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1064887

A model of inflammatory arthritis highlights a role for oncostatin M in pro-inflammatory cytokine-induced bone destruction via RANK/RANKL

Oncostatin M is a pro-inflammatory cytokine previously shown to promote marked cartilage destruction both in vitro and in vivo when in combination with IL-1 or tumour necrosis factor alpha. However, the in vivo effects of these potent cytokine combinations on bone catabolism are unknown. Using adenoviral gene transfer, we have overexpressed oncostatin M in combination with either IL-1 or tumour necrosis factor alpha intra-articularly in the knees of C57BL/6 mice. Both of these combinations induced marked bone damage and markedly increased tartrate-resistant acid phosphatase-positive multinucleate cell staining in the synovium and at the front of bone erosions. Furthermore, there was increased expression of RANK and its ligand RANKL in the inflammatory cells, in inflamed synovium and in articular cartilage of knee joints treated with the cytokine combinations compared with expression in joints treated with the cytokines alone or the control. This model of inflammatory arthritis demonstrates that, in vivo , oncostatin M in combination with either IL-1 or tumour necrosis factor alpha represents cytokine combinations that promote bone destruction. The model also provides further evidence that increased osteoclast-like, tartrate-resistant acid phosphatase-positive staining multinucleate cells and upregulation of RANK/RANKL in joint tissues are key factors in pathological bone destruction.

Introduction Bone is an important skeletal extracellular matrix, and bone erosions are a major characteristic in rheumatoid arthritis (RA). The cytokines IL-1 and tumour necrosis factor (TNF) alpha play key roles in promoting joint inflammation, synovitis and cartilage/bone resorption [ 1 , 2 ]. These cytokines are overexpressed in RA cartilage and synovial membranes, and raised levels are found in synovial fluid and sera that correlate with disease activity and cartilage/bone destruction in RA [ 3 - 5 ]. Anti-IL-1 and TNF-α therapies in animal arthritis models and anti-TNF-α in humans with RA have been shown to significantly reduce arthritis incidence, inflammation and joint destruction [ 1 , 6 - 8 ], suggesting that the mediating pathways of joint damage are, at least in part, mediated by IL-1 and/or TNF-α. Oncostatin M (OSM), a cytokine produced by activated T cells and macrophages, is structurally and functionally related to the IL-6 cytokine family. Raised levels of OSM are detected in synovial macrophages and synovial fluids of RA patients [ 9 - 11 ], and the levels correlate with markers of joint inflammation and destruction [ 3 , 10 ]. OSM causes joint inflammation, synovitis and structural damage in experimental animals [ 12 , 13 ]. Blockade of OSM ameliorates joint inflammation and cartilage damage in collagen-induced arthritis [ 14 ]. OSM has been found to enhance the differentiation and proliferation of osteoblasts during bone development and also induces the formation of osteoclasts and bone erosions [ 15 - 17 ]. These data indicate an important role for this cytokine in chronic joint inflammation and cartilage/bone damage. Furthermore, growing evidence from in vitro and in vivo studies suggests that OSM appears to be an important cofactor with other pro-inflammatory cytokines such as IL-1, TNF-α and IL-17 in mediating cartilage/bone destruction [ 9 , 18 , 19 ]. When these pro-inflammatory cytokines are overexpressed in combination with OSM in murine joints, a marked increase in damage to the joint tissues is observed [ 20 , 21 ]. RANKL is a TNF superfamily member and an essential mediator of osteoclastogenesis. It is produced from osteoblastic-stromal cells, synovial fibroblasts, chondrocytes and activated T lymphocytes [ 22 , 23 ]. This TNF-related cytokine and its receptor, RANK, are considered key factors in osteoclast differentiation, and RANK signalling is vital for osteoclast activation and survival [ 24 , 25 ]. RANKL binds directly to RANK on pre-osteoclasts and osteoclasts, initiating signal transduction that results in the differentiation of osteoclast progenitors as well as activation of mature osteoclasts, and therefore is implicated in the