of those sequences that showed signs of IGHV gene replacement or the utilization of multiple D genes. Five sequences utilized two D gene segments, as identified using strict criteria as previously described \[[@B21]\] (EMBL:U97246, L12190, L29154, AJ519292, AJ245025). Evidence of IGHV gene replacement, in the form of V gene \'footprints\', was seen in 7 sequences (EMBL:L29154, AJ245008, AJ245280, AJ519296, AY003831, X54445 Kabat:AL311). The footprints were unique sequences of 6 or more nucleotides derived from V gene ends containing a cryptic recombination signal sequence (cRSS) which is thought to be essential for replacement events \[[@B23]-[@B26]\]. The final dataset contained 294 sequences. Within the final dataset of 294 sequences there were 245 sequences for which IGHV, IGHD, IGHJ, N1 and N2 regions could be defined. A further 49 sequences, lacking determinable D, N1 and N2 region but possessing identifiable IGHV and IGHJ genes were also included in the dataset. For these 49 sequences, it was not possible to confidently determine the utilized IGHD gene within the junctional nucleotides, however, the ends of the V and J regions could be determined accurately. All IGHV, IGHD and IGHJ gene subgroups were represented within the dataset. Details of the dataset can be seen in the Appendix \[see Additional File [1](#S1){ref-type="supplementary-material"}\]. Exonuclease removals from genes and gene subgroups -------------------------------------------------- Exonuclease removal was evident in each of the 245 IGH V-D-J rearrangements examined, with 25% of IGH V-D-J rearrangements displaying removal from all four gene ends. A further 48% of IGH V-D-J sequences had removals from three of the four gene ends. The average number of nucleotides lost from each of the gene ends is presented in Figure [1](#F1){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Average exonuclease removal from IGH genes.**The average nucleotide removal from of the gene ends was examined for 294 IGHV and IGHJ genes and 245 IGHD genes. For the IGHD genes, removals were considered from each end of the gene; 5\' (V-D side) and 3\' (D-J side). Bars represent standard error. :::  ::: Examination of the 294 IGHV and IGHJ segments revealed 41% of IGHV ends lacked removals, compared to just 18% of IGHJ ends. Sixteen percent of the 245 IGHD genes lacked removals from the 5\' end of the D gene, and 17% had no removals from the 3\' end of the D region. Exonuclease removals ranged from 0 to 13 nucleotides at the 3\' V-REGION and 0 to 14 nucleotides at the 5\' D-REGION end. At the D-J junction, 3\' D-REGION and 5\' J-REGION removals both ranged from 0 to 20 nucleotides. A significant difference in the extent of exonuclease removals was observed between the IGHV, IGHD and IGHJ gene ends (p \< 0.0001, Kruskal-Wallis Test). Average removals from IGHV region ends were significantly lower than removals from IGHD and IGHJ region ends (p \< 0.001, Dunn\'s Multiple Comparison Test). On average, only 1.2 nucleotides were lost from IGHV region ends while average removals of 4.7 nucleotides were evident from each of the IGHD region ends as well as from the IGHJ region ends. The average number of nucleotides removed from each gene subgroup within the three genes was calculated to identify differences in processing of sequences at the gene subgroup level (Figure [2](#F2){ref-type="fig"}). It was necessary to exclude IGHV7 from the analysis as only a single sequence from this subgroup was in the dataset. Average removals differed significantly among the six IGHV subgroups analyzed (p = 0.03, Kruskal-Wallis Test) (Figure [2A](#F2){ref-type="fig"}). Comparison testing was, however, unable to identify the source of the difference. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Average exonuclease removal from gene subgroups.**The average exonuclease removals from gene ends was investigated for each IGHV, IGHD and IGHJ subgroup. Significant differences were seen among the 294 IGHV genes (A). No significant difference between the 245 IGHD genes were seen at the 5\' end (B). The 3\' IGHD end does show significant differences for the IGHD subgroups (C) as do the six IGHJ subgroups (D). Bars represent standard error. :::  ::: Removals from D gene subgroups were examined at each of the region ends. The removals from the 5\' D end did not reveal significant differences, but significant differences were seen between the subgroups at the 3\' end (p \< 0.0001, one-way ANOVA) (Figure [2B](#F2){ref-type="fig"}, Figure [2C](#F2){ref-type="fig"}). More extensive removals, of 6.0 and 7.5 nucleotides respectively, were observed from IGHD2 and IGHD3 subgroup members (p \< 0.0001, Tukey\'s Multiple Comparison Test). The remaining 5 IGHD subgroups experienced average deletions of 2.1 nucleotides at their 3\' ends. Comparison of average nucleotide loss for each of the six IGHJ subgroups revealed significant differences between the average removals (p \< 0.0001, one-way ANOVA). The low level of removals from IGHJ3 was notable. On average just 1.3 nucleotides were removed from IGHJ3 sequences, while an average of 6.4 nucleotides were removed from IGHJ6 sequences (p \< 0.002, Tukey\'s Multiple Comparison Test) (Figure [2D](#F2){ref-type="fig"}). Influence of W and S motifs --------------------------- A more detailed examination of exonuclease removals from the IGHJ genes was undertaken, to investigate the influence of W and S motifs. The presence of these motifs in the first 15 5\' nucleotides of the IGHJ ends was considered. IGHJ ends containing 5\' S motifs showed significantly lower average removals than those lacking a 5\' S motif (p \< 0.0001, Kruskal-Wallis Test). The IGHJ genes whose sequences did not possess an S motif within the first 15 5\' nucleotides had, on average, three more nucleotides removed (Figure [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Influence of W and S motifs on nucleotide loss.**IGHJ genes were grouped by the presence of W and S motifs within the first 15 nucleotides of the IGHJ subgroup sequence. Average exonuclease loss was examined for the three sets; 5\' W only, 5\' S only and S then W. :::  ::: Contribution of P nucleotides to diversity ------------------------------------------ Putative P nucleotides were identified among those gene ends that remained untrimmed by exonuclease activity during the process of IGH V-D-J rearrangement. Examples of nucleotides that satisfied the P nucleotide criteria were observed at 3\' V, 5\' D, 3\' D and 5\' J gene ends. The identified nucleotides ranged in length from 1 to 4 nucleotides. The observed P nucleotides fell into twenty-three sets based upon unique sequences, and the gene end at which they were observed (Table [1](#T1){ref-type="table"}). Each of the 23 sets was analyzed to determine the likelihood that apparent P nucleotides were actually the result of N additions. The p-values for each of the P nucleotide sequences are shown in Table [1](#T1){ref-type="table"}. Correction of the significance level for the comparison of the 23 sets using the Bonferroni adjustment resulted in a required alpha value of 0.003. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Putative P Nucleotides in a dataset of 294 human IgM sequences ::: Gene Segment Putative P Sequence Observed Total Junctions p-value^1^ -------------- --------------------- ---------- ----------------- ------------ IGHV C 10 111 0.99 T 25 111 0.023 CC 2 105 0.99 TC 10 105 0.049 TG 2 105 0.98 TCT 3 100 0.045 TGT 1 100 0.55 TCTC 1 86 0.21 IGHD 5\' A 1 37 0.99 C 5 37 0.99 AC 1 36 0.86 CA 1 36 0.86 CC 2 36 0.96 CCC 1 33 0.77 IGHD 3\' G 11 40 0.88 GT 2 36 0.57 TC 1 36 0.86 GTT 3 33 0.0022 IGHJ T 11 42 0.042 GT 3 34 0.26 AGC 1 32 0.45 ACT 2 32 0.026 ^1^α equal to 0.003 ::: A single case of significance was observed among the putative P nucleotide sequences. This was for a sequence of 3 nucleotides (GTT) which was associated with the 3\' end of the IGHD region. The occurrence of 3 \'GTT\' sequences in the dataset remains the only significant putative P nucleotides even if the alpha value is increased to 0.01. Using a 0.05 significance level, 6 sets out of 23 appear significant, however this conclusion carries a 69% chance of being incorrect and that the results occurred by chance. Although putative P nucleotide sequences are present in 10% of sequences most of these are likely to have arisen as the result of N nucleotide addition. P nucleotides can only be confidently attributed to less than 1% of sequences with three sequences from the IgM dataset contained statistically significant P nucleotides out of the 245 IGH V-D-J rearrangements examined. The overall contribution of P nucleotides to junctional nucleotides was 9 nucleotides out of 2899 junctional nucleotides, or 0.3% of junctional nucleotides, within the IgM dataset. The probability of \'GTT\' occurring within an N region is 0.007875, therefore, the sequence could be expected to occur twice at the observed position in the 245 junctions examined, by chance alone. Of the three identified P nucleotides it is therefore possible that only one is a true P nucleotide. The contribution of P nucleotides to junctional nucleotides could therefore be as low as 0.1%, with P nucleotide inclusions occurring in less than 0.5% of sequences. Discussion ========== Investigation of the role played by nucleotide loss and addition in the generation of immunoglobulin diversity has been limited by the ability of researchers to accurately determine gene ends. The development of a statistically based partitioning method has allowed this study to gain insights into the nature of nucleotide loss and addition in the expressed human immunoglobulin repertoire. Analysis of 294 human IgM sequences revealed significant differences between average nucleotide losses from different heavy chain genes segments. IGHV genes suffer less removal in comparison to other genes, suggesting that a process or processes may act to prevent the removal of critical components or to select against sequences in which such removals have occurred. Critical components may include the conserved TGT that defines the start of the CDR3 \[[@B27]\] and the internal heptamer site utilized in V~*H*~gene replacement \[[@B23]-[@B25]\]. The extension of the analysis to the gene subgroup level showed significant differences among removals from the IGHV gene subgroups, among the IGHD subgroups at the 3\' end of the D gene and between the six IGHJ gene subgroups. The most striking contrast was observed for the IGHJ gene subgroups, specifically for IGHJ3 and IGHJ6. Removals from IGHJ3 averaged only a single nucleotide, while IGHJ6 on average lost in excess of 6 nucleotides. The differences observed as part of this study suggest that the loss of nucleotides during the creation of human heavy chain immunoglobulin sequences is not random. Unique \'patterns\' of exonuclease removal between gene subgroups have previously been reported in murine immunoglobulins \[[@B12],[@B28]\], however we are not aware of any such reports from studies of the human repertoire. Murine IGHJ4 genes have been reported to undergo an average removal of 2 nucleotides more than any other murine IGHJ subgroup \[[@B28]\]. Comparison of the murine IGHJ4 sequence to that of human IGHJ6 shows these two sequences to be identical for the first 7 nucleotides. The common sequence, ATTACTA, is unique to these IGHJ subgroups. This suggests that the common sequence may be linked to the high levels of nucleotide loss experienced by these gene subgroups, relative to the other IGHJ genes. The nucleotide composition of gene has previously been stated to influence exonuclease processing in the murine system \[[@B12]\]. The presence of two \'motifs\' was thought to be the determining factor in the outcome of exonuclease processing. One motif involves stretches of two or more G or C nucleotides and is referred to here as the S motif. The other motif is composed of stretches of three or more consecutive A or T nucleotides and is referred to here as the W motif. S motifs in murine sequences were associated with low average removals from gene region ends, while the presence of W motifs correlated with high average removals \[[@B12]\]. Similar results were seen for human immunoglobulins in this study, with average removals from IGHJ genes containing 5\' S motifs being significantly lower than from those containing 5\' W motifs. Interestingly, the average position of the first S motif within the IGHJ genes coincided with the average level of removal from IGHJ genes. The correlation between S motif position and average exonuclease removal suggests that the S motif may act to block continued exonuclease removal from the gene region end. This may explain the high removals from the human IGHJ6 and murine IGHJ4 gene segments, as these sequences lack S motifs which may prevent such extensive exonuclease processing. Consideration of the W and S motif composition of the 3\' D gene segments showed the IGHD2 and IGHD3 gene subgroups to be rich in W motifs and to lack S motifs (data not shown). These two subgroups showed higher average removals compared to other IGHD subgroups. The relationship between nucleotide composition and exonuclease activity could therefore explain the significant differences observed at the 3\' end of the IGHD gene subgroups. IGHD2 and IGHD3 are both long D genes. A relationship between D gene length and exonuclease activity may have therefore been acting to influence exonuclease processing. Examination of exonuclease activity of D genes grouped by length did reveal significant differences (data not shown), however, these differences were only evident at the 3\' end of the D genes and as the analysis was confined to IGHD2 and IGHD3 sequences, it is difficult to conclude whether sequence length has a role. The influence of nucleotide composition on exonuclease removals from heavy chain gene is easily examined in the IGHJ genes, due to the small number of alleles and the clear division of sequences based on the presence or absence of S and W motifs. Significant differences in the V genes were not further examined as the larger number of alleles made sample groups too small to allow for meaningful statistical analysis. The absence of significant differences at the 5\' end of the IGHD genes may result from the lack of distinct differences in the nucleotide composition of these sequences. This would make any variations in exonuclease processing more subtle, and thus a larger sample size would be necessary to observe any differences. P nucleotide addition has been reported to contribute to diversity in between 10% \[[@B7],[@B8],[@B29]\] and 41% \[[@B9]\] of immunoglobulin sequences. Initial analysis of putative P nucleotides in the dataset of 294 human IgM sequences in this study revealed the frequency the presence of putative P nucleotides to be around ten percent of sequences. This is consistent with previous reports \[[@B7],[@B10],[@B29]\]. Statistical analysis of the putative P nucleotides sequences, however, revealed that only one \'P nucleotide sequence\' was observed at a frequency that was significantly above the frequency that would be expected from N nucleotide addition alone. This suggests that the true contribution of P nucleotides to diversity in the expressed human IgM repertoire is much lower than previously reported, with P nucleotides present in less than 1% of sequences and accounting for approximately 0.3% of junctional nucleotides in the IgM dataset. It should be noted that even those P nucleotides accepted on the basis of statistical analysis carry a degree of uncertainty. Accounting for the possible misidentification of TdT additions among P nucleotides suggests that the contribution of P nucleotides to junctional nucleotides may be even lower than 0.3%. The identification of P nucleotides during partitioning of immunoglobulin sequences introduces a greater margin of error than would result from their exclusion from partitioning processes. For example, the rare nature of P nucleotide inclusion in rearranged immunoglobulins means that in a dataset of 1000 sequences less than 10 sequences may possess P nucleotides. Arbitrary identification of putative P nucleotides in the same dataset could however lead to 100 of the sequences being identified as having P addition. The error is therefore smaller if P nucleotides are not allocated as part of the partitioning process. The statistical demonstration of the phenomenon of P nucleotides by Meier and Lewis utilized altered recombination substrates, where the IGHV, IGHD and IGHJ gene regions were replaced by restriction sites on a plasmid vector \[[@B8]\]. The recombinant substrates were then transfected into murine cell lines and the processing of the substrates was then examined. Meier and Lewis observed that putative P nucleotides occur at a significant frequency among the processed recombinant substrates and this has been used as the basis for the allocation of P nucleotides in subsequent immunoglobulin studies \[[@B8]\]. Examination of the frequency of P nucleotides among the recombination substrates, however, revealed a five fold greater frequency of P nucleotide inclusions than was seen among adult murine T cell receptors and immunoglobulin sequences \[[@B8]\] and adult human immunoglobulins \[[@B7],[@B29]\]. The applicability of Meier and Lewis\' statistical analysis of the altered recombination substrates to demonstrate the contribution of P nucleotides must therefore be questioned, as the reporting of statistical significance is likely to be a direct result of the elevated frequency of putative P nucleotides among the recombinant substrates. Examination of putative P nucleotides at five-fold lower frequencies eliminates the significance observed in the original studies and supports the figure reported here of a contribution to junctional diversity in less than 1% of sequences (data not shown). Conclusions =========== Substantial *in vitro*evidence in support of the formation of hairpin loops as part of the immunoglobulin rearrangement mechanism \[[@B6],[@B30],[@B31]\] and for the creation of P nucleotides as part of the process of hairpin loop opening exists \[[@B32],[@B33]\]. The results reported here suggest that the P nucleotides generated by the loop opening do not, however, contribute significantly to the diversity of the final rearranged immunoglobulin. Exonuclease processing of IGH genes is not random and the nucleotide composition of the gene end appears to be influential. Further investigations into factor(s) influencing the exonuclease processing of gene ends will be required in order to elucidate the exact nature of the relationship between the gene end and exonuclease processing. Methods ======= Dataset creation ---------------- Human IgM sequences were obtained from public nucleotide databases; IMGT/LIGM-DB available through the IMGT, The IMmunoGeneTics Information System^*R*^, <http://imgt.cines.fr>\[[@B18]\], the Entrez nucleotide database from the National Center for Biotechnology Information (NCBI) \[[@B34]\] and the Kabat Database of Sequences of Immunological Interest \[[@B35]\]. The sequences obtained were screened to exclude those sequences of fetal origin, those associated with diseases and those of a non-productive nature. Screening was necessary to avoid the introduction of any biases that may be associated with particular disease states or stages of immunological development. Sequences that contained in excess of 5 mutations within the V gene were also excluded from the final dataset. Sequences displaying higher levels of mutation were excluded from the analysis as the partitioning method utilised is most accurately applied to sequences with low levels of mutation. Partitioning of rearranged immunoglobulin sequences --------------------------------------------------- The determination of genes and N regions of the sequences within the dataset was performed with the aid of a statistical analysis of point mutations \[[@B21]\]. This method uses the number of mutations in the core region of the V genes to predict the level of mutation within other regions of the immunoglobulin sequence. The approach is based upon the mutability of trinucleotides, while also factoring in the exponential decay of somatic point mutations \[[@B36]\] and the effects of antigen selection \[[@B21]\]. The key to the analysis is the calculation of mutability scores for the various genes. Mutability scores can be used to indicate the likelihood that mutations will be distributed in a particular way between two or more parts of an immunoglobulin sequence. The focus of the study upon exonuclease removal required careful definition of rules for the identification of nucleotide losses. Preliminary alignment of sequences were performed using IMGT/V-QUEST \[[@B18]\]. Where identical IMGT/V-QUEST alignment scores were achieved to different alleles of a germline gene, the allele first allele reported was recorded. IMGT/V-QUEST compares input sequences to the IMGT reference sets which are the most complete sets of sequences that are available for IGHV, IGHD and IGHJ functional genes, their alleles and open reading frame genes. Alignments produced by IMGT/V-QUEST give an overall indication of similarity between a rearranged sequence and germline genes. D gene determination was performed using previously described criteria \[[@B21]\], where the level of required similarity to a germline sequence was dictated by the length of the junction and the likelihood of N nucleotides being misidentified as IGHD segments by chance. To aid in the allocation of D genes, a D Gene Alignment Utility was developed. This web based tool allowed alignments to be performed between a junctional sequence and all germline D genes, including inverted D gene sequences \[[@B37]\] obtained from the IMGT Reference Directory \[[@B19]\]. The program utilized an altered Smith-Waterman algorithm that did not allow for gaps \[[@B38]\]. Difficulties with immunoglobulin partitioning are often experienced, especially in the determination of gene ends. In this study, runs of consecutive nucleotide differences between a IGH V-(D)-J rearrangement and a germline sequence at a gene end were always attributed to exonuclease removal, rather than mutation of the gene end. The portion of a IGH V-(D)-J rearrangement that contained such differences, with respect to the germline sequence, was allocated to the N region, and the gene was considered to have undergone exonuclease processing at the gene end. Situations where IGHV or IGHJ ends revealed a series of differences and similarities to the germline sequence required the consideration of each possible combination of exonuclease removal and point mutation that could have led to the creation of the observed region end. Probabilities were calculated for each \'path\' to the observed segment end. Mutability scores for the region end were used to determine the likelihood of point mutations contributing to the region end. TdT addition probabilities were used to calculate the likelihood of N additions generating particular nucleotide sequences. The TdT probabilities used were p(G) = 0.35, p(C) = 0.35, p(A) = 0.15, p(T) = 0.15 \[[@B5]\]. The path that displayed the greatest likelihood was used to allocate nucleotides to either an N region or a region end. Sequences that lacked exonuclease removals from gene region end(s) were examined for the presence of P nucleotides \[[@B2]\]. Self-complementary repeats of the gene end located in the neighboring N region were designated as putative P nucleotides. For example, if the V gene ended with the nucleotides GA, then CT was sought at the start of the N1 region. All possible lengths of P insertions were considered as part of the identification process. Examination of P nucleotides ---------------------------- The contribution of P nucleotides to immunoglobulin diversity was investigated through data generated by the investigations reported here. Unique putative P nucleotides were identified and their appearance was tallied within the dataset. The number of junctions that displayed a lack of exonuclease activity at one or more gene ends was also calculated. The cumulative binomial probability of observing a given number of P nucleotides or greater was then calculated for each putative P nucleotide sequence in an attempt to estimate the contribution of P nucleotides to immunoglobulin diversity. Role of W and S Motifs in exonuclease processing ------------------------------------------------ The effect of W and S motifs upon exonuclease activity was investigated within the IGHJ genes. The IGHJ genes were grouped based upon the relative location of such motifs. W motifs were defined as sequences of 3 or more consecutive A or T nucleotides. S motifs were defined as sequences of 2 or more consecutive G or C nucleotides. The presence of these motifs was considered within the first 15 5\' nucleotides of the IGHJ genes. Three sets were established, based upon the observed configurations of the motifs in the IGHJ subgroups; S motif followed by W motif, S motif only and W motif only (Table [2](#T2){ref-type="table"}). Analysis of exonuclease removals for each of these groups was then performed by calculating the average number of nucleotides removed from the IGHJ end for each set. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Grouping of IGHJ genes by relative location of W and S motifs ::: J Gene Sequence^1^ Group ----------- ----------------------------------------------- -------------- IGHJ1\*01 *GC*TGAATACTT*CC*AG 5\' S only IGHJ2\*01 TGCTACT*GG*TACTTG 5\' S only IGHJ3\*01 TGAT*GC*[TTTT]{.underline}GATGT 5\' S then W IGHJ3\*02 AT*GC*[TTTT]{.underline}GA[TAT]{.underline}CT 5\' S then W IGHJ4\*01 ACTAC[TTT]{.underline}GACTACT 5\' W only IGHJ4\*02 ACTAC[TTT]{.underline}GACTACT 5\' W only IGHJ4\*03 *GC*TAC[TTT]{.underline}GACTACT 5\' S then W IGHJ5\*01 ACAACT*GG*TT*CG*ACT 5\' S only IGHJ5\*02 ACAACT*GG*TT*CG*ACC 5\' S only IGHJ6\*01 [ATTA]{.underline}CTACTACTACT 5\' W only IGHJ6\*02 [ATTA]{.underline}CTACTACTACT 5\' W only IGHJ6\*03 [ATTA]{.underline}CTACTACTACT 5\' W only ^1^W motifs shown [underlined]{.underline}, S motifs shown in *italics* ::: Statistical analysis -------------------- The extent of nucleotide deletion was calculated as the average number of nucleotides removed for a given dataset. Significant differences between average removals were determined using one-way ANOVA for normally distributed datasets, and Kruskal-Wallis Test for other datasets. Where significant differences were found, multiple comparison testing was carried out using Tukey\'s Multiple Comparison Test, for normally distributed datasets, and Dunn\'s Multiple Comparison Test for non-normally distributed datasets. All analysis of exonuclease removals was carried out using GraphPad Prism (Version 3.00, 1999, GraphPad Software) with an alpha value of 0.05. An analysis of P nucleotides was performed by calculating the probability that putative P nucleotides may actually have resulted from N nucleotide addition by TdT. Probabilities were calculated as described by Meier and Lewis \[[@B8]\]. The probability of the presence of the observed or a greater number of P nucleotides was calculated as follows \[[@B8]\]:  where, *n*is the observed number of P nucleotides and *N*is the total number of sequences containing junctional inserts equal to or greater than the length of the P nucleotide(s) being examined, and *p*is the expected frequency of the observed P nucleotide sequence, which was calculated using reported TdT frequencies \[[@B5]\]. Probabilities were calculated for each observed putative P nucleotide sequence, and the alpha value was adjusted for the number of comparisons made, using the Bonferroni correction. Authors contributions ===================== KJ carried out the dataset creation and partitioning, performed the statistical analysis and drafted the paper. BG aided in the design of the study and the development of the statistical analysis. WS advised on study design and development. AC devised the study, aided in study design and co-ordination and revised the manuscript. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 **294 Partitioned human IgM Sequences**294 human IgM sequences were partitioned using a statistically based model and used in the examination of exonuclease activity and P nucleotide addition in the expressed human repertoire. The file is in Microsoft Excel format. ::: ::: {.caption} ###### Click here for file :::

PubMed Central

2024-06-05T03:55:47.723265

2004-9-2

PMC517711

Background ========== The first somatotopic maps of the homuncular organisation of the primary somatosensory cortex (S1) were established in 1937 by using intra-operative electrical stimulation of the brain surface \[[@B1]\]. Subsequent, non-invasive investigations in humans on the hand representation in S1 have described a somatotopic organisation along the central sulcus with the thumb located laterally, anteriorly and inferiorly to the little finger \[[@B2]-[@B6]\]. Studies in non-human primates have revealed the cytoarchitectonic subdivisions of S1, namely areas 3a, 3b, 1 and 2, that outline the cortex in the postcentral gyrus \[[@B7]\]. Area 3a occupies the fundus of the central sulcus, area 3b the anterior wall of the postcentral gyrus, area1 its crown and area 2 its posterior wall. Each area contains a fairly complete map of the body surface and is the cortical representation of different somatosensory receptors. In area 3b the neurons are predominantly responsive to stimulation of cutaneous receptors. As opposed to neurons in area 1, that also receive input from cutaneous receptors, those in area 3b possess discrete receptive fields with a homuncular organisation that may be more distinct \[[@B8]\]. Previous studies on somatotopy in the hand area using functional Magnetic Resonance Imaging (fMRI) have yielded varying results. Gelnar et al. failed to show a somatotopy in S1 when applying vibratory stimuli to three of the fingers of the right hand \[[@B5]\]. Maldjian et al. demonstrated somatotopy in 3 out of 5 subjects \[[@B3]\]. Similarly, Kurth et al., using electrical stimulation of two fingers, found somatotopically arranged activation patterns in 5 out of 20 subjects \[[@B9]\]. In a follow-up study where activation of all fingers in area 3b was found in 7 out of 10 subjects, the same authors reported a general somatotopy, without further specification \[[@B4]\]. Methods differ considerably between these three studies with regard to anatomical considerations, number of subjects studied, and the field strength used. Maldjian et al. used the highest field strength, 4 Tesla (T), while both Kurth et al. and Gelnar et al. used 1.5 T \[[@B3]-[@B5]\]. However, Maldjian et al. did not contribute activations to any area in S1. Also Maldjian et al. included the smallest number of subjects (5); data from one were discarded due to motion artefacts and group analysis was based on data from the remaining 4. In the present study we readdressed the issue of somatotopy in the hand area as assessed with fMRI. Our aim was to optimise results by including a larger number of subjects, by focussing on area 3 b, where homuncular organisation expectedly is most distinct and by performing fMRI at 3 Tesla (T). Results ======= Tactile stimulation of the fingers of the dominant hand yielded activation in contra-and ipsilateral S1, contra- or bilateral secondary sensory cortex (S2), ipsilateral cerebellum, and in some subjects the contralateral thalamus. Significant activation for all five fingers in area 3b was present in seventeen subjects and these data were further analyzed. In the one volunteer excluded from the analysis, activation was present for four fingers. The spatial distribution of the activations in the contralateral S1 for tactile stimulation versus rest in one subject is shown in Figure [2](#F2){ref-type="fig"}. A somatotopic organisation with the representation of the thumb located laterally to the little finger was present in 16 out of 17 subjects, with the thumb located anteriorly to the little finger in 14 out of 17 subjects and with the thumb located inferiorly to that of the little finger in 16 out of 17 subjects. Group averages of the distances from D2 to D1 (D2-D1), D3 to D1(D3-D1), D4 to D1 (D4-D1) and D5 to D1 (D5-D1) are presented in the Table and shown as graphs in Figure [3](#F3){ref-type="fig"}. Combined these indicate a strict somatotopy with the distance to D1 increasing for every finger in each of the three directions. Distances to D1 were compared for neighbouring fingers. In the medial-lateral direction, the distance D4-D1 was different from D3-D1. In the anterior-posterior direction a significant difference was observed between D4-D1 and D3-D1. Finally, in the superior-inferior direction the distances D2-D1 and D3-D1 as well as D3-D1 and D4-D1 differed; the location of D2, as determined by its distance to D1, was different from the location of D1 \[0, 0, 0\]; no difference was found between the distances D5-D1 and D4-D1. Considering that the three coordinates \[x, y and z\] together define one point in the 3D Cartesian space, the coordinates of D3, D4 and D5, differed from those of D1, p now \<0.05/3, corrected for multiple comparisons (not in Table). The Euclidian distance from D1 to D2 was 10.6 mm (SEM ± 1.5). The distance from D2 to D3 was 5.5 mm (± 0.9), from D3 to D4 7.4 mm (± 1.1) and from D4 to D5 6.8 mm (± 1.2), resulting in an average for D2-D3, D3-D4 and D4-D5 of 6.6 mm. The spatial extension of the representation of the hand in area 3b, defined as the Euclidian distance between D1 and D5 was 17.2 mm (± 2.0 mm). Discussion ========== In the group average from the present study the strict somatotopic organisation in the primary sensory cortex known from studies using a variety of modalities, was reproduced \[[@B2]-[@B6]\]. The fingers\' average activations were laid out on the body map, with the thumb located most laterally, anteriorly and inferiorly and the little finger most medially, posteriorly and superiorly and the remaining fingers in between, the distance to the thumb increasing for every finger in each of the three directions. In individual subjects the arrangement in the hand representation with the thumb located laterally, anteriorly and inferiorly to the little finger is frequently found, while the remaining fingers may or may not display the orderly lateral-to-medial, anterior-to-posterior and inferior-to-superior organisation \'D1-D2-D3-D4-D5\' \[[@B2],[@B3],[@B10],[@B11]\]. We chose to present group averages as it is our belief our results would have greater significance if a regular somatotopy was present at the group level. Body maps in non-human primates demonstrating the regular sequence mentioned above, were established with cortical single unit recordings and are supposedly the golden standard. The present study is based on data from 20 subjects, generating results available for analysis from 18 of these; the spatial representation of all fingers in area 3b of the primary somatosensory cortex was localised in 17 subjects. The average extension of the hand representation in area 3b of 17 mm with a somatotopic arrangement of fingers 1-5 as described above is consistent with results from previous studies using a range of modalities \[[@B2]-[@B4],[@B7]\]. Also the mean distance between D2-D3, D3-D4 and D4-D5 of 6.6 mm and 6.3 mm for main and differential effects, respectively, is in good agreement with human electrophysiological and fMRI data \[[@B2],[@B4]\]. The larger distance between the thumb and index finger as compared to distances between subsequent fingers suggests a larger representation for the thumb. This finding is in agreement with results from a study using electrocorticography with subdural electrodes in three patients \[[@B1],[@B12]\]. With the spatial resolution used in this study, 3 × 3 × 3 mm^3^, resampled to 1.5 × 1.5 × 1.5 mm^3^, activation for all five fingers was found in 17 of 18 subjects. For comparison, Kurth et al. used a resolution of 1.7 × 1.7 × 1.7 mm3 and electrical stimulation with ring electrodes and found activation in area 3b for all five fingers in 7 out of 10 subjects (70%) \[4\]. The present study showed activations for all fingers in 94 % of subjects. This difference might be due to the higher field strength used in this study as the higher magnetisation vector and sensitivity to changes in susceptibility increase the signal-to-noise ratio. In one subject excluded from the analysis, activation was present for four fingers and another was excluded due to general lack of activation. Lack of activation may be due to subjects being \'low-activators\' in fMRI experiments, due to inadequate stimulation or to some other, unknown factor. The type of stimulation used is decisive of what area in S1 can be expected to be activated. For example, neurons in area 3a are responsive to deep receptor and proprioceptive stimulation and in one study punctate tactile stimulation did not activate area 3a \[[@B13]\]. Also, receptive fields are maximally focused in area 3b, while in area 1 receptive fields become larger and more complex. In area 2, receptive fields are even more complex with reduplications. This combined knowledge made area 3b the area of our choice to study somatotopy and explains why we chose not to report on activations in areas 3a, 1 and 2. We found activation in the anterior wall of the postcentral gyrus, defined as area 3b according to our operational definition during tactile stimulation (Fig [1](#F1){ref-type="fig"}). More pronounced activation was noticed frequently in the crown or posterior wall of the postcentral gyrus, defined as areas 1 and 2 (Fig [2](#F2){ref-type="fig"}). Similar observations have been made in other studies using both fMRI and PET \[[@B5],[@B14]\]. According to studies in non-human primates the representation of the distal fingertips in area 1 points posteriorly, a finding confirmed in a recent fMRI-study on humans \[[@B15]\]. Area 2 then is the mirror-image of area 1 with the fingertips pointing anteriorly. The activation in the posterior wall might represent activation in both areas 1 and 2 localised at their meeting point, i.e. the fingertips. The larger cluster size of this activation is explained by the clusters arising from area 1 and 2 being contiguous and therefore additive. In the present study the activation of area 1 and 2 is probably due to hierarchical processing in the rostrocaudal direction within S1. A previous observation that electrical stimulation of the cutaneous afferents of the median nerve resulted in evoked potentials in area 3b after 30 msec while a potential in area 1 was seen after another 5 msec lends support to this assumption \[[@B16]\]. Conclusion ========== In the group analysis, a somatotopic organisation for all the fingers in the hand representation of area 3b could be demonstrated using fMRI; the Euclidian distance between the thumb and the little finger was well comparable to that determined in previous studies. On the subject level the cortical somatosensory representation of the thumb was located laterally, anteriorly, and inferiorly to that of the little finger in 14 out of 17 subjects. The spatial localisation of the remaining fingers showed a less stringent somatotopic order when compared individually. Methods ======= Subjects -------- Twenty healthy, self-reportedlly right-handed volunteers (6 male and 14 female, age 21--43 years, mean 29.4 years) were included in the study. The protocol was approved by the local ethics committee and written informed consent was obtained. All volunteers had normal images on a fluid-attenuated inversion recovery (FLAIR) sequence. Stimulation ----------- Tactile stimulation consisted of brushing the glabrous skin of the two distal phalanges of each finger continuously forwards and backwards with a commercially available tooth brush. During the experiment, volunteers were positioned on the MR table with their right arm from the elbow down in a padded cast, that also provided support for the dorsal part of the hand. They were instructed to rest their arm against the magnet bore so that both arm and hand were relaxed. Pieces of soft cloth were placed between the fingers in order to avoid that stimulation also involved a neighbouring finger. The frequency was 1 Hz; no forced pressure was exerted. Consistency was tested on a finger model firmly taped onto a computerized electronic scale (Biopac Systems, DA 100B, MP 100A; Macintosh Powerbook G3 with software AcqKnowledge 3.5): the intra-examiner error was 18% based on a mean pressure of 6.64 g, standard deviation 1.199 g. Imaging ------- MRI was performed using a 3 T head scanner (Siemens Allegra) with a quadrature birdcage coil. Morphological T1-weighted images with a resolution of 1 × 1 × 1 mm^3^using a magnetisation prepared gradient echo sequence (MPRAGE) were acquired. Functional echo-planar image volumes of the whole brain (number of slices = 49, thickness = 3 mm, voxel size = 3 × 3 × 3 mm^3^) sensitised to the Blood Oxygenation Level Dependent (BOLD)-effect (echo time = 30 ms) were acquired. Five scanning sessions were performed. Each session included 92 functional volumes with a temporal resolution of 3 seconds. The first two volumes in each session were discarded from further analysis to allow for initial T1-equilibrium effects. Experimental protocol --------------------- Fingers 1 to 5 (D1 = thumb, D5 = little finger) of the right hand were stimulated sequentially in separate sessions according to a block design that included four periods of stimulation and five of rest for each finger. The epoch length for both stimulation and rest periods was 30 seconds. Postprocessing -------------- Image processing and analysis were carried out using the SPM99 soft ware package \[[@B17]\]. All functional images were resliced to a voxel size of 1.5 × 1.5 × 1.5 mm^3^and then realigned to the first image and coregistered to the T1-weighted image volume. All data were spatially filtered using an isotropic 4 mm, full-width, half-maximum Gaussian kernel. A high pass filter (cut off frequency 0.008 Hz) was applied to eliminate low frequency signal fluctuations. In order to preserve each subject\'s somatotopic arrangement in area 3b no normalization to a common brain atlas was performed. Data analysis ------------- Functional data from two subjects were excluded due to major motion artefacts (subject no. 7) and global lack of activation (subject no.18). Task specific effects were estimated using the general linear model (GLM) that included a box car function convolved with the canonical hemodynamic response function in SPM99. The effect of sensory stimulation of each finger versus rest was determined using a one-sample t-test of pertinent linear contrasts of parameter estimates in each subject with a significance level of p \< 0.001 (uncorrected). Then the spatial coordinates of the peak activation voxel in area 3b were determined. Due to lack of neuroanatomical landmarks, exact delineation of the cytoarchitectonically defined areas within S1 cannot be achieved in MR images. Therefore we used an operational definition based on cytoarchitectonic studies of S1 on 10 post-mortem brains. In \>50 % of these brains area 3a was located in the fundus of the central sulcus, area 3b in the rostral bank of the postcentral gyrus and area 1 on its crown reaching down into the postcentral sulcus \[[@B18],[@B19]\]. Fig [1](#F1){ref-type="fig"} illustrates these locations, that continue along the central sulcus. As interareal borders vary across brains, the same authors constructed probability maps for each area by superimposing histological volumes of the individual brains on a computerized reference brain. Volumes of interest (VOIs) were defined for each area in which \>50 % of the brains had a representation of that area. Despite close relationship of areas 3a, 3b and 1 in the postcentral gyrus, the three VOIs overlapped by \<1% of their volumes. These probability maps were at hand when the spatial coordinates of the peak activation voxel in area 3b were determined. Activated peak voxels were labelled as belonging to area 3b when they were located within the anterior wall of the postcentral gyrus (Figure [1](#F1){ref-type="fig"}). The spatial coordinates of the peak voxel for the thumb (D1) was defined as being at origo \[0, 0, 0\] in a 3D Cartesian coordinate system.When the spatial coordinates for all fingers were known in all subjects, somatotopy was assessed by determining the average distances for the whole group from each finger to the thumb. Euclidian distances between fingers as well as from each finger to the thumb (D1) were calculated as:  with x~DI~, y~DI~and z~DI~representing the coordinates of the finger DI and x~DII~, y~DII~and z~DII~representing the coordinates of DII in the three directions x, y, z in a system where the coordinates of D1 are at origo \[0, 0, 0\]. Statistics ---------- Each finger\'s distance to the first finger was compared to that of the directly neighbouring fingers using the Wilcoxon matched pairs test with a significance level of p \< 0.05. The Euclidian distances to the first finger were compared for each finger using the Wilcoxon matched pairs test with a significance level of p \< 0.05. Authors\' contributions ======================= DvW conceived of the study, performed the image analysis and drafted the manuscript. PF guided the image analysis. JO designed the MR-parameters. EML participated in the design of the study. BR and GL initiated and conducted the stimulation consistency study. All authors read and approved the final manuscript. Acknowledgments =============== The authors thank dr S Geyer, Research Center Jülich, Germany for probability maps delineating areas 3a, 3b and 1. This study was supported by the Swedish Medical Research Council, the Swedish Brain Foundation, the Medical Faculty at Lund University, the Skåne County Council Research and Development Foundation and the Royal Physiographic Society in Lund.. Part of this work was presented at the annual meeting of the European Society for Magnetic Resonance in Medicine and Biology, Rotterdam 2003. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Areas of S1 as defined in cytoarchitectonic studies on 10 post-mortem brains \[18, 19\]: area 3a occupies the fundus of the central sulcus (dark blue), area 3b the anterior wall of the postcentral gyrus (red), area1 its crown (light blue) and area 2 its posterior wall (green). The black arrow indicates the central sulcus. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Activation in the contralateral somatosensory cortex during tactile stimulation of the fingers of the right hand versus rest in a single subject. The first column shows transverse anatomical image with z-coordinate indicated. Subsequent columns show the activation patterns in S1 overlayed on magnified T1-weighted images for each finger. The location of the peak voxel in area 3b is indicated by blue crosshairs. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Distance to the thumb (D1) for each finger (mm), mean (diamonds) and SEM (errorbars), as presented in Table. The coordinates for D1 are defined as origo \[0, 0, 0\]. Distances to D1 are shown in the medial-lateral (M-L) direction, in the left panel also in the posterior-anterior (P-A) direction and in the right panel also in the superior-inferior (S-I) direction. The fingers are sequentially positioned more medially, posteriorly and superiorly. :::  ::: ::: {#T1 .table-wrap} Table **Distance to finger 1** -------- -------------------------- ------------------------- ------------------------- -------------------------- **D1** 0 (0) 0 (0) 0 (0) 0 (0) **D2** 2.9 (1.6) 0.4 (1.8) [4.8 (1.0)]{.underline} [10.6 (1.5)]{.underline} **D3** 3.6 (1.4) 1.4 (1.5) [6.8 (1.2)]{.underline} 11.2 (1.5) **D4** [6.9 (1.4)]{.underline} [4.6 (1.7)]{.underline} [9.4 (1.4)]{.underline} [15.4 (1.7)]{.underline} **D5** 7.4 (1.7) 6.8 (1.2) 10.4 (2) 17.2 (2.0) Distance from each finger (D2, D3, D4, D5) to the thumb (D1) ± standard error of the mean (SEM) (mm). The lateral-to-medial direction is named \'x\', the anterior-to-posterior direction \'y\' and the inferior-to-superior direction \'z\'. Underlined are those distances to D1 that differ significantly from the distance of the previous finger to D1 (Wilcoxon matched pairs test, p \< 0.05). :::

PubMed Central

2024-06-05T03:55:47.726587

2004-8-20

PMC517712

Background ========== Scrapie is a contagious prion disease \[[@B1]\] of sheep and goat. In contrast to BSE, there is no evidence for a transmission to humans. Nevertheless, BSE is experimentally transmittable to sheep and the resulting disease can not be distinguished from scrapie \[[@B2],[@B3]\]. Even though BSE has not been found in farmed sheep yet, the possibility that BSE occurs in \"scrapie\" diseased sheep can not be excluded. Therefore, scrapie control programs were implemented in many countries. Since no vaccine or therapeutic means is currently available, these programs rely on selective breeding for scrapie resistance. Susceptibility to scrapie is largely controlled by three polymorphic amino acid positions (136, 154, 171) of the ovine prion protein gene (*prnp*) \[[@B4]\] and reliable genotyping of the corresponding DNA polymorphism is required as a basis for selection decisions. For determination of *prnp*alleles or haplotypes, there are several typing techniques applied today. Direct sequencing covering the region of exon 2 encoding amino acid positions 136, 154 and 171 is the most accurate method, that enables the typing of additional and detection of hitherto unknown polymorphisms. Other methods are classical PCR-restriction fragment length polymorphism (RFLP)-typing \[[@B5],[@B6]\], DNA strand conformation polymorphism detection, denaturing gradient gel electrophoresis (DGGE) \[[@B7]\], PCR-single-strand conformational polymorphism (PCR-SSCP) \[[@B8]\], hybridisation with allele-specific oligonucleotides \[[@B9],[@B10]\], primer extension \[[@B11]\], and so on. For high-throughput genotyping matrix assisted laser desporption/ionisation -- time of flight (MALDI-TOF) systems (e.g. for bovine *prnp*\[[@B12]\] and the LGC-web page \[[@B13]\]), taqman^®^(Applied Biosystems, CA) \[[@B14]\], and pyrosequencing (Biotage AB, Uppsala, Sweden) are used, but methodologically details of these methods are not very well documented in the literature. The amplification refractory mutation system (ARMS) method is well established \[[@B15]\] and has been applied not only to single nucleotide polymorphism (SNP) detection but also to haplotype determination \[[@B16]\]. In the present work we combined the use of fluorescence labelled oligonucleotides with ARMS technology for the simultaneous detection of 4 SNPs without using common primers. Based on this PRNP ARMS method we developed a cost-effective, well-reliable and low- to high-throughput technology for *prnp*typing that can easily be adopted by a wide range of other laboratories. Methods ======= DNA-Samples ----------- EDTA-blood or Typifix^®^-tissue samples were collected from half-sib families from three common sheep breeds in Bavaria. All lambs were born in the years 2001 and 2002. DNA was isolated by using the E.Z.N.A. Blood DNA Mini Kit (\#12-3482-03, PEQLAB Biotechnologie GmbH, Erlangen, Germany) or the NucleoSpin^®^Multi-96 Tissue kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany), respectively. PRNP-ARMS --------- ### Design of Primers An overview of primer locations is given in Fig. [1](#F1){ref-type="fig"}. Initially, allele-specific primers were designed that differed only at the 3\'-nucleotide (given in bold letters in Table [1](#T1){ref-type="table"}). Using these primers it was not possible to obtain reliable discrimination of alleles. Especially the simultaneous use of primers for alleles Q-171 and R-171 resulted in cross-amplification products. Therefore, a number of additional primers were tested to optimise allele discrimination. Destabilizing mismatches were introduced at the first or second penultimate base in addition to the allele-specific base at the 3\' termini of the primers (ARMS principle, indicated by small letters in Table [1](#T1){ref-type="table"}). The sense primers specific for the alleles at amino acid position 136 were labelled with two different fluorochromes, 4, 7, 2\', 7\'-tetrabetweenchloro-6-carboxyfluorescein (TET) and 6-carboxyfluorescein (FAM) (Table [1](#T1){ref-type="table"}, Fig. [1](#F1){ref-type="fig"}). That allows the detection and discrimination of the alleles by standard gel electrophoresis on a fluorescent sequencer. Pieces of neutral sequence \[[@B17]\] were added to the 5\'-end (underlined in Table [1](#T1){ref-type="table"}) of the unlabeled antisense primers for amino acid positions 154 and 171. These enable the differentiation of the alleles by different lengths. Mismatches between primers were deliberately introduced at the 5\' region in these neutral sequences to avoid jumping amplification products. For a delineation of primer sequences with a reference sequence (Fig. [3](#F3){ref-type="fig"}). ### PCR-reaction For the ARMS reaction haplotypes were amplified from 2 μl of DNA solution with standard buffer conditions, 1.5 mM MgCl~2~, dNTP\'s (25 nM each), and 0.75 units of HotStar-taq polymerase (Qiagen, Hilden, Germany) in a final volume of 10 μl on a t-gradient 96-well thermocycler (Biometra, Göttingen, Germany). Primer concentrations were 10 nmol each. Cycling was for 15 min at 95°C, \[0.5 min at 94°C, 1 min at 62°C, 1 min at 74°C\]33× without final extension. ### Fragment analysis Fragment lengths of ARMS amplification products were analysed on an ABI PRISM^®^310 genetic analyser (Applied Biosystems, Foster City, CA) with 5 sec injection time, 15 kV, 60° and 18 min runtime using 36 cm capillaries. Allele designations were generated automatically using the Genotyper^®^software (V. 2.5, Applied Biosystems) as described \[[@B18]\]. The data were imported into an Microsoft Access database. Standard DNA samples representing different alleles were included in the assay to control each PCR mix and reaction. As an additional control a sql-query was used to flag all genotypes transferred to the database that were not compatible with the 15 known standard genotypes (e.g. peaks from FAM in the range of 153 bp) for retyping. Comparative sequencing ---------------------- Part of *prnp*exon 3 coding for amino acids 47 to 246 of the prion protein was amplified in 10 μl reaction volume using 2 μl of genomic DNA, 0.05 μM of each primer (5\'-tcc tgg agg caa ccg cta tc-3\' and 5\'-gga gga tca cag gag ggg aag-3\'), 50 μM of each dNTP, 0.5 units of HotStar-taq polymerase (Qiagen, Hilden, Germany), and reaction buffer containing 1.5 mM MgCl~2~. Cycling-conditions on a Biometra T gradient 96-well thermocycler were: 15 min at 95°C, \[0.5 min at 95°C, 1 min at 60°C, 0,5 min at 72°C\]35× and 10 min, 60°C final extension. Before direct sequencing PCR-reactions were purified using the 96-well MultiScreen-PCR plates (Millipore, Bedford, MA). Sequencing was performed using BigDye V 2.0 terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). Sequencing analysis was run on an ABI PRISM^®^310 genetic analyser. Results ======= Development of the PRNP ARMS method ----------------------------------- We have established a multiplex \`amplification refractory mutation system\' (ARMS) based method for the identification of *prnp*136-154-171 genotypes in sheep. The PRNP ARMS method was developed using standard DNAs representing all five main haplotypes. In a first stage, different primer pairs were tested for allele specific amplification using an anneal-temperature gradient. By redesign of primers for amino-acid positions 154 and 171 and introducing deliberate mismatches close to the 3\' end an optimised primer set was established (Table [1](#T1){ref-type="table"}) allowing multiplex, allele-specific amplification. \"False positive fragments\" (peaks above the background signal generated by primers complementary to alleles that were not present in the corresponding sample) were not observed using the optimised primer set. Nevertheless, the average peaks heights differed between alleles. For example the average peak heights of the allele Q-154 was 1.5 times higher than that of R-154. The ratio of peak heights in heterozygous individuals differed from 0.5 to 2.5 fold (compare Fig. [2](#F2){ref-type="fig"}). These were observed when different PCR assays with low number of samples and small volumes of master-mix are compared. The peak height differences depend mainly on the relative primer concentrations and can be avoided e.g. by using premixed primer solutions containing all ARMS primers. For automated \"base calling\" the minimum peak height was set to 100 (in practice most samples gave peak heights well above 1000). Proof-of-principle ------------------ To provide a proof-of-principle the same 140 sheep DNAs were analysed by direct PCR sequencing and by PRNP ARMS. Typing results were identical with both methods. In a second step, 420 sheep from half-sib families and 20 samples from the international sheep and goat DNA typing comparison test 2003 of the International Society of Animal Genetics were analysed by the PRNP ARMS method. All genotypes followed the rules of Mendelian inheritance and no deviation from the main 5 haplotype patterns was observed. Allele frequencies derived from these animals from three different Bavarian breeds are shown in Table [2](#T2){ref-type="table"}. Paternity of all lambs was checked with a set of 9 microsatellites (data not shown). Typical electropherograms representing different haplotype combinations are shown in Fig. [2](#F2){ref-type="fig"}. Allele length determination was highly reproducible (Table [3](#T3){ref-type="table"}). The range of individual peaks from each individual allele was well below +/-1 bp allowing unambiguous allele-calling and automatic generation of results tables using the genotyper software. Furthermore, the PRNP ARMS reaction worked fairly stable with different DNA qualities. The quality and concentration of the DNAs that were isolated from tissue varied widely (5 -- 50 ng/μl, various degrees of fragmentation). Failed PCR reactions (e.g. one primer missing) were identified by analysing the standard samples. Failed individual samples (e.g. due to low DNA concentrations or failure of the fragment analyses run) were easily identified by missing or very low and out-of-range peaks. Nevertheless, the proportion of samples that had to be retyped was well below 1%. Discussion ========== The PRNP ARMS genotyping method has several advantages. It is based on a one step reaction using competitive allele discrimination. The fragments can be analysed on an automated sequencer that allows data to be directly transferred to a database. The reaction proved to be highly specific and no false negative or positive results were observed yet. Furthermore, it is robust with respect to variations of DNA quality and, since no further purification or reaction is necessary, the costs for consumables are low. The ARMS method allows the determination of partial (136-154 and 136-171, compare Fig. [1](#F1){ref-type="fig"}) *prnp*haplotypes, thereby facilitating the detection of \'complex\' prnp genotypes that do not match one of the 15 standard genotype patterns. If necessary, it can easily be extended to complete haplotype determination by adding additional labelled forward primers for position 154 (for haplotype 154-171). All methods currently available for SNP typing are inherent sensitive to nucleotide changes within the primer attachment or restriction sites. The resulting mismatches can cause inconclusive typing results or null alleles, as frequently observed with microsatellite loci. Therefore, it is crucial to be aware of this phenomenon and to take measures to reduce the risk of mistyping. The primers for PRNP ARMS were carefully checked against a *prnp*in-house database containing 16 published and 11 unpublished (mainly from rare German and Spanish breeds) alleles. No known polymorphism interfere with the attachment of the selected ARMS primers. Nevertheless, the recently described rare polymorphism K-171 \[[@B19]\] with unknown effect to scrapie resistance is not included in the standard PRNP ARMS set (that gives Q-171 as typing result for this allele) and requires one additional specific oligonucleotide. This oligonucleotide should be added if the respective breeds (mainly hair breeds \[[@B19]\]) are genotyped. When further polymorphisms associated with resistance to scrapie will be identified appropriate oligonucleotides can easily be added. Unlabelled additional oligonucleotides can be used when SNPs upstream of the amino acid position 136 shall be typed. Nevertheless, the inclusion of positions downstream of position 136 would require additional labelled oligonucleotides. A third fluorescent dye might be useful especially when the lengths of the fragments interfere with on of the other alleles. The PRNP ARMS method is highly flexible with respect to scale and instrumentation. It is easily adoptable from low- to medium-throughput typing system using identical reaction conditions. In principle, all primer combinations can even be performed as single reactions that can be analysed on agarose gels (data not shown). Nevertheless, the reliability of genotyping results improves when competitive reaction conditions and commonly available DNA sequencing machines are used. Since all reactions can be performed in microtiter plates, the ARMS reaction can easily be adopted for a pipetting robot. Together with the one step reaction and the automated data transfer, the risk of cross-contamination or interchanging of samples is minimized. The runtime of 18 min results in 25 min analyses time per sample on the ABI 310 and could be optimised by using shorter capillaries. When using the standard run conditions on the smallest, single capillary sequencer the throughput is limited to about 55 samples per working day but can be scaled up to more than 5000 samples per day by using a 96-well capillary sequencer. That should be sufficient even for large, existing breeding programs. PRNP ARMS can be multiplexed with other typing systems (microsatellite or SNP), since it is analysed using standard fragment analysis technique. Moreover, it is suitable for further applications as e.g. allele frequency estimation from pooled DNA \[[@B20]\] for investigating rare breeds. Conclusions =========== An easy and robust one-step *prnp*typing method has been established that is universally suited for a broad range of typing projects with different requirements. This typing method was developed by optimising ARMS primers and combining these with standard fragment length analyses. The method provides an efficient and inexpensive diagnostic mutation analysis that can be implemented by most molecular genetic laboratories using standard equipment. It should contribute to reliable and economic genotyping of the ovine *prnp*by the many smaller labs throughout Europe. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= J.B. designed the project and protocols involved, performed analysis of results and drafted this manuscript. J.S. conceived the ARMS reaction, fragment analysis and DNA sequencing. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/30/prepub> Acknowledgements ================ We would like to thank Georg Mendel, Albert Steiner and Max Wagenpfeil for collecting sheep samples. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Scheme of PRNP ARMS-PCR design.**PrP amino acid positions are given above. The allele-specific up-primers (discrimination of A-V at codon 136) are fluorescence labelled, whereas down primers (discrimination of R-H and Q-R-H at codon 154 and 171, respectively) are of different lengths (tails are shown as red lines). :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Electropherograms from 5 *PRNP*genotypes.**The \"green\" lanes correspond to TET labelled fragments, the \"blue\" lines to FAM labelled products. The *PRNP*genotype of each individual is given above each lane. Only the informative lanes (no peaks were observed in the \"blue\" lane of A136 and no peaks were observed in the \"green\" lane of V136 homozygotes, respectively) are shown. The scheme at the bottom shows how the peaks relate to the alleles. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Location of ARMS Primers within the reference sequence.**Primer sequences are given above the sequence representing the \"wild-type\" allele (Genbank accession number AJ000739). The 5 prime and 3 prime ends are indicated. Small letters indicate deliberately introduced mismatches; underlining indicate pieces of neutral sequence that were deliberately added to elongate the primer. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### ARMS primers used to type the ovine PRNP ::: Name Sequence of Primer 5\'-3\' Allele Length/Label Fragment Length ------------ ------------------------------------------------------------------ -------- -------------- ----------------- PRNP\_136A [A]{.underline}TACATGCTGGGAAGTG**C** A^136^ 18 bp/TET \- PRNP\_136V CTACATGCTGGGAAGTG**T** V^136^ 18 bp/FAM \- PRNP\_154R [AC]{.underline}TAACGGTACATGTTTTCA**C** R^154^ 21 bp/- 92 bp PRNP\_154H [AGTTT]{.underline}GTAACGGTACATGTTTTgA**T** H^154^ 25 bp/- 96 bp PRNP\_171Q [GG]{.underline}AAGTTGTTCTGGTTACTATc**CT** Q^171^ 24 bp/- 146 bp PRNP\_171R T[CAGTT]{.underline}AAGTTGTTCTGGTTACTATt**CC** R^171^ 28 bp/- 150 bp PRNP\_171H [TTCTGA]{.underline}GC[AT]{.underline}AAAGTTGTTCTGGTTACTATA**A** H^171^ 32 bp/- 154 bp Small letters indicate deliberately introduced mismatches (ARMS principle); underlining indicate pieces of neutral sequence that were added to elongate the primer to allow allele-discrimination by capillary electrophoresis. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### *PRNP*allele frequencies (%) as observed in three sheep breeds common in Bavaria ::: breed ----- ------- ------- ------- AHQ 7.68 0.00 1.96 ARH 0.18 0.00 0.00 ARQ 82.14 24.58 27.45 ARR 9.82 69.49 66.67 VRQ 0.18 5.93 3.92 ML, Merinolandschaf; SK, German Blackheaded Mutton; SU, Suffolk. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Statistics for peak lengths of the PRNP ARMS system ::: Calculated peak sizes^\#^ ----- ------------ -------- --------------------------- ------ -------- -------- TET PRNP\_154R 92 bp 89.16 0.12 88.91 89.70 PRNP\_154H 96 bp 92.13 0.09 91.98 92.45 PRNP\_171Q 146 bp 142.91 0.12 142.67 143.48 PRNP\_171R 150 bp 148.05 0.18 146.72 148.42 PRNP\_171H 154 bp 153.13 0.72 152.31 153.74 FAM PRNP\_154R 92 bp 89.56 0.13 89.36 89.84 PRNP\_154H 96 bp np np np np PRNP\_171Q 146 bp 143.29 0.11 143.13 143.48 PRNP\_171R 150 bp np np np np PRNP\_171H 154 bp np np np np ^\#^peak lengths as calculated using the genotyper software from all genotype results in the database; np: not present in the dataset :::

PubMed Central

2024-06-05T03:55:47.728385

2004-9-2

PMC517713

Background ========== Hepatitis B is an important health problem and a major cause of acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Approximately 30% of the world population (1.8 billion people) have the serologic evidence of HBV infection of whom 350 million are estimated to suffer from chronic HBV (hepatitis B virus) infection; at least 500,000 chronically infected people die of liver malignancy and cirrhosis each year \[[@B1]\]. According to Iranian studies, about 22% to 37% of general population in Iran are HBcAb positive \[[@B2],[@B3]\] (e.g. previous exposure to HBV) and about 1.3% to 8.69% of the population are chronic HBV carriers \[[@B2]-[@B6]\]. Compared to the United States where HBV is the cause of 25% of chronic hepatitis cases, HBV accounts for up to 70% to 80% of chronic hepatitis cases in Iran \[[@B7]\]. Therefore, HBV alone is the leading cause of chronic liver disease (CLD) in Iran and it is evident that HBV transmission prevention can be one of the health priorities in the country. We believe that major routes of HBV transmission, local epidemiological factors and the already performed prevention programs of each region are among important factors in identifying the populations at risk of the infection and planning the region-specific prevention strategies. In Iran, universal neonatal vaccination against HBV started in 1993 according to WHO recommendations. It means that Iranians 10 years of age or older at the time of this study received no prevention services against HBV and so most of them could contract the infection if attacked by the virus. For this at-risk population, several preventive strategies can be suggested which of course should have economic justification. Premarriage transmission prevention can be considered as one of the possible solutions to protect this population though it should not be regarded as the only or the best solution available. Sexual contact is one of the common routes of HBV transmission. In Iran, due to a particular cultural and religious background, homosexuality is not known as a common phenomenon compared to the western countries. For the same reasons, it is very unlikely for an individual to have sexual contact (especially in the form of intercourse) with his/her would-be spouse. On the other hand, almost all premarriage individuals (those considering legal marriage) are obliged by Iranian law to undergo a predefined battery of screening tests in government-designated laboratories; this can make premarriage individuals an accessible group for a preventive intervention. Finally, since most premarriage individuals are in young age groups, they have a rather long life expectancy, which allows enough time for them to suffer from chronic complications of HBV infection in productive years of life. We decided to perform this study to provide health policy makers in Iran and those countries with similar demographic conditions (especially in the Middle East) with an economic analysis of premarriage prevention of hepatitis B transmission. Methods ======= Model ----- The economics of performing two rather similar strategies in addition to no intervention strategy were compared using a decision tree (Fig. [1](#F1){ref-type="fig"}). The software used for analysis was Decision Analysis by TreeAge (DATA™, Williamstown, MA, USA). As shown in Fig. [1](#F1){ref-type="fig"}, the overall options available for premarriage individuals can be one of the major strategies mentioned below: 1\. Screening all premarriage individuals for HBsAg and then performing the following prevention protocol (marked as P.P. in Fig. [1](#F1){ref-type="fig"}) for HBsAg negative individuals whose would-be spouse is HBsAg positive: **a.** Three-dose HB vaccine (0, 1, 6 mo) **b.** Single dose HBIG injection **c.** Using condoms (2 boxes/mo) during all intercourses for 7 months **d.** Measurement of HBsAb (hepatitis B surface antibody) 1 month after the 3rd dose of the vaccine **e.** An extra dose vaccine and additional condom protection for another month for the persons whose HBsAb is not in protective ranges (lower than 10 IU/l) No protection protocol is considered for those couples who are both HBsAg positive or negative. 2\. Screening all premarriage individuals for HBsAg followed by rescreening of the HBsAg negative spouses of HBsAg positive persons for HBcAb and finally performing the prevention protocol (e.g. the **a to e** steps above) only for HBcAb (and HBsAg) negative individuals whose would-be spouse is HBsAg positive. No protection protocol is considered for those couples who are both HBsAg positive or negative. 3\. No screening and no prevention. The main analysis considered the perspective of health care system. However, in the final analysis (including the sensitivity analysis), a threshold analysis was performed from societal perspective. The results were expressed by the cost per chronic infection (e.g. more than 6 months HBsAg positive) averted (e.g. average cost effectiveness). Assumptions ----------- The assumptions in the model included: 1\) HBV vaccination is not harmful for the individual or community; 2\) the efficacy of preventive methods such as 3-dose HB vaccine, HBIG injection and condoms in preventing sexual transmission of HBV do not significantly vary by geographic region; 3\) the compliance of the population for receiving the preventive methods is 100%; 4\) the preventive methods are available throughout the country and can cover 100% of the population; 5\) HBcAb positive persons are not at risk of HBV infection and this group will not be considered for receiving preventive measures; 6\) all HBsAg positive individuals are HBcAb positive too; 7\) the sensitivity and specificity of the screening tests (for HBsAg and HBcAb) are 100%; 8\) the average age at marriage is 25 years of age for both sexes; 9\) the costs associated with CLD are incurred during a 10-year period starting at the age of 50 \[[@B1],[@B24]\]; 10\) the transmission rate of HBV from men to women and vice versa are equal; 11\) premarriage individuals do not have intercourse before marriage; and 12\) Iran is located in a region with intermediate prevalence of hepatitis B. Probabilities and costs ----------------------- The probabilities included in the decision analysis model (Table [1](#T1){ref-type="table"}) were assumed to be of 2 types: 1) the probabilities that do not significantly vary by geographic location (e.g. the efficacy of 3-dose HB vaccine, HBIG injection and condom in preventing sexual transmission of HBV) and 2) the ones that seem to be significantly different in various geographic locations (e.g. HBsAg or HBcAb prevalence rates in the society). To have preliminary estimates of the latter category probabilities, all available and accurate Iranian medical literature (published from 1993 to 2003) were reviewed. For the former category probabilities, international resources (PubMed) were included in addition to the Iranian sources mentioned above. If a probability was not found in medical literature, the consensus of an expert team including 5 gastroenterologists collaborating with our research center was considered as the base case value. A review of Iranian studies showed that the prevalence of HBsAg in general population varied from 1.2% to 8.69% in different parts of Iran \[[@B2]-[@B6]\]. In this study, we considered an average rate of 2% as the baseline in a range of 1% to 9% (Table [1](#T1){ref-type="table"}). The Iranian studies also showed that the prevalence of HBcAb varied from 15% to 37% in different parts of Iran. Therefore, we assumed an average rate of 20% as the baseline in a range of 15% to 40% (Table [1](#T1){ref-type="table"}) \[[@B2],[@B3]\]. The review of international and Iranian medical literature revealed that the probability of being HBsAg positive for an HBsAg positive person\'s spouse (P3) is about 4% to 15% \[[@B8]-[@B11]\]. We assumed a baseline probability of 5%. (Table [1](#T1){ref-type="table"}) The probability of becoming HBsAg positive for an HBsAg positive person\'s spouse after receiving the prevention protocol (P4) was indirectly calculated by the formula below: P~4~ = P~3~ (1 - efficacy of preventive protocol) The baseline value for the efficacy of the prevention protocol used in our model to protect against spouse-spouse HBV transmission (3-dose HB vaccine, HBIG injection and condom protection up to complete immunity) was assumed to be 90%. The figure was reached by considering the values found in the literature for the efficacy of 3-dose HB vaccine \[[@B12],[@B13]\], HBIG injection \[[@B14]-[@B19]\] and condom protection \[[@B20]-[@B23]\] which was finally modified by consensus from the expert team described above. The most pessimistic and optimistic estimations for the efficacy of the prevention protocol were assumed to be 75% and 100%, respectively. Therefore, the baseline value for P~4~ was assumed to be 0.05% in a range of 0% to 1.25%. The direct medical costs of interventions (Table [2](#T2){ref-type="table"}) were extracted from the resources and tariffs of Iranian Health Ministry, Iran Pasteur Institute and Iranian Transfusion Organization in 2003 (unpublished data) and were used as baseline costs in the model. The indirect medical costs of the intervention such as transportation and time costs for the recipients of the preventive methods (to receive the services) were assumed to be zero and were not included in the model. The costs of the averted morbidity (HBV infection especially chronic liver disease) were not directly put in the model because of unavailability of relative Iranian studies. However, as a solution to better analysis, the latter costs were calculated as a final variable in a threshold analysis, considering the fact that of the adults in chronic carrier state, 15% will eventually develop chronic liver disease (CLD) \[[@B1],[@B24],[@B25]\]. In most economic analyses, the costs are modified for the outcomes occurring in the future, a process called discounting. Considering the average age at marriage and the age at which CLD starts (see assumptions) the cost of CLD calculated through the threshold analysis was discounted by a discount rate of 3% to the beginning of the 10-year period of CLD development. The costs are expressed in the text and tables in US \$ and Iranian Rials (1US \$ = 8300 Iranian Rials). The currency conversion rate reported here is the one for mid-2003 when the study was performed. Sensitivity analysis -------------------- Uncertainty management, which is one of the central processes in decision-making, usually involves working with probabilities that usually vary in different circumstances. Therefore, the outcome values and final decision is prone to change when the value of probabilities change. Sensitivity analysis is a method in which the final decision and the value of outcomes are estimated while each probability (univariate) or combinations of probabilities (multivariate) are varied in a reasonable range. This will reveal the variables whose change the model is sensitive to. In this study, univariate sensitivity analysis was performed for prevalence of HBsAg positivity in general population (P~1~), prevalence of HBcAb positivity in general population (P~2~), probability of becoming HBsAg positive for an HBsAg positive person\'s spouse (P~3~) and probability of becoming HBsAg positive for an HBsAg positive person\'s spouse after receiving the prevention protocol (P~4~). Multivariate sensitivity analysis was performed for P~1~ and P~2~, considering the fact that the two probabilities were dependent. All of the direct medical costs of the intervention (Table [2](#T2){ref-type="table"}) were assumed to be constant in the sensitivity analyses. The results of the above sensitivity analyses were evaluated in respect of their impact on the value of the cost per chronic HBV infection averted and the preference of strategies 1 and 2. Results ======= Having run the model for baseline values (Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}), the average cost effectiveness of strategies 1 (without additional screening for HBcAb) and 2 (including additional screening for HBcAb) were 1,675,500 Rials (202 \$) and 1,633,200 Rials (197 \$) for each chronic HBV infection prevented, respectively. The worst-case analysis (e.g. setting all input probability values so that they would act to decrease effectiveness and increase the costs) was performed setting P~1~, P~2~ and P~3~ at their minimum values and P~4~ at its maximum value. It showed that the average cost-effectiveness ratio of strategy 1 would be 2,460,204 Rials (296 \$) and that of strategy 2 would be 2,440,015 Rials (293 \$) in the worst case. It is noteworthy that the costs were not varied and were kept at their baseline values in this sensitivity analysis. A threshold analysis was performed to find the threshold value for the lifetime cost of CLD for one individual (from a societal perspective) below which the interventions in the model were not cost saving (e.g. the net benefit was negative). The analysis was performed keeping all other variables at their baseline values. The preliminary (non-discounted) threshold value for the lifetime cost of CLD was found to be 11,170,000 Rials (1346 \$) and 10,888,000 Rials (1312 \$) using strategies 1 and 2, respectively. After discounting, the threshold figures for CLD costs were 23387500 Rials (2818 \$) for strategy 1 and 22797054 Rials (2747 \$) for strategy 2. This showed that for the cost of CLD higher than the thresholds above, the respective strategies used for HBV transmission prevention would be cost saving. Sensitivity analysis -------------------- Sensitivity analysis showed that when the prevalence of HBsAg positivity in general population (P~1~) varied from the minimum to maximum, the cost per chronic HBV infection averted varied from 1,503,440 Rials (183 \$) to 2,740,560 Rials (330 \$) in strategy 1 and from 1,482,620 Rials (179 \$) to 2,568,310 Rials (309 \$) in strategy 2 (Fig. [2](#F2){ref-type="fig"}). When the prevalence of HBcAb positivity in general population (P~2~) increased from the lowest to highest, the cost per chronic HBV infection prevented did not vary in strategy 1; but in strategy 2, it decreased from 1,645,720 Rials (198 \$) to 1,561,530 Rials (188 \$). Therefore, the higher rates of HBcAb positivity made the cost of strategy 2 become remarkably lower than that of strategy 1 (Fig. [3](#F3){ref-type="fig"}). Changing the probability of becoming HBsAg positive for an HBsAg positive person\'s spouse after marriage (P~3~) from minimum to maximum varied the cost-effectiveness ratio from 2094370 Rials (252 \$) to 558,500 Rials (67 \$) in strategy 1 and from 2,041,500 Rials (246 \$) to 544,400 Rials (66 \$) in strategy 2. It shows that higher spouse-to-spouse transmission rates significantly increase the cost of both strategies (Fig [4](#F4){ref-type="fig"}). When the probability of becoming HBsAg positive for an HBsAg positive person\'s spouse after receiving prevention protocol (P~4~) changed from *the highest to lowest*, the cost-effectiveness ratio decreased from 2,010,600 Rials (242 \$) to 1,507,950 Rials (182 \$) in strategy 1 and from 1,959,840 Rials (236 \$) to 1,469,880 Rials (177 \$) in strategy 2. It shows that higher efficacy of the preventive protocol results in lower costs-effectiveness ratios (Fig. [5](#F5){ref-type="fig"}). Strategy 2 was always cheaper than strategy 1 for all values of P~1~, P~2~, P~3~ and P~4~ in the univariate sensitivity analyses (explained above). The results of multivariate sensitivity analysis of the two variables P~1~(the prevalence of HBsAg positivity in population) and P~2~ (the prevalence of HBcAb positivity in population) revealed that the strategy 2 was always cheaper than strategy 1 while the two probabilities varied. Discussion ========== Preventing sexual transmission of HBV is not a new issue; however, the authors did not encounter any studies directly addressing the economic aspects of premarriage prevention of hepatitis B in their literature review. The reason can be the particular cultural backgrounds of Iranian community in which extramarital sexual relationships with the would-be spouse is expected to be rare due to strong traditional and religious bans against it. Consequently, the model used in this study may not be an appropriate one for countries with remarkable cultural difference in terms of extramarital sexual relationship such as Western countries. On the other hand, the cultural similarities between Iran and some other Eastern countries can increase the external validity of our model for policy makers in such countries. One of the important challenges in our study was the lack of precise data regarding some probabilities. Most of the studies in our literature review contained the prevalence of HBsAg positivity (chronic HBsAg carrier state) in the spouses of HBsAg positive people and the extent to which it was different from the rate in general population \[[@B8]-[@B11]\]. Because such a rate existed in literature, we preferred to use this prevalence rate representing the initial outcome of sexual contact with an HBsAg positive spouse after marriage. Thus, what is seen in the model (Fig. [1](#F1){ref-type="fig"}) as HBsAg+ final outcome does not mean getting infected with HBV; it means getting into a chronic HBsAg carrier state. In our model, we assumed all such cases to be chronic HBsAg carriers that entered the chronic carrier state asymptomatically or following an acute infection; 15% of such carriers could finally develop CLD during their lifespan \[[@B1],[@B24],[@B25]\]. Therefore, it is obvious that our model have primarily focused on more chronic outcomes of HBV infection (e.g. we ignored the costs of acute infections). However, this will not endanger the data robustness in our study; instead, it will always guarantee that all of the cost-effectiveness ratios calculated here are actually higher than that would be resulted with including the costs for acute cases which usually comprise a considerable portion of the symptomatic cases in adults \[[@B1],[@B24],[@B25]\]. The costs of acute and chronic liver disease (CLD) due to HBV infection in Iran were other variables for which data was lacking. One of the important reasons we did not use a Markov model to calculate CLD costs for Iran was the lack of important necessary data for running a Markov model (e.g. lack of data on age specific mortality rates, etc.). We first calculated cost-effectiveness ratios ignoring the costs of acute and chronic liver disease (the costs of the morbidity averted); this surely exaggerated the calculated cost-effectiveness ratios in the study. Thus, one should judge them from a more optimistic point of view. In the second step, to compensate for the lack of lifetime CLD costs in Iranian literature, we performed a threshold analysis using the baseline values for input probabilities and determined a threshold value for the lifetime costs of CLD in Iran, above which the preventive interventions were cost saving. The threshold levels for CLD lifetime costs estimated above do not seem high costs compared with the costs that CLD can impose on the society in terms of direct and indirect medical costs and productivity losses due to time spent sick or years of life lost because of premature death. The relevant medical costs or the costs associated with the productivity losses due to CLD was not accessible at the time of this research, so we discussed the point through some indirect comparisons considering the threshold we calculated for lifetime CLD costs. Though comparing the costs of CLD in Iran with that in the United States does not seem a standard approach, the large differences between the threshold figure we calculated for CLD costs in Iran and the CLD costs estimations (including productivity losses) mentioned in the studies for the United States in 2001 (64,382 \$) \[[@B25]\] may partially reveal some facts \[[@B1],[@B24],[@B25]\]. To perform a more realistic comparison, we used Purchasing Power Parity (PPP) rates instead of exchange rates to convert the threshold cost in Rials into PPP dollars. PPP is defined as the numbers of units of a country\'s currency needed to buy in the country the same amounts of goods and services as, say, one US dollar would buy in the United States. The PPP rate for Iranian Rial was extracted from the National Health Account 2002 by Planning and Management Organization of Iran (unpublished data) and from figures reported by the World Bank Group. The threshold cost using PPP rates would be equal to about 9615 to 9863 PPP \$ (for 2 strategies). Therefore, even if the total CLD costs in a country like Iran were 7-fold smaller than that in the United States, the strategies 1 and 2 mentioned in the model could still be cost saving. To give a sketchy view of some of CLD costs in Iran, the costs of a liver biopsy and those of a pharmacotherapy regimen related to CLD was retrieved by contacting discharge and accounting departments of a state-run hospital and a major drug distributor in Tehran. The discounted cost for an uncomplicated liver biopsy needing 1 to 2 days of hospitalization, blood coagulation serial tests and a special liver biopsy needle turned out to be about 2,093,778 Rials (252 \$) in all state-run hospitals in Iran. Pharmacotherapy with new antiviral and immunomodulatory drugs is employed for treatment of active chronic liver disease in patients with hepatitis B \[[@B26]\]. The discounted cost of lamivudine, a typical example of these drugs, can be another instance of costs CLD imposes on many patients. A 12-month course of lamivudine (Iranian brand) in Iran can cost a patient 2,521,956 Rials (304 \$). If the same calculation is performed for the foreign brand of lamivudine available in Iran (Zeffix), the discounted cost will be 12,991,892 Rials (1565 \$). In addition, these costs may be much higher when some more expensive drug regimens are used or more prolonged regimens are repeated due to chronicity or intractability of disease. The costs mentioned here can not directly give a clue to total CLD costs and mentioning them was to help the reader get a view of the scale of some familiar CLD costs in Iran. In a different approach, we converted the threshold value into the number of productive months for a national of a country having a GDP (Gross National Product) per capita like Iran. GDP per capita shows the amount that an individual contributes to domestic income every year. According to the World Bank Group, Iran had a GDP per capita of 1641 \$ in 2002, and the average annual growth of GDP per capita in Iran for 2002 to 2006 was 4.5%. From here, the GDP per capita in 2003 comes to be 1790 \$. Assuming that the average growth of GDP per capita for Iran will remain at 5% in the coming 25 years (the period used for discounting the CLD threshold costs), the GDP per capita will be 6062 \$ at the end of the period. Considering such figures, the threshold calculated above would be equal to about 6 months of productivity based on a GDP view. The average number of Quality Adjusted Life Years (QALY) that CLD can deduct of an Iranian\'s life is not yet determined. Nevertheless, a 6-month period does not seem a long time compared with the life years lost due to premature mortality and the QALYs lost due to sickness in the proportion of CLD patients with cirrhosis and hepatocellular carcinoma only. Finally, the fact that the strategies would be cost effective can be further emphasized when taking into account the costs of acute cases of HBV infection that comprise the majority of symptomatic cases in adults and were ignored in the model due to lack of Iranian data and for sake of simplicity. Another similar topic that merits discussion here is the topic of mother-to-child vertical transmission of HBV. If a female gets into a chronic HBsAg carrier state and remains positive for HBeAg during pregnancy, it is very likely that her child is infected with HBV. Since a considerable proportion of infected infants will get chronic HBV carriers, this can lead to newer CLD cases further increasing the costs of CLD. Considering this fact, the cost of averted morbidity owing to the preventive strategies will increase even more and the model would seem more cost-effective. On the other hand, the prevention protocol in our model might seem a bit extravagantly designed when looking at the final HBsAb test and the extradose of HB vaccine administered when HBsAb serum levels are insufficient. In addition, one may argue that condoms may be more available while being nearly as protective against HBV transmission as HBIG injection. This can be useful in modifications poor countries can make to the model to get similar results in lower price or with more flexible/available choices. The average cost-effectiveness ratios associated with the two preventive strategies shown in the model did not differ much. The strategy 2 was always slightly cheaper than strategy 1. When the prevalence of HBcAb positive people in the general population (P2) rose, strategy 2 would get remarkably cheaper. Finally, it is noteworthy to mention the issue of compliance. As explained in methods, we assumed all recipients of the preventive interventions (strategies 1 and 2) would be 100% compliant and there was a full coverage of such services in the country. The reason for such assumption was the strict regulations set by Iranian government for all premarriage individuals to undergo a battery of screening tests in which the strategies in our model could be integrated. Nevertheless, we can assume that compliance variations can affect the efficacy of our prevention protocol (e.g. with lower rates of compliance for accepting preventive strategies, we will have lower efficacy of the prevention protocol). As stated in results, we performed sensitivity analysis for P4 (probability of becoming HBsAg positive for an HBsAg positive person\'s spouse after receiving the prevention protocol), which is a variable dependent on the efficacy of the prevention protocol (see the formula in methods). Therefore, we indirectly incorporated compliance into our model\'s sensitivity analysis. Considering compliance issue, we may prefer strategy 1 because it includes fewer steps (e.g. it does not include screening for HBcAb) and may be easier to administer in a low compliance population especially that it is negligibly more expensive than strategy 2. Conclusions =========== Finally, we conclude that applying the preventive strategies in our model for HBVsexual transmission prevention before marriage in the countries with cultural backgrounds similar to Iran seems cost saving. Further investigations in the country for precise calculation of costs of HBV infection especially the costs associated with CLD is necessary for more accurate economic evaluations. Competing Interests =================== Nond declared. Author\'s Contributions ======================= **PA**: Proposing the main idea, supervising the project and counseling the methodology development **MR**: Developing methodology, critical appraisal, rewriting the final article from draft, literature review, and responding to reviewers **DR**: Literature review, methodology development, decision tree development, calculations and results, and writing the first draft of the article **NB**: Contributing to decision tree development, searching literature for costs **SA**: Review literature for probabilities, contributing to decision tree **MHS**: Counseling the scenarios of the decision tree model **SS**: Counseling the methodology, model development and discounting problems **MRZ**: Senior supervisor of the research project All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/31/prepub> Acknowledgements ================ The research project was accomplished in and financially supported by Research Center for Gastroenterology and Liver Disease, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. The authors wish to express their special gratitude to Dr Ahmad Shavvakhi for his kind assistance and counseling in this study. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The decision tree model for premarriage prevention of HBV sexual transmission. Two similar intervention strategies start with HBsAg screening of all premarriage individuals. HBsAg negative individuals whose would-be spouse is HBsAg positive are considered for further intervention. Strategy 2 contains additional screening for HBcAb and applying the preventive protocol (P.P.) -see text- only to those negative for HBcAb (assumed to be serosusceptible) whereas in strategy 1, no HBcAb screening occurs and the prevention protocol (P.P.) is immediately performed. Only important probabilities are depicted in the figure. P~1~, P~2~, P~3~, P~4~ are defined in Table 1. P.P. stands for the preventive protocol explained in methods section in text. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### While the prevalence of HBsAg positivity in general population (P~1~) varied from the minimum to maximum in sensitivity analysis, the cost per HBV chronic infection averted varied from 1,503,440 Rials (183 \$) to 2,740,560 Rials (330 \$) in strategy 1 and from 1,482,620 Rials (179 \$) to 2,568,310 Rials (309 \$) in strategy 2; strategy 2 remained slightly cheaper throughout the range. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### When the prevalence of HBcAb positivity in general population (P2) increased from the lowest to highest limit, the cost per chronic HBV infection prevented did not vary in strategy 1, but in strategy 2, it decreased from 1,645,720 Rials (198 \$) to 1,561,530 Rials (188 \$). Therefore, the higher rates of HBcAb positivity made the cost of strategy 2 become more remarkably lower than that of strategy 1. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Changing the probability of becoming HBsAg positive for an HBsAg positive person\'s spouse (P3) after marriage from minimum to maximum varied the cost per HBV chronic infection averted from 2094370 Rials (252 \$) to 558,500 Rials (67 \$) in strategy 1 and from 2,041,500 Rials (246 \$) to 544,400 Rials (66 \$) in strategy 2. It shows that higher spouse-to-spouse transmission rates significantly increase the cost of both strategies. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### When the probability of becoming HBsAg positive for an HBsAg positive person\'s spouse after receiving prevention methods (P4) changed from the highest to lowest, the cost per HBV chronic infection averted decreased from 2,010,600 Rials (242 \$) to 1,507,950 Rials (182 \$) in strategy 1 from 1,959,840 Rials (236 \$) to 1,469,880 Rials (177 \$) in strategy 2. It shows that higher efficacy of the preventive intervention results in lower costs-effectiveness ratios. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Input values for the probabilities in the model. Min. and Max. values were used in sensitivity analysis. ::: **Variable** **Definition** **Baseline** **Min.** **Max.** **References** -------------- --------------------------------------------------------------------------------------------------------- -------------- ---------- ---------- ---------------- P~1~ Prevalence of HBsAg positivity in general population 0.02 0.01 0.09 2--6 P~2~ Prevalence of HBcAb positivity in general population 0.20 0.15 0.40 2,3 P~3~ Probability of becoming HBsAg + for an HBsAg + person\'s spouse 0.05 0.04 0.15 8--11 P~4~ Probability of becoming HBsAg + for an HBsAg + person\'s spouse after receiving prevention protocol ^§^ 0.005 0 0.0125 12--23 ^§^ See text for details. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Input values for the costs in the model.^§^ ::: **Variable** **Definition** **Cost Rials (US \$)**\* -------------- -------------------------------------- -------------------------- C1 Cost of HBsAg screening 30000 (3.6) C2 Cost of HBcAb screening 30000 (3.6) C3 Cost of HB vaccination (one dose) 40000 (4.8) C4 Cost of HBIG injection (single dose) 120000 (14.5) C5 Cost of Condoms (one box) 8000 (1) C6 Cost of HBsAb screening 30000 (3.6) ^§^ The direct medical costs of the interventions were extracted from the resources and tariffs of Iranian Health Ministry, Iran Pasteur Institute and Iranian Transfusion Organization (unpublished data). \* 1US \$ = 8300 Iranian Rials :::