osteoclastogenic process in erosive arthritis [ 22 , 24 ]. The biological activity of RANKL is regulated by the soluble decoy receptor osteoprotegrin (OPG), a TNF-receptor superfamily member that is secreted by stromal cells and osteoblasts [ 26 ]. OPG competitively inhibits RANKL binding to RANK on the cell surface of osteoclast precursor cells and mature osteoclasts, thus inhibiting the osteoclastogenic actions of RANKL [ 27 ]. The levels of OPG and RANKL in osteoblastic and stromal cells are often reciprocally regulated in vitro and in vivo by bone active cytokines and hormones [ 28 ]. Excessive production of RANKL and/or deficiency of OPG may therefore contribute to the increased bone resorption typified by the focal bone erosions and peri-articular bone loss in RA. We have recently shown in a murine model that OSM in combination with IL-1 or TNF-α synergistically promoted inflammation and cartilage degradation and increased matrix metalloproteinase expression [ 20 , 21 ]. Since bone erosions are also a major pathological feature of RA, we examined the effects of these cytokine combinations on bone in this model. In the present study we confirm that OSM exacerbates the effects of both IL-1 and TNF-α with respect to bone breakdown, osteoclast formation and the expression of RANK/RANKL, and further confirm that this rapid model of inflammatory arthritis is suitable for studies of RA. Materials and methods Adenoviral vectors and delivery of cytokines Replication-defective recombinant adenoviruses engineered to overexpress murine IL-1β, TNF-α and OSM were as described previously [ 11 , 20 , 21 ], as was the empty control vector (Add170) [ 11 ]. Previous studies have validated these adenoviruses as an effective means of cytokine overexpression in synovial tissues [ 13 , 15 , 20 , 21 ]. All animal studies were compliant with the Canadian Council on Animal Care guidelines and were approved by the Animal Research Ethics Board at McMaster University, Canada. C57BL/6 mice were purchased and housed until 12–14 weeks old. Mice were injected intra-articularly with adenovirus (5 × 10 6 plaque-forming units [pfu]/vector/joint) or PBS as previously described [ 13 , 20 ]. Briefly, anaesthesia was maintained with isofluorane, knees were swabbed with 70% ethanol and a 5 μl volume (treatment) was injected into the synovial space. The contralateral knee was treated with control vector or with PBS. One knee ( n = 4 mice per treatment) was injected with combinations of vectors or with each vector alone combined with control vector to ensure that the total dose of vector was equivalent for each knee (1 × 10 7 pfu/joint). The animals were sacrificed at day 7 after administration. Histology and histopathological scoring The whole knee joints were dissected away from the limbs and were fixed with 7% formaldehyde in phosphate buffer (pH 7.4) overnight. Subsequently, joints were decalcified in 10% EDTA in phosphate buffer (pH 7.4) for 10 days at 4°C, and were then processed for paraffin embedding and sectioning (5 μm). Sections were stained with H&E. Bone damage was rated 0–5 (0 = normal, to 5 = severely affected) according to the following semiquantitative rating scale [ 29 ]: 0, none; 1, minimal (not readily apparent on low magnification); 2, mild (more numerous areas of resorption but not readily apparent on low magnification); 3, moderate (obvious foci of resorption, numerous osteoclasts); 4, marked (large erosions extending into bone cortices, more numerous osteoclasts); and 5, extensive erosions (markedly disrupted joint architecture). All scoring was performed blind with respect to the specific treatment. Immunohistochemistry and tartrate-resistant acid phosphatase staining Immunohistochemistry was performed with anti-RANK and anti-RANKL polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), using the VECTASTAIN Elite ABC Kit PK 6101 (Vector, Burlingame, CA, USA). Formalin-fixed paraffin sections were deparaffinized, rehydrated and incubated with 10 mM sodium citrate, pH 6.0, for 2 hours at room temperature, and then with 3% H 2 O 2 for 15 min. Thereafter, the sections were blocked with 1.5% normal sheep serum for 30 min, and were then incubated with primary antibody directed against RANK (rabbit polyclonal antibody raised against the epitope corresponding