PubMed Central

2024-06-05T03:55:47.730212

2004-9-4

PMC517714

Background ========== The Severe Acute Respiratory Syndrome (SARS) was first reported in November 2002 and rapidly spread to a number of distant global regions by early 2003. A new coronavirus, the SARS-CoV, was identified to be the cause of SARS \[[@B1],[@B2]\] and was rapidly sequenced and characterized \[[@B3],[@B4]\]. SARS-CoV is an enveloped, positive strand RNA virus with a wide host range. Recombination and mutation rates of RNA viruses are high, several orders of magnitude higher than DNA based microbes and in eukaryotes, and have been the cause of rapid changes in antigenicity, virulence, and drug sensitivity. Thus, the direct estimate of the mutation rates of the SARS-CoV in human populations and the analysis of the mutational spectrum would aid in developing strategies for monitoring and therapy. Previously, our analysis of 14 SARS sequences (five of which originated from Singapore) in May 2003 indicated that there are two different genotypes circulating in the world \[[@B5]\]. Recently, there has been a substantial increase in the number of SARS-CoV genomes sequenced. A total of 54 SARS-CoV genomic sequences (37 from the public database prior to October 14, 2003 and 17 sequenced within our institute) are used in our current analysis. This large dataset coupled with the availability of clinical data for cases related to Singapore patients and our molecular observations during in vitro cell passage presents an opportunity for a comprehensive analysis of the SARS-CoV mutational behavior. Methods ======= Viral RNA genome isolation and sequencing ----------------------------------------- SARS-CoV from the primary patient tissues were isolated by homogenizing the tissues in PBS buffer followed by a low speed centrifugation to obtain the viral particle containing supernatant. The virus-containing samples were also inoculated into Vero cell E6. The cells were maintained at 37°C using the usual viral cell culture media, and repassaged after 7 days of incubation. The virus-containing supernatants of homogenize or different passages of Vero cell E6 showing CPE were centrifuged at 23,000 RCF for 2.5 hours to pellet the viral particles and followed by RNA extraction using the QiAmp viral RNA mini kit (Qiagen, <http://www.qiagen.com>). The RNA genome templates were converted into double strand cDNA and sequenced as previously described \[[@B5]\]. The processing of raw sequence reads (base calling, assembly, and editing) was done using PHRED/PHRAP/CONSED (University of Washington, Seattle, WA, USA, <http://www.phrap.org>). Genotype determination using MassArray technology ------------------------------------------------- A number of single nucleotide variations (SNVs) were further confirmed using a sensitive Mass Spectrometry based genotyping assay that was developed within our institute \[[@B6]\]. The RNA of the virus was first isolated using QiAmp viral RNA mini kit and then reverse-transcribed into cDNA (using the RNA as template, SuperScript kit from Invitrogen, and sequence specific primers), which were further purified. Primer extension assays were carried out for the SNVs of interest. The extension products were then detected in the MassARRAY (from Sequenom) to determine the genotypes. Data and statistical analysis ----------------------------- We aligned the 54 SARS-CoV genomes using CLUSTALW \[[@B7]\]. To minimize the effect of sequencing errors and other artefacts to our analysis, we employed a filtering scheme where only SNVs shared by more than two different isolates are kept. The phylogenetic trees were reconstructed using the filtered variations. The reconstruction was done using PAUP\* \[[@B8]\] with Maximum Likelihood criterion, keeping the other parameters to the default. The significance of the variations that pass the proposed mutation filter (where only mutations shared by more than 2 out of 54 isolates are considered real) can be assessed by calculating the probability that a random noise would meet the filtering criterion. The null hypothesis is that the noisy variations are generated independently between genomes. Let *q*be the rate of noisy mutation in a genome (based on our findings, as reported earlier in the text, we conservatively set , i.e. about two per SARS-CoV genome). The probability that, at a given nucleotide, a noisy mutation is shared by exactly *i*out of *m*different isolates is . Thus, the probability that a given nucleotide position has an erroneous mutation shared by more than *k*isolates is . In a genome with *n*bases, applying the Bonferroni inequalities, the probability that at least one position is corrupted by noise more than *k*times is *p*(*k*,*n*,*m*) ≤ *n*× *s*(*k*,*m*). In the case of 54 SARS-CoV genomes analyzed in this paper, , *m*= 54, *k*= 2, *n*= 30000, and hence the *p*-value of mutations that satisfy the filter is ≤ 2.2 × 10^-4^. In the estimation of SARS-CoV daily mutation rate, we employed the conservative constant mutation rate model \[[@B9]\], where the number of mutations *d*found in an isolate from its ancestor is proportional to the mutation rate *k*and the temporal difference *t*between the isolate and its ancestor, so that *d*= *k*× *t*. Based on the Singapore patients contact tracing information (see Figure [4](#F4){ref-type="fig"}), we obtained 6 pairs of isolates with known definite ancestor-descendant relationship, calculated the number of mutations (*d*) and the time difference (*t*) for each pair, and estimate the mutation rate *k*for the model using least square fitting. The goodness-of-fit were measured using the adjusted R-square statistics. Another pertinent question in the analysis of SARS-CoV evolution is prediction of the possible date of origin of the human SARS-CoV. Based on the animal-origin hypothesis of SARS-CoV, we assumed the SARS-CoV isolated from palm civet cat as the putative principal isolate that infected the human population. Adhering to the constant mutation rate model, we fit the following model: *d*~*x*~= *d*~0~+ *kx*, where *k*is the daily rate of mutation, *x*is the sampling date measured relative to 1^st^November 2002, and *d*~*x*~is the number of mutations, as compared to the civet cat isolate, of the isolate sampled at date *x*. Twelve data points were calculated and used to fit the model. The date of origin can be solved by solving *x*for *d*~*x*~= 0. The goodness-of-fit was measured using the adjusted R-square statistic. Results and discussion ====================== SARS-CoV mutations *in vitro* ----------------------------- First, we sought to determine the rate of mutation of the SARS-CoV in Vero cell culture. To achieve this, we subjected SIN2774 isolate to 5 passages in Vero cells. At the appearance of cytolysis at each passage samples were withdrawn and their genomes completely sequenced. Any ambiguities by capillary sequencing were clarified by mass spectroscopic validation \[[@B6]\]. Our results showed that the Vero cell passages are actually comprised of two coexisting quasispecies bearing either an A or G at position 18372. No mutations emerged upon passage, and the ratio of A/G at 18372 remained constant over the passages (Table [1](#T1){ref-type="table"}). This suggests that the mutation rate in culture of SARS-CoV is very low at \<1 in 5 passages. Thus artificial mutations from limited *in vitro*cell culture are negligible. Mutations associated with human-to-Vero and Vero-to-human transition -------------------------------------------------------------------- Next, we asked whether the transition from human tissue to growth in Vero cell culture engendered either mutations or clonal selection. The SARS-CoV were sequenced from three human tissue-Vero cell culture pairs of viral samples from Singapore and one pair was obtained from the public domain (see supplemental information, Table S1). The results shown in Table [2](#T2){ref-type="table"} showed that mutations emerged in only one case of human to Vero cell passage posted in Genbank (AS → HSR1) but in none of the Singapore pairs. However, the viral sequence from SIN3725V isolated from a lung sample showed evidence for co-infection by two distinct genotypes of SARS CoV. This was manifested by the simultaneous presence of T and C at positions 548, 1727, 13347, confirmed by genotyping using MALDI-TOF MassARRAY technology. Subsequent deconvolution by tracing the \"haplotypes\" at these loci in different Singaporean isolates revealed probable sequence signatures of T, T, C at these locations in one isolate and C, C, T in the other (see Table S2). Though tissue-derived SIN3725V has two SARS-CoV quasispecies, the isolate after subsequent Vero cell culture showed only one (bearing the T, T, C haplotype, supplemental information, Table S2). These results again show that coinfection by multiple quasispecies is not uncommon in human tissues, and that passage to Vero cells may either generate new mutations at a low rate, or titrates out one quasispecies in the transition. Singapore encountered an unusual incident where a stable lab SARS-CoV isolate commonly used for in vitro experimentation accidentally infected a laboratory worker \[[@B10]\]. We sequenced both the originating laboratory isolate (SIN\_WNV; see Table S1) and the viral sample directly from the patient\'s sputum (SIN0409; see Table S2) and found no sequence difference between the two viruses. This reconfirms that the mutation rate from a single point source of virus has a low mutation rate when expanded during human infection. Sequence variation filter ------------------------- Inferring phylogenetic relationships between the known SARS-CoV isolates using existing public data has been problematic because of the potential for sequencing errors. Moreover, the rate of SARS-CoV mutation in culture was not previously known and was thought to be significant given the mutation rates in other RNA viruses. Our experiments provided information as to the potential causes and rates of sequence variations of the SARS-CoV in culture. Based on our Vero cells passages and human-to-Vero transition data, we estimated that, at most, one sequence variation from the original tissue virus can be accounted for by in vitro culture artifacts. The average base-calling error probability (as reported by PHREP \[[@B11]\]) of our sequences is about 7.5 × 10^-5^, or 2.25 errors per SARS-CoV genome. Accordingly, we suspected that sequencing errors in the reported SARS sequences would be approximately 1--2 bases per reported genome. We used this information to assess the true sequence variants reported in the public SARS sequence databases employing a \"mutation filter\" \[[@B5]\]. This mutation filter identifies a sequence variant as a probable mutation if it appeared in more than one isolate. Higher filter stringency can be applied by demanding a sequence variant to be present in two, three, or more isolates. A total of 54 isolates were analyzed, including 22 from Singapore. Our results show that the number of mutations appearing in only one isolate is high at 349 (see supplementary Table S3); however, those mutations present in more than two isolates are much lower and appeared relatively stable (Figure [1](#F1){ref-type="fig"}). Statistical analysis confirmed that the probability of finding any false mutation shared by more than two isolates out of 54 is very low (*p*≤ 2.2e - 4) as compared to the probability of finding a false mutation shared by more than one isolate (*p*≤ 0.19). These results are consistent with our error estimates as outlined above. We tested the biological validity of this approach by examining the mutational frequency of known genes in the SARS genome. Because of the importance of the 3CL protease and the polymerase for viral replication, we suspected that true non-synonymous mutations in the SARS-CoV present in clinical samples might be rare in these two ORFs in comparison to other structural genes such as those encoding the S, M, and N proteins. Without a mutation filter, sequence variations are commonly observed in the 3CL protease and the polymerase genes. However, when mutations are identified only as variants seen in two or more isolates, then no mutations are detected in the critical 3CL protease and polymerase genes, whereas mutations are noted in the S, M, and N genes regardless of the filter stringency (Figure [2](#F2){ref-type="fig"}). Therefore, we determined that the most effective mutation filter is presence of a sequence variant in more than two isolates. Molecular history of the viral isolates --------------------------------------- Using this filter stringency, we assessed the phylogenetic relationship between all 54 isolates describing the recent SARS epidemic. TOR2, Urbani, SIN2500, HKU-39849, CUHK-Su10 formed the core of the early isolates. Employing these as \"root\", four major clusters appeared: two Singaporean branches, one Taiwan branch, and one North China branch (Figure [3](#F3){ref-type="fig"}). Validating these clusters, the two sequences from Germany, \'Frankfurt\' and FRA, which grouped with the Singaporean branch, were actually derived from the Singaporean doctor who treated the patient SIN2774 and was later hospitalized in Frankfurt. Of the Singaporean cases, SIN2500, SIN2677, SIN2748, and SIN2774 formed one molecular sub-branch which matched with the contact tracing (Figure [4](#F4){ref-type="fig"}). The clinical contact tracing data was ambiguous as to the direct source of SIN2679\'s exposure. Intriguingly, however, SIN2679 was the root of a second sub-branch within Singapore that had its origins most probably from the Hotel M cluster. This suggested a potential direct infection of SIN2679 from a Hotel M source other than SIN2500. Using the same contact tracing information, we calculated the average mutation rate during human transmission to be about two mutations per human transmission. Estimation of the mutation rate of the SARS-CoV ----------------------------------------------- We obtained the precise dates of symptom onset of 13 Singaporean cases (Table S1). Using the common mutations identified through application of the mutation filter, we employed the constant mutation rate model and estimated the mutation rate of the SARS-CoV during this recent epidemic. We estimated the mutation rate to be 0.1722 nucleotides per day, or 5.7 × 10^-6^nucleotide substitutions per site per day (adjusted R-square value of the fitted model = 0.4014). The rates for synonymous and non-synonymous mutations were equivalent at 2.5 × 10^-6^and 3.2 × 10^-6^nucleotide substitutions per site per day respectively. Using the Singapore isolates with known date of onset, and using the SZ3 and SZ16 genomes isolated from palm civet cat \[[@B12]\] as the putative \"original\" SARS-CoV that jumped from animal to human, we calculated the daily substitution rate to be 0.1303 nucleotides per day, or 4.3 × 10^-6^nucleotide substitutions per site per day, (adjusted R-square = 0.5880) and the estimated possible \"date\" of SZ3/SZ16 emergence was Oct 21, 2002. Overall, the mutation rate of SARS-CoV appears to be consistent with the reported rate of other viruses \[[@B13],[@B14]\]. The mutational analysis also revealed 5 separate deletions and one insertion that distinguished the different isolates (Figure [5](#F5){ref-type="fig"}). Intriguingly, they all clustered within a short 200 bp region in the 3\' end of the viral genome spanning putative ORFs sars 7b to sars 8b. Despite the overlapping nature of some of the deletions, there was no descendent relationship amongst them and the addition of the insertion/deletion information did not add to the clustering. Our assumption therefore is that this is a region of relative instability that is dispensable for viral replication. Conclusions =========== The focus of this investigation was to measure the mutational frequency and dynamics both in vitro and in vivo of the SARS-CoV. Our findings suggest that the overall SARS-CoV\'s rate of mutation in culture is low. Inoculation of Human SARS-CoV into Vero cell introduces, on the average, less than one nucleotide mutation. Subsequent culturing of SARS-CoV infected Vero cells induced less than one nucleotide mutation in the five consecutive Vero cell passages. No mutations were also observed during the infection of SARS-CoV cultured in Vero cell to human. This would be consistent with the notion that the SARS-CoV isolates from the patients that gave rise to the in vitro lines are well adapted for in vitro growth. Our proposed mutation filter, which is based on these observations, seems to be stable and effective. Using this filter, reconstruction of molecular phylogenetic relations of the 54 SARS-CoV genomes revealed at least three major branches composed of cases related to Hotel M (Hong Kong), cases reported in North China, and cases found in Taiwan. Moreover, we show that these molecular sequence associations can be effectively used to more precisely reconstruct contact tracing. Our estimated the daily substitution rate of SARS-CoV to be 0.1722 nucleotides, or 5.7 × 10^-6^nucleotide substitutions per site, a mutation rate similar to other RNA viruses \[[@B13],[@B14]\]. Taking the SARS-CoV isolated from palm civet cat as the putative originating SARS-CoV, our calculations suggest that the earliest possible date for SARS emergence is predicted to be Oct 21, 2002. During the final preparation of this manuscript two reports were published addressing the mutation rate of the SARS-CoV in human populations. Yeh *et. al.*\[[@B15]\], examining Taiwanese SARS samples estimated the CoV mutation rate to be about 1.83 × 10^-6^nucleotides per site per day. The Chinese SARS Molecular Epidemiology Consortium \[[@B16]\], examining a larger number of viral isolates, recently determined the mutation rate to be 8.26 × 10^-6^nucleotides per site per day using samples from China. These estimates were very close to ours. In addition, The Chinese Consortium \[[@B16]\] projected the time of emergence of the SARS CoV epidemic to be November 2002. The remarkable consensus of these three studies using different patient populations on the mutational dynamics of the SARS CoV suggests that these results are bona fide. Competing interests =================== None declared. Authors\' contributions ======================= VBV and ETL performed most of the data analysis and prepared the draft manuscript. STSY and LAE provided SARS-CoV samples from patients and Vero cells. KFT and OEE provided continue SARS-CoV containing Vero cell passage samples. YR and LWS designed the experiments and coordinated sample acquiring and the viral genome sequencing. CLW and TZ generated all DNA sequence data. WHL processed all DNA sequence and assembled the viral genomes. JL verified all sequence variations by conducting MALDI-TOF mass spectrometry analysis. PK assessed the effects of nucleotide variations to ORFs and their proteins. VBV and PML carried out the mathematical and statistical analysis. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/32/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table S1 List of the sequences used in the analysis. This list is available at http://giscompute.gis.a-star.edu.sg/sars\_mut\_dyn/ ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Table S2 Single nucleotide variations (SNVs) detected initially in capillary sequencing (second row) and subsequently confirmed by MALDI-TOF MS-based genotyping (first row). This table is available at http://giscompute.gis.a-star.edu.sg/sars\_mut\_dyn/ ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Table S3 Complete list of single nucleotide variations (SNVs) observed in the 54 SARS-CoV isolates. The Singapore sequences used were all based on capillary sequencing. This data is available at http://giscompute.gis.a-star.edu.sg/sars\_mut\_dyn/ ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We want to express their appreciation to Mr. Thoreau Herve, Mr. Landri Lim, Ms. Carine Bonnard, Mr. Meah Wee Yang, and Ms. Lin Su for providing technical support, and Mr. Chia Jer Ming for assisting the ORF analysis. This study was supported by the Agency for Science, Technology, and Research of Singapore, and the Biomedical Research Council of Singapore. The authors wish to express their appreciation to Mr. Thoreau Herve, Mr. Landri Lim, Ms. Carine Bonnard, Mr. Meah Wee Yang, and Ms. Lin Su for providing technical support, and Mr. Chia Jer Ming for assisting the ORF analysis. This study was supported by the Agency for Science, Technology, and Research of Singapore, and the Biomedical Research Council of Singapore. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Effects of applying mutation filter to number of substitutions observed.**To exclude nucleotide variations arising from sequencing error or other artifacts, we include only variations that are present in at least a number of different isolates. The graph shows an exponential decrease in the number of nucleotide variations detected with increasing filter stringency. The curve essentially flattens around 3, which corresponds to the filtering scheme that considers a sequence variation as a real mutation if it is shared by more than two isolates. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Non-silent variations in five key ORFs.**Nucleotide variations that lie within a coding region might result in amino acid variations in the corresponding protein product. Such non-silent variations could play a significant role in determining the survivability of SARS-CoV variants. Application of the proposed variation filter reveals conservation of the polymerase (RdRp) and 3CLpro, which is consistent with previous studies of other Corona viruses. :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Molecular relationship between 54 SARS-CoV genomes.**The phylogenetic tree reconstructed using PAUP\* on nucleotide variations shared by more than two isolates. The tree was re-rooted on the earliest reported case, i.e. TOR2. Four major branches can be observed in the tree, each largely belonging to a certain geographical origin. Two of which encompass the Singapore cases, indicating the possibility of separate infection sources of the Singapore cases. :::  ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Relations between the Singapore patients and others related to Hotel M, Hong Kong.**Contact tracing information of the Singapore patients and the two early reported SARS cases. Contact information revealed a single source of infection for Singapore patients, but molecular analysis, reported here and in (6), suggests the existence of an additional infection source (represented by dashed arrows and box) from the Hotel M case to Singapore. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Map of significant mutations, insertions and deletions in the SARS-CoV genome.**Map of the SARS genome, plotted with substitutions appearing in more than two isolates and other major insertion and deletion regions. Golden bars signify the SARS-CoV genome, with the approximate nucleotide positions shown in the scale on top of it. Arrows drawn on top of the genome indicate nucleotide mutations (i.e. variations shared by more than two isolates) observed in the SARS-CoV genome. Amino acid changes in SARS-CoV\'s proteins are reflected as arrows on top of the protein bars (blue bars). Significant multiple-nucleotide deletions (pink bar) and insertions (light-blue bar) were also observed (denoted as arrows under the genome) and appear to cluster around position 27000nt to 28000nt. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Table 1: Single nucleotide heterogeneity (SNH) observed in the six passages of Vero cells culture. Along the six Vero cell passages, nucleotide heterogeneity was observed (initially through capillary sequencing and confirmed using MassARRAY genotyping) at nucleotide position 18356. Presence of single nucleotide heterogeneities (SNHs) indicates coexistence of multiple SARS-CoV isolate in the Vero cell culture. ::: **Genomic Location (Based on SIN2774)** **SIN2774\_P1** **SIN2774\_P2** **SIN2774\_P3** **SIN2774\_P4** **SIN2774\_P5** **ORF** ----------------------------------------- ----------------- ----------------- ----------------- ----------------- ----------------- ------------------------------------------ 18356 A/G A/G A/G A/G A/G 135aa of nuclease ExoN homolog *\[R/G\]* ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Quasispecies fluctuations and mutations during the transition from human tissue to Vero cell culture. Nucleotide variations observed between primary human tissue isolates and their respective subsequent Vero cell culture. Both quasispecies selection (SIN3275V → SIN849M) and new emergence (AS → HSR1) are observed during the transmission of SARS-CoV from human tissue sample into Vero cell culture. ::: **Source of viral sequence: Human Tissue** **Source of viral sequence: Passage to Vero cell culture** **Nucleotide Heterogeneity** **Position (Urbani)** **ORF (based on NC\_004718.3)** -------------------------------------------- ------------------------------------------------------------ ------------------------------ ----------------------- ----------------------------------- Sin3408L Sin842M \- \- \- Sin3725V Sin849M Y \[t/C\] → T 548 95aa of Leader protein (I, T → I) Y \[T/C\] → C 13347 131aa of NSP10 (SILENT) Sin3765V Sin852M \- \- \- AS HSR 1 G → R \[G/A\] 27254 637aa of sars6 (D → D, N) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Nucleotide variations during the transition from Vero cell culture to human. No nucleotide variations were observed between isolate from laboratory-acquired SARS patient and its infection source. ::: **Source of viral sequence: Vero cell culture** **Source of viral sequence: Passage to Human Tissue** **Number of stable nucleotide substitutions** ------------------------------------------------- ------------------------------------------------------- ----------------------------------------------- SinWNV Sin0409 (sputum) 0 :::