to amino acids 317–616 mapping at the carboxy terminus of RANK of human origin [H-300]) or against RANKL (rabbit polyclonal antibody raised against the epitope corresponding to amino acids 46–317 of RANKL of human origin [FL-317]) for 90 min at room temperature. After rinsing, sections were incubated with biotinylated secondary antibody for 30 min followed by avidin–biotin complex for 30 min according to the manufacturer's instructions (Vector). The signals were developed by 3,3'-diaminobenzidine tetrahydrochloride chromogen solution (DAKO Ltd, Ely, UK) and were counterstained with hematoxylin. A rabbit IgG antibody (X0936; Dako, Carpinteria, CA, USA) was used as a specificity control that gave no positive staining (data not shown). Tartrate-resistant acid phosphatase (TRAP) enzyme was detected in paraffin sections (5 μm thick) using a commercial acid phosphatase leukocyte kit (Sigma, St Louis, MO, USA) according to the manufacturer's protocol. Statistical analysis All data are presented as the mean ± standard error of the mean. Statistical significance was assessed by the two-tailed unpaired Student's t test for comparisons between the means of two groups. P ≤ 0.05 was considered significant. Results Intra-articular overexpression of OSM in combination with either IL-1 or TNF-α induces bone damage The morphology of H&E-stained sections from all treated joints (5 × 10 6 pfu/vector/joint) was assessed, and indicated that the contralateral joints treated with the control vector showed no evidence of bone damage (Fig. 1a ). Administration of each cytokine alone caused a moderate synovial hyperplasia and bone erosions (Fig. 1b,1c,1d ). For the OSM + IL-1 combination (Fig. 1e,1f ), pronounced bone destruction was observed with severe synovial hyperplasia and soft tissue inflammation. Prominent features of this arthritic lesion were the marked bone erosion, especially in the area of the bone and cartilage junction where marked synovial proliferation was seen with evidence of significant angiogenesis (Fig. 1e ). Some areas of bone erosion and disconnection were seen with evidence of marked synovial invasion and multinucleated cells (Fig. 1e,1f ). Similar results were obtained for the OSM + TNF-α combination (Fig. 1g,1h ). A number of channels developing links between the synovial tissue and the bone marrow were seen (Fig. 1g ) with focal bone erosions (Fig. 1h ). Semiquantitative evaluations of bone damage [ 29 ] indicated an increase in the severity and extent of bone erosions with both of the cytokine combinations compared with the individual cytokines alone ( P < 0.05) (Fig. 2 ). Intra-articular delivery of OSM with either IL-1 or TNF-α leads to an increase in TRAP-positive cells No TRAP-positive staining cells were found in the control joints (Fig. 3a ), but there was evidence of TRAP staining at the synovium–bone interface in joints treated with OSM, IL-1 or TNF-α (Fig. 3b,3c,3d , respectively). When OSM was combined with IL-1 a substantial increase in the number of TRAP-positive staining cells was found at the leading edge of the synovium–bone interface (Fig. 3e ), at sites within the synovium (Fig. 3f ) and at the pannus–subchondral bone junction (Fig. 3g ). These cells were often interposed between the bone surface and the 'erosive front' of the synovium and bone (Fig. 3h ). At many sites of focal bone erosion (as in Fig. 1 ), TRAP-positive multinucleated cells were seen at the erosion front and in the synovium (Fig. 3e,3f ), as well as in erosion pits in the bone (Fig. 3h ). Furthermore, TRAP-positive staining cells were also seen in the area of cartilage/bone junctions and also in the pannus and synovium away from the eroded bone surface (Fig. 3f,3g ). Similar results were seen in the joints treated with OSM + TNF-α where TRAP-positive cells were aligned on the bone surface (Fig. 3i,3j,3k,3l ). Multinucleated cells were also seen at the bone surface (Fig. 3i,3j ) and in deep pockets where bone was eroded (Fig. 3k ). TRAP-positive cells were also seen within the synovial tissue and the area of cartilage/bone junctions (Fig. 3l ). Intra-articular delivery of OSM with IL-1 or TNF-α elevates RANK and RANKL expression There was little or no RANK-positive staining in synovial tissues taken from control joints (Fig. 4a ). Increased RANK expression