PubMed Central

2024-06-05T03:55:47.732840

2004-9-6

PMC517715

Background ========== Bile duct dilatation and increasing jaundice are often onset symptoms of a number of either malignant or benign pathological conditions. In most cases, common cross-sectional imaging techniques such as ultrasonography (US) or spiral-CT or cholangio-pancreatico-biliary magnetic resonance (MRCP) are highly capable of depicting the causes of obstruction by simply showing extrinsic masses in case of infiltrating tumors or benign causes of jaundice, like stones \[[@B1]\]. These non invasive techniques, however, can still yield uncertain results in some particular benign or malignant pathological conditions. When stones are too small for example, or when bile duct dilatation is limited. For final confirmation or exclusion of endobiliary stones ERCP is unavoidable before the subsequent therapeutic steps. There are also some pathological conditions where an \"intrinsic\" parietal alteration is the cause of biliary obstruction and it is very difficult to distinguish benign from malignant lesions using cross-sectional imaging procedures alone (figure [4](#F4){ref-type="fig"} - [5](#F5){ref-type="fig"}). A biliary dilatation may also occur after surgical interventions involving the biliary system, like bilioenteric anastomoses for gastric or pancreatic neoplasms or cholecystectomies. It is not always possible to distinguish between a recurrent disease or a fibrotic post-surgical stenosis, especially if it occurs early and if inflammatory alterations were already present during surgery. A number of obstructions remains therefore unexplained because the aforementioned imaging modalities do not show any extrinsic compressing or infiltrating mass, nor a calcolous disease. In these cases, only the intrahepatic bile ducts dilation and the level of the obstruction can be determined, but additional diagnostic procedures should be performed for a diagnosis of the real nature of the obstruction \[[@B2]\]. Patients with obstructive jaundice often undergo percutaneous biliary cholangiography and drainage (PBD) for decompression of the biliary tree. Although relatively invasive, not even PBD allows to achieve a definitive differential diagnosis between malignant and benign pathologies. Tissue sampling becomes therefore essential for the histological characterization of parietal alterations and for planning their appropriate treatment \[[@B3]\]. Bile cytology, although easily feasible either through a transhepatic or an endoscopic route, does not accomplish adequate pathological diagnoses in most cases \[[@B5]-[@B7]\]. In our study, three different endoluminal sampling techniques for the characterization of biliary strictures of uncertain nature have been reviewed and comparatively evaluated with the purpose of determining which one of them is the most accurate for differentiating between benign or malignant pathology. Methods ======= Our investigation was performed on a population of 80 patients who, from January 1992 to September 1999, underwent endoluminal brushing and/or biopsy for biliary stenosis or obstructions of uncertain nature. A retrospective evaluation of the efficacy of these techniques was carried out by comparing the pathological findings on one hand and radiological findings, clinical long-term follow ups, and/or post-surgical specimens, on the other. Patient population included 43 men and 37 women, with a mean age of 62 years (range 37--87 years). All patients presented jaundice as a common symptom, weight loss was present in 20 cases (25%), and itching in 12 cases (15%). At admission, all patients presented biliary dilation, but no masses were identifiable by imaging procedures such as US, CT, MRCP. ERCP had been performed in 23 patients with middle-low common bile duct stenosis but the nature of obstruction could not be determined nor a drainage could be placed. Obstruction were located at the biliary bifurcation in 18 cases (22.5%), the common extrahepatic bile duct in 50 (62.5%), the right (2) and left (2) hepatic duct in 4 cases (5%) Eight patients with bilioenteric anastomoses (10%) were also evaluated in order to determine the nature of the stricture. The patients came to the interventional unit needing for a biliary drainage. PBDs were performed using standard interventional techniques.\[[@B5]\] The percutaneous approach was right lateral in all patients but 25 of them required also an anterior subxyphoid approach. The procedures for cytological and/or histological samples were performed in any case 3--5 days after placement of the biliary drainage in order to operate on a decompressed biliary system. One hundred and two endoluminal procedures were included in this study. Twenty-one patients underwent cytologic brushing only, 25 endoluminal biopsy only, and 22 both brushing and biopsy. A total number of 43 brushings and 47 biopsies were performed. In 12 additional cases, we obtained cytological and tissue samples from an angioplastic balloon used for the bilioplasty of strictures of uncertain nature (balloon surface sampling) with techniques that will be described. Sixteen patients underwent brushing only, because the cholangioscope was not available yet and five because the diagnosis of malignancy achieved with brushing was considered sufficient, thus not requiring further diagnostic workups to planning the appropriate therapy. All the specimens were collected after PBD thus after translesional advancement of a 10 French catheter; in 12 particularly heavy stenosis a preliminary balloon dilatation was also accomplished. Brushing was performed according to the following technical steps: \- the indwelling biliary drainage catheter was exchanged over a guide-wire with a 7/9-F. introducer sheath without valves at the proximal end (\"peel-away\", William Cook, Europe) \- a flexible probe (Fig. [3](#F3){ref-type="fig"}) with a cilindrical brush at the tip, 5 mm in diameter 10 mm in length (Olympus Italia srl. Code number BC20295010), was advanced through the sheath until it was beyond the lesion; the sheath was withdrawn to expose the brush, which was then pulled back and forth and rotated across the lesion several times under fluoroscopic control ::: {#F3 .fig} Figure 3 ::: {.caption} ###### A metallic flexible probe with a cilindrical brush at the atraumatic tip, 5 mm in diameter 10 mm in length(Olympus Italia srl. Code number BC2029501) brush (approximately 1 cm in length and 5 mm in diameter) :::  ::: \- the brush was then carefully pulled back to be removed from the patient into the sheath, to avoid malignant spreading through the transhepatic tract -samples were then placed on a glass slide, fixed with Sprayfix (Surgipath Medical Ind.; Illinois, USA), immediately submitted for cytological examination and stained by the standard Papanicolau technique. Endoluminal forceps biopsy was performed under direct visualization with a 5-mm. cholangioscope (Olympus URF. Type P, Japan) (Fig. [1](#F1){ref-type="fig"}). Sometimes, the site of the lesion endoscopically visible did not correspond to the site of the stenosis fluoroscopically detectable; additional samples were therefore collected under fluoroscopic guidance, according to the following technique: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flexible cholangioscope (5 mm in diameter) (Olympus, URF, type P, Japan) :::  ::: \- the biliary catheter was replaced, over a guide, by a 18-F \"peel-away\" introducer sheath through which the cholangioscope was advanced until it was close to the lesion; \- a flexible forceps (5-F. Olympus, FB 185X Fig. [2](#F2){ref-type="fig"}) was passed through the working channel of the cholangioscope to obtain 3--4 specimens for each patient; ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Alligator forceps (Olympus, FB 195X, Japan). The wire tip is open 25 :::  ::: \- the specimens were fixed in a 10%-solution of formaline and sent to the pathologist, included in paraffin, and dissected in slices of 2--4 microns at microtome; subsequently, they were stained by hematoxilyn-eosin, PAS and Mallory techniques; \- after completion of the biopsy procedures, all the patients underwent cholangiographic control checking for contrast material extravasation at the biopsy site and monitored for symptoms of hemobilia and/or bacteriemia. All the patients were submitted to antiobiotic therapy before and during the three days following endobiliary procedures. Twelve patients underwent preliminary bilioplasty because of the difficulties in advancing the drainage catheter across the biliary stricture. In this group, a different sampling was performed, based on the examination of those cells and/or tissue remained stuck to the angioplastic balloon after dilatation (balloon surface sampling): \- once deflated, the balloon (8--10 mm., Meditech, Boston, USA) was pulled back into the sheath and removed from the patient; \- the balloon was then placed in a saline solution with 10% of formalin, inflated and agitated several times to facilitate detachment of samples, which were then immediately delivered to the pathology laboratory. Pathological specimens from 14 patients were compared with open surgery findings and post-surgical pathological reports. The pathologists, hystologists and cytologists, were blinded as to corresponding results. The follow-up of the patients undergone to surgery stopped after the operation. Patients not candidate to surgery were treated with interventional procedures only such as bilioplasty, biliary drainage, interstitial radiotherapy and/or with chemotherapy or just a supportive therapy. This group of unoperated patients was followed-up on the basis of clinical and radiological data obtained by case-record reviews and by correspondence with their referring clinicians and general practitioners. The follow-up period ranged from 3 to 48 months. A minimum of 12 months of healthy negative cytological-histological diagnosis was necessary because either a clinically benign stenosing lesion or a cytohistological diagnosis negative for malignancy must be confirmed by a prolonged survival, as well as by clinical and radiological findings. Calculations of success rate, sensitivity, specificity, positive predictive value, and negative predictive value for each technique were based on the number of biopsy procedures (n = 102) rather than on the number of patients (n = 80), (Table [1](#T1){ref-type="table"}) since each biopsy was considered as a separate event The success rate is the percentage of biopsy procedures resulting in sufficient material for microscopic evaluation. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Differentiation of biliary obstruction with different percutaneous endoluminal techniques ::: **Technique** **N. PTS** **SR** **TP** **TN** **FP** **FN** ------------------ ------------ -------- -------- -------- -------- -------- Brushing 43 \* 39/43 11 16 0 12 Biopsy 47 \* 47/47 35 9 0 3 Balloon Brushing 12 12/12 7 4 0 1 SR= Success Rate; TP= True Positive; TN= True Negative; FP= false positive; FN= False Negative \*22 patients underwent either brushing and forceps biopsy. ::: Confidential Intervals (CIs) were determined for brushing and biopsy groups assuming a P value of .01 by using the Geigy Scientific Table (Geigy, Florence, 1984). The agreement coefficient between biopsy or brushing and final diagnosis was calculated using the Cohen\'s \"K\" value using SPSS 8.0 for Windows (SPSS, Chicago, Illinois, 1997). Results and discussion ====================== A final diagnosis of malignant disease was confirmed in 56 (70%) cases and a final diagnosis of benign disease in 24 (30%) cases for an overall of 80 patients. Final diagnoses in the malignant group included: cholangiocarcinoma (n = 31), adenocarcinoma (n = 6), metastatic adenocarcinoma (n = 5), pancreatic carcinoma (n = 13) and malignant endocrine tumor (n = 1). Final diagnoses in the benign group included: iatrogenic stenosis (n = 9), sclerosing cholangitis (n = 12) and primary (N = 2) and secondary biliary cirrhosis (N = 1). Thirty-nine of the 43 brushing biopsies procedures were technically adequate for the diagnostic evaluation. Four cases were considered \"poorly cellular\" by the pathologists, with an overall success rate of 90.7% (Table [1](#T1){ref-type="table"}). These 4 cases underwent endoluminal forceps biopsy within 15 days from the brushing. Malignant cells were detected by brushing in 11 cases including: adenocarcinoma (n = 2), cholangiocarcinoma (n = 6), pancreatic carcinoma (n = 2), and metastatic adenocarcinoma (n= 1) (table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### True positives ::: **Type of tumour** **Brushing** **Biopsy** **Balloon-brushing** ---------------------- -------------- ------------ ---------------------- Cholangiocarcinoma **6** **18** **5** Adenocarcinoma **2** **1** **1** Metastatic adenoca. **1** **5** **-** Pancreatic carcinoma **2** **10** **1** Neuroendocrine tumor **1** **-** ::: Of the 32 patients with negative findings for malignant cells, the 4 cases in whom the samples were considered \"acellular\" by the pathologists were excluded by the statistical analysis, as just mentioned. Hence, 16 out of 28 had a final diagnosis of benign disease confirmed by long-term clinical follow-up (true negatives) (Table [3](#T3){ref-type="table"}) and 12 patients had a final diagnosis of malignancy (false negatives) (Table [4](#T4){ref-type="table"}). Therefore, 11 true positives, 16 true negatives and 12 false negatives were obtained by cytological brushing, with an overall sensitivity, specificity, accuracy, PPV, and NPV in the detection of malignant diseases of 47.8, 100, 69.2, 100, and 57.1 % respectively (Table [1](#T1){ref-type="table"},[5](#T5){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### True negatives ::: **Type of tumor** **Brushing** **Biopsy** **Balloon-brushing** ------------------------ -------------- ------------ ---------------------- Sclerosing cholangitis **10** **4** **-** Biliary cirrhosis **3** **2** **-** Iatrogenic strictures **3** **3** **4** ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### False negatives ::: **Type of tumor** **Brushing** **Biopsy** **Balloon-brushing** ---------------------- -------------- ------------ ---------------------- Cholangiocarcinoma **7** **-** **1** Adenocarcinoma **2** **1** **-** Pancreatic carcinoma **3** **2** **-** ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Statistical analysis ::: **Technique** **Sensitivity** **Specificity** **Accuracy** **PPV** **NPV** ------------------ ---------------------- ---------------------- -------------- --------- --------- Brushing 47.8% 28.10--69.66\* 100% 87.30--100.00\* 69.2% 100% 57.1% Biopsy 92.1% 75.56--98.53\* 100% 89.34--100.00\* 93.6% 100% 75% Balloon brushing 87.5% 52.30--99.96\* 100% 64.31--100.00\* 91.7% 100% 80% \* 99% C.I. (Geigy scientific tables) ::: Endoluminal forceps biopsy was performed in 47 cases. Thirty-five of these were interpreted as containing malignant cells (Table [2](#T2){ref-type="table"}): cholangiocarcinoma (n = 18), pancreatic carcinoma (n = 10), metastatic adenocarcinoma (n = 5), adenocarcinoma (n = 1), and neuroendocrine tumor (n = 1). Nine of the 12 cases interpreted as containing inflammatory cells were confirmed by clinical and radiological follow-up as follows: sclerosing cholangitis (n = 4), iatrogenic stenosis (n = 3) and biliary cirrhosis (n = 2). Three cases were erroneously interpreted as benign (false negative) but the clinical follow-up revealed 2 pancreatic carcinomas and 1 adenocarcinoma (Table [4](#T4){ref-type="table"}). With 35 true positives, 9 true negatives and 3 false negatives, endoluminal forceps biopsy showed a sensitivity of 92.1%, a specificity of 100%, an accuracy of 93.6%, and a PPV and NPV of 100 and 75%. CIs were reported in Table [5](#T5){ref-type="table"}. \"K\" values for biopsy and brushing vs clinical/surgical final diagnosis were 0.613 and 0.404, respectively (Table [6](#T6){ref-type="table"}). ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Cohen\'s Kappa value ::: **Cohen\'s Kappa** **Significance** ----------------------- -------------------- ------------------ Brushing vs follow-up 0.404 0.001 Biospy vs follow-up 0.613 0.019 ::: As previously discussed, 12 samples were obtained in as many patients with an angioplastic balloon after dilation of biliary strictures (balloon surface sampling). This technique demonstrated malignant cells in 7 (58.3%) cases and benign cells in 5 (41.7%). The final clinical diagnosis of the malignant group included adenocarcinoma (n = 1), cholangiocarcinoma (n = 5) and pancreatic carcinoma (n = 1) (Table [2](#T2){ref-type="table"}). The final clinical diagnosis of the benign group included four iatrogenic stenosis (Table [3](#T3){ref-type="table"}). In one case a sclerosing cholangitis was diagnosed by balloon brushing, but clinical follow-up and further investigations revealed a cholangiocarcinoma (Table [4](#T4){ref-type="table"}). With this technique, we therefore had 1 false negative, 7 true positives and 4 true negatives, with a sensitivity, specificity, accuracy, positive predictive value and negative predictive value of 87.5, 100, 91.7, 100, 80%, respectively (Table [5](#T5){ref-type="table"}). Twenty-two patients underwent brush cytology together with endoluminal forceps biopsy. By excluding the 4 patients whose specimens were considered \"acellular\" and comparing each procedure with the clinical follow-up and surgical specimens, we obtained 12 concordant and 6 discordant diagnoses. In the group of 12 concordant diagnoses, 6 benign diseases, such as primary sclerosing cholangitis (n = 3), iatrogenic stricture (n = 1) and biliary cirrhosis (n = 2) and 6 malignant diseases, such as cholangiocarcinoma (n = 3), pancreatic carcinoma (n = 2), and metastatic adenocarcinoma (n = 1) were included. All concordant diagnoses were confirmed by clinical or surgical follow-up. In the group of 6 discordant diagnoses, cholangiocarcinoma (n = 4) adenocarcinoma (n = 1) and pancreatic carcinoma (n = 1), were included. All the 6 malignancies misdiagnosed by brushing were correctly diagnosed by biopsy. Transient hemobilia (spontaneously reversed from 1 to 12 hours after the procedure), was observed in 5/47 (10.6%) patients who had undergone biopsy. In 4 of them, particular angulations of the access route was present and there were difficulties in negotiation of the stricture. No major complications directly related to the brush cytology or the endoluminal forceps biopsy procedures occurred. Discussion ========== The evaluation of patients with biliary tract obstruction without evidence of any intrahepatic or extrahepatic growing mass has traditionally involved a variety of diagnostic imaging techniques \[[@B1]\]. Ultrasound and CT are often the initial diagnostic investigation to be performed and provide good information about the presence of biliary obstructions and the degree of ductal dilatation \[[@B8]\]. These imaging modalities, are however limited in depicting intraductal anatomy and, sometimes, in exactly determining the level and the cause of the obstruction \[[@B1],[@B9]\]. Magnetic resonance cholangiography (MRC) is a non-invasive imaging modality providing a good visualization of the biliary. system. The sensitivity of MRC in the detection of choledocholithiasis has been reported as 90--100%, a comparable rate with that of endoscopic retrograde cholangiopancreatography (ERCP) \[[@B10]-[@B15]\]. The assessment of the level of obstruction also has been reported as highly accurate \[[@B16]-[@B19]\]. However, low-grade strictures or lesions causing biliary dilation may be missed by MRC \[[@B20]\]. The pathological characterization of presumed malignant strictures can be therefore, difficult, if not impossible, using noninvasive imaging studies alone in some intrinsic lesions causing biliary dilation. As a final diagnosis could radically affect further therapeutic choices histological, an histological characterization is required in the management of patients with biliary strictures \[[@B1]\]. Fine-needle percutaneous biopsy (FNPB) and fine-needle aspiration (FNA) has been reported poorly valuable in absence of a lesion clearly identifiable \[[@B21],[@B22]\]. The tumor most frequently not identifiable as a true growing mass is cholangiocarcinoma and the differential diagnosis with primary sclerosing cholangitis is of somewhat importance \[[@B23]\]. The differential diagnosis between some carcinomas of the pancreatic head or small submucosal tumors of the ampulla and cholangiocarcinoma or inflammatory diseases, such as sclerosing cholangitis, can be very difficult in many cases. Patients with bilioenteric anastomoses after tumoral mass resection have a very complex local anatomic alteration that makes extremely difficult any radiological investigation searching for small recurrent neoplastic infiltrations \[[@B27]\]. Treatment protocols require pathological diagnoses for palliative or possibly curative therapy in almost all these types of conditions. In addition, the surgical technique itself can be different if a malignancy is present or not. Some Authors, when liver transplant is still indicated, suggest a large excision associated with gastrectomy, pancreatectomy and a transverse ascending colectomy \[[@B25]\]. Although the efficacy of this surgical approach is still under debate, the role of a preoperative diagnosis is extremely important. Malignant cells surrounding the biliary ducts may continuously exfoliated into the bile, especially in the case of tumors which breack through the mucosa, and become available for the cytological examination when the bile is collected either percutaneously or by ERCP. Cytodiagnosis is easy to be performed, atraumatic in nature, with less potential risk and associated with relatively low charges. This technique has shown low sensitivity (15--28%) and accuracy (48--58%) rates, due to an early cellular degeneration after bile collection and to \"poorly cellular\" specimens. In addition, some pathological aspects can affect the efficacy of the procedure, such as in case of lesions extrinsically compressing the bile duct wall without a complete transmural infiltration or an adequately wide mucosal disruption \[[@B22]\]. Endoluminal brush cytology was successfully performed either percutaneously, by Radiologists, or endoscopically, by Gastroenterologists \[[@B26]-[@B28]\]. In our experience, brush cytology showed a high specificity but low sensitivity and accuracy rate in the detection of malignant diseases. Technical limits were mostly represented by \"poorly cellular samples\". In our study, in fact, 4 cases (9.3%) were considered \"poorly cellular\" by the pathologists. In addition to these technical problems, similarly to bile cytology, we have to consider some morphological aspects that can negatively influence the sensitivity of this technique. According also with our cholangioscopic experience, biliary tumors may remain intramural causing an annular constriction of the biliary duct \[[@B21],[@B29]\] without complete transmucosal infiltration, and this condition can mimic sclerosing cholangitis and render brush cytology ineffective. In light of the fact that there is a frequent relationship between sclerosing cholangitis and malignant strictures, the cytological differentiation between inflammatory and malignant changes can be extremely difficult. In addition, these tumors are frequently so well differentiated that their identification as \"malignant\" can be, even histologically, difficult \[[@B29],[@B30]\]. These are the theoretical explanations that we can give to the fact that cytological diagnosis of cholangiocarcinoma yielded a relatively low sensitivity, verified in our study as well as in the literature \[[@B1],[@B21]-[@B23],[@B26],[@B30]\]. At endoscopic sampling, cholangiocarcinoma has a higher sensitivity \[[@B28]\] In this review only patients not suitable for ERCP or coming from a failed drainage or other type of retrograde endoscopic intervention were evaluated, thus the percutaneous and endoscopic techniques can not be compared. Whenever possible especially if skilled endoscopists are available, retrograde approach could still to be considered the first step, for its potentially high sensitivity especially if repeated sampling are performed \[[@B27],[@B28]\] Percutaneous brush cytology, if compared with bile transendoscopic cytodiagnosis, has the potential risk of a malignant spreading of cells through the transhepatic tract. To overcome this risk, in our opinion, the brush should be pulled back and removed from the patient into the introducer sheath. With this technique, in fact, no spreading along the transhepatic tract was observed in our malignant patients at imaging follow-up. Brush cytology is a easy, safe and at relatively low-cost procedure, similarly to bile collection. A single sampling however has a low possibility of detecting a malignancy. The results can improve with multiple samples. Three consecutive negative samples decrease the probability of a malignancy from more than 55% to less than 5% \[[@B30]\]. The absence of false positives in our and others, series \[[@B2]\] means that an intraductal biopsy has no purpose when an exfoliative cytology is positive. Meanwhile, in presence of a negative cytology, other techniques such as percutaneous FNA and endoluminal forceps biopsy should be mandatory. Fine needle aspiration performed either percutaneously or endoscopically has some technical advantages over endoluminal brushing in those lesions extrinsically compressing the biliary duct without deeply infiltrating the ductal wall, since the inner epithelial layer is not involved \[[@B21]\]. Most of these patients with biliary obstruction, especially with high lesions, however, in our Hospital, usually undergoes percutaneous biliary drainage in the early phases of their clinical work-up. A percutaneous FNA would represent an adjunctive interventional procedure that could be avoided by using the transhepatic route, already available. We had adequate percutaneous biopsy specimens in epithelial lesions, such as cholangiocarcinoma, and inadequate specimens in those lesions with inflammatory and/or necrotic changes. The dense fibrotic and scirrus reaction associated with pancreatic carcinoma may result in poor biopsies specimens \[[@B31]\]. In addition, pancreatic carcinoma is often associated with pancreatitis, necrotic cellular debris and a dense fibrotic reaction which can further contribute to the disappointing results obtained with even aggressive percutaneous biopsy techniques \[[@B32]\]. The differentiation degree of a tumor can affect the accuracy of the histological classification. In those cases of extrahepatic and periampullary biliary tumors, in fact, usually highly undifferentiated, the histological characterization can be difficult, although a generic diagnosis of malignancy can be made. Potential complications due to forceps biopsy, such as disruption of the ductal wall with consequent bile leak and bleeding, cholangitis and pancreatitis, are reported \[[@B33]\]. Endoluminal forceps biopsy especially in patients with hemobilia or cholangitis, should therefore be performed in the remission phase of the disease, and, in any case, at least 5 days after biliary drainage, to avoid the complications related to manipulations into the biliary tree. No major complications were observed in this series, neither from percutaneous brushing nor from forceps biopsy. A transient hemobilia was observed in 5 patients who had undergone biopsy (10.6%). This series, including part of a previously analyzed smaller population \[[@B33]\] confirms that percutaneous endoluminal forceps biopsy has a very high sensitivity (92.1%), specificity (100%), accuracy (93.6%), PPV (100%), and NPV (75%) in the detection of malignant diseases (Table [2](#T2){ref-type="table"}). The higher accuracy of biopsy over brushing is very clear, especially analyzing the data obtained in those patients in whom both techniques were performed. On the other hand, biopsy presents some disadvantages, such as the higher costs of the equipment and the difficult trackability across tight strictures or acute angles of the biliary ducts. A high diagnostic value was proven by the simple examination of tissue samplings coming from balloon dilatation of biliary or bilio-enteric anastomotic strictures (balloon surface sampling). Although the number of cases reported in this series is relatively small, it should be considered that it was possible to distinguish benign from malignant diseases in almost all the cases. Our suggestion, therefore is to associate a tissue collection from the balloon to bilioplasty, as a standard procedure when screening for malignant pancreatobiliary diseases is required. In this way. it is possible to save time and avoid risks related to further endobiliary procedures. Conclusion ========== Patients with obstructive jaundice, who are candidates for biliary drainage, often come to the interventional radiologist still without a definitive diagnosis of the real nature of their disease, even after high-level cross-sectional imaging procedures, such as abdominal MR, MRCP or abdominal spiral-CT. When the clinical diagnosis needs to be histologically confirmed for further therapeutic choices, the transhepatic route can be successfully used for the intraductal sampling. Forceps biopsy is highly accurate under cholangioscopic guidance. As an alternative, repeated brushings can be performed under fluoroscopy. If a balloon blioplasty, for any reason, is performed, it is advisable to collect the tissue fragments on the balloon surface and send them for pathological evaluation. Competing interests =================== None declared. Authors\' contributions ======================= MR carried out the interventional procedures, participated in the design of the study and drafted the manuscript, FMS carried out the interventional procedures, VC defined the design of the study and performed the statistical analysis, LG collected the results of the procedures, LG participated in the histological analysis, AR participated in the design of the study and drafted the manuscript,, GG participated in the imaging evaluations, EP performed the patients follow-up, VD supervised the drafting of the study ::: {#F4 .fig} Figure 4 ::: {.caption} ###### A 58 year old man, who 8 years ago underwent left hepatectomy and cholecistectomy, for complicated intrahepatic biliary stones, presented with jaundice and weight loss. Enhanced CT scan showed marked intrahepatic biliary dilation. :::  ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Same patient as fig 4, at lower level, although, an extrinsic mass was not detected, the lumen of CBD appeared replacement by soft tissue mass density (arrows). :::  ::: Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2342/4/3/prepub> Acknowledgments =============== The study was supported by funds of La Sapienza University.

PubMed Central

2024-06-05T03:55:47.735548

2004-8-25

PMC517716

Background ========== Cervical cancer makes up about 10--12% of total women cancers \[[@B1],[@B2]\] with the level of mortality in Russian population 5.0 per 100000 \[[@B1]\]. The tendency is being observed for the past decades towards reduction of both incidence and mortality. It is mainly due to the population-wide screening protocols in developed countries which allow identifying early asymptomatic forms of cervical carcinomas. However some problems remain to be solved concerning early detection of this type of cancer. The main screening test for cervical cancer is the cytological smear staining technique developed by G. Papanicolaou \[[@B3]\] and known as Pap test. Despite evident success this test gives a substantial rate of both false-positive and false-negative results. Histological analysis of a biopsy sample, more laborious in preparation and study as compared with that of a cytological smear, is also not absolutely efficient owing to a substantial rate of interobserver discrepancies among expert pathologists examining the same material \[[@B4]\]. Infection with human papilloma viruses (HPV) belonging to so-called high-risk group is the main risk factor of cervical cancer incidence \[[@B2]\]. To detect high risk HPVs in epithelial cells of a patient polymerase chain reaction (PCR) has been applied during the past two decades \[[@B5]\]. However this highly sensitive technique cannot resolve the problem of early cervical carcinoma detection also so far as many early stage lesions regress and epithelial dysplasia (i.e. cervical intraepithelial neoplasms, CINs) and carcinomas appear only in a minor part of the persons in whose epithelium (on a smear) high risk HPVs had been detected \[[@B2]\]. For the recent years several research groups \[[@B4],[@B6]-[@B16]\] including us \[[@B11]\] have dwelt on the protein p16^INK4a^for a possible supplementary marker of dysplastic and neoplastic cervical epithelium lesions. This protein belongs to the group of cyclin-dependent kinase Cdk4/6 inhibitors \[[@B17]\] and is encoded by tumor suppressor gene *INK 4a*(synonyms: *MTS 1*, *CDKN2*, *INK4a/ARF*). Gene *INK4a*plays an important role in the regulatory pathway Cdk-Rb-E2F. The product of this gene p16^INK4a^prevents pRb phosphorylation by inactivating Cdk4/6; pRb keeps on binding E2F transcription factors and as a result cells stay in G1 phase not passing to DNA replication. In various tumor types *INK4a*as a bona fide tumor suppressor undergoes homozygous deletions, is inactivated by point mutations, LOH or hypermethylation; p16^INK4a^expression is reduced or ceases under such conditions or the protein function may be impaired \[[@B18]\]. Peculiarity of cervical carcinomas is due to the ability of HPV oncoprotein E7 to interact with pRb and inactivate it \[[@B19]\]. As a result, the regulatory pathway Cdk-Rb-E2F is disrupted and the status of gene suppressor *INK4a*and its protein becomes of no importance for a cell so far as they function upstream of the site of breakage. Cells with thus inactivated pRb pass cell cycle checkpoint G1/S without any obstacle. Reciprocallity between status of pRb and that of p16^INK4a^commonly found in human permanent cell lines (including cervical cell line cultures) \[[@B17],[@B20],[@B21]\] as well as in primary tumor cells \[[@B9],[@B22],[@B23]\] served for a logical prerequisite of utilizing p16^INK4a^protein as a marker of premalignant and malignant cervical epithelium cells. Functionally active gene *RB*was shown to be able to negatively regulate the expression of *INK4a*on a transcriptional level, but details of this negative feed-back loop remain obscure \[[@B24]\]. To estimate the applicability of p16^INK4a^as a marker of dysplastic and neoplastic alterations in cervical epithelium cells we analyzed the expression of this protein utilizing a highly p16^INK4a^-- specific monoclonal antibody. We examined 197 samples in total. The materials studied included: 1) samples of normal epithelium of healthy women (cytological vaginal smears), 2) diagnostic biopsy samples from patients with cervical ectopia, 3) samples of CINs of various stages, 4) samples of invasive cervical cancer, 5) samples from different normal utery body and cervix tissues from women with gynecological diseases not associated with dysplastic lesions of epithelium (diagnostic biopsies and surgical materials) and 6) samples of cells from 3 cervical carcinoma cell lines. We also compare our data with the results presented by other groups working in similar directions \[[@B4],[@B6]-[@B10],[@B12]-[@B16]\]. We demonstrate in the present work that overexpression of the protein p16^INK4a^is typical for some samples of dysplastic and neoplastic epithelium of cervix uteri. We have not found at least a single sample overexpressing p16^INK4a^among control samples. The portion of p16^INK4a^-positive samples increases in the following row: CIN I -- CIN II -- CIN III -- invasive carcinoma. However p16^INK4a^-negative CINs and carcinomas have also been found. All stages of CINs and carcinomas analyzed turn out to be heterogeneous with respect to p16^INK4a^expression: side by side with the samples which express p16^INK4a^in 25% of cells or more we detect samples which are stained poorly or lack any staining. So p16^INK4a^-negativity does not seem to be a sufficient reason to exclude a patient from the high risk group when results of Pap-test, HPV detection by PCR or histological investigation warn about possibility of poor prognosis. As far as normal cervical epithelium is p16^INK4a^-negative and the ratio p16^INK4a^-positive/ p16^INK4a^-negative samples increases at the advanced stages of CINs and carcinomas application of immunohisto-/cytochemical test for p16^INK4a^may be regarded as a supplementary (optional) test for early diagnostics of cervical cancer. Methods ======= Immunohisto-/immunocytochemical study was performed on 197 samples in total. Those included 6 samples from normal epithelium of healthy women (cytological vaginal smears taken during regular examination), as well as the following surgical and diagnostic biopsy materials: 37 samples from patients with cervical ectopia, 113 samples of CINs of different stages including cancer in situ, 26 samples of invasive cervical cancer (21 squamous cell carcinomas and 5 adenocarcinomas), 12 samples of normal tissues from uterus body (myometrium) and cervix from patients with different gynecological diseases, 3 cervical cell line samples taken for positive control (see below). Apart from 9 smears (which included 6 samples from healthy women and 3 samples taken from cell cultures which served as controls) the rest 188 materials were paraffin-embedded histological blocks. The quality of smears turned out to be satisfactory for immunochemical analysis (Fig. [1a](#F1){ref-type="fig"} and [1d](#F1){ref-type="fig"}). Neutral formalin-fixed paraffin embedded samples of biopsies and surgery materials were received from archives of N.N.Blokhin Cancer Research Center (Moscow), P.A. Gertzen Institute of Oncology (Moscow), Central Clinical Hospital (Moscow) and Fourth Clinical Hospital (Poltava, Ukraine). For immunohistochemical analysis 4--5 μm serial sections were transferred on Histobond slides with adhesive layer (SMT Geraetehandel GmbH, Germany). The first section was stained with hematoxylin-eosin for traditional morphological analysis and verification of diagnosis by not less than two independent pathomorphologists. Presence of high risk HPV in cervical CIN and carcinoma cells was verified by PCR \[[@B25]\]. Some of the squamous cell carcinomas were tested by Southern blot hybridization in addition to PCR. The results of both methods coincided completely. To detect high-risk HPV in adenocarcinomas Hybrid Capture 2 test (enabling to detect but not to discern HPV 16 and HPV 18) was used. The following portions of samples (out of those studied immunochemically) were analysed for high-risk HPV genetic material: CINs I -- 9/51, CINs II -- 9/32, CINs III -- 19/24, invasive squamous cell carcinomas -- 21/21, adenocarcinomas -- 5/5. This study did not cover all CINs which we studied immunochemically due to the tiny size of most of those samples. Vaginal normal epithelium smears obtained during regular examination from healthy women were transferred on HistoBond slides. Absence of abnormal cells was confirmed in Cytology Department of Cancer Research Center on a parallel slide. Smears were fixed for 5 min in 10% formaldehyde, washed with flowing water, and processed for further development as described for deparaffinized histological slices \[[@B9]\]. Cells of cervical cell lines were cultured under standard conditions in DMEM supplemented with 10% of foetal bovine serum. For immunocytochemistry cells were taken during culture receeding, dropped in a culture medium on a Histobond slide, air-dried and then processed identically to smears from healthy women. Immunohistochemical staining was carried out using p16^INK4a^-specific monoclonal antibodies E6H4 (MTM Laboratories AG, Germany) according to protocol by Klaes et al \[[@B9]\]. Controls in the course of immunohistochemical studies were as follows --------------------------------------------------------------------- ### Positive controls 1). In the beginning of the study as a whole cells of three cervical cell lines were stained first. They were SiHa (HPV16-positive), C33a and HT-3 (both HPV-negative); all of them had been characterized as expressing the protein p16^INK4a^in earlier immunocytochemical studies with the same antibody \[[@B9]\]. 2). One HT-3 cell slide was stained in parallels with the first series of biopsy materials. 3). Every next group of slides intended for staining compulsorily included the slide with one of the serial sections of the CINIII or invasive carcinoma sample which had been characterized as a p16^INK4a^-positive one in our previous studies (on preceeding cuts). The analysis of the given slide series was carried out only if the positivity of the control sample was confirmed. Thus neither slide series in the present study was completely p16^INK4a^-negative. ### Negative controls 1). One more serial cut made from p16^INK4a^-expressing material was included into every slide series meant for staining (as in variant 3 of positive controls, -- see above) which was processed in a usual way but PBS was applied instead of p16^INK4a^-specific antibody. The results were regarded as valid if this slide was negative. 2). As an intrinsic negative control served adjacent to CIN or carcinoma normal tissues including stromal elements. In neither case did we find any staining in these tissues (fig. [1c](#F1){ref-type="fig"}). In addition to four categories of staining defined by Klaes et al. \[[@B9]\],- poor (less than 1% of stained cells,- figure [1b](#F1){ref-type="fig"}), sporadic (1--5% of stained cells, -- figure [1b](#F1){ref-type="fig"}), focal (cell clusters were stained but not more than 25% of cells were positive, -- figure [2a](#F2){ref-type="fig"}) and diffuse (more than 25% of cells were stained, -- figures [1c](#F1){ref-type="fig"} and [2c,2f,2g,2h](#F2){ref-type="fig"}), we formed one more group (negative) of those samples which totally lacked any stained cells (Figures [1a](#F1){ref-type="fig"}, [2b,2d](#F2){ref-type="fig"} and [2e](#F2){ref-type="fig"}). We regarded those cells as stained in which p16^INK4a^was expressed in nuclei and/or in a cytoplasm. Results ======= Various types of p16^INK4a^-specific staining of cervix uteri normal, dysplastic and cancer cells are presented in Fig. [1a,1b,1c,1d](#F1){ref-type="fig"} and Fig. [2a,2b,2c,2d,2e,2f,2g,2h](#F2){ref-type="fig"}. We did not register any staining in either of 6 smear samples taken from healthy women (Fig. [1a](#F1){ref-type="fig"}). An example of a very poor cytoplasmic staining in some separate CIN I cells with a more pronounced (sporadic, both nuclear and cytoplasmic) staining in the adjacent cancer in situ cells is shown in Fig. [1b](#F1){ref-type="fig"}. In one invasive carcinoma we detected cytoplasmic staining in the predominant majority of cancer cells while but sole nuclei turned out to be stained (Fig. [1c](#F1){ref-type="fig"}); the boundary between cancer and normal tissues coincided with the line at which the staining discontinued. As to the cervical cells cultivated in vitro taken for positive controls, in HT-3 cells the specific staining was strongly manifested both in nuclei and in a cytoplasm (Fig. [1d](#F1){ref-type="fig"}) while in SiHa and C33a cells it was exclusively cytoplasmic. It is not clear yet why in one and the same cervical cancer sample the protein p16^INK4a^(normally showing its activity in nuclei) may be detected but in a cytoplasm in the majority of cells while in a number of cells both in nuclei and cytoplasm, why in some cervical cell cultures it is found solely in cytoplasm and in other cervical cell lines -- in nuclei also. We did not find at least a single sample with an exceptionally nuclear staining; similar were the results by other investigators \[[@B9]\]. With keeping in mind that subcellular location of p16^INK4a^-specific staining varies to such a degree we scored as positive every sample in which the staining was expressed either in a cytoplasm or in both nuclei and cytoplasm. The data of the analysis of the cervical epithelium preparations stained with monoclonal antibodies E6H4 are summarized in Table [1](#T1){ref-type="table"}. In all 37 samples from patients with cervical ectopia p16^INK4a^-positive cells were observed with low frequencies: 34 samples were negative and 3 ones (8,1%) were stained poorly. In the group of CINs I 63% of the samples were p16^INK4a^-negative. In 7 samples (about 14%) sporadic or focal (Fig. [2a](#F2){ref-type="fig"}) type of staining was observed. Group of CINs II turned out to be highly heterogeneous in terms of the ratio of p16^INK4a^-positive cells as well. Most of these samples (68%, 26 out of 38, Fig. [2b](#F2){ref-type="fig"}) did not differ from the samples of normal epithelium, but the rest 12 samples we attributed to poor, sporadic or focal types. In neither sample of CIN I or CIN II did we observe diffuse staining for p16^INK4a^. Among the samples of CIN III about 54% (13 out of 24) were attributed to sporadic (Fig [1b](#F1){ref-type="fig"}), focal or diffuse (Fig. [2c](#F2){ref-type="fig"}) types. However every third CIN III sample was found to be p16^INK4a^-negative (Fig. [2d](#F2){ref-type="fig"}). As to invasive cancers, only 1 out of 26 samples (3.8%) lacked p16^INK4a^-positive cells (Fig. [2e](#F2){ref-type="fig"}). One sample expressed the marker poorly. In 24 cases sporadic, focal or diffuse staining was observed. Diffuse staining was registered in about 50% of these samples (Figs. [1c](#F1){ref-type="fig"}, [2f,2g,2h](#F2){ref-type="fig"}). Among 14 samples of invasive carcinomas which had been stored in paraffin blocks for 7 years 1 (7.1%) turned out to be p16^INK4a^-negative, 1 (7.1%) stained poorly, 3 (21.4%) stained sporadically, 4 (28.4%)-focally and 5 (35.8%) expressed diffuse staining. We have examined several cases with more than one type of lesion on the same slide. Examples are presented on Figures [1b](#F1){ref-type="fig"} and [2c](#F2){ref-type="fig"}. As a rule in combined cases p16^INK4a^expression was more pronounced in cells belonging to a more advanced lesion (Fig. [1b](#F1){ref-type="fig"}). All the HPV-tested samples of CINs and invasive squamous cell carcinomas were high-risk HPV-positive (data on CINs I and CINs II are shown in the additional file, results with CINs III and squamous cell carcinomas -- in Table [2](#T2){ref-type="table"}). High-risk HPVs were also found in all the samples of adenocarcinomas. In reference group composed of samples from normal uterus body and cervix uteri tissues obtained from patients with gynecological diseases (stromal and glandular tissues of the cervix from patients with cervical ectopia from which squamous cell epithelium had been fully cut off; myometrium of uterus body from the patient after surgery for cervical carcinoma) the results of staining with p16^INK4a^-specific antibodies were negative in all 12 cases (Table [3](#T3){ref-type="table"}). Discussion ========== Immunochemical detection of p16^INK4a^by monoclonal antibodies shows that the overwhelming majority of invasive cervical carcinoma samples differ from normal cervical epithelium of healthy women. We found no cases of sporadic, focal or diffuse staining with E6H4 antibodies among 6 vaginal smears from healthy women as well as among 37 samples from patients with cervical ectopia including those in which ectopically localized cervical epithelium koilocytes or condyloma had been registered. In addition, in 11 samples of normal glandular and stromal cervical tissues and one sample of uterus body normal myometrium tissue which were obtained from patients with gynecological disorders p16^INK4a^was not expressed at all. The predominant majority of invasive carcinoma samples were both high-risk HPV-positive and p16^INK4a^-expressing. However in 2 samples not expressing any p16^INK4a^or expressing it poorly high-risk HPV DNA sequences were detected by both PCR and Southern blot hybridization. These data are in a good agreement with those by Klaes et al., who described two samples of p16^INK4a^-negative but HPV-positive cervical cancer \[[@B9]\]. Among CIN III samples which were stained in our experiments poorly or lacked any staining (table [2](#T2){ref-type="table"}, samples 1--6) high risk HPVs were found in 6 out of 6 tested. As to CIN I and CIN II samples expressing p16^INK4a^in less than 1% of cells ([Additional file](#S1){ref-type="supplementary-material"} data, samples 1--5 and 10--14, respectively), we did not find any high-risk HPV-negative case in those groups either. Thus there seem to exist dysplastic and neoplastic lesions of cervix uteri which do not overexpress the protein p16^INK4a^but harbor high-risk HPV DNA. We confirm the data by Klaes et al. \[[@B9]\] that prolonged (for 7 years in our case) preservation of cervical carcinoma samples in paraffin blocks does not preclude the material from diffuse staining. Monoclonal antibodies E6H4 originally described by Klaes et al \[[@B9]\] had been tested by those authors among a number of clones of p16^INK4a^-specific monoclonal antibodies. The following commercially available clones had been taken: 1). DCS-50.1/H4-NA29 (Oncogene Research Products, Cambridge, MA), 2). 375P (Biogenex Laboratories, San Ramon, CA), 3). ZJ11 and JC8 (NeoMarkers, Freemont, CA), 4) 05--418 (Upstate Biotechnology, Lake Placid, NY) and 5). J175--405 (PharMingen, San Diego, CA). The results of the comparative experiments by Klaes at al \[[@B9]\] had demonstrated that clone E6H4 had turned out to be the most specific as inferred on the lowest level of unspecific staining on different tumor cell lines, both HPV-negative and positive. As in our experiments Klaes et al. \[[@B9]\] registered mainly cytoplasmic staining both in cells of permanent cell lines and in preneoplastic and carcinoma samples; an exclusive nuclear staining was not detected. We observed strong p16^INK4a^-positivity (sporadic or focal staining) in a comparatively small number of CIN I and CIN II samples (13--14 %). Among CINs III the portion of such samples increased up to 54,2%, with diffuse staining in every sixth case. The data we have obtained for CINs of all stages rather differ from the results by Klaes et al. \[[@B9]\]. All of CINs II and CINs III were stained as diffuse by Klaes et al. \[[@B9]\]. In the present study the group of CINs as a whole is much more heterogeneous. The reasons for those discrepancies are not quite clear yet. The population of Russian and Ukrainian patients whose materials were used in the present study was extremely heterogeneous with respect to age, nationality, etc. We cannot exclude that the discrepancies mentioned may be due to these factors as had been discussed earlier \[[@B26]\]. Nevertheless the coincidence between our results and the data by Klaes et al \[[@B9]\] concerning the general trend seems much more important: the increase of p16^INK4a^expression in dysplastic and neoplastic cervical epithelium in the course of progression. This trend is especially evident in our study when negative cases only are taken into consideration. All 6 samples were p16^INK4a^-negative in control group. Similar was the situation in the reference group: 12/12 negative. Among CINs I, CINs III and invasive carcinomas these indices made up 32/51 (63 %), 8/24 (33%) and 1/26 (4 %) respectively. The data presented herein allow us to conclude that hyperexpression of p16^INK4a^when detected immunohistochemically may be regarded as a marker of dysplastic and neoplastic lesions in cervical epithelium. Positive correlation between p16^INK4a^expression and morphological stage of the disease on the same slide (when combined cases with both dysplastic and neoplastic lesions were found) also favors this inference. In a number of recent communications immunochemical staining for p16^INK4a^was suggested to be performed on cytological smears \[[@B9],[@B13]-[@B15]\]. Cytological approach has some advantages, so far as it enables a patient to avoid surgical intervention. That is why one may expect in the nearest future immunocytochemical version for detecting p16^INK4a^to become rather widely used not only as an addition to surgery but also for regular examinations in outpatient clinics. In this connection it seems important to estimate frequencies of a feasible false-negativity of the test. Keeping in mind that dysplastic lesions of all stages frequently regress and do not convert into invasive cervical carcinomas \[[@B2]\], we summarized the data by different groups on the frequencies of p16^INK4a^-negative invasive carcinoma samples (Table [4](#T4){ref-type="table"}). We found that p16^INK4a^-negative cervical adenocarcinomas had been detected with frequencies which had varied from 0% up to 42,5% while p16^INK4a^-negative squamous cell carcinomas -- with frequencies that had not exceeded 10,1%. Substantial variability of the data may be due to small numbers of samples analyzed (in some of the studies), to utilization of different types of monoclonal antibodies, to different criteria used by different research groups for the results interpretation, etc. It seems important to realize the proper place of immunocyto-/ immunohistochemical analysis of p16^INK4a^expression among previously developed tests for early detection of cervical carcinomas. In this connection the following points deserve mentioning. A pathologist in the course of common histological investigation usually registers some morphologic features such as zones of active mitotic divisions, multipolar mitoses, vicinity of condilomas and other formations. However when performing an immunocytochemical analysis on a smear a cytologist cannot bring in a correlation with morphological structure results of staining with p16^INK4a^-specific antibodies in separate cells. According to the results of the present study about 33% of CINs III as well as about 5% of invasive squamous cell carcinomas of cervix uteri do not differ from normal cervical epithelium with respect to p16^INK4a^expression. Thus immunochemical staining for p16^INK4a^(in both cyto- and histochemical versions) does not seem to be the approach that can help to fully overcome absolutely all existing ambiguities of cervical cancer early diagnostics. As to possible sources of false positivity of immunochemical detection of the protein p16^INK4a^the study by Agoff et al \[[@B16]\] deserve mentioning. According to this communication in 10 out of 10 endometrial biopsy samples studied endometrial cells were positively stained with the p16^INK4a^-- specific antibody E6H4. So far as endometrial cells may occur on vaginal smears the test does not seem to enable one to fully avoid false positivity. Conclusions =========== Overexpression of the protein p16^INK4a^encoded by tumor suppressor gene *INK4a*is a characteristic of displastic and neoplastic alterations of cervical epithelium. The portion of p16^INK4a^-positive samples increases in the following row: CIN I -- CIN II -- CIN III -- invasive carcinoma. All stages of CINs and carcinomas analysed are heterogeneous with respect to p16^INK4a^expression: side by side with the samples which expressed p16^INK4a^in 25% of cells or more we detected samples which were stained poorly or lacked any staining. According to the data we present herein p16^INK4a^-negative cervical neoplasms and carcinomas do exist. Thus the lack of p16^INK4a^-positive cells in samples from different types of cervical lesions should not be regarded as a sufficient reason for excluding a patient from the high-risk group. Despite this as far as normal cervical epithelium is p16^INK4a^-negative and the ratio p16^INK4a^-positive/ p16^INK4a^-negative samples increases at the advanced stages of CINs and carcinomas application of immunohisto-/cytochemical test for p16^INK4a^may be regarded as an additional (optional) test for early detection of precancerous lesions in cervical epithelium. Competing interests =================== none declared Author\'s contributions ======================= F.K., D.S., G.V. and G.F. contributed to study conception and design; E.K. and A.B. delt with sample obtaining and preliminary diagnosis; Yu.A., G.F. and V.E. were independent pathologists who confirmed the diagnosis; G.V., L.Z. and D.G. performed immunohistochemical staining and analysis; A.B. carried out HPV typing; G.V. drafted the manuscript. All authors read and approved the final version. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/4/58/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Data on high risk HPV genome detection by PCR in CIN I and CIN II samples. the data are presented on high-risk HPV genetic material detection in 9 CIN I and 9 CIN II samples. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The authors thank Drs N.P.Kisseljova and N.N.Mazurenko for promoting discussion of the results. We appreciate assistance of Drs. A.Petrov and A.Petrenko in preparing the manuscript. The work was supported in part by FIRCA grant NIH 0600-070-c223 as well as by the Russian Foundation of Basic Science (grant No 02-04--48271) and Russian Ministry of Science (grant N 1877). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Different types of cervical cell staining with the p16^INK4a^-specific antibodies. a. Normal epithelium (smear): negative staining. b. CIN I (indicated with a dotted arrow) and cancer in situ (solid arrows): a very poor cytoplasmic staining in separate CIN I cells and sporadic staining in cancer in situ. c. Invasive squamous cell carcinoma. Diffuse cytoplasmic staining with the sole cell expressing p16^INK4a^in the nucleus (solid arrow). The boundary with adjoining normal tissue is marked with a dotted arrow. d. HT3 cells (smear) with both nuclear and cytoplasmic subcellular location of the positive staining. :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Cervical tissue samples after immunohistochemical staining with p16^INK4a^-specific antibodies. a. CIN I. Focal staining. b. CIN II. Negative staining. c. Cancer in situ (indicated with an arrow) and CIN III. Diffuse staining. d. CIN III. Negative staining. e. Squamous cell carcinoma. Negative staining. f. Squamous cell carcinoma. Diffuse staining. g. Squamous cell carcinoma embol. Diffuse staining. h. Adenocarcinoma. Diffuse staining. :::  ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### p16^INK4a^EXPRESSION IN NORMAL, DYSPLASTIC AND NEOPLASTIC EPITHELIAL CELLS OF CERVIX UTERI ::: STAINING\* ------------------------------------------------------------------------ ---- ------------ ----------- ---------- ---------- ----------- Normal epithelium (smears) 6 6 (100) \- \- \- \- Cervical ectopia including 37 34 (91,9) 3 (8,1) \- \- \- C.e. + coilocytosis 10 9 1 \- \- \- C.e. + condyloma 2 1 1 \- \- \- Dysplasia: CINs I 51 32 (62,7) 12 (23,5) 6 (11,8) 1 (2,0) \- CINs II 38 26 (68,4) 7 (18,4) 4 (10,5) 1 (2,7) \- CINs III including cancer in situ 24 8 (33,3) 3 (12,5) 7 (29,2) 2 (8,3) 4 (16,7) Invasive carcinomas including squamous cell carcinomas adenocarcinomas 26 1 (3,8) 1 (3,8) 4 (15,5) 7 (26,9) 13 (50,0) Squamous cell carcinomas 21 1 (4,8) 1 (4,8) 3 (14,3) 6 (28,5) 10 (47,6) Adenocarcinomas 5 \- \- 1 (20,0) 1 (20,0) 3 (60,0) \* percent is given in parenthesis ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Data on high risk HPV genome detection by PCR in CIN III and invasive squamous cell carcinoma samples ::: Sample No Type of immunochemical staining HPV type --------------------------------------- --------------------------------- ---------- **CINs III** 1 negative 16 2 negative 18 3 negative 16 4 poor 16 5 poor 16 6 poor 16 7 sporadic 16 8 sporadic 16 9 sporadic 16 10 sporadic 16 11 sporadic 16 12 sporadic 16 13 sporadic 16 14 focal 16 15 focal 16 16 diffuse 16 17 diffuse 16 18 diffuse 18 19 diffuse 18 **invasive squamous cell carcinomas** 20 negative 16\* 21 poor 16\* 22 sporadic 16\* 23 sporadic 16 24 sporadic 16\* 25 focal 16\* 26 focal 16 27 focal 16 28 focal 18 29 focal 16 30 focal 16 31 diffuse 16 32 diffuse 16 33 diffuse 16 34 diffuse 66 35 diffuse 16 36 diffuse 16 37 diffuse 16 38 diffuse 16 39 diffuse 18 40 diffuse 16 + 18 \* confirmed by Southern blotting ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### STAINING OF THE REFERENCE MATERIALS WITH p16^INK4a^-SPECIFIC MONOCLONAL ANTIBODIES ::: MATERIALS number of samples negative poor sporadic focal diffuse ----------------------------------------------- ------------------- ---------- ------ ---------- ------- --------- Normal tissues, including 12 12 \- \- \- \- Uterus body (myometrium) 1 1 \- \- \- \- Stromal and glandular tissues of cervix uteri 11 11 \- \- \- \- ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### THE SHARE OF p16^INK4a^-NEGATIVE SAMPLES AMONG CERVICAL CARCINOMAS (LITERATURE DATA) ::: Research group (reference) The share (per cent) of p16^INK4a^-negative samples -------------------------------- ----------------------------------------------------- among adenocarcinomas: Lu et al, 1998 (7) 17/40 (42.5%) Sano et al, 1998 (8) 4/15 (26.7%) Milde-Langosch et al, 2001(10) 8/58 (13,8%) Klaes et al, 2001(9) 1/7 (14.3%) Saqi et al, 2002 (13) 0/2 (0%) Murphy et al, 2003 (14) 0/2 (0%) Negri et al, 2003 (15) 0/18 (0%) Agoff et al, 2003 (16) 2/7 (28.6 %) Present study 0/5 (0%) among squamous cell carcinomas: Wong et al, 1997 (6) 8/79 (10.1%) Sano et al, 1998 (8) 1/39 (2.8%) Klaes et al, 2001 (9) 1/53 (1.9%) Sano et al, 2002 (12) 0/34 (0%) Klaes et al, 2002 (4) 0/46 (0%) Saqi et al, 2002 (13) 0/1 (0 %) Murphy et al, 2003 (14) 0/8 (0%) Agoff et al, 2003 (16) 4/46 (8.7%) Present study 1/21 (4.8%) :::

PubMed Central

2024-06-05T03:55:47.738199

2004-8-31

PMC517717

Background ========== Cervical carcinoma is a leading cause of death in women of reproductive age worldwide, particularly in developing countries. While curable in early stages, the treatment results of locally advanced disease are unsatisfactory. The current standard of treatment -cisplatin-based chemoradiation- fails to cure at least 15% to 45% of bulky IB to IIIB patients, and in addition, multimodality treatment incorporating chemotherapy, surgery and radiation at its best is unlikely to substantially increase the cure rate. Because of this, the logical step to follow is the testing of molecular targeted therapies trying to improve the prognosis of cervical cancer patients \[[@B1]\]. Human papillomavirus infection is recognized as the stronger etiological factor for the development of this tumor; however, overexpression of the epidermal growth factor receptor family members is also common and seems to play an important oncogenic role \[[@B2]\]. HER2 (also known as c-erbB-2) is a transmembrane receptor protein with tyrosine kinase activity that belongs to this family and it is overexpressed in a number of solid tumors. Its overexpression and prognostic significance in breast cancer led to the development and approval of the use of trastuzumab (Trastuzumab, Genentech, South San Francisco, CA), a recombinant monoclonal antibody to HER2, for the treatment of patients with metastatic breast carcinomas overexpressing HER2 \[[@B3]\]. Until more recently, poor standardization in HER2 status evaluation precluded reliable comparison of overexpression rates in different tumors. A source of variability in results not only comes from methodological variations in tissue processing (time to fixation, duration of fixation, denaturation, heating, antigen retrieval, the staining procedure) and grading scores but also from the antibody used. This issue was addressed by Press et al., who showed extremely variable results in 187 breast cancer specimens evaluated with 7 polyclonal and 21 monoclonal antibodies \[[@B4]\]. However, standardized methodologies have been introduced recently for these analyses, and have identified frequencies of 51%, 44%, 26% and 25% in Wilm\'s tumor, bladder, pancreatic and breast carcinoma, respectively. Other tumors tested had frequencies below 20% \[[@B5]\]. Before the introduction of the Hercep Test, it was known that a variable subset of cervical carcinomas ranging from 8% to 77% express HER2 as evaluated by diverse methods \[[@B6]-[@B14]\] and that in some studies its overexpression has shown to confer a worse prognosis \[[@B7]-[@B9],[@B13]\]. Because these results on HER2 expression in cervical cancer were obtained before the standardization required in breast cancer, we wanted to investigate the expression status of HER2 using the Hercep Test in a series of cervical carcinoma cell lines, primary tumors of locally advanced cervical cancer cases and in four recurrent tumors of these patients. Methods ======= Tumor specimens --------------- Thirty-five paraffin-embedded tumor tissues from patients FIGO staged as IB2 to IIIB, treated with standard radiation concurrent with weekly cisplatin. Diagnosis was made on the basis of routine hematoxilin-eosin examination under light microscopy according to the World Health Organization criteria. Tumor specimens at diagnosis were taken before any treatment was instituted whereas the tumors samples from the four recurrent cases were also taken before patients received any second line therapy. Cell lines and reagents ----------------------- DMEM culture media and Fetal Calf Serum were purchased from Gibco BRL Life Technologies (Grand Island, New York). HeLa, CasKi, SiHa and C33A carcinoma cell lines were obtained from the ATCC. The cell line ViBo established from a Mexican patient with cervical cancer was kindly provided by Dr. Monroy (FES Zaragoza, UNAM, Mexico City). Cells were grown in DMEM supplemented with 10% FCS at 37°C and 5% CO~2~. Cell lines were grown on two-chamber polysterene vessel Falcon^®^(Becton Dickinson, NJ.) and subsequently formalin-fixed for 24 hrs at room temperature, then rehydrated in graded ethanol. Afterwards immunochemistry was performed as below described. Hercep test ----------- Hercep Test was performed following the manufacturer\'s guidelines of HER2 protein expression as follows. Sections were deparaffinized in xylene and rehydrated through graded ethanols to distilled water. The sections were immersed in Dako Epitope Retrieval Solution (10 mM citrate buffer, pH6) that had been preheated to 95°C in a water bath and then heat-treated at 95°C for 40 min. After a 20-minute cooldown period at room temperature, the sections were washed with Dako Wash Buffer, a procedure that followed every subsequent incubation. Endogenous peroxidase was blocked with Dako Blocking Buffer (0.3% hydrogen peroxide containing 15 mM sodium azide) for 5 min at room temperature. The sections were incubated with the primary polyclonal antibody, an affinity-purified rabbit antihuman HER2 antibody supplied in the kit, for 30 min at room temperature. Bound primary antibody was labeled by incubating the slides with the Dako Visualization reagent (horseradish peroxidase-labeled dextran polymer conjugated to affinity-purified goat antirabbit immunoglobulins in Tris-HCl) for 30 min. Color development was achieved with 3,3\'-diaminobenzidine (DAB) for 10 min. The sections were counterstained with hematoxylin and eosin. To confirm validation of the staining run, control cell slides, which were provided in the kit and consisted of three pelleted, formalin-fixed, paraffin-embedded human breast cell lines with known HER2 positivity (MDA-231: 0; MDA-175: 1+; SK-BR-3: 3+), were also stained simultaneously. In the negative controls, the primary antibody was replaced by normal rabbit serum (Dako Negative Control Reagent) for the HER2 primary antibody. The antibody used in Hercep Test did not exhibit cross-reactivity to HER3 and HER4 in western blot analysis. Following the FDA scoring guidelines for breast carcinomas, only membrane staining intensity and pattern were evaluated using the 0--3+ scale as illustrated in the Hercep Test kit scoring guidelines (0 for no staining at all or membrane staining in less than 10% of the tumor cells; 1+ for only partial, weak staining of the cell membrane of more than 10% of the tumor cells; 2+ for moderate staining of the complete cell membrane in more than 10% of the tumor cells; 3+ for intense staining of the complete membrane in more than 10% of the tumor cells). The analysis was performed by a pathologist (VP-S) familiarized in the use of Hercep Test for breast cancer patients. In accordance with the Hercep Test kit guide, HER2 overexpression was assessed as negative for scores of 0 or 1+ and positive for scores of 2+ and 3+. FISH in the four recurrent cases -------------------------------- Amplification of *Her-2/neu*was evaluated using the Path-Vysion DNA Probe Kit (Vysis), which uses a dual-color probe for determining the number of copies of both *Her-2/neu*(orange) and the chromosome 17 centromeres (green). The kit was used following the manufacturer\'s instructions with a few minor modifications. Slides containing 5μ thick paraffin-embedded tissue sections of studied cervical tumor cases and a known Her-2/neu amplified breast tumor were placed on a slide warmer overnight at 58°C, followed by deparaffinization in Xilol, dehydration in 100 ethanol, and drying on a slide warmer at 45 to 50°C. Slides were then pretreated with 0.2 N hydrochloric acid for 20 minutes, followed by washes in purified water and immersion in Vysis wash buffer. They were subsequently immersed in Vysis protease solution at 37°C for 10 minutes, washed in Vysis wash buffer, and dried on the slide warmer. The slides were then immersed in 10% buffered formalin at room temperature for 10 minutes, immersed in Vysis wash buffer, and dried on the slide warmer. Sections were denatured by placing the slides in formamide for 5 minutes at 72°C followed by dehydration in 70, 85 and then 100% ethanol. Slides were then dried on a slide warmer, and 10 μl of probe was applied. They were then coverslipped, sealed and placed in a prewarmed humid incubation chamber at 37°C for 21 hours. This was followed by immersion in prewarmed postwash solution at 72°C for 2 minutes. The slides were air-dried, and a 4\',6-diamidino-2-phenylindole (DAPI) counterstain was applied. The scoring system used is described in detail in the manufacturer\'s instruction. A minimum of 60 nuclei were scored by each of 2 observers using a Zeiss Axioskop-2 fluorescent microscope with V.2 filter. The ratio of *Her-2/neu*signals (orange) to chromosome 17 centromere signals (green) was determined with ratios of \<2.0 considered nonamplified and those ≥ 2.0 amplified. Results ======= The immunohistochemical expression of HER2 in the primary tumors of 35 patients at diagnosis was evaluated. Accordingly, these patients had no received any previous anticancer therapy; their mean age was 40.8 years; 5 were staged as IB2-IIA, 14 as IIB and 16 as IIIB; 31 and 4 were histologically classified as squamous and adeno/adenosquamous respectively. Overexpression of HER2 was demonstrated in only one out of 35 cases, which had a score of 3+ (not shown). The remaining cases were interpreted as negative \[score of 0\]. The case with HER2 overexpression at diagnosis was a 56-year old woman diagnosed with a FIGO stage IIB large cell poorly differentiated squamous cell carcinoma, who received treatment with 6 weekly courses of cisplatin concurrent with external beam radiation and brachytherapy. She is currently free of disease at 44 months of follow-up. At a median follow-up time 40 months, seven patients have relapsed. HER2 expression at recurrence could only be analyzed in four of these seven relapsed patients in whom whose recurrent disease was histopathologically confirmed. Two of these four tested positive with a staining intensity of 2+ and 3+ respectively, (Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}), both cases were squamous cell carcinomas and tested negative in the pretreatment surgical specimen The five cervical cancer cell lines were negative. None of the four recurrent cases tested by FISH were HER2 amplified (Figure [3](#F3){ref-type="fig"}). Discussion ========== Molecular targeted therapies are currently being tested in a variety of tumor types with promising results. Because HER2 overexpression occurs and is related to a worse prognosis in cervical cancer, \[[@B7]-[@B9],[@B13]\], its blockade with trastuzumab could potentially have therapeutic value. This monoclonal antibody is currently widely used in metastatic breast cancer and is being evaluated in an adjuvant setting as well as in a variety of tumor types \[[@B3],[@B15]-[@B17]\], Based on the fact that the efficacy of this antibody is strongly associated to the level of HER2 expression in the primary tumor, the FDA approved the Hercep Test in the aim to grade the expression level so that only those patients whose tumors exhibit a 2+/3+ levels are candidates to trastuzumab therapy, though currently in most centers, tumors expression of 2+ is considered undeterminate therefore these cases are also evaluated by FISH analysis \[[@B18]\]. Previous reports on cervical cancer using non-standardized methods for HER2 expression showed that up to 77% of cases express the receptor and that in general HER2 expression predominates in adenocarcinoma and adenosquamous carcinoma histologies \[[@B6]-[@B14]\] In this work, using the Hercep Test with its corresponding guidelines for evaluation, we found contrastating results as none of the cell lines expressed HER2 and only a single tumor of squamous histology (1 out of 35) expresses this oncoprotein at a level of 3+. Such a discrepancy does not seem to be limited to this tumor type. For instances, in ovarian clear cell carcinoma a 43% of overexpression was reported utilizing systems other than Hercep Test \[[@B19]\], however, when evaluated with this standardized test, only 1 out of 17 tumors expressed 3+ \[[@B20]\]. Likewise, the proportion of patients with 2+/3+ expression level of HER2 with the Hercep Test is uniformly low in tumor such as lung \[[@B21]\], colorectal carcinomas \[[@B22]\], gallbladder \[[@B23]\], and melanoma \[[@B24]\]. A variety of factors such as the kind of antibody used, the technique per se, and scoring criteria may explain such phenomenon and its clarification requires further studies. A recent paper by Bellone et al., have reported that a substantially higher proportion of cervical cancer cells lines either established from fresh tumors or commercial ones (including CasKi, SiHa, HeLa and C33A which are negative by immunochemistry) express the receptor when evaluated by flow-cytometry and are growth inhibited when incubated with trastuzumab or trastuzumab plus IL-2 \[[@B25]\]. These data led them to suggest the targeting the HER in cervical cancer could be more effective than the indicated by low immunohistochemical expression \[[@B25]\]. However, it is largely known that in breast \[[@B26]\] and more recently in lung cancer \[[@B27]\], tumor responses are almost confined to those with a 3+ level of expression. For instances, in a recent published study in 111 assessable breast cancer patients, the response rate to single agent trastuzumab for those expressing 3+ versus 2+ was 35% and 0% respectively \[[@B26]\], while in lung cancer, a phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive patients, yet there was not overall differences in response between both arms, the benefit was limited to those with 3+ of expression with the Hercep Test. Accordingly, five out of six patients with such level of expression receiving trastuzumab plus gemcitabine-cisplatin showed response \[[@B27]\]. These data argue against the potential usefulness of trastuzumab in cervical cancer patients with HER expression that can only be detected by flow cytometry \[[@B25]\]. On the other hand, the HER2 expression in breast cancer is relatively stable, with 95% concordance between the HER2 status of primary and metastatic lesions, being rare a shift from positivity in the primary to negativity in the metastases \[[@B5]\]. Conversely, 6% of breast cancer patients whose primary tumors are HER2 negative, convert to high expression (Hercep Test 3+) in their metastases \[[@B28]\]. Such behavior is in line with experimental observations that receptor activation potentiates tumor cell motility, protease secretion and invasion, and also modulates cell cycle checkpoint function, DNA repair, apoptotic responses and multidrug resistance \[[@B29],[@B30]\]. The findings of \"conversion to positive\" in cells of recurrent cervical tumors showed by us and other authors \[[@B25],[@B31]\] strongly suggest that expression of HER2 may have a role in tumor resistance and progression as shown in experimental models, and therefore its targeting in recurrent cervical cancer could have therapeutic value. It is remarkable the finding that none of the four recurrent cases, including the two that converted to IHC positive analyzed by FISH showed HER2 gene amplification. This result is unlikely to be a false negative as the green signal was perfectly observed in most of the cells. Previous studies in cervical cancer have shown that the frequency of gene amplification as determined by FISH is low irrespective of tumor histology. Mark et al., reported only 2 out of 23 cases amplified using the Her-2/neu FISH probe (Vysis, Inc., Downers Grove, IL) both of which were adenocarcinomas \[[@B32]\]. In a more recent study looking at DNA copy number of cervical adenocarcinomas, it was found that despite more than 50% of patients had chromosome 17q copy number gains, only 9% (2 out of 22) of these tumors showed an HER-2/neu protein over-expression at the level of 2+ with the Hercep test. These findings suggest that amplification of HER-2/*neu*is rare in cervical adenocarcinomas and that low level chromosome 17q copy number gains are not associated with HER-2/*neu*overexpression \[[@B33]\]. Such overexpression without gene amplification could result from transcriptional deregulation leading to increased receptor expression \[[@B34]\] and is not a rare phenomenon in breast carcinoma \[[@B35]\]. Conclusions =========== In conclusion, our study suggest that the clinical usefulness of anti-HER2 antibodies in the primary treatment of cervical cancer patients may be limited due to the low frequency of HER overexpression, nevertheless, it is desirable to further test a larger number of recurrent cervical cancer patients by IHC and FISH analyses regardless of the histological type, as a start point for clinical trials design using trastuzumab. Competing interests =================== None declared. Authors\'contributions ====================== A C-B, AG-F, carried out the tissue culture work and immunohistochemical analysis; VP-S and T V-C interpreted the histological data; MC critically analyzed and participated in manuscript; CP, contribute with the clinical data; SV performed the FISH analysis; and AD-G conceived the study and wrote the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/4/59/prepub> Acknowledgements ================ We thank to Virginia Enriquez for her technical support. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Photomicrography of a recurrent tumor positive at a intensity of 2+. (200X). :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Photomicrography of a recurrent tumor positive at a intensity of 3+. (200X). :::  ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Photomicrography of the FISH analysis of a negative case of a recurrent tumor that was Hercep Test-positive, and a control positive case of breast carcinoma. :::  :::