associated with inflammatory and synovial cells was observed following treatment with TNF-α (Fig. 4b ), and similar levels of expression were observed for OSM and IL-1 (data not shown). RANK expression was increased further, especially at the bone erosion fronts, when OSM was combined with IL-1 or TNF-α (Fig. 4c,4d ). Interestingly, RANK appeared to be expressed as a gradient from the synovial tissue where increased numbers of RANK-positive cells were observed close to the cortical bone and periosteum of the patella, the femur and the tibia. Diffuse RANK staining in the superficial layer of cartilage was seen for the joints treated with each of the vectors separately (see Fig. 4b ; some data not shown), and this staining was more intense in the joints treated with OSM + IL-1 or with OSM + TNF-α (Fig. 4e,4f ). No RANKL staining was evident in the control joints, either in the cartilage (Fig. 4g ) or in the synovium (data not shown). Treatment with all the individual vectors alone induced a similar positive staining for RANKL in synovial cells and in the infiltrating (inflammatory) cells (Fig. 4h ). There was a marked increase in RANKL expression, consistent with the increase in inflammatory cells and synovial inflammation, in the joints treated with the combinations of OSM + IL-1 (Fig. 4i ) or OSM + TNF-α (Fig. 4j ). Within the articular cartilage there was very diffuse RANKL staining for both the control and each individual cytokine vector alone (data not shown), while strong RANKL expression was seen for both cytokine combinations close to the articular surface (Fig. 4k,4l ). Discussion In the present study, we demonstrate for the first time that overexpression of OSM in combination with either IL-1 or TNF-α causes profound bone damage with osteoclast formation and activation, and increased expression of RANK/RANKL in inflammatory cells, in inflamed synovium, in articular cartilage and at the invading front of bone erosions. It has been long recognized that pro-inflammatory cytokines are intimately associated with bone destruction during RA. IL-1, TNF-α and OSM have all been reported to induce joint inflammation and cartilage/bone destruction in vitro and in vivo [ 1 , 2 , 9 , 13 ]. Elevated levels of these cytokines are found in the synovial fluid of RA patients, and these levels correlate with disease activity and cartilage/bone destruction [ 2 , 5 , 9 , 12 ]. Recent studies indicate that cytokines such as TNF-α and IL-1 are likely to synergize with RANKL to promote bone loss [ 30 - 33 ]. Indeed, TNF-α stimulates differentiation of osteoclast precursors after priming by <1% of the amount of RANKL normally required to induce osteoclast formation [ 31 ]. TNF-α induces IL-1 production, and both IL-1 and RANKL function as osteoclast survival/activation factors [ 33 ]. In the inflammatory arthritides such as RA, TNF-α and IL-1 may therefore promote bone loss by amplifying RANKL effects. This is exemplified in transgenic mice overexpressing the human TNF-α gene; these mice exhibit an erosive arthritis, which is improved by administration of OPG, of neutralizing anti-TNF-α antibodies, or of the bisphosphonate pamidronate [ 34 ]. OSM-induced expression of RANK/RANKL in a murine arthritis model has also been reported [ 15 ]. In the present study, the combination of OSM with TNF-α significantly induced RANKL expression in inflammatory cells, in inflamed synovium and in articular chondrocytes. A number of factors contribute to arthritic cartilage/bone destruction in RA, including the proliferation of synovial cells, the influx and interaction of inflammatory cells, and the maintenance of a destructive fibroblastic phenotype, which result in the final loss of cartilage and bone. Indeed, CD14 + monocytes/macrophages have been shown to be osteoclast precursors within the inflamed synovium that promote bone resorption following differentiation [ 35 ]. In support of this we found evidence of high numbers of TRAP-positive multinucleate cells in the synovial tissues of mice treated with OSM + IL-1 or with OSM + TNF-α combinations. As well as inducing marked synovial hyperplasia, angiogenesis and inflammation as described previously [ 20 , 21 ], marked bone erosions were also evident. Active synovial cells can cause bone erosions as well as produce factors