PubMed Central

2024-06-05T03:55:47.741281

2004-9-1

PMC517718

Background ========== Enteral feeding of very low birth weight (VLBW) infants is a challenge, since metabolic demands are high and administration of enteral nutrition is limited by immaturity of the gastrointestinal tract. In particular, small for gestational age VLBW infants may have impaired gut function, as fetal blood flow to heart, brain and adrenals is compensatory increased, while other organs including the gastro-intestinal tract are relatively hypoperfused in intrauterine growth retardation \[[@B1]\]. Experimental studies have shown that the amino acid glutamine plays an important role in maintaining functional integrity of the gut \[[@B2]-[@B5]\]. Glutamine serves as fuel for enterocytes \[[@B2]\] and provides nitrogen for the synthesis of amino sugars, involved in maintenance of tight junctions \[[@B3]\] and mucin synthesis \[[@B4]\]. Moreover, glutamine has a stimulatory and regulatory effect on mucosal cell proliferation and differentiation \[[@B5]\]. Glutamine is not only utilised at a high rate by intestinal epithelium but also by cells of the immune system. In vitro studies have shown that increasing availability of glutamine stimulates proliferation of T-lymphocytes in response to T-cell mitogens \[[@B6]\], phagocytosis and antigen presentation by monocytes \[[@B7]\] and Th1 cytokine response \[[@B8]\]. In critically ill patients, endogenous glutamine synthesis cannot meet increased demand and for this reason glutamine is considered a conditionally essential amino acid \[[@B9]\]. VLBW infants may be especially susceptible to glutamine depletion as placental supply suddenly ceases at birth, tolerance of enteral nutrition is limited and parenteral nutrition does not contain glutamine for solubility and stability reasons. Glutamine depletion has negative effects on the functional integrity of the gut \[[@B10]\] and leads to immunosuppression \[[@B11]\]. Studies in adults have shown that glutamine supplementation decreases mortality in critically ill adults \[[@B12]\], infectious morbidity in recipients of bone marrow transplantation \[[@B13]\] and multiple trauma patients \[[@B14]\] and length of hospital stay in surgical patients \[[@B15]\]. In VLBW infants, only two studies have investigated efficacy of glutamine supplementation \[[@B16],[@B17]\]. In the study of Lacey *et al*\[[@B16]\], 78 VLBW infants at high risk of developing necrotising enterocolitis (birth weight 530--1250 g) were randomised to receive either standard or glutamine-supplemented parenteral nutrition. After exclusion of 34 infants, 22 treated and 22 control infants were compared for length of stay, days on total parenteral nutrition, days on the ventilator and infectious morbidity. In infants with a birth weight ≥ 800 g no effect of glutamine supplementation was found. However, in infants with a birth weight \<800 g glutamine supplementation was associated with shorter time to full enteral nutrition, fewer days on parenteral nutrition, fewer days on ventilatory support and reduced length of stay. Incidence of positive blood cultures and rate of weight gain were not different in glutamine and control groups. Neu *et al*\[[@B17]\] performed a randomised controlled trial of glutamine-enriched enteral nutrition in 68 VLBW infants with a gestational age of 24--32 weeks and a birth weight of 500--1250 g. Analysis was performed in 66 infants on an intention to treat basis. Feeding tolerance (as measured by number of days on which feeding had to be withheld) was better in the glutamine group compared to the control group. In addition, after adjusting for birth weight the odds ratio of developing sepsis was 3.8 for the control group compared to the glutamine group. Average weight at different time intervals and length of stay were not different between the groups. Although some methodological concerns can be raised (the sample size of both studies is small; Lacey *et al*\[[@B16]\] did not perform analysis on an intention to treat basis), these studies suggest that glutamine supplementation enhances feeding tolerance and decreases infectious morbidity in VLBW infants. The current double-blind randomised controlled trial is designed to determine the effect of glutamine-enriched enteral nutrition on feeding tolerance in a sufficient large population VLBW infants. We hypothesise that time to full enteral feeding is shorter in infants who receive glutamine-enriched nutrition compared to infants in the control group. Furthermore, infectious morbidity and short-term outcome are evaluated. To elucidate the effect of glutamine-enriched enteral nutrition on the functional integrity of the gut, intestinal permeability for macromolecules is measured. As part of the postnatal adaptation of the gut, the intestinal permeability decreases during the first days of life \[[@B18]\]. We hypothesise that glutamine-enriched enteral nutrition stimulates postnatal adaptation of the gut, reflected by a larger decrease in intestinal permeability. Another aspect of postnatal adaptation of the gut is the development of intestinal microflora. In VLBW infants, the colonisation by bacteria (including beneficial *Bifidobacterium*and *Lactobacillus*species \[[@B19],[@B20]\]) commonly present in healthy breast fed infants is delayed \[[@B21]\]. Intestinal mucin is an important site for bacterial adhesion and colonisation \[[@B22]\]. Glutamine may improve mucin quality \[[@B4]\] and consequently influence bacterial colonisation. We hypothesise that glutamine-enriched enteral nutrition stimulates the presence of *Bifidobacterium*and *Lactobacillus*species in the intestinal microflora. Intestinal microflora is investigated by determining faecal microflora with a culture independent technique. The effect of glutamine-enriched enteral nutrition on the immune response is investigated by determining plasma T-helper type 1 (Th1) and T-helper type 2 (Th2) cytokine concentrations following in vitro whole blood stimulation. As pregnancy is associated with skewing towards Th2 immunity \[[@B23]\], Th2 cytokine responses dominate the neonatal immune response \[[@B24]\]. Exposure to microbes stimulates Th1 cytokine responses and deviates the neonatal immune response towards balanced Th1/Th2 cytokine responses \[[@B24]\]. We hypothesise that glutamine-enriched enteral nutrition contributes to balanced Th1/Th2 cytokine responses by stimulating the Th1 cytokine response \[[@B8]\]. To assess safety of glutamine-enriched enteral nutrition, plasma amino acid profiles are determined. We hypothesise that plasma amino acid profiles in glutamine and control groups will not differ during the study period. In addition, to exclude negative effects of glutamine-enriched enteral nutrition on neurodevelopmental outcome, neuromotor development at the corrected age of 1 and 2 years and mental/motor development at the corrected age of 2 years are assessed. In conclusion, this double-blind randomised controlled trial aims to determine the effect of glutamine-enriched enteral nutrition on feeding tolerance, infectious morbidity and short-term outcome in VLBW infants. In addition, an attempt is made to elucidate the role of glutamine in postnatal adaptation of the gut and modulation of the immune response. Methods ======= The study is designed as a double-blind randomised clinical trial. The national central committee on research involving human subjects and the medical ethical review board of our hospital approved the study protocol. Study population ---------------- Infants with a gestational age \<32 weeks and/or birth weight \<1500 g admitted to the level III neonatal intensive care unit (NICU) of the VU University Medical Center, Amsterdam, are eligible for participation in the study. Written informed consent is obtained from all parents. Exclusion criteria are: major congenital or chromosomal anomalies, death \<48 h after birth, transfer to another hospital \<48 h after birth and admission from an extraregional hospital. Treatment allocation and blinding --------------------------------- To balance birth weight distribution into treatment groups, each infant is stratified to one of three birth weight groups (\<799 g, 800--1199 g, ≥ 1200 g) and randomly allocated to treatment \<48 hours after birth. An independent researcher uses a computer-generated randomisation table based on blocks of four (provided by Nutricia Nederland BV, Zoetermeer, The Netherlands) to assign infants to treatment A or B, which correspond to batch numbers on the nutrition products. Investigators, parents, medical and nursing staff are unaware of treatment allocation. The code for the batch numbers is broken after data analysis is performed. Treatment --------- Glutamine powder contains 82% L-glutamine and 18% glucose (nitrogen 15.5 wt/wt%; 371 kcal/100 g), whereas the isonitrogenous control powder contains 100% L-alanine (nitrogen 15.7 wt/wt%, 435 kcal/100 g). The two powders are indistinguishable by appearance, colour and smell. During the study period, glutamine and control powder are monitored for stability and microbiological contamination. Between days 3 and 30 of life, supplementation is administered in increasing doses to a maximum of 0.3 g/kg glutamine per day in the glutamine group. Initially, the supplementation dose is based on birth weight. After 2 weeks the dose is adjusted to actual weight. Two members of the nursing staff daily add supplementation to breast milk or to preterm formula (Nenatal^®^, Nutricia Nederland B.V., Zoetermeer, The Netherlands), according to the parents\' choice. Per 100 ml, Nenatal^®^provides 78 kcal, 2.1 g protein (casein-whey protein ratio 40:60), 4.4 g fat and 7.5 g carbohydrate. Nenatal^®^does not contain free L-glutamine. When infants are transferred to other hospitals before the end of the study, the protocol is continued under supervision of the principal investigator. Nutritional support ------------------- Protocol guidelines for the introduction of parenteral and enteral nutrition follow current practice at our NICU. Administration of parenteral nutrition starts at day 2 and will be advanced gradually until amino-acid intake reaches 3 g/kg/day at day 6. Parenteral nutrition is discontinued if enteral feeding reaches a volume of approximately 150 mL/kg/day. Parenteral nutrition, an all-in-one mixture provided by the hospital pharmacy, contains per 100 mL 54 kcal, 8.5 g glucose, 1.7 g amino acids and 1.7 g lipids. If necessary, glucose, amino acids and lipids are given in separate solutions. Guidelines for the introduction of enteral nutrition are as follows: 1. minimal enteral nutrition starts at day 1 (6--12 mL daily); 2. enteral nutrition is advanced either from day 3 or from day 5 in case of complications: BW \<p10, GA \<26 weeks, Apgar score at 5 minutes \<6, umbilical artery pH \<7.10 or base deficit \>10 mmol/L; 3. feeding is advanced at a dose of 15--20 mL/kg/day to a maximum of 150 mL/kg/day (based on actual weight). Furthermore, guidelines for reduction/withholding of enteral feeding are: 1. enteral feeding is reduced/withheld in case of gastric residuals (\> total volume of past 2 feedings), bilious residuals, emesis, ileus or necrotising enterocolitis Bell\'s stage ≥ II \[[@B25]\]; 2. when signs of feeding intolerance resolve, feeding is advanced in the volume given before reduction/withholding within 2 days. For each infant in the study a feeding schedule is proposed, based on birth weight and the guidelines as mentioned above. However, the staff of our NICU has final responsibility for the administration of parenteral nutrition and advancement of enteral feeding. Study outcome measures ---------------------- ### Study outcome measures Primary outcome of the study is time to full enteral feeding, defined as a feeding volume ≥ 120 mL/kg/day. Furthermore, other parameters of feeding tolerance, infectious morbidity, and short-term outcome are evaluated (Table [1](#T1){ref-type="table"}). In addition to clinical outcome, intestinal permeability, faecal flora, plasma Th1/Th2 cytokine concentrations and plasma amino acid profiles are determined during the 30 day study period (Table [2](#T2){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Clinical outcome measures ::: ---------------------------------------------------- -------------------------- **Remarks** **Feeding tolerance** Enteral feeding \>120 mL/kg/day Primary outcome Age at finishing parenteral nutrition Days of no enteral feeding during study period Necrotising enterocolitis Bell *et al*\[25\] **Infectious morbidity** Serious infections Number of infectious episodes Cultured micro-organisms **Short-term outcome** Weight *z*scores at birth, day 30 and at discharge Usher *et al*\[33\] Patent ductus arteriosus Ventilatory support Use of oxygen at postmenstrual age of 36 weeks Jobe *et al*\[34\] Intraventricular hemorrhage Papile *et al*\[35\] Retinopathy of prematurity Committee for ROP \[36\] Death Age at discharge from NICU and at discharge home ---------------------------------------------------- -------------------------- ROP = retinopathy of prematurity; NICU = neonatal intensive care unit. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Study schedule ::: -------------------------- ------------- ----------- ------------ ------------ **\< 48 h** **day 7** **day 14** **day 30** Amino acid profile x x x x Intestinal permeability x x x x Faecal flora x x x x Th1/Th2 cytokine profile x x x \- -------------------------- ------------- ----------- ------------ ------------ ::: ### Clinical outcome measures The following perinatal characteristics are registered to assess prognostic similarity: maternal age and race, obstetric diagnosis, administration of antenatal steroids and antibiotics, mode of delivery, sex, gestational age, birth weight, birth weight \<p10, Apgar scores, pH of the umbilical artery, clinical risk index for babies \[[@B26]\] and administration of surfactant. During the study period actual intake of enteral and parenteral nutrition, powder supplementation and type of feeding (breast milk or preterm formula) are recorded daily. Evaluation of medical records for the presence of serious infections is performed by one investigator/neonatologist, unaware of treatment allocation. Serious infections include sepsis, meningitis, pyelonephritis, pneumonia, and arthritis. Sepsis work-up consists of blood, cerebrospinal fluid and urine (suprapubic bladder tap) culture. Sepsis is defined as the combination of a positive blood culture and the presence of at least two clinical signs (body temperature \<36.5°C or \>37.5°C, hypotension, tachycardia, apnoeic attacks, feeding problems, irritability or apathy). Meningitis is diagnosed when micro-organisms are cultured in the cerebrospinal fluid. Pyelonephritis is diagnosed when both urine culture and dimercaptosuccinic acid (DMSA) renal scan are positive. Pneumonia is defined as the combination of a positive culture of tracheal aspirate, bronchial secretion or sputum and the presence of at least one clinical sign in ventilated infants (purulent sputum, changed sputum characteristics or deterioration of ventilation settings) or at least two clinical signs in non-ventilated infants (tachypnea, cyanosis, wheezing/rales/crepitation or purulent sputum/changed sputum characteristics). Arthritis is defined as the combination of a positive culture of intra-articular fluid and the presence of signs of articular inflammation. ### Postnatal adaptation of the gut The effect of glutamine-enriched enteral nutrition on postnatal adaptation of the gut is studied by measuring intestinal permeability and by determining faecal flora. Intestinal permeability is measured by the sugar absorption test, as previously described \[[@B18]\]. After instillation of the test solution, 2 ml/kg by nasogastric tube, urine is collected for 6 hours. After collection, 0.5 ml chlorohexidine digluconate 20% (preservative) is added to the urine and samples are stored at -20°C until analysis. Lactulose and mannitol concentrations (mmol/mol creatinine) are measured by gas chromatography as previously described \[[@B27]\]. The lactulose/mannitol ratio is used as a measure of intestinal permeability. Faecal samples are stored at -20°C until analysis by fluorescent in situ hybridisation (FISH) using specific 16S rDNA-targeted probes as described by Harmsen *et al*\[[@B28]\]. ### Immune response The effect of glutamine enriched-enteral nutrition on the immune response is investigated by determining plasma Th1/Th2 cytokine concentrations following in vitro whole blood stimulation. Heparinized blood (0.5 mL), diluted 1:1 in sterile medium (RPMI 1640 without L-glutamine, Gibco, Paisley, United Kingdom) is stimulated for 24 h at 37°C in the presence of anti-CD3/anti-CD28 (Central Laboratory of the Netherlands Red Cross Blood Transfusion service, Amsterdam, the Netherlands) and *Escherichia coli*lipopolysaccharide (concentration 1:1000 both). After incubation, blood is centrifugated, supernatant is collected and stored at -20°C until analysis. Th1 cytokines IFN-γ, TNF-α, IL-2 and Th2 cytokines IL-4, IL-5 and IL-10 are measured by cytometric bead array (BD biosciences, Alphen aan den Rijn, the Netherlands). ### Safety Safety of enteral glutamine supplementation in a dosage of 0.3 g/kg/day is investigated by determination of plasma amino acid profiles. Immediately after withdrawal, heparinized blood (0.5 mL) is centrifuged at 10000 rpm for 4 minutes. Plasma is deproteinized by sulfosalicylic acid (2mg/100 μL) and stored at -70°C until analysis. Amino acid profiles are determined by high-performance liquid chromatography as described by Teerlink *et al*\[[@B29]\]. To investigate neurodevelopmental outcome, neuromotor development at the corrected age of 1 and 2 years \[[@B30]\] and mental/motor development at the corrected age of 2 years are assessed \[[@B31]\]. Sample size ----------- We have calculated that a sample size of 102 infants is necessary to detect a difference of at least 2.5 days in time to full enteral feeding, assuming a SD of 4.5 days (two-tailed α = 0.05, β = 0.20). The SD value is based on an retrospective analysis of time to full enteral feeding in infants with GA \<32 weeks and/or BW \<1500 g admitted to our NICU in 1998. Statistical analysis -------------------- To determine whether randomisation is successful, prognostic similarity (perinatal and nutritional characteristics) between treatment groups is assessed. The Students\' t-test, Mann-Whitney U test, and chi-square test or Fisher\'s exact test are used to compare continuous normally distributed data, nonparametric continuous data and dichotomous data respectively. Cox regression is performed to examine the effect of glutamine-enriched enteral nutrition on time to full enteral feeding. Logistic regression is performed to examine whether glutamine-enriched enteral nutrition influences the incidence of serious infections. In an additional analysis, adjustments are made for possible confounding factors as administration of antenatal corticosteroids, birth weight \<p10, administration of breast milk and other prognostic factors that may be different between treatment groups. Analyses of secondary outcomes (only crude) is performed by Mann-Whitney U test, chi-square test or Fisher\'s exact test and log rank test for nonparametric continuous data, dichotomous data, and time-dependent data respectively. Generalised estimated equations for longitudinal analysis \[[@B32]\] are used to analyse changes over time in intestinal permeability, faecal microflora, plasma Th1/Th2 cytokine concentrations and plasma amino acid profiles. Distribution of optimal and non-optimal neuromotor development and normal and abnormal mental/motor development in glutamine and control groups is examined by logistic regression with adjustments for possible confounding factors as gestational age and birth weight. All statistical analyses are performed on an intention to treat basis. In addition, alternative per protocol analyses are performed, excluding all patients who are not treated according to protocol, defined as more than 3 consecutive days or a total of 5 days on minimal enteral feeding or without supplementation. A p value \<0.05 is considered significant (two-tailed). SPSS 9.0 (SPSS Inc., Chicago, IL, USA) and STAT 7.0 (StatCorp LP, College Station, TX, USA) are used for data analysis. Competing interests =================== Nutricia Nederland B.V. (Zoetermeer, the Netherlands) provided Nenatal^®^, glutamine and placebo supplementation. Authors\' contributions ======================= Ruurd van Elburg and Willem Fetter formulated the research question and wrote the study protocol. Anemone van den Berg, Ruurd van Elburg and Willem Fetter contributed to the development of the protocol. Jos Twisk gave advice on data analysis. Anemone van den Berg wrote the draft for this manuscript and the other authors reviewed the manuscript. All authors approved the final version of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2431/4/17/prepub> Acknowledgements ================ We would like to thank HN Lafeber, AM van Furth and J Knol for their contribution to the study design.

PubMed Central

2024-06-05T03:55:47.743413

2004-9-1

PMC517719

Background ========== Diarrheal diseases in the developing world continue to cause significant morbidity and mortality, especially when associated with malnutrition \[[@B1]\]. Dehydration, potassium depletion and acidosis are the main life-threatening complications of the acute losses during watery diarrhea. More than 25 years of extensive use has demonstrated that these complications could be effectively treated or prevented by oral rehydration therapy \[[@B2]\]. However, the oral glucose-electrolyte rehydration solution such as that recommended by WHO and Unicef neither shortens the duration of the illness nor reduces the stool loss and may cause an increase in stool volume at least during the first hours in children with acute diarrhea \[[@B3]\]. Optimization of the standard WHO-ORS solution, by reducing its osmolarity, has been shown to reduce diarrhea duration, total stool output, and the need for unscheduled intravenous therapy \[[@B4]\]. Adjuvant therapy to ORT, based on oral administration of live probiotic bacteria aimed to improve recovery of infants from acute watery diarrhea, has been under active investigation \[[@B5]\]. These studies, done in the developed world, have shown a benefit only for viral mild gastroenteritis. Isolauri et al have shown that oral bacterial therapy with *Lactobacillus casei*strain GG (LGG) reduces both the severity and duration of acute non dehydrating rotavirus enteritis \[[@B6]\]. Another trial in Karelia republic, LGG was shown to decrease the duration of acute diarrhea in children with viral acute diarrhea but not in those with bacterial diarrhea \[[@B7]\]. In a multicenter study carried out in Europe, LGG was administrated in a hypotonic oral rehydration solution to children with acute diarrhea, showing that it was safe and resulted in shorter duration of diarrhea, less chance of a protracted course, and faster discharge from the hospital \[[@B8]\]. Limited information is available on the potential role of LGG in infants living in the developing world, with more severe acute watery diarrhea \[[@B9]\], where etiologic agents other than rotavirus are involved more frequently and where mixed infections are more prevalent \[[@B10]\]. We conducted this study aimed to assess the effect of a milk formula containing *Lactobacillus casei*strain GG (LGG) on the duration and severity of diarrhea in children with acute watery diarrhea from our hospital. The study was conducted in the Rehydration Unit of Cayetano Heredia Hospital in Lima, Peru from September 1991 to June 1992. The study protocol and Consent Form were reviewed and approved by the Ethical Committee of Cayetano Heredia University in compliance with the Helsinki Declaration. Methods ======= Study population ---------------- Male infants 3--36 months of age brought for treatment because of acute diarrhea to the Rehydration Unit of Cayetano Heredia Hospital, Lima during September 1991 to June 1992 were eligible for the study if they met the following criteria: (a) a history of three or more watery stools per day for less than 48 hours, (b) no bloody stools at the moment of first examination, (c) clinical signs of dehydration, (d) no clinical features of hypovolemic shock, (e) no clinical signs of a coexisting acute systemic illness (i.e., meningitis, sepsis, pneumonia) or a recognized chronic disease such as pulmonary tuberculosis, and (f) no history of current antibiotic or antidiarrheal medication use. Patients 3--6 months old were considered for the study only if they were not on exclusive breastfeeding. Patients were not selected if their weight for age was less than 60% of the median weight for age established by the National Center for Health and Statistics (NCHS) \[[@B11]\]. Only patients whose parent or legal guardian was willing and able to give written informed consent were admitted to the study. Only male patients were selected in order to facilitate separate collection of stools and urine. The study size was calculated expecting a 33% reduction in duration of diarrhea and mean volume of diarrhea stool for a significance level of 0.05 and a power of 80%. Study design ------------ Infants fulfilling the admission criteria were randomly assigned to a double-blind comparison of a milk formula containing 10^9^cfu/ml of *Lactobacillus casei*strain GG (LGG) or a similar milk formula not containing LGG. Both formulas were of identical looking and tasting. Both milk formulas were provided by the manufacturer (Valio Ltd, Helsinki, Finland) as milk powder to reconstitute with water. They were given in bottles, appropriately labeled and number-coded for each child to secure the double-blind design. The actual assignment of children to their study numbers was made at the time of their enrollment into the study. Each child admitted into the study was allocated to either treatment group by restricted randomization using random permuted blocks. The codes were prepared in Finland and kept in sealed envelopes in Lima. Baseline assessment ------------------- A clinical history and physical examination was completed upon admission to identify the patient, to determine the duration, type and severity of his diarrhea, to assess associated clinical features (fever, vomiting, dehydration, abdominal pain, distension), and to establish the nutritional status and previous dietary regimen of the subject. Blood for determination of serum sodium, potassium, chloride, glucose and bicarbonate, blood urea nitrogen, microhematocrit and plasma specific gravity was drawn from each patient before starting treatment. A routine urine analysis was also made. Fecal samples or rectal swabs taken at admission were transported in Cary-Blair transport medium to Cayetano Heredia University Microbiology Laboratory for primary isolation and identification of bacterial enteropathogens. A separate vial containing phosphate buffer saline was used to transport fecal specimens for rotavirus examination. Microbiologic methods --------------------- Specimens were cultured for *Salmonella*, *Shigella*, *E. coli*and Vibrios by standard direct and enrichment enteric media. *Campylobacter*was selectively cultured on sheep blood agar containing Butzler\'s antibiotic supplement and *Aeromonas*on ampicillin blood agar preceded by overnight enrichment in alkaline peptone water. Five lactose fermenting colonies of *E. coli*isolates per patient were serotyped according to standard methods using polyvalent sera for the recognition of enteropathogenic *E. coli*. Detection of Rotavirus was made by ELISA method using Rotazime^®^. Identification of enterotoxigenic strains of E. coli was not done. Details of the microbiologic methods used in this study are described in detail elsewhere \[[@B10]\]. Rehydration ----------- The degree of dehydration was assessed clinically. Dehydration was corrected and then fluid balance maintained using the oral rehydration salts (ORS) in its standard WHO\'s recommended formulation, following the WHO Guidelines \[[@B12]\]. Briefly, each child was given approximately 100 ml/kg of ORS during the first four hours. The ORS was administered in frequent sips using a spoon or by nasogastric tubes if vomit or stool output rates were high. Children were not given other fluid or foods during this period. Upon completion of this period, on-going fecal losses were replaced with the same solution, on a volume to volume basis until diarrhea ceased. Administration of the study formula ----------------------------------- Once dehydration was corrected, feeding, including administration of the assigned study formula was initiated. The first dose of the assigned study formula was administered as soon as rehydration was completed and subsequent doses were given every four hours until cessation of diarrhea or for a maximum of five days. The formula was prepared immediately before administration of each dose by a specially trained nurse that was exclusively working for the study. Measuring spoons provided by the manufacturer were used and the formula powder was kept refrigerated all the time once the container was opened. Before use, the sachets with the study formulas were kept in a -20°C refrigerator. The formula powder was diluted in warm boiled water as to provide 670 Kcal per liter. The amount of formula given to each child was calculated based on the child\'s body weight, each child being given 150 ml/kg/day to a maximum of 1000 ml/day. On average, each serving of 100 ml will supply 10^11^cfu of LGG for those receiving the formula containing LGG. Administration of the formula was by bottle and not compulsive. If the child refused to drink the formula after reasonable attempts, no extraordinary methods, like delivery via nasogastric tubing, were used; in this situation the formula was offered again four hours later. Other foods given ----------------- Patients under six months of age were fed only the randomly assigned study formula to provide a daily caloric intake of at least 100 Kcal/kg (in a volume of 150 ml/kg/day). No other foods were given for these under six months patients. Older patients were fed the assigned formula *ad libitum*for a maximum of 1000 ml/day. Caloric requirements were completed with a blended soft baby food prepared from fresh ingredients (chicken breast, rice, carrots, squash, potatoes, and vegetable oil), by the Hospital\'s Nutrition Department. This soft baby food provides 1 Kcal per gram (approximately, 10% of its calories as protein, 45% as carbohydrates and 45% as fats). A total of at least 100 Kcal/kg/day (including the calories provided by the study formula) were offered to these children. Measurement of clinical outcomes -------------------------------- All oral intake (ORS, study formula, breast milk, plain water, soft baby food) as well as urine, stool and vomitus volume were strictly measured and recorded during hospitalization. For this purpose patients were put on metabolic beds (which have a hole in the mattress were a container was placed to allow direct stool collection). A urine collection bag was fitted to avoid contamination of stools with urine and to measure the urine volume. After the first 72 hours, differential weight of dry and soiled diapers was used instead the metabolic bed to determine stool weight if the previous observed purging rate was below 5 ml/kg/hour. Blood analyses taken at admission were repeated at 24 hours. Stool examination for pH, glucose, reducing substances, leukocytes and microscopic fat were performed at admission, 24, 72 hours and at discharge. Urine specific gravity was determined every 24 hours until discharge. Criteria for stopping treatment and discharging the patient ----------------------------------------------------------- Patients were discharged from the study 24 hours after cessation of diarrhea, or at the end of five days from admission, or at the time treatment failure occurred. At discharge each patient was categorized as have completed the trial, as a treatment failure, as an unresolved diarrhea situation, or as a withdrawal. Primary outcomes ---------------- • Rate of treatment failure: proportion of patients in each study group who have recurrence or continued presence of more than 5% dehydration, worsening electrolyte abnormalities, no weight gain since admission, developing of ileus or severe diarrhea defined as a purging rate in excess of 10 ml/Kg/hr in two consecutive 4-hour periods. • Rate of unresolved diarrhea after five days of treatment: proportion of patients in each study group with continuing diarrhea after five days of treatment. • Duration of diarrhea: time in hours from admission until cessation of diarrhea • Duration of hospitalization: time in hours from admission until discharge from hospital. Patients with treatment failure are excluded. • Total stool output: volume of diarrheic stools collected from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in milliliters per kilogram of body weight. • Total intake of oral rehydration solution: volume of ORS taken from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in milliliters per kilogram of body weight. Secondary outcomes ------------------ • Total study formula intake: volume of study formula taken from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in milliliters per kilogram of body weight. • Total energy intake: energy from all sources (ORS, formula) taken from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in kilocalories per kilogram of body weight. • Total volume of vomitus: volume of vomitus collected each day from admission until cessation of vomitus or for a maximum of 120 hours, expressed in milliliters per kilogram of body weight. • Total volume of urine: volume of urine collected each day from admission until cessation of diarrhea or for a maximum of 120 hours, expressed in milliliters per kilogram of body weight. Working definitions ------------------- The following definitions were used for this study: • Carbohydrate malabsorption: stool sample giving a positive reaction for reducing substances (0.75 g % or above). • Cessation of diarrhea: passage of formed stool or passage of no stool for 12 consecutive hours. • Early withdrawal: patient who (a) developed a complicating illness, (b) presented bloody diarrhea after admission and before five days of treatment, (c) was prematurely discharged under request of his parent or legal guardian, or (d) had any protocol violation or non-compliance. Statistical analysis -------------------- The data, initially collected in pre-coded forms, were entered in a database organized with a relational model under Fox Pro^®^v. 1.02 (Dbase^®^compatible formats) using a data entry program with on-line checking. The data was checked again in batch mode and finally transferred to EpiInfo^®^for Windows^®^v. 3.01 (November 2003) where the tabulations and statistical analysis were performed. Letters (A or B, maintaining blindly the treatment group assignment) identified treatment groups until all statistical analysis was completed. Only after completing the analysis investigators unblinded the treatment assignment. Baseline and outcome two-way comparisons were made between the two treatment groups. A 5% level of significance for statistical tests was set in advance for all comparisons. Chi-square Yates corrected was used to compare discrete variables. One way analysis of variance was performed for continuous variables. Data from patients prematurely withdrawn from the study was included in the analysis up to the time of withdrawal (intention-to-treat analysis). Results ======= A total of 179 patients were admitted into the study, 90 in the LGG group and 89 in the control group. Nineteen patients, eight from the LGG group and 11 from the control group, were prematurely withdrawn from the study (figure [1](#F1){ref-type="fig"}) because of either one of the following reasons: bloody stools within the first 24 hours after admission (11 patients); parental non-compliance (four patients); no diarrheal stools passed within the first 24 hours since admission (two patients); typical severe cholera-like diarrheal disease improperly included (one patient); and severe systemic infection present but not recognized at admission (one patient). Baseline characteristics ------------------------ Table [1](#T1){ref-type="table"} presents the baseline characteristics of patients upon admission. The treatment groups were comparable in age, nutritional status, and other clinical and laboratory variables. Marginal differences were found, however, in stool output during the first four hours of the rehydration phase, before administration of the study formula. The LGG group was thus comprised by slightly more severe cases. Table [2](#T2){ref-type="table"} shows stool microbiology and parasitology at admission. The distribution of enteropathogens was similar between both groups, except for rotavirus which was found more frequently in the placebo group (39.3%) than in the LGG group (24.4%) with a marginal statistical significance for the difference (p = 0.052). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Clinical and laboratory features on admission ::: **Placebo group** **N** **LGG group** **N** **P** ------------------------------------------------------ ------------------- ------- --------------- ------- ------- Age (months) 14.7 ± 6.4 89 14.9 ± 7.5 90 0.878 Duration of diarrhea before admission (h) 29.5 ± 14.9 89 28.8 ± 14.9 90 0.733 N° of stool motions 24 h before admission 6.6 ± 3.2 89 7.5 ± 3.9 90 0.101 N° of patients with vomitus 24 h before admission 64 (71.9%) 89 66 (73.3%) 90 0.963 N° of patients with fever 24 h before admission 39 (43.8%) 89 43 (47.8%) 90 0.703 Clinical dehydration 89 90 0.165  Mild 48 (53.9%) 36 (40.0%)  Moderate 40 (44.9%) 52 (57.8%)  Severe 1 (1.1%) 2 (2.2%) \% of weight gain after rehydration 3.0 ± 2.2 89 3.1 ± 2.3 90 0.763 Fever at admission (≥ 37.5°C) 16 (18.0%) 89 14 (15.9%) 88 0.868 Weight for height\* 94.8 ± 9.3 89 94.1 ± 7.6 90 0.551 Height for age\* 88.9 ± 12.1 89 97.2 ± 3.7 90 0.546 Weight for age\* 88.9 ± 12.1 89 88.9 ± 10.8 90 0.952 Stool output (ml/kg) first 4 h (rehydration phase) 14.4 ± 12.4 89 18.1 ± 16.1 90 0.085 Hematocrit (%) 35.0 ± 4.2 89 35.5 ± 3.8 90 0.426 Plasma specific gravity 1.028 ± 0.002 89 1.028 ± 0.002 90 0.769 Serum Na^+^(mEq/l) 136.6 ± 4.7 89 136.4 ± 4.6 87 0.770 Serum K^+^(mEq/l) 4.0 ± 0.7 89 4.0 ± 0.7 87 0.985 Serum Cl (mEq/l) 102.5 ± 5.2 89 102.6 ± 5.4 87 0.874 Serum total CO2 (mmol/l) 14.0 ± 3.9 88 14.0 ± 3.9 87 0.971 Blood urea nitrogen (mg/dl) 13.1 ± 10.5 70 11.4 ± 6.6 73 0.253 Blood glucose (mg/dl) 107.5 ± 33.4 88 106.5 ± 35.9 88 0.848 N° of patients with fecal leukocytes (\> 20 PMN/hpf) 30 (34.5%) 87 21 (23.6%) 89 0.154 N° of patients with microscopic fat 56 (64.4%) 87 52 (58.4%) 89 0.513 Fecal pH 6.3 ± 1.1 87 6.6 ± 1.1 89 0.126 N° of patients with CHO malabsorption 39 (45.9%) 85 36 (41.4%) 87 0.659 \* % of NCHS median Values are mean ± SD or n (%) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Stool microbiology and parasitology at admission ::: **Placebo group** **N** **LGG group** **N** **P** ---------------------------------------- ------------------- ------- --------------- ------- ------- Rotavirus 33 (39.3%) 84 22 (24.4%) 90 0.052 EPEC 12 (17.6%) 68 9 (13.2%) 68 0.635 *Vibrio cholerae* 7 (8.0%) 88 11 (12.4%) 89 0.471 *Campylobacter sp*. 6 (6.8%) 88 9 (10.1%) 89 0.605 *Shigella sp*. 9 (10.2%) 88 4 (4.5%) 89 0.241 *Aeromonas sp*. 2 (2.3%) 88 1 (1.1%) 89 0.992 *Salmonella sp*. 0 (0.0%) 88 1 (1.1%) 89 0.995 Negative microbiology 25 (36.8%) 68 32 (47.1%) 68 0.452 *Cryptosporidium parvum* 6 (7.0%) 86 2 (2.2%) 89 0.256 *Giardia lambliae* 2 (2.3%) 86 3 (3.4%) 89 0.969 *Strongyloides stercolaris* 1 (1.2%) 86 0 (0.0%) 89 0.986 *Trichomonas sp*. 0 (0.0%) 86 2 (2.2%) 89 0.492 Negative parasitology 77 (89.5%) 86 82 (92.1%) 89 0.738 Negative microbiology and parasitology 22 (35.5%) 62 30 (44.8%) 67 0.371 No patients were found positive for the following parasites: Ascaris, Enterobius, Trichuris, *E. hystolytica*, Ancylostoma, Necator. ETEC identification was not done Values are n (%) ::: Stool purging reduction ----------------------- The mean total stool output (Table [3](#T3){ref-type="table"}) was lower in the placebo group (195.0 ml/kg) than in the LGG group (247.8 ml/kg) with a marginal statistical significance for the difference (p = 0.047). The difference between means in total stool output was -52.8 ml/kg, with 95% confidence limits between -105.4 to -0.2 ml/kg. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Clinical outcomes ::: **Placebo group** **N** **LGG group** **N** **P** **Difference between means** ------------------------------------------------ ------------------- ------- --------------- ------- ------- ------------------------------ Discharge condition 89 90  Early withdrawal 11 (12.4%) 8 (8.9%) 0.609  Treatment failure 16 (18.0%) 19 (21.1%) 0.734  Cessation of diarrhea 51 (57.3%) 52 (57.8%) 0.931  Unresolved diarrhea 11 (12.45%) 11 (12.2%) 0.842 Mean duration of diarrhea (hours)\* 50.4 ± 28.0 51 58.5 ± 30.2 52 0.157 -19.6 to 3.4 Mean duration of hospitalization (hours)\*\* 74.7 ± 33.7 62 81.2 ± 32.6 63 0.280 -18.4 to 5.4 Total stool output (ml/kg) 195.0 ± 171.9 89 247.8 ± 180.2 90 0.047 -105.4 to -0.2 Total oral rehydration solution intake (ml/kg) 236.5 ± 195.6 89 272.8 ± 188.1 90 0.208 -93.7 to 21.1 Total study formula intake (ml/kg) 215.5 ± 136.3 89 231.5 ± 136.6 90 0.435 -56.8 to 24.8 Total energy intake (Kcal/kg)^§^ 220.7 ± 140.4 89 247.4 ± 143.0 90 0.209 -69.1 to 15.7 Total volume of vomitus (ml/kg) 27.6 ± 45.5 89 27.4 ± 33.9 90 0.968 -11.8 to 12.2 Total volume of urine (ml/kg) 81.5 ± 65.2 89 87.1 ± 69.4 90 0.579 -25.7 to 14.5 \* For patients who ceased diarrhea before 120 hours. \*\* Patients with treatment failure or early withdrawals are excluded. ^§^Calculated from calories provided by study formula, soft foods, breastmilk, and oral rehydration solution. Values are mean ± SD, n (%), or 95% confidence limits. ::: Duration of diarrhea -------------------- The proportion of patients with unresolved diarrhea after 120 hours was similar in both treatment groups (table [3](#T3){ref-type="table"}). For those patients that ceased diarrhea during the study period the mean duration of diarrhea was similar between groups (table [3](#T3){ref-type="table"}). The difference between means in duration of diarrhea was 8.1 hours with 95% confidence limits between -19.6 hours to 3.4. Blood chemistry --------------- No patient had to be withdrawn from the study because of worsening blood chemistry abnormalities. Carbohydrate malabsorption -------------------------- The proportion of patients with carbohydrate malabsorption 24 hours after admission into the study was high although not different between the treatment groups (table [4](#T4){ref-type="table"}). However, the rate of carbohydrate malabsorption after 24 hours of treatment increased significantly in both treatment groups with respect to admission (table [4](#T4){ref-type="table"}). The pH of stools at 24 hours after admission is also significantly lower in both groups compared to the pH at admission, a result consistent with worsening carbohydrate malabsorption (table [4](#T4){ref-type="table"}). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Comparison of stool characteristics at admission and 24 hours after ::: **Admission** **N** **24 h later** **N** **P** ------------------- --------------- ------- ---------------- ------- ---------- Fecal pH  Placebo group 6.3 ± 1.1 87 5.9 ± 0.8 76 0.014  LGG group 6.6 ± 1.1 89 6.1 ± 0.9 74 0.004 CHO malabsorption  Placebo group 39 (45.9%) 85 51 (68.0%) 75 0.008  LGG group 36 (41.4%) 87 52 (72.2%) 72 \< 0.001 Values are mean ± SD or n (%) ::: No adverse effects due to the study formula were notice in either group during the study. Discussion ========== Children included into this study probably represent the type of patients with diarrheal disease most frequently seen at a health service in Peru before the cholera epidemic of 1991. They were infants under two years of age, with an episode of acute watery diarrhea, presenting with signs of mild to moderate dehydration and a slight compromise of the nutritional status. Thus, we can conclude that the results of this study can be applied to most cases of acute watery non-cholera diarrhea seen in this population setting. The comparison of baseline characteristics showed no significant differences between groups. Marginal differences (not statistically significant) were observed in two variables that measure severity of illness: stool frequency 24 hours before admission and stool output during rehydration phase. Both were higher in the LGG group. Other indicators of severity, like clinical estimate of dehydration, percentage of weight gain after rehydration and proportion of patients with vomitus before admission, were similar between both groups. On the other hand, there were more cases (not statistically significant) of rotavirus in the placebo group than in the LGG group. In order to make sure that the groups were in fact comparable, we perform the statistical adjustment for these variables using multiple regression analysis and found no association with the principal outcomes (results of this analysis are not shown in the report). In more than 55% of patients an enteropathogen could be identified. This figure could be higher because studies for ETEC were not done. The most prevalent agents were rotavirus (30.7%), EPEC (11.7%), *Vibrio cholerae*(10.1%) and *Campylobacter sp*. (8.4%), with an important proportion of patients having mixed infections. These findings differ considerably from those of the Finnish clinical trial in which LGG was also tested for efficacy in reducing acute infantile diarrhea \[[@B6]\]. In the Finnish trial, rotavirus accounted for more than 80% of diarrheal cases, with no cases of bacterial diarrhea. A setting of greater diversity of etiologic agents and frequently co-pathogen association is perhaps more representative of developing countries \[[@B10]\]. The first topic to be discussed here is the high proportion of patients that did not recovered optimally during the trial, a condition that did not depend on the assigned treatment group. About 12% of patients had an unresolved diarrhea and an additional 20% were classified as treatment failures, mostly due to severe diarrhea. In two clinical trials conducted at our unit similar rates were found. For instance, in a clinical trial designed to test the clinical efficacy of a citrate-based ORS in children with acute watery diarrhea, patients who received standard therapy (WHO-ORS) had a treatment failure rate of about 24% \[[@B13]\]. This rate was 40% if only rotavirus associated diarrhea was considered. In another study, in which a chicken soup-based ORS was evaluated for children with similar characteristics (not published), 15.4% treatment failure rate for all subjects standard and 20% for those with rotavirus was observed. In the Finnish study, all children recovered before 5 days and no treatment failures were reported \[[@B6]\]. This striking difference could be explained in part by dissimilar etiologies as was mentioned above. Bacterial diarrhea and infection with more than one pathogen might produce a more severe illness. The nutritional status is also important to explain the difference. It has been well established from studies in developing countries that diarrhea lasts longer in poorly nourished children \[[@B14]\]. This difference seems to be important even when only slight malnutrition is present, as is the case for the patients admitted into this study. Except for total stool output (which showed a marginal statistical significance), no significant differences in the principal outcomes between groups were found. Duration of diarrhea, duration of hospitalization, and total ORS intake were not significantly different between the experimental and placebo group. The mean volume of formula (and of lactose) administered to children was the same for both groups. The confidence limits of the difference between the two groups indicate that a maximum reduction on duration of diarrhea of 6.7% attributable to the LGG formula could have been detected with the sample size of this study. These numbers indicate that the study does have a sample size sufficient to reject a type II error. No positive effects of treatment could be demonstrated as opposed to Isolauri\'s experience in Finland \[[@B6]\]. The dose factor could not explain the difference between the two studies. The dose of LGG in Isolauri\'s study was 2 × 10^10--11^cfu per day \[[@B6]\]. The dose of LGG in our study was 6--8 × 10^11^cfu per day, well above the threshold dose of 10 billion cfu suggested as most effective \[[@B15]\]. Viability of the *Lactobacillus GG*in the sachets while stored in Lima was not confirmed bacteriologically. There were slightly fewer cases of rotavirus in the LGG group than in the placebo group (24.4% vs. 39.3%), although the difference was not statistically significant. LGG appears to be more effective in viral than in bacterial diarrhea \[[@B8]\]. These facts could explain in part the lack of beneficial effect of LGG in our study. It is possible that LGG was not able to colonize the gut of our patients in the presence of bacterial pathogens. Although colonization has been documented during rotavirus diarrhea in children \[[@B16]\], to our knowledge this ability has not been demonstrated when other intestinal pathogens are involved. Colonization properties of LGG in patients with bacterial enteritis would deserve further studies. Our study clearly shows that the rate of carbohydrate malabsorption raised in both treatment groups from 43.6% at admission to 70.1% 24 hours later. The amount of lactose offered to patients in this study was much higher than in the usual feeding regimen at our unit (10.5 g/kg/day vs. 3.7 g/kg/day, respectively). Although to some investigators lactose intolerance is becoming a decreasing problem \[[@B17]\], it is clear from these data that lactose malabsorption as a factor cannot be dismissed. We had consistently found a high proportion of patients with carbohydrate malabsorption in all similar studies conducted in our unit. Thus, one possible explanation for the lack of efficacy of LGG in our patients is that worsening diarrhea due to lactose malabsorption could have masked any beneficial effect of the LGG. If this is true, then reducing the lactose content of the formula might reveal a positive effect. Another option is to deliver the LGG in suitable non-lactose containing infant food that could be given to diarrheic children as part of their treatment. Carefully conducted studies might prove this hypothesis. Conclusions =========== Both groups were comparable in their baseline characteristics. Marginal differences were found in variables that measured severity of illness before treatment. Total stool output was slightly higher in the LGG group reaching a marginal statistical significance. No significant differences in the other principal outcome variables were found between treatment groups. The proportion of patients with unresolved diarrhea after 120 hours was similar in both treatment groups. For those patients that ceased diarrhea during the study period the mean duration of diarrhea was similar between groups. The rate of carbohydrate malabsorption after 24 hours of treatment increased significantly in both treatment groups. Competing interests =================== None declared. Authors\' contributions ======================= ES-L participated in the design of the study, organized and coordinated the study, participated in the statistical analysis and drafted the manuscript. PM-L carried out the clinical work, participated in the statistical analysis and the drafting of the manuscript. MC-S carried out the statistical analysis. EC-W participated in the clinical work. RBS conceived of the study, and participated in its design and drafting of the manuscript. All authors read and approved the final manuscript ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Diagram showing the flow of study subjects throughout the study :::  ::: Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2431/4/18/prepub> Acknowledgements ================ The study was supported by a grant from Valio Ltd, Helsinki, Finland to the Child Health Foundation, Columbia, Maryland.