that can themselves induce synovial proliferation, inflammation, osteoclast formation and activation. Angiogenesis contributes to synovial growth and leukocyte recruitment, thus potentiating disease progression [ 36 ]. The matrix metalloproteinases released by active synovial cells are also involved in angiogenesis, tissue invasion and inflammatory cell migration [ 37 ], as well as in osteoclast activation, migration and bone resorption [ 38 , 39 ]. The high levels of RANK/RANKL expression, the synovial hyperplasia, the angiogenesis and the osteoclast activity that the OSM + IL-1 or OSM + TNF-α treatments induced was associated with pronounced bone damage in this murine model with a similar pathology to that of active RA. Our previous studies have shown that these cytokine combinations upregulate matrix metalloproteinases [ 20 , 21 ]. TRAP is used as a molecular marker enzyme for chondroclast/osteoclast differentiation, the function of which is considered to relate to cartilage/bone resorption [ 40 ]. The cytokine combinations used in this study induced an increased number of TRAP-positive staining multinucleated cells at the pannus and cartilage/bone junctions compared with joints injected with the cytokines alone. The expression of TRAP activity by the multinucleated cells located within erosion pits and bone disconnection sites provides strong evidence of their osteoclast-like nature. The mechanism of induction of TRAP-positive multinucleated cells could be related to the marked induction of RANK/RANKL expression and the interactions between osteoblast and osteoclast precursor cells that is crucial for osteoclast development [ 15 ]. We also found that treatment with OSM increased the numbers of TRAP-positive cells at the invading front of bone erosion sites and at the bone surface, as well as in the synovium. This differs from a recent report that OSM induced synovial inflammation and increased the expression of IL-6, RANK and RANKL, but did not stimulate osteoclast activity [ 15 ]. The same authors also found marked growth plate damage, and determined that OSM-induced inflammation and proteoglycan depletion were IL-1 dependent [ 41 ]. Conclusion Using gene transfer technology we have provided evidence of a murine model with an aggressive pathological phenotype that closely resembles RA in terms of inflammation, angiogenesis, and cartilage and bone destruction. These data further highlight the pro-inflammatory nature of OSM and confirm a potential role for these potent cytokine combinations in the joint destruction characteristic of inflammatory arthritic diseases. Abbreviations H&E = haematoxylin and eosin; IL = interleukin; OPG = osteoprotegrin; OSM = oncostatin M; PBS = phosphate buffered saline; pfu = plaque-forming units; RA = rheumatoid arthritis; RANK = receptor activator of nuclear factor kappa B; RANKL = receptor activator of nuclear factor kappa B ligand; TNF = tumour necrosis factor; TRAP, tartrate-resistant acid phosphatase. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CDR supplied and administered the adenoviral vectors. WH performed the tissue sectioning and staining experiments. ADR drafted the manuscript and, with TEC, conceived of the study and participated in its design and coordination.

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1064888

Serum cathepsin K levels of patients with longstanding rheumatoid arthritis: correlation with radiological destruction

Cathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix by cleaving proteins such as collagen type I, collagen type II and osteonectin. Cathepsin K therefore plays a role in bone remodelling and resorption in diseases such as osteoporosis, osteolytic bone metastasis and rheumatoid arthritis. We examined cathepsin K in the serum of 100 patients with active longstanding rheumatoid arthritis. We found increased levels of cathepsin K compared with a healthy control group and found a significant correlation with radiological destruction, measured by the Larsen score. Inhibition of cathepsin K may therefore be a new target for preventing bone erosion and joint destruction in rheumatoid arthritis. However, further studies have to be performed to prove that cathepsin K is a valuable parameter for bone metabolism in patients with early rheumatoid arthritis.

Introduction Progressive bone and cartilage destruction in arthritic joints leads to irreversible joint destruction, and subsequently to functional declines and work disability [ 1 , 2 ]. New biomarkers such as cartilage oligomeric matrix protein [ 3 , 4 ], osteoprotegerin [ 5 - 7 ] or receptor activator of NF-κB ligand [ 8 - 10 ] have been developed to describe the local bone and cartilage process in affected joints. Cathepsin K is a cysteine protease that plays an essential role in osteoclast function and in the degradation of protein components of the bone matrix. It is produced by bone resorbing macrophages and synovial fibroblasts, and it cleaves proteins such as collagen type I, collagen type II and osteonectin [ 11 ]. Cathepsin K therefore plays a role in bone remodelling and resorption in diseases such as osteoporosis, osteolytic bone metastasis and rheumatoid arthritis (RA) [ 12 , 13 ]. Cathepsin K is a tissue-specific protease associated with pycnodysostosis, a rare genetic disorder that manifests itself in bone abnormalities such as short stature, acroosteolysis of distal phalanges and skull deformities [ 14 , 15 ]. Cathepsin K knockout mice develop an osteopetrosis. Inhibition of cathepsin K may therefore prevent bone resorption, as could be demonstrated in bone metastasis from breast cancer [ 16 ]. Osteoprotegerin has been shown to inhibit the expression of cathepsin K, the main enzyme involved in bone resorption. The aim of this study was to measure serum levels of cathepsin K in RA and to prove that cathepsin K is a parameter of bone remodelling and resorption in a nonselected cohort of patients with longstanding RA. This patient group shows a variation of age, inflammatory level and Larsen score. We divided this cohort into different groups, according to age, inflammatory level, disease-modifying antirheumatic drug (DMARD) therapy, radiological progression and disease activity, to verify cathepsin K as an age-independent and laboratory inflammatory parameter-independent protease. Materials and methods Serum levels of cathepsin K were measured in the sera of 100 patients suffering from RA according to the criteria of the American Rheumatism Association [ 17 ]. Clinical and laboratory data are presented in Tables 1 and 2 . The control group consisted of nonselected healthy blood donors from a central blood bank ( n = 50; 21 female, 29 male) aged 18–65 years. Most of the patients received DMARDs. The most frequently used DMARD was methotrexate, followed by leflunomide, sulfasalzopyrine and gold. Furthermore, azathioprine and chloroquine but no biological therapy were prescribed (Table 3 ). Each examination consisted of a full interview, the assessment of functional disability and a standardised physical examination, which included a joint examination for tenderness (Ritchie score), pain on motion, soft tissue swelling, 44-swollen joint count and swollen proximal interphalangeal score [ 18 , 19 ]. The disease activity of RA was measured by the disease activity score (≤ 2.4, low activity; > 2.5 and ≤ 3.7, mean activity; > 3.7, high activity). The radiological progression in RA was calculated by the Larsen score [ 20 ]. The blood examination at each visit consisted of the determination of cathepsin K, the erythrocyte sedimentation rate, the haemoglobin level, the thrombocyte count, the serum rheumatoid factor (RapiTex ® RF; Dade Behring, Liederbach, Germany), antinuclear antibodies (indirect immunfluorescent technique, ANA Fluor Kit 240 ® ; Diasorin, Stillwater, MN, USA) and C-reactive protein (CRP) (Rheumajet CRP ® ; Biokit, Barcelona, Spain). The variables of age, sex, duration of disease, visual analogue scale of general health and morning stiffness, treatment with DMARDs and reason for their discontinuation, and the Steinbrocker stage [ 21 ] were also recorded. Serum was obtained in the morning from the routinely taken blood samples and was centrifuged immediately. The samples were kept at -80°C prior to