PubMed Central

2024-06-05T03:55:47.745055

2004-9-2

PMC517720

Background ========== Vitamin D is an essential element for establishing and maintananing bone structure. Vitamin D deficiency results in rickets and osteomalacia. Even slight vitamin D deficiency results in secondary hyperparathyroidism and increased bone resorption \[[@B1],[@B2]\]. In addition, there has been increased attention to the physiologic importance of vitamin D in non-skeletal tissues \[[@B3]\]. Vitamin D is supplied by consumption of vitamin D-rich foods and by vitamin D synthesis in skin. Natural nutrient materials are not a sufficient source of vitamin D to supply the body requirements; therefore where there is no supplementation of foodstuffs, the main source for vitamin D is produced by UV light \[[@B4],[@B5]\]. Regarding the significant role of sunlight in vitamin D synthesis, it is quite logical to suggest low prevalence of vitamin D deficiency in tropical countries. However the studies carried out in the preceding two decades have shown a high prevalence of vitamin D deficiency in tropical countries such as China, Turkey, India, Iran and Saudi Arabia \[[@B6]-[@B14]\]. The prevalence of vitamin D deficiency varied between 30% and 93%. However, the majority of these studies were limited to specific age and sex groups. Therefore, elucidation of vitamin D status at the community level and in different climates of a country seems essential. The present study is a part of a national project of prevention, diagnosis and treatment of osteoporosis that investigates the prevalence of vitamin D deficiency and its influencing factors in the population of Tehran. Methods ======= 1272 healthy men and women aged 20--69 years were selected based on randomized clustered sampling from 50 blocks in Tehran. Exclusion criteria were known hepatic or renal disease, metabolic bone disease, malabsorption, sterility, oligomenorrhea, type I diabetes, hypercortisolism, malignancy, immobility for more than one-week, pregnancy, lactation, and medications influencing bone metabolism. The study protocol was approved by research ethics committee of Endocrinology & Metabolism Research Center (EMRC). Sampling was performed after taking informed consent at the beginning of 2001 in the subjects place of residence. 1210 of 1272 selected subjects participated in this study (response rate was 95%). One fasting blood sample was taken from each participant in his/her place of residence. Sample centrifuge and serum extraction were done in the field. Then samples were sent to the EMRC laboratory for analysis and were frozen immediately. 25-hydroxy vitamin D (25(OH) D) level was measured with RIA method (Biosource Europes.A,Ò). Normal range for serum vitamin D (25(OH) D) was 23 to 113 nmol/l. Serum PTH measurement was done using RIA method (Diasorin,Ò). Normal range for PTH is 13 to 54 nmol/l. Interassay and Intrassy for 25(OH) D were 8%, 6.8% and for PTH were 8.9% and 6.1% respectively. The subjects were asked to complete a questionnaire at the time of bone mineral densitometry analysis. The questionnaire included details of duration of exposure to sun light in previous month (less than 30 minutes/day; between 30 to 60 minutes/day; between 60 to 120 minutes/day; more than 120 minutes/day), sunscreen cream usage, clothing (exposure of hand and face or more than). In order to quantify the level of vitamin D and calcium consumption in the previous month, a food frequency questionnaire which was designed and standard by the Iranian Nutrition Institute was completed. Height and weight were measured at this stage. 25(OH)D equal or less than 12.5 nmol/l was considered as severe vitamin D deficiency or group 1 and vitamin D more than 12.5 nmol/l and less than 25 nmol/l was considered as moderate deficiency or group 2 \[[@B15]\]. PTH changes in various vitamin D serum levels were applied to detect mild vitamin D deficiency which has 25 (OH)D more than 25 nmol/l and less than or equal to 35 nmol/l. Threshold for mild vitamin D deficiency was measured by applying PTH changes in different serum levels of 25(OH) D. SPSS software (version 10) was used for data analysis. In descriptional statistics 5, 50, 95 percentiles were used. Results were expressed as mean ± SD or median. To find any significant difference between groups, X^2^test Kruskal-Wallis were used. Results ======= In order to quantify serum levels of vitamin D and other biochemical parameters, serum samples were taken from 1210 subjects (response rate was 95%). 41% of subjects were male and 59% were female. Age and sex distribution of participants are shown in table [1](#T1){ref-type="table"}. In the second part of study (recall for bone mineral densitometry) for 666 subjects the questionnaire was completed. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Age and Sex distribution of participants ::: **Age(year)** **Total number** **Female** **Male** --------------- ------------------ ------------ ---------- **20--29** 241 128 113 **30--39** 308 203 105 **40--49** 294 191 103 **50--59** 209 116 93 **60 \>** 158 77 81 ::: Figure [1](#F1){ref-type="fig"} demonstrates vitamin D levels histogram in the study population. Total prevalence of severe, moderate and mild vitamin D deficiency was 9.5 %, 57.6% and 14.2 % respectively (Figure [2](#F2){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Histogram of Vitamin D serum levels in study population :::  ::: Figure [3](#F3){ref-type="fig"} demonstrates 95, 50 and 5 percentiles of vitamin D according to age and sex. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Median, 5, and 95 percentile of Vitamin D in variable age and sex groups :::  ::: Serum levels of vitamin D in females above 60 years was higher than in other age groups (P \< 0.001: Kruskal-Wallis test). Vitamin D serum levels in females between 20--29 years and 30--39 years was less than other age groups (P \< 0.001). Median vitamin D level in females in age range of 20--29 years and above 60 years was 17 nmol/l and 39 nmol/l, respectively. Prevalence of high level of vitamin D (more than 150 nmol/l) in 60--69 years old female age group was significantly more than other age and sex group (P \< 0.01). In recalling for bone densitometry, 666 returned (55% of study population) in whom the effect of influencing factors was evaluated. Table [2](#T2){ref-type="table"} shows mean BMI and daily calcium intake in different vitamin D groups. BMI was not significantly different in vitamin D groups but calcium intake in normal vitamin D group was significantly higher than other groups. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Mean BMI and daily calcium intake in variable vitamin D groups ::: --------------------------------------------------------------------------------------------------------------------------------------------- **Groups**\ **Vitamin D ≤ 12.5 (nmol/l)** **12.5\<vitamin D ≤ 25** **25\<vitamin D ≤ 35** **35.1\<vitamin D ≤ 150** **Parameters** ----------------------------- ------------------------------- -------------------------- ------------------------ --------------------------- **BMI (kg/m^2^)** 27.32 ± 5.02 26.44 ± 4.52 27.66 ± 5.16 26.99 ± 4.93 **Calcium intake (mg/day)** 503.39 ± 303.1\* 577.93 ± 304.9\* 595.84 ± 313.6\* 714.67 ± 330.8 --------------------------------------------------------------------------------------------------------------------------------------------- \*Significant difference with normal group (35.1\<vitamin D ≤ 150) P \< 0.05 ::: Discussion ========== In our study the prevalence of severe and moderate vitamin D deficiency was 9.5 % and 57.6%, respectively. Mild vitamin D deficiency had a prevalence of 14.2%. Multiple studies have been carried out about the prevalence of vitamin D deficiency but they were mostly limited to a small sample size or assessed a specific age group (especially elderly). In countries where vitamin D fortified foodstuffs are available (USA and some Scandinavian countries), prevalence of vitamin D deficiency is between 1.6--14.8% in different age groups \[[@B16]-[@B18]\]. In other European countries where there is no vitamin D supplementation, deficiency is more prevalent. The studies which assessed middle-aged and elderly people showed vitamin D deficiency prevalence of 14% to 59.6% in these age groups \[[@B19]-[@B22]\]. Vitamin D deficiency prevalence is much higher in Asian countries. Fonseca and colleagues, demonstrated vitamin D level above 10 ng/ml in only 3 saudian females out of 31 \[[@B13]\]. Sedrani and colleagues showed vitamin D deficiency prevalence of 44%--100% in Saudian young females with different coverage and race \[[@B9],[@B10]\]. Azizi & colleagues showed vitamin D level less than 18 ng/ml in half of the study population. Vitamin D deficiency prevalence in 10--19, 20--24, 30--41 was 47.4%, 59.5%, 44.8% respectively \[[@B11]\]. In the present study 81.3 % of subjects had vitamin D deficiency. Most studies have shown higher prevalence of vitamin D deficiency in the elderly \[[@B15]-[@B18]\]. Elderly females demonstrated statistically significant higher serum levels of vitamin D compared with young and middle aged females. Parenteral vitamin D intake by elderly was the major differentiating factor between various age groups that could explain high prevalence of a high level of vitamin D in elderly females. Subjects who took vitamin D in the sampling period were excluded from the study, but those who had taken vitamin D in the preceding months were not omitted. Vitamin D has a long half-life and its frequent prescription especially in elderly women with musculo-skeletal complaints can explain differences in serum vitamin D. Regarding the essential role of sunlight in vitamin D synthesis, it is quite unexpected to see a high prevalence of vitamin D deficiency in countries such as Saudi Arabia. Different hypotheses can be made such as insufficient sun exposure, clothing habits, hyper pigmentation, air pollution, insufficient intake of vitamin D and special dietary habits \[[@B27]\]. Although sunlight plays an essential role in vitamin D synthesis, its\' role in vitamin D deficiency of Asians is not obvious. Tehran, which is located in 36° 21\'\'N, has a mean sun exposure of 8 hours per day \[[@B28]\]. In the present study sun exposure was not significantly different between subjects with vitamin D deficiency and those with normal vitamin D status. Although there is sufficient sunlight in all seasons in Saudi Arabia, Sedrani showed that half of people who had more than 30 minutes of sun exposure had vitamin D less than 8 ng/ml (20 nmol/l) \[[@B10]\]. Holick & colleagues showed similar rate of vitamin D synthesis in Asians as of Europeans; but Asians required greater duration of exposure \[[@B29]\]. Other studies showed the same degree of increase in 25 (OH) D in summer months in Asians compared with Europeans \[[@B30]\]. In our study, there was no difference in clothing habits of vitamin D deficient group and normal group. Sedrani showed 70% vitamin D deficiency in males compared with30 % in young females in spite of greater clothing in females \[[@B10]\]. Another hypothesis says that air pollution prevents enough UV exposure to skin. Insufficient vitamin D intake is another hypothesis for high prevalence of vitamin D deficiency in Asians. Insufficient dietary supplies of vitamin D in countries where foodstuffs are not supplemented, leads to generally low dietary intake of vitamin D. In the Omdahl study, daily vitamin D intake in elderly healthy women was 54 units \[[@B16]\]. Our study does not assess daily dietary vitamin D intake. Decreased dietary calcium level induces increased serum PTH level and increased catabolism of 25 (OH) D, therefore decreased 25(OH)D is induced by dietary calcium deficiency \[[@B15]\]. Average calcium intake was 660 ± 350 mg/day in this study. There was no significant difference in dietary calcium intake among the different vitamin D groups. Although consumption of phytates and animal-derived proteins was not investigated in present study, high dietary consumption of phytates and low dietary intake of animal proteins is one of the suggested hypothesis for vitamin D deficiency \[[@B24],[@B26],[@B27]\]. There are other hypotheses to explain vitamin D deficiency among Asians. Awumey et al showed higher activity level of 24-hydroxylase in fibroblasts of Indian-Americans compared with controls \[[@B31]\]. Therefore, increased vitamin D catabolism may cause vitamin D deficiency in Asians. In order to elucidate specific etiologies responsible for high prevalence of vitamin D deficiency in Asians further studies should be carried out. It is possible that vitamin D deficiency is induced by combination of above mentioned etiologies. In order to clarify the significance of each etiologic factor, randomized controlled trials are necessary. Conclusions =========== Given the high prevalence of vitamin D deficiency in Iran, effective solution to overcome its consequences seems indispensable. Competing interest ================== None declared. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Frequency of variable Vitamin D groups :::  ::: Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/4/38/prepub> Acknowledgments =============== The authors are thankful to Dr. Fatemeh Bandarian and Dr. Seyed Mohammad Akrami for their efforts in preparing this article and to EMRC laboratory staff for their continuous co-operation.

PubMed Central

2024-06-05T03:55:47.748499

2004-8-25

PMC517721

Background ========== Bronchodilators are the primary agents used to manage chronic obstructive pulmonary disease (COPD). They modestly improve forced expiratory volume in one second (FEV1) and reduce dynamic hyperinflation; breathlessness may lessen and exercise tolerance increase despite little improvement in spirometric measurements \[[@B1]\]. The bronchodilators currently available for COPD include β2 agonists (e.g., salbutamol and salmeterol), anticholinergics (e.g., ipratropium bromide) and methylxanthines (e.g., theophylline). According to the Canadian guidelines for the treatment of stable COPD \[[@B2]\] first line treatment consists of ipratropium, two to four doses three to four times daily, plus a short acting β2 agonist administered on an \"as needed\" basis. If the patient uses substantial amounts of short acting β2 agonists, or if the symptoms are greater at night than in the early morning, a long acting β2 agonist (salmeterol or formoterol) is added twice daily. However, recently some have recommended the latter as first line agents for stable COPD \[[@B3]\], \[[@B4]-[@B7]\] potentially replacing the less expensive ipratropium \[[@B4]\], \[[@B5]-[@B8]\]. Several trials have demonstrated the usefulness of salmeterol and formoterol for the management of COPD\[[@B8]\], \[[@B9]-[@B11]\]. According to a 1998 meta-analysis \[[@B12]\], in patients with non-reversible COPD these agents produce small increases in FEV1; however, these changes alone may not correlate highly with symptom relief \[[@B13]\]. The authors of the meta-analysis suggested that these drugs be prescribed only for patients who find they provide definite clinical improvement: reduced breathlessness or better exercise capacity. All three trials in the meta-analysis \[[@B10],[@B14]-[@B16]\]. compared long acting β2 agonists with placebo. Since then, other studies of these agents in COPD, including comparisons with anticholinergics \[[@B8],[@B11]\] have appeared in the literature. Canadian provincial drug plan managers have noted a substantial increase in the use of salmeterol and formoterol in recent years, an observation supported by data from International Medical Services Canada, which collects information on Canadian patterns of drug prescribing and estimates use: between 1997 and 2001, the use of salmeterol and formoterol for COPD increased 1,150% and 1,975%, respectively, whereas the use of ipratropium for COPD decreased by 37% \[[@B17]\]. In light of the new trials and the recent changes in prescribing practices, we undertook a systematic review to evaluate the efficacy and safety of long acting β2 agonists when compared with placebo or anticholinergic agents in patients with stable, poorly reversible, COPD. Methods ======= Searching --------- We obtained published literature and conference abstracts for this document from two separate sources: (1) search results from the CCOHTA\'s published health technology review \"Long-acting β2-agonists for maintenance therapy of stable chronic obstructive pulmonary disease: a systematic review\"; and, (2) search results from CCOHTA\'s ongoing clinical review on long-acting β~2~-agonists for maintenance treatment of stable chronic obstructive pulmonary disease in mixed population. The first search was performed on MEDLINE^®^, EMBASE^®^, HealthSTAR, BIOSIS Previews^®^in June 2001 using a sensitive search strategy. The second search performed in December 2002 had a more focused search strategy and included PASCAL, SciSearch and ToxFile databases in addition to MEDLINE^®^, EMBASE^®^and BIOSIS Previews^®^databases. As designed in the search strategy, this search captured all the studies included in CCOHTA\'s published review as well as some additional trials published since previous search date. Search details for both searches can be found in Appendix 2 \[see Additional file [2](#S2){ref-type="supplementary-material"}\]. Regular alerts have been established on these databases to capture new studies and are ongoing in 2004. Parallel searches were performed and updated in PubMed and the Cochrane Library. In addition, we periodically searched Web sites of clinical trial registries and health technology assessment (HTA) and related agencies. Google™ and other search engines were used to retrieve conference abstracts of major respiratory associations. We also hand searched selected journals and documents in the library of the Canadian Coordinating Office for Health Technology Assessment and the bibliographies of retrieved reports. As well, we contacted the Canadian offices of the manufacturers of salmeterol and formoterol for nonconfidential information on unpublished studies. Selection --------- Two reviewers (D R H and V K S) worked independently on these phases of the study. Disagreements were resolved by discussion and consensus; a neutral third party (M B) was consulted when necessary. The reviewers evaluated the 504 unique citations by reviewing titles and abstracts, discarding those deemed irrelevant (e.g., case reports, review articles, and studies unrelated to the use of β2 agonists for maintenance treatment of stable COPD). They then selected all reports of randomised controlled trials (RCTs) comparing salmeterol or formoterol with placebo or an anticholinergic agent, with or without the additional use of short acting β2 agonists. No restrictions were placed on dosage, but the trials had to be of either parallel or crossover design, have lasted four weeks or longer, and have included patients that met each of the following criteria. • Non-asthmatic. • Stable COPD: no infections, exacerbations, or hospitalizations in the past month. • FEV1 ≤ 75% of predicted. • Ratio of FEV1 to forced vital capacity (FVC) ≤ 88% of predicted. • After a dose of a short or long acting β2 agonist \< 15% improvement in FEV1. Since bronchodilators are much more efficacious in asthma than in COPD, including patients with asthma would have influenced the findings. It may be difficult to determine whether chronic airflow obstruction with relatively large responses to short acting β2 agonists represents COPD with reversibility or asthma with incomplete reversibility. A suggestive feature in the differential diagnosis of COPD is irreversible airflow limitation.\[[@B18]\] To better reflect this and to minimize the chance of including patients with asthma, we excluded those trials in which the average FEV1 response to a bronchodilator was greater than or equal to 15%. In addition, the trials had to have investigated one of the following outcomes. • Lung function, including FEV1 and peak expiratory flow rate (PEFR). • Exercise capacity: six minute or shuttle walking test. • Health related quality of life (QoL). • Dyspnea, including symptom diary scores. • Exacerbations of COPD. • Rescue use of salbutamol, a short acting β2 agonist. • Adverse effects, including tachycardia, hypokalemia, and dry mouth. Validity assessment ------------------- The reviewers independently scored the quality of the included trial reports using a five-point scale described by Jadad \[[@B19]\], which assigns zero to two points each for appropriateness of randomization and double blinding and zero to one point each for reporting on withdrawals and dropouts; low scores are associated with exaggerated estimates of benefit. Concealment of allocation to treatment was also categorized as adequate, inadequate, or unclear. Data abstraction ---------------- The reviewers independently recorded characteristics of the trials and patients, as well as details of the interventions and outcomes. When outcome data were available only graphically, each reviewer estimated values, and the means of the two estimates were reported. Quantitative data synthesis --------------------------- When possible, we calculated mean differences with 95% confidence intervals (CIs) for continuous outcomes and odds ratios (ORs) with 95% CIs for binary outcomes for individual trial data using Statistics with Confidence software \[[@B20]\], We used intention-to-treat data when available and otherwise end point data for patients completing the trials. Qualitative data were recorded descriptively. We had intended to do a meta-analysis, pooling data on outcomes of interest. This approach is useful when the samples of individual studies are too small for detection of an effect and when results from several trials disagree in magnitude and direction of effect \[[@B21]\]. However, it is only appropriate when the trials are clinically homogeneous. We found that even commonly measured outcomes, such as FEV1, could not be combined by meta-analysis because of differences in how they were reported. For example, in the six trials comparing salmeterol with placebo, FEV1 was reported as a mean change in percent predicted\[[@B16]\], a mean change overall\[[@B15]\], a mean difference between trial arms\[[@B10]\], no difference (without data)\[[@B22]\], baseline and overall FEV1 (after 24 hrs without medication)\[[@B8]\] and as an 0 to 12 hour area-under-the-curve (FEV1-AUC) function\[[@B23]\] We were not successful in obtaining more data from study authors. We also had concerns about the meta-analysis of data from trials of parallel and crossover design\[[@B24]\] and differences in spirometry protocols including allowable medications. Therefore, we decided on a best evidence synthesis approach \[[@B25]\] instead. Results ======= Trial Flow ---------- Both reviewers agreed to tentatively accept 35 of the 58 potentially relevant reports. After further evaluation one reviewer disagreed with including 14 of the 35, which resulted in a moderate level of agreement (Kappa = 0.58; 95% CI 0.39 to 0.78). Discussion revealed that this difference related primarily to confusion surrounding interpretation of one of the criteria for eligibility, and ultimately the other reviewer agreed to reject the disputed reports. The reviewers then independently selected the same nine reports \[[@B8],[@B10],[@B14]-[@B16],[@B22],[@B23],[@B26],[@B27]\] for final inclusion. Figure [1](#F1){ref-type="fig"} illustrates the study selection process. The updated search strategy identified 24 additional potentially relevant reports. Of these, four reports were independently selected based on the inclusion criteria. There were no disagreements. Appendix 1 \[see Additional file [1](#S1){ref-type="supplementary-material"}\] presents all of the 69 reports excluded with reasons. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Flow diagram of RCT screening and selection procedure.**Process through which reports were selected from those potentially relevant. RCT = randomised controlled trial; ROAD = reversible obstructive airways disease; FEV1 = forced expiratory volume in one second. :::  ::: Study characteristics --------------------- The 13 reports were of 12 trials, all funded by manufacturers of the drugs. One report \[[@B14]\] describes outcomes in a subset of patients fully described in another report \[[@B10]\]. One reports was a conference abstract \[[@B22]\]; the other twelve reports were journal articles. Duplicate reports were used as a source of supplementary information. Based on the reports, eight of the trials \[[@B8],[@B15],[@B16],[@B23],[@B26]-[@B29]\] were of high quality (score \> 2) and four\[[@B10],[@B22],[@B30],[@B31]\] of low quality (score ≤ 2). Concealment of the allocation sequence was unclear from all of the trial reports. The reviewers agreed completely about quality. Table [1](#T1){ref-type="table"} presents details of the trials and patients. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of included randomised, double blind, controlled trials of long acting β~2~agonists in maintenance therapy for chronic obstructive pulmonary disease (COPD) ::: First author, year of publication, design Trial quality Patients meeting inclusion criteria Interventions Outcomes investigated Notes ------------------------------------------- --------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------- Ulrik, 1995\[16\] Crossover 3 66 current smokers with FEV~1~of 1--2 L (\< 60% of predicted) and FEV~1~/FVC \< 60% of predicted. FEV~1~of \<15% or 300 ml after salbutamol Salmeterol (50 μg twice daily) or placebo for 4+4 weeks; no crossover washout. FEV~1~, PEFR, daytime and night-time symptom scores, rescue use of salbutamol. Two week run in. Methylxanthines, corticosteroids (short oral courses) allowed. Newman, 1996\[22\] (abstract) Crossover 2 42 patients with mean FEV~1~of 0.93 L (35% of predicted) and no response to oral steroids. Salmeterol (100 μg twice daily) or placebo for 8+8 weeks. FEV~1~, FVC, six minute walk test and Borg dyspnoea assessment,\[26\] daytime and night-time symptom scores, rescue use of salbutamol, proportion of days unable to perform normal activity, incidence of adverse events and COPD exacerbations. Two week run in. Salbutamol rescue allowed. Grove, 1996\[15\] Crossover 3 29 patients with FEV~1~25%--75% of predicted and 5%--15% reversibility with 200 μg of salbutamol. Salmeterol (50 μg twice daily) or placebo for 4+4 weeks; one1 week crossover washout. FEV~1~, FVC, TLC, RV, 6 minute walk test and exertion on Borg scale, oxygen uptake. At least one week run in. Inhaled corticosteroids, anticholinergics, oral theophylline allowed. Boyd, 1997\[10\] Parallel 2 674 patients with FEV~1~≤ 70% and FEV~1~/FVC ratio ≤ 60% of predicted and 5%--15% reversibility of FEV~1~with 400 or 800 μg of salbutamol. Salmeterol (50 or 100 μg twice daily) or placebo for 16 weeks. FEV~1~, six minute walk test and Borg dyspnoea assessment, daytime and night-time symptom scores, rescue use of salbutamol. Two week run in. Medications other than β~2~agonists allowed. Jones, 1997\[14\] Parallel 2 283 patients with FEV~1~≤ 70% and FEV~1~/FVC ratio ≤ 60% of predicted; 5%--15% reversibility of FEV~1~with 400 or 800 μg of salbutamol. Salmeterol (50 or 100 μg twice daily) or placebo for 16 weeks. HRQoL with SGRQ27 and SF-36\[28\]. Two week run in. Medications other than β~2~agonists allowed. Mahler, 1999\[8\] Parallel 3 145 patients with FEV~1~≤ 65% and FEV~1~/FVC ratio ≤ 70% of predicted; ≤ 15% reversibility of FEV~1~with short acting β~2~agonist; grade 1 baseline severity of breathlessness. Salmeterol (42 μg twice daily) or ipratropium bromide (36 μg four times daily) or placebo for 12 weeks. FEV~1~AUC, six minute walk test, daytime and night-time symptom scores, dyspnoea on BDI and TDI,\[29\] supplemental use of salbutamol, HRQoL on CRDQ,\[30\] COPD exacerbations. Run in six hours to three days. Prednisone (≤ 10 mg) or equivalent or inhaled corticosteroids allowed. Rennard, 2001\[23\] Parallel 3 179 patients with FEV~1~≤ 65% and FEV~1~/FVC ratio ≤ 70% of predicted; ≤ 12% reversibility of FEV~1~with salbutamol; score ≥ 1 on MMRC five point dyspnoea scale. Salmeterol (42 μg twice daily) or ipratropium (36 μg four times daily) or placebo for 12 weeks. FEV~1~and FVC AUC, dyspnoea on BDI and TDI, six minute walk test and Borg dyspnoea assessment, symptom scores, QoL on CRDQ, COPD exacerbations. Corticosteroids, inhaled and oral (\< 10 mg/d), allowed. Rossi, 2002\[27\] Parallel 3 418 patients with FEV~1~\< 70% and FEV~1~/FVC ratio ≤ 88% of predicted; \< 15% reversibility of FEV~1~with short acting β~2~agonist; grade 1 baseline severity of breathlessness. Formoterol (12 or 24 μg twice daily) or placebo or oral slow release theophylline for 12 months. FEV~1~AUC. Inhaled corticosteroids and rescue use of salbutamol allowed. Stahl, 2002\[26\] Parallel 3 183 patients with FEV~1~\< 60% and FEV~1~/FVC \< 70% of predicted; \< 12% reversibility of FEV~1~after single dose of formoterol. Formoterol (18 μg twice daily) or ipratropium (80 μg three times daily) or placebo for 12 weeks. FEV~1~, FVC, PEFR, shuttle walking test, morning and evening symptom scores, HRQoL on SGRQ. Inhaled corticosteroids at constant doses and rescue use of short acting β~2~agonists allowed. Gupta, 2002\[29\] Parallel 4 33 patients with FEV~1~\< 60 % predicted and FEV~1~/FVC ≤ 70%; reversibility \<12 % improvement of FEV~1~after 400 μg salbutamol Salmeterol (50 μg twice daily) or placebo twice daily for 8 weeks FEV~1~, FVC, six minute walk test, HRQoL on SF-36\[28\], dyspnoea on BDI, patient self-assessment, and rescure inhaler usage Two week run in period. Patients required to take beclomethasone 400 μg twice daily and ipratropium 20 μg four times daily. Mahler, 2002\[30\] Parallel 2 158 patients with FEV~1~\< 65 % predicted and FEV~1~/FVC ≤ 70%; reversibility \<12 % improvement of FEV~1~after 400 μg salbutamol Salmeterol (50 μg twice daily) or placebo twice daily for 24 weeks FEV~1~, morning PEF, dyspnoea on BDI and TDI; rescue salbutamol use; HRQoL on CRDQ \[30\]; symptoms on CBSQ Randomization stratified by reversibility. Calverly, 2003\[28\] Parallel 5 733 patients with FEV~1~25--70% predicted and FEV~1~/FVC ≤ 70%; reversibility \<10 % of predicted FEV~1~after salbutamol Salmeterol (50 μg twice daily) or placebo twice daily for 52 weeks FEV~1~, FVC, relief medication, symptom scores, night-time awakenings, exacerbation rates, HRQoL on SGRQ Two week run in and two week follow up Hanania, 2003\[31\] Parallel 2 163 patients with FEV~1~\< 65% predicted but \> 700 ml (or if ≤ 700 ml \> 40 % predicted) and FEV~1~/FVC \< 65%; reversibility \< 12 % of predicted FEV~1~after salbutamol Salmeterol (50 μg twice daily) or placebo twice daily for 24 weeks FEV~1~, morning PEF, dyspnoea on BDI and TDI; rescue salbutamol use; HRQoL on CRDQ \[30\]; symptoms on CBSQ, exacerbation rates (all severities) Randomization stratified by reversibility AUC = area under the curve; BDI = baseline dyspnoea index;\[29\] CBSQ = chronic bronchitis symptom questionnaire; \[42\] CRDQ = chronic respiratory disease questionnaire;\[30\] FEV1 = forced expiratory volume in one second; FVC = forced vital capacity; HRQoL = health related quality of life; MMRC = Modified Medical Research Council; PEFR = peak expiratory flow rate; RV = residual volume; SF-36 = Medical Outcomes Study Short Form 36;\[28\] SGRQ = St. George\'s Respiratory Questionnaire;\[27\] TDI = transition dyspnoea index;\[29\] TLC = total lung capacity. ::: Data synthesis -------------- ### Comparative efficacy of long acting β2 agonists and anticholinergic agents Three trials \[[@B8],[@B23],[@B26]\] that compared long acting β2 agonists and anticholinergic agents were identified. Two 12 week trials compared salmeterol, ipratropium, and placebo \[[@B8],[@B23]\].; however, only one trial \[[@B8]\] reported data for FEV1 and transition dyspnea index (TDI) scores for the subset of patients that met our inclusion criteria, and the data were presented graphically. No significant differences (p \> 0.05) between the salmeterol and ipratropium groups were observed in the change in FEV1 from baseline, in TDI scores, or in the rescue use of salbutamol \[[@B8]\]. In a 12 week trial \[[@B26]\] formoterol produced significantly greater improvement in morning PEFR from baseline to endpoint than ipratropium (15.3 versus 7.1 l/min, p = 0.040). However, the differences between the active treatment groups were not significant (p \> 0.05) for percent predicted FEV1 (13% versus 7%, p \> 0.05), percent predicted FVC (8% versus 8%, p \> 0.05), improvement in breathlessness score (-0.21 versus -0.29, p \> 0.05), or improvement in the St. George\'s Respiratory Questionnaire (SGRQ) total score (0.0 versus -0.5, p \> 0.05). Data on adverse effects of interest, including tachycardia, hypokalemia, and dry mouth, were not available from the reports. ### Comparative efficacy of long acting β2 agonists and placebo Ten trials \[[@B8],[@B10],[@B14]-[@B16],[@B22],[@B23]\], \[[@B28]-[@B31]\] had salmeterol and placebo treatment arms; the other two \[[@B26],[@B27]\]. had formoterol and placebo arms. Table [2](#T2){ref-type="table"} and the following text summarize outcome data only for the patients that met our inclusion criteria. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Selected results ::: ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **First author** **FEV**~1~ **Symptom scores (lower is better)** -------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ***Salmeterol versus placebo*** Ulrik\[16\] No significant differences in reversibility of percent predicted FEV~1~with treatment. Mean (SE): 2.7% (0.4) versus 3.4% (0.4). Significant differences in median (range) symptom scores during treatment.\ Daytime (scale 0--5): 1.0 (0--3.4) versus 1.8 (0.1--4.0).\ Night-time (scale 0--4): 0.9 (0--3.4) versus 1.6 (0.1--4.0). Newman\[22\] No significant differences in measurements with treatment (data not reported). Symptoms significantly reduced during salmeterol compared with placebo treatment.\ Scale and scores not reported. Grove\[15\] Significant differences one and six hours after single dose and six hours after four weeks of treatment. Mean change: 120 versus 10 ml after four weeks. Boyd\[10\] Significant differences in improvement with treatment. Mean difference (95% CI): for salmeterol 50 μg versus placebo 97.80 (55.6 to 139.99) ml; for salmeterol 100 μg versus placebo 117.60 (67.88 to 167.32) ml. Significant difference in distribution of median daytime and night-time symptom scores between active treatment and placebo groups (CI 0.0 to 0.0 in all cases) but not between active treatment groups.\ Daytime (scale 0--5): baseline, 2 in each group; from week 5, 1 in active treatment groups and 2 in placebo group.\ Night-time (scale 0--4): baseline, 1 in placebo and salmeterol 50 μg groups and 0 in salmeterol 100 μg group; from week 1, 0 in salmeterol 50 μg group and no change in other groups. Jones\[14\] (Presented QoL results for subset of patients described in Boyd\[10\].) Gupta\[29\] A mean increase in predose FEV~1~of 170 ml (distibution not reported) for salmeterol vs. a mean decrease of 20 ml (distribution not reported) for placebo after 8 weeks. Both salmeterol and placebo produced significant improvemnts in BDI scores, however the magnitude of increase was greater vs. placebo (3 vs. 1); 100% patients treated with salmeterol reported decreased cough and dyspnea vs. 69% (11/16) of placebo recipients Mahler 2002 \[30\] A mean increase of 80 ml (95%CI 35 to 125) for salmeterol vs. mean decrease of -8 ml (95%CI: -53 to 37) for placebo. Two-hour post-dose FEV~1~mean increase of 175 ml (95%CI: 116 to 234) vs. mean increase of 28 ml (95%CI: -17 to 73) Mean increase of 0.5 (SE 0.4) in TDI for salmeterol recipients and 0.4 (SE 0.3) for placebo recipients. Not clinically or statistically significant. Calverly \[28\] A mean increase in predose FEV~1~of 25 ml vs. a mean decrease of -38 ml (P \< 0.05) in salmeterol and placebo recipients. Smaller difference for two-hour post-dose FEV~1~(data not reported). Mean scores for cough (scale 0--3); breathlessness (scale 0 to 4); sputum production (scale 0 to 3); sputum colour (scale 0 to 4): salmeterol: cough 1.36 (SE0.03); breathlessness 1.59 (0.03); sputum production 1.30 (0.03) and colour 1.35 (0.03) vs. placebo: cough 1.44 (0.03); breathlessness 1.66 (0.03), sputum production 1.34 (0.03) and colour 1.36 (0.03). Hanania\[31\] A mean increase of 26 ml (95%CI: -27 to 79) for salmeterol vs. mean increase of 19 ml (95%CI: -26 to 64) for placebo. Two-hour post-dose FEV~1~mean increase of 119 ml (95%CI: 70 to 168) vs. mean increase of 71 ml (95%CI: 24 to 118) The magnitude of TDI responses was less in non-reversible vs. reversible patients. (Data are not reported) **Salmeterol versus ipratropium versus placebo** Mahler\[8\] Significant differences between active treatment and placebo groups but not between active treatment groups. Peak improvements with treatment: 155, 165, and 24 ml, respectively. No significant differences in change of mean daytime symptom score with treatment. No significant differences in TDI except between ipratropium and placebo groups at week 8.\ After 12 weeks, mean TDI 0.35, 0.98, and 0.48, respectively. Rennard\[23\] Significant differences between active treatment and placebo groups but not between active treatment groups. FEV~1~AUC 0--12 hour responses significantly greater with salmeterol and ipratropium than with placebo (data not reported). **Formoterol versus placebo** Rossi\[27\] Significant differences in estimated difference in FEV~1~AUC 0--12 hour responses: between formoterol 12 μg and placebo groups, 145 ml; between formoterol 24 μg and placebo groups, 141 ml. (Individual values for treatment groups not available.) **Formoterol versus ipratropium versus placebo** Stahl\[26\] Significant differences in improvement in percent predicted FEV~1~between active treatment and placebo groups but not between active treatment groups: 13%, 7%, and 6%, respectively. Significant differences between active treatment and placebo groups in change from baseline in breathlessness (scored 0 to 4 morning and evening). Means: -0.21, -0.29, and 0.0, respectively. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- CI = confidence interval; SE = standard error. ::: #### Lung function ##### FEV1 As table [2](#T2){ref-type="table"} shows, the changes in FEV1 from baseline to endpoint differed significantly (p \< 0.05) between the salmeterol and placebo groups in eight of ten trials and between the formoterol and placebo groups in two trials. ##### FVC Five trials\[[@B15],[@B23],[@B26],[@B28],[@B31]\] reported on this outcome. In one 4+4 week (4 weeks then crossover then 4 additional weeks) trial \[[@B15]\] the increase in FVC was significantly greater with salmeterol than with placebo six hours after a single dose (200 versus 30 ml, 95%CI for difference: 40 to 290) but not after four weeks of treatment (150 versus 130 ml, 95%CI for difference: -180 to 220). In one 12 week study \[[@B23]\] the change in FVC was significantly greater (p \< 0.001) for salmeterol (and ipratropium) than for placebo on day 1, there was no loss of response during treatment, and after four weeks the morning predose values were significantly greater in the patients treated with either active drug (data not reported). In the other 12 week trial \[[@B26]\] the percent predicted FVC was significantly increased by the end of formoterol treatment, compared with placebo treatment, by 8% versus -0.4% (p = 0.02). In one 52 week trial).)\[[@B28]\], the change in mean FVC measured 12 hours after treatment was 86 ml greater (p = 0.004) in salmeterol recipients. The difference in mean change in FVC at 52 weeks was 200 ml between groups. In an 8 week trial\[[@B29]\], the mean increase in FVC was 280 ml in the salmeterol group compared to a fall of 8 ml in the placebo group (p \< 0.05). ##### PEFR Three trials\[[@B16],[@B26],[@B28]\].).) reported on this outcome. In one four week trial \[[@B16]\] salmeterol treatment compared with placebo treatment produced a mean treatment difference in morning values of 12 l/min (238 compared with 226 l/min, 95%CI for difference: 6 to 17; p \< 0.001); a statistical difference for evening values was not detected \[242 (95%CI: 222 TO 262) and 237 (95% CI: 217 to 257) l/min for salmeterol and placebo, p \> 0.1\]. The diurnal variation was significantly lower during salmeterol treatment, at 3 (95%CI: -0.9 to 6.9) versus 11 (95%CI: 7.1 to 14.9) l/min; however, the mean treatment difference was only 7 (95%CI: 3 to 11) l/min. In the other trial, lasting 12 weeks \[[@B26]\], the change in morning PEFR was significantly greater by the end of formoterol (or ipratropium) treatment compared with placebo treatment: 15.3 versus -0.9 l/min (p \< 0.001). In one 52 week trial).)\[[@B28]\], the change in mean PEF values differed significantly (p \< 0.0001) for salmeterol treatment, at 257 l/min (95%CI: 253 to 261) versus placebo, at 242 l/min (95%CI: 238 to 246). #### Exercise capacity Results (but not always data) for six minute walk tests were reported from six trials \[[@B8],[@B10],[@B15],[@B22],[@B26],[@B29]\]. None of the trials found statistically significant differences between salmeterol and placebo therapy, although one 12 week trial\[[@B8]\] found that at week 10 the patients receiving ipratropium walked a mean of 14 (95%CI: 0.3 to 27.7) yards farther in six minutes than those receiving placebo; there were no differences in prewalk or postwalk breathlessness between the treatment groups. The only other trial reporting data \[[@B15]\] found a median (interquartile range) distance in six minutes of 450 (371--491) m for placebo recipients and 425 (392--473) m for salmeterol recipients; the difference was not reported to be significant, but the patients receiving salmeterol (50 μg twice daily) perceived significantly less exertion by the end of treatment, as measured on the Borg scale \[median (interquartile range) 0.5 (0--1) for salmeterol versus 1 (0--2) for placebo, p = 0.004\] \[[@B32]\]. A 16 week trial \[[@B10]\] found a significant (p \< 0.05) reduction in postwalk breathlessness (three or more points on the 10 point Borg scale) after eight and 16 weeks of 50 μg but not 100 μg of salmeterol twice daily, compared with placebo (OR 0.62 \[95% CI 0.42 to 0.91\]). Similarly, an 8+8 (8 weeks then crossover then 8 additional weeks) week trial \[[@B22]\] did not detect a significant (p \> 0.05) difference in postwalk breathlessness between patients receiving 100 μg of salmeterol and those receiving placebo. One study comparing formoterol, ipratropium, and placebo \[[@B26]\] reported mean changes in walking distance from baseline to endpoint, measured with the shuttle walking test, of 19.2, 17.5 and 5.1 m, respectively; the differences were not significant (p \> 0.05). #### Dyspnea In several trials \[[@B10],[@B16],[@B22],[@B26],[@B28]-[@B31]\] the patients assessed symptom severity every day, generally using ordinal scales. One 12 week trial comparing salmeterol, ipratropium, and placebo \[[@B8]\] measured the severity of dyspnea at baseline with a multidimensional baseline dyspnea index (BDI) and changes in severity every two weeks with a TDI \[[@B33]\]. As table [2](#T2){ref-type="table"} shows, some differences during treatment with an active drug as compared with placebo were significant and others were not. #### Rescue use of a short acting β2 agonist In five of six trials salmeterol treatment was associated with less salbutamol use than was placebo treatment \[[@B10],[@B16],[@B22],[@B28],[@B29]\] In one trial (4+4 weeks) \[[@B16]\] the median numbers (range) of daytime rescue doses were 1.7 (0--6.1) and 2.6 (0--7.9), respectively, and the median numbers of night-time doses 0 (0--4.2) and 0.3 (0--5.0). In a 52 week trial.\[[@B28]\], the median number of rescue inhalations per day was 2 for both salmeterol and placebo recipients, but these groups were statistically different (p = 0.028). Another trial\[[@B29]\] reported the mean number of doses of rescue salbutamol was significantly lower during treatment in salmeterol recipients (0.59, 95%CI: 0.30 to 0.88) versus placebo recipients (1.75, 95%CI: 1.33 to 2.17). In one 12 week trial comparing salmeterol, ipratropium, and placebo \[[@B8]\] no significant difference was observed in additional bronchodilator use between the placebo and active drug groups. #### Quality of life HRQoL was evaluated in four trials\[[@B14],[@B26],[@B28],[@B29]\] In a salmeterol study \[[@B14]\] a subset of a larger patient group was asked to complete the disease-specific SGRQ \[[@B34]\] and the Medical Outcomes Study Short Form 36 (SF-36) \[[@B35]\] at baseline and after 16 weeks of treatment. The SGRQ has three components: distress due to respiratory symptoms, effects of disturbances on mobility and physical activity, and psychosocial impact of the disease; negative changes represent improvement. Data from 283 patients (95 in the placebo group and 94 in each salmeterol group) were analysed; data for others were excluded because of noncompletion of one or both questionnaires at 16 weeks or inability to meet quality control criteria or both. Salmeterol 50 μg (but not 100 μg) twice daily was associated with significantly greater improvement in mean (standard deviation) SGRQ scores from baseline to endpoint than was placebo: -6.8 (13.2) versus -1.4 (11.7) for total score and -8.0 (17.6) versus 0.0 (15.7) for impact score. No significant differences between placebo and either dose of salmeterol were observed in any of the domains of the SF-36 except for \"role-emotional\": these scores were significantly worse for recipients of salmeterol 100 μg than for recipients of placebo. In the 52 week study).)\[[@B28]\], health status was assessed with the SGRQ. The adjusted mean score was not statistically different in salmeterol recipients, at 45.2 (95%CI: 44.4 to 46.0) versus placebo recipients, at 46.3 (95%CI: 45.3 to 47.2). In an 8 week study\[[@B29]\] the magnitude of improvement for salmeterol versus placebo recipients rated on an SF-36 scale was significantly greater for the dimensions of \"general health\" (p = 0.008), \"health change\" (p = 0.026); physical functioning\" (p = 0.008) and \"vitality energy and fatigue\" (p = 0.008) In the trial comparing formoterol, ipratropium, and placebo \[[@B26]\] HRQoL was also evaluated with the disease specific SGRQ. Of the 183 patients, 144 completed the assessment; reasons for not doing so were not reported. The changes from baseline to endpoint in total score were negligible in all three groups, at 0.0, -0.5, and 1.5, respectively. #### COPD exacerbations Three trials\[[@B10],[@B22],[@B28]\] reported on this outcome; only one trial).)\[[@B28]\] defined \"COPD exacerbation\" as episodes that required antibiotics or corticosteroids but not hospital admission; these occurred at a mean rate of 0.54 exacerbations/patient/year in salmeterol recipients and 0.76 exacerbations/patient/year in placebo recipients (p = 0.0003). In one 16 week trial \[[@B10]\] the numbers (and proportions) of patients having exacerbations among those receiving salmeterol 50 or 100 μg twice daily or placebo were 75 (33%), 91 (42%), and 98 (43%), respectively. In an 8+8 week trial \[[@B22]\] there were fewer exacerbations during treatment with salmeterol 100 μg twice daily than during treatment with placebo (p = 0.065); data were not presented. #### Adverse Effects Data on adverse effects of interest were not available from the reports. Discussion ========== We identified thirteen reports of twelve randomized controlled trials describing the effect of administering the long acting β2-agonists, salmeterol and formoterol, to patients with poorly reversible COPD. It is not clear from the reports whether the twelve selected trials had sufficient power to detect significant differences between treatment and control groups in the various subjective and objective outcome measures. Since data were not pooled for meta-analysis, we were not able to conduct a sensitivity analysis based on the quality of trial reporting. Accordingly, we cannot comment on the possible influence of quality on the effect size of the outcome measures. Clinical heterogeneity among the trials limited assessment of the overall effect of the interventions. Since we did not perform a meta-analysis, statistical heterogeneity was not an issue. We selected reports that met our inclusion criteria, regardless of publication status, language and trial quality using a systematic research methodology; this approach has been shown to minimize potential selection and publication bias and lead to more reliable conclusions\[[@B36]\] We made every effort to conduct our review and report its results with the highest rigour. A potential limitation of our research is that we did not seek trials comparing long acting β2-agonists marketed outside of Canada (e.g., bambuterol) or those trials comparing long acting β2-agonists to agents other than ipratropium and placebo (e.g., short-acting β2-agonists, methylxanthines). Similarly, we excluded those trials in which the FEV1 response to a bronchodilator was not reported or greater than 15%. Thus, our results may not be generalisable to the greater population of patients who can be currently defined as having COPD\[[@B18]\]. We plan to include a greater number of comparators and a broader population in an upcoming analysis\[[@B37]\] Our results are similar to those of an earlier review \[[@B12]\] that identified three placebo controlled trials included in our review, but there are two important differences. In the earlier review FEV1 endpoint data from the placebo and salmeterol groups in two crossover trials \[[@B15],[@B16]\] were pooled; the weighted mean differences were not significant. We preferred to analyse net improvement in FEV1 (the difference from baseline to endpoint), as we felt that it more accurately reflected the impact of maintenance therapy. In addition, no trials comparing long acting β2 agonists and ipratropium were available at the time of the earlier review. Another review has recently been published.\[[@B38]\] However, these authors restricted their search to MEDLINE and failed to identify a clinical trial comparing formoterol with ipratropium.\[[@B26]\] As a consequence, the evidence describing the use of formoterol versus ipratropium is limited to a single trial.\[[@B39]\] In contrast, we opted to exclude this trial after identifying two trials because roughly 40% of patients exhibited partial reversibility of FEV1 (15% to 80%) to an inhaled dose of 200 mcg salbutamol at baseline. We are in agreement with the authors\' summary of the evidence surrounding salmeterol versus ipratropium. We believe our findings are in accord with current guidelines, such as the GOLD guidelines, that suggest bronchodilators should be prescribed according to individual patient responses. However, policymakers with limited health service resources need to be aware of an identifiable sub-population of patients with poorly reversible COPD for which long acting β2-agonists may result in reduced efficiency (cost-effectiveness). Our research also suggests clinical investigators of COPD trials should stratify trial participants into groups for which outcomes may consistently differ. Of the trials identified, four\[[@B23],[@B30],[@B31],[@B40]\] used this approach. Outcome information from patients with poor reversibility was also analyzed in an abstract\[[@B41]\] of an excluded trial but not in the published report.\[[@B39]\] We were unable to ascertain sufficient details surrounding this analysis to add it to our findings. Conclusions =========== In terms of clinical outcomes and safety, we could not find convincing evidence that salmeterol and formoterol have demonstrated advantages to ipratropium, a less expensive drug, for patients with stable COPD and poor reversibility. Compared to placebo, we found evidence of reduced rescue inhaler usage and improved spirometric outcomes without a significant impact on quality of life or exercise capacity. Competing interests =================== Donald Husereau, Vijay Shukla, Michel Boucher, and Shaila Mensinkai have no competing interests to declare. CCOHTA is an independent, nonprofit health research agency funded by the federal, provincial, and territorial governments of Canada. Robert Dales sits on the advisory committees for GlaxoSmithKline (makers of the long acting β2 agonist, salmeterol) and Boeheringer Ingelheim (makers of the anticholinergic agent, ipratropium bromide). Authors\' contributions ======================= DH edited and prepared the final manuscript for publication. VS led development of the research protocol, supervised the literature review, and summarized results. DH and VS were responsible for reviewing articles, judging their relevance, assessing their quality, and extracting data. MB assisted in developing the research protocol and in conflict resolution during study selection. RD assisted in developing the research protocol and provided clinical expertise. SM designed and conducted the electronic searches and provided expertise in the area of information science. All authors either wrote sections or critically reviewed drafts of this article. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2466/4/7/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Appendix 1. A list of reports considered in this review but excluded. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Appendix 2. Search strategies including databases, time horizons and subject headings/keywords used to locate trials. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank Dorothy Rhodes, IMS, for providing drug utilization data and Ann Bolster for assistance in editing the manuscript.

PubMed Central

2024-06-05T03:55:47.750021

2004-8-31

PMC517722

Background ========== The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune skin diseases\[[@B1]\]. Reliable diagnosis by DIF not only requires an experienced observer, but first of all proper skin (or mucosal) biopsies with well-preserved immunoreactants. For the latter purpose biopsies are usually snap-frozen in liquid nitrogen or, alternatively, placed in Michel\'s fixative that facilitates transport of biopsies from outside hospitals \[[@B2]-[@B6]\]. But even representative and optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate desired specific staining (DSS) of the epidermal basement membrane zone (BMZ) is involved \[[@B7]\]. In such cases, specific IgG fluorescence is easily masked by the relatively high dermal \"background\" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates. The background fluorescence, consisting of both undesired specific staining (USS) and non-specific staining (NSS), largely determines the signal-to noise ratio \[[@B7]\]. This ratio in turn determines the detection threshold and thereby the diagnostic sensitivity of the DIF technique. A low ratio for IgG resulting from weak DSS and high USS plus NSS will undoubtedly yield false negative cases. So far, the signal- to noise ratio in diagnostic IF has received little attention. The present study was initiated by the unexpected finding of significant increase of the signal-to noise ratio in a skin biopsy submitted for DIF and accidentally kept overnight in normal saline. The biopsy, obtained from a patient suspect of pemphigoid, showed substantial reduction of IgG background fluorescence and relatively bright specific IgG fluorescence along the BMZ. This finding encouraged us to compare diagnostic results of DIF in matched skin biopsies using standard snap-freezing, Michel\'s fixative and normal saline. Methods ======= Patients -------- The 25 patients included in this study were selected on the basis of previously confirmed positive direct immunofluorescence (IF) in skin biopsies transported in liquid nitrogen. The final diagnosis was reached by clinical, routine laboratory, histological and direct IF findings. In case of bullous autoimmune diseases, serum samples were characterized by indirect IF on 1.0 M NaCl-split skin \[[@B8],[@B9]\], immunoblotting \[[@B10]\], and ELISA (desmoglein 1 and 3) \[[@B11]\]. The patients had one of the following diagnoses: bullous pemphigoid (BP; n = 5); mucous membrane pemphigoid with skin involvement (MMP; 1); linear IgA dermatosis (LAD; 1), anti-epiligrin cicatricial pemphigoid (AECP; 1); epidermolysis bullosa acquisita (EBA; 1); dermatitis herpetiformis (DH; 1); pemphigus vulgaris (PV; 3); pemphigus foliaceus (PF; 3); subacute and systemic lupus erythematosus (LE; 5); and small vessel IgA vasculitis (4). Skin biopsies and processing ---------------------------- From each patient four skin specimens were obtained by punch biopsy (4 mm) using lidocaine as the local anaesthetic. The biopsies were taken from perilesional skin within an area of 2 cm^2^to minimize the risk of local variation of immunoreactants. The matched skin specimens from each patient were immediately placed in one of the following transport media: (a) liquid nitrogen, (b) Michel\'s fixative 48 hours with appropiate pH, (c) saline 24 hours and (d) saline 48 hours. We used 5 ml screw-capped polypropylene vials for transporting biopsies in Michel\'s fixative and saline. Freezing -------- Biopsies were placed in aluminum vials, snap-frozen in liquid nitrogen and stored at -80°C until further processing within two weeks for DIF (see below). Fixative -------- Michel\'s fixative and buffer solution were prepared monthly according to the original description.^1^Biopsy specimens were kept in 5 ml Michel\'s fixative for 48 hours (Mi48) at room temperature, followed by washing for 30 minutes in Michel\'s buffer solution. The specimens were then blotted on filter paper to remove excess moisture, and stored at -80°C until further processing. Saline ------ We used normal saline solution (0.9% NaCl in aqua dest.) without addition of calcium or magnesium. Skin specimens were kept in 5 ml saline solution for 24 hours (S24) and 48 hours (S48) at room temperature. Preliminary experiments with saline time of 6 hours did not result in improvement of the signal-to noise ratio. The specimens were then blotted on filter paper and stored at -80°C until further processing for DIF. Direct immunofluorescence microscopy ------------------------------------ For comparative purposes, matched skin specimens of each patient were processed for direct IF microscopy at the same occasion. Cryosections of 4 μm thickness were mounted on polysine™ glass slides, air-dried for 30 min before a fan, and encircled with a hydrophobic emulsion (PAP-pen; DAKO; Glostrup). The sections were then stained for 30 min in a moist chamber at room temperature, using fluorescein (FITC)-labeled, Fc-specific goat F(ab\')~2~antibodies against human IgG, IgA and IgM (Protos Immunoresearch, Burlingame CA), and rabbit antibodies against human C3c and fibrinogen/fibrin (DAKO; Glostrup). Proper conjugate dilutions were made in phosphate-buffered saline (0.01 M PBS, pH 7.3) supplemented with bovine serum albumin 1%. After washing in 1000 ml PBS for 30 min, the sections were coverslipped under fresh PBS/glycerol (50% v/v). The slide preparations were kept at 4°C until microscopic examination within two days. The sections were examined with a Leica DMRA microscope (Leica, Wetzlar, Germany) for selective incident light fluorescence using a xenon arc (XBO 75W) as light source and PL Apo ×40/0.80 dry objective. The fluorescent staining was graded as follows: - (negative), ± (doubtful), + (weak), ++ (moderate), +++ (bright). The desired specific staining (DSS) of the following target structures was scored^12;13^.: (a) epidermal in vivo ANA, (b) epidermal intercellular spaces, (c) basement membrane zone (dermo-epidermal junction), and (d) blood vessel walls. In addition, the background staining (USS plus NSS) of the upper dermis was scored. All sections were read blindly by the same experienced observer (MCJMdJ). The diagnosis made by direct IF was regarded as conclusive if the DSS score of the relevant target structure was at least weak but definite (Table). The diagnosis was regarded as tentative in case of weak staining that was not consistently distributed at the relevant target structure. A case was regarded as non-diagnostic if target structures showed negative to doubtful DSS scores. Statistical analysis of IgG and IgA background fluorescence in matched biopsies was done by the McNemar test. Results ======= A total of 95 biopsies from 25 patients were examined; three biopsies were missing, and two biopsies proved unsuitable at cryosectioning. Cutting of 4 μm cryosections was easiest with biopsies transported in saline and hardest with biopsies transported in Michel\'s fixative. Biopsies kept in saline for 24 and 48 hours showed statistically significant reduction of background fluorescence in the dermis, especially with IgG, as compared with fresh-frozen and fixed biopsies (p \< 0.01). After 24 hours, this resulted in enhanced signal-to noise ratios and accordingly more easy detection of immunoreactants at target structures, in particular the epidermal basement membrane zone (BMZ) and subepidermal blood vessel walls (Fig. [1](#F1){ref-type="fig"}). In comparison, fresh-frozen and fixed biopsies showed a relatively poor signal- to noise ratio for IgG (and IgA). In these biopsies, weak (+) specific fluorescence of IgG at the BMZ or IgA in vessel walls was found to be masked easily by relatively high background staining. Biopsies kept for 48 hours in saline showed a variable degree of diminution of specific staining, and tended to become negative in case of weak (+) specific fluorescence. The IgG fluorescence of epidermal in vivo ANA, present in fresh-frozen and fixed biopsies of two cases with (S)LE, became negative in saline biopsies (both 24 h and 48 h). In general, biopsies with moderate to bright (++/+++) specific fluorescence remained positive in all transport media. The reduced background fluorescence in saline biopsies occasionally revealed doubtful to weak focal IgG fluorescence of the BMZ that was regarded as non-relevant. Furthermore, we observed dermo-epidermal split formation in saline biopsies, not present in matched fresh-frozen and fixed biopsies. The extent of split formation varied among saline biopsies and increased with time (48 h \> 24 h). In cases with pemphigoid, IgG was found predominantly at the epidermal side of splits, in contrast with e.g. SLE where IgG was found at the dermal side (Fig. [2](#F2){ref-type="fig"}). In case an artificial split is induced because of saline incubation a BMZ signal stand more out because of the dark background of the blister cavity (Fig. [2](#F2){ref-type="fig"}). The overall morphology in saline biopsies was quite fair and largely sufficient for the purpose of diagnostic IF microscopy. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Comparison of direct immunofluorescence in cryosections of matched skin biopsies transported in liquid nitrogen, Michel\'s fixative or saline. Note the substantially reduced background fluorescence in saline-transported biopsies. Pemphigus foliaceus showing characteristic IgG fluorescence at the epidermal intercellular space. Additional granular IgG staining at the basement membrane zone (arrow) stands out most clearly in saline transported biopsy. (obj. ×20) Mucous membrane pemphigoid with skin involvement showing weak linear IgG fluorescence at the basement membrane zone that is only visible in the saline transported biopsy (arrow). (obj. ×20) Lupus erythematosus showing granular IgG fluorescence at the dermo-epidermal junction. Additional IgG staining of subepidermal vessel walls is best visible in the saline-transported biopsy. (obj. ×20) Vasculitis showing fine-granular IgA fluorescence in subepidermal capillary walls (arrows) which is most distinct in the saline-transported biopsy. (obj. ×40) :::  ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Direct immunofluorescence (IgG, combined with transmitted light) in saline transported skin specimen of lupus erythematosus. After 48 hours in saline there is subepidermal split formation, not present in fresh-frozen (N~2~) and fixed (Mi48) skin. Note the still obvious granular IgG fluorescence at the dermal side of the split. (obj. ×40) :::  ::: The diagnostic results of direct IF in matched biopsies are summarized in Table [I](#T1){ref-type="table"}. By interpreting these data it should be realized that the minority of cases (20%) showed bright (+++) specific fluorescence in standard frozen biopsies, whereas the majority (52%) showed only weak to moderate (+/++) specific fluorescence of the relevant target structure(s). Two originally positive cases, one pemphigus and one IgA vasculitis became doubtful or negative (non-diagnostic) in all transport media. The highest rate of conclusive cases by direct IF was obtained in biopsies kept in saline for 24 hours (S24, 84%), and the lowest in fixed biopsies (Mi48, 50%). The case of mucous membrane pemphigoid with weak IgG (+) and IgA (+) fluorescence at the BMZ was obvious in saline biopsies (S24, S48) but non-diagnostic in matched fresh-frozen and fixed biopsies. The highest non-diagnostic percentage was obtained in fixed biopsies (32%) and the lowest in S24 saline biopsies (8%). In one case, classified as misdiagnosed, the fresh-frozen biopsy led to the diagnosis of LAD as IgA (+) and C3c (+) were the only immunoreactants observed, whereas the matched fixed and saline biopsies showed additional linear IgG (+/++) fluorescence at the BMZ (suggestive of mixed IgG/IgA pemphigoid). After correction for the missing biopsies, the results were only statistically significant comparing Mi and S24 (p \< 0.05). Other comparisons (N2 versus Mi, S24 and S48 or Mi versus S48 or S24 versus S48) were not significant. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Results of direct immunofluorescence (DIF) of matched skin biopsies transported in different media. ::: **N2** **Mi48** **S24** **S48** -------------------- --------- ---------- ---------- ---------- ---------- a\. conclusive +/+++ 14 (56%) 11 (50%) 21 (84%) 16 (70%) b\. tentative **±/+** 5 (20%) 4 (18%) 2 (8%) 3 (13%) c\. non-diagnostic **-/±** 5 (20%) 7 (32%) 2 (8%) 4 (17%) d\. mis-diagnosed 1 (4%) \- \- \- N~2~, snap-frozen specimens in liquid nitrogen; Mi48, specimens in Michel\'s fixative for 48 hours; S24 and S48, specimens in saline for 24 and 48 hours respectively; DSS, desired specific staining; n, number of cases ^§^Two S48 and three Mi48 matched biopsies were either lacking or considered unsuitable. ::: Discussion ========== Perilesional skin biopsies kept in saline for 24 hours yielded a higher diagnostic rate in direct IF than fresh-frozen biopsies in liquid nitrogen or biopsies kept in Michel\'s fixative. Several authors have described the positive effect of saline in increasing the sensitivity of IF analysis. Judd and Lever showed that skin biopsies stored for 24 hours in 0.15 M phosphate buffered saline prior to freezing gave a very high incidence of positive readings in direct IF \[[@B14]\]. Similarly, the use of 1.0 M NaCl split skin as a diagnostic tool for direct and indirect IF has been reported to increase the sensitivity of these methods \[[@B9],[@B15]-[@B17]\]. It has been suggested that the increase of IF sensitivity by saline incubation is due to improved exposure of epitopes and/or by a decrease of background staining \[[@B16]\]. Our data suggest that the improved DIF sensitivity in saline biopsies is primarily due to decreased background staining resulting in better image contrast (signal-to noise ratio). We know that our visual perception is sensitive for light contrast and not for intensity \[[@B18]\]. The staining intensity perceived of a given specific fluorescent signal is strongly correlated with the intensity of the background (compare looking at the stars in daylight and at night; the intensity is the same, but seems multiplied at night). In this respect, IgG (and IgA) fluorescence in skin tissue is hampered by relatively strong background fluorescence in the dermis that may mask weak specific fluorescence at the BMZ and in vascular walls. Saline appears to reduce this background staining, resulting in relative increase of desired specific staining and thereby enhanced diagnostic sensitivity. A disadvantage of saline is the limited time of transport (24 h) for consistently reliable results. If biopsies are kept longer than 24 hours in saline, decreased fluorescence of tissue-bound immunoreactants may be encountered, although we have observed bright specific fluorescence of the BMZ in biopsies kept in saline for at least 5 days. Michel\'s fixative seems to have a similar limitation: Skeete and Black found that biopsies stored in this fixative should be received within 1 day of biopsy for consistently reliable results \[[@B4]\]. The second disadvantage of saline is the loss of at least some epidermal *in vivo*ANAs, possibly due to extraction or degradation of nuclear antigens. Another disadvantage of saline might be, from a histopathological point of view, morphological disturbances such as hydropic degeneration \[[@B19]\], and splitting at the dermo-epidermal junction, not found with Michel\'s fixative \[[@B6]\]. That is why saline is not suitable for antigen mapping in the diagnosis of genetic diseases \[[@B20]\]. Neither are biopsies kept in saline suited for immuno-electron microscopy \[[@B5],[@B6],[@B20]\]. However, for the diagnosis of autoimmune and immune complex-mediated diseases by DIF, it is not optimal morphology that counts, but the low detection threshold of immunoreactants. In this regard, (artificial) dermo-epidermal split-formation may add value to the method, rather than being a problem, by darkening of the juxtaposed background and mapping of the linear epidermal BMZ deposition \[[@B21]\]. Besides the diagnostic benefit, the preference would also go to the use of saline because it is a ready available, inexpensive, and convenient transport medium, and it certainly improves cutting properties of skin biopsies compared to biopsies fixed in Michels\' medium. Saline can be used as standard medium at room temperature for express postal delivery of IF biopsies to the laboratory, if delivery is guaranteed within 24 hours. Transport of biopsies by express postal delivery or in-house airtube post is much more cost-effective than courier delivery necessary for biopsies transported in liquid nitrogen. If it is expected to tide over a longer period we advise to store the biopsy in saline for 24 hours and then to place it in Michel\'s fixative for further transport. Practically, place the biopsy in a screw-capped 5 ml polypropylene tube filled to the top with saline. The saline does not have to be sterile. If the specimen arrives in the laboratory the same day after biopsy, we advise to keep it overnight in saline at room temperature, followed by snap-freezing and (optional) storage at -80°C the next day. Conclusions =========== The use of normal saline offers an attractive alternative to liquid nitrogen and Michel\'s fixative for diagnostic IF in autoimmune and immune complex-mediated dermatoses, but is only consistently reliable if the specimens are received within 24 hours after biopsy. Competing interests =================== None declared. Abbreviations ============= BMZ: basement membrane zone BP : bullous pemphigoid DH: dermatitis herpetiformis DIF: direct immunofluorescence microscopy DSS: desired specific staining EBA: epidermolysis bullosa acquisita IF: immunofluorescence microscopy LAD: linear IgA dermatosis LE : lupus erythematosus MMP: mucous membrane pemphigoid NSS: non-specific staining PF : pemphigus foliaceus PV : pemphigus vulgaris USS: undesired specific staining Authors\' contributions ======================= RV carried out the studies, participated in the design and data analysis and drafted the manuscript. MdJ participated in patient selection and carried out the immunofluorescence microscopy and imaging. HM performed the immunofluorescence technical work up. MW participated in material harvesting. HP participated in patient diagnosis. MJ conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-5945/4/10/prepub>

PubMed Central

2024-06-05T03:55:47.754311

2004-8-27