determination of cathepsin K. The serum used for the measurement of cathepsin K was the remainder from routinely drawn blood examinations on the day of hospitalisation; no examination was performed only for quantification of cathepsin K. Clinical data were used from a database developed for the long-term observation of patients with RA in our clinic. An enzyme immunoassay for cathepsin K developed by Biomedica Austria (Vienna, Austria) was used. The Cathepsin K test kit is an enzyme immunoassay designed to determine cathepsin K directly in biological fluids (serum, plasma, cell culture supernatants). The ELISA used in this study is based on antibodies specific for amino acids 1–20 and amino acids 196–210 of the mature enzyme. The antibodies were produced by immunisation of sheep with peptides of that amino acid sequence coupled to Keyhole Limpet Hämocyanine (primary immunisation, 0.5 mg; boost, 0.25 mg). Antisera were purified using the biotinylated peptides coupled to streptavidine sepharose (Amersham-Pharmacia Biotech Ltd, Little Chalfont, UK). A synthetic cathepsin K (Pichem GmbH, Graz, Austria) was used as the calibrator. Signal generation was accomplished by labelling with horseradish peroxidase. Briefly, the assay procedure consisted of incubating 50 μl sample with 200 μl horseradish peroxidase-labelled detection antibody on capture antibody precoated plates overnight at room temperature. After a washing step to remove unbound detection antibody, tetramethyl benzidine was added as the substrate. The reaction was stopped after 30 min by adding 50 μl of 0.9% H 2 SO 4 . The yellow colour that is directly proportional to the amount of cathepsin K present in the sample was measured on a standard microplate reader at 450 nm with 620 nm as the reference. The detection limit of the assay was calculated as 1.1 pmol/l (0 standard + 5 × standard deviation). No cross-reactivity to cathepsin E, cathepsin D, cathepsin B and cathepsin L or rheumatoid factors was detected. Statistical methods included Spearman correlation analysis, the Wilcoxon two-sample test the Kruskal–Wallis test and analysis of variance, if appropriate. P < 5% was considered statistically significant. Results The cathepsin K serum levels of the patients with RA (median first–third quartile range, 54.8 pmol/l) compared with the healthy control group (median first–third quartile range, 12.7 pmol/l) were significantly elevated ( P = 0.0003) (Table 4 ). The Larsen score ranged from 0 to 164 (median score, 39). The Spearman rank correlation showed a statistically significant correlation between cathepsin K and the Larsen score ( P = 0.004). The highest levels of cathepsin K were observed in patients with the highest Larsen scores. We divided the cohort into three Larsen groups with equal numbers of patients (Larsen score < 18 points, Larsen score between 19 and 74 points, and Larsen score ≥ 75 points). Cathepsin K levels showed an increase with the augmentation of radiological destruction ( P = 0.035) (Table 5 ). Cathepsin K seems to be independent or only weakly correlated with laboratory inflammation parameters. It was not associated with CRP ( P = 0.27), but weak correlations were found with the erythrocyte sedimentation rate ( P = 0.03) and the disease activity score of the whole cohort ( P = 0.04). However, the division of the disease activity score into three groups with low activity, medium activity and high activity did not show any difference. We could not find any correlation with sex and age (whole group/division into two patient groups ≤ 65 years and ≥ 66 years, P = 0.32), whereby the two groups were comparable in disease activity (3.53 versus 3.12), laboratory parameters (CRP, 25.4 mg/l versus 25.9 mg/l), clinical score (Ritchie score, 14 versus 9) and radiological score (Larsen score, 47 versus 62). The most frequently used DMARD was methotrexate ( n = 42), followed by leflunomide ( n = 10) and sulfasalzine ( n = 10). Twenty-two patients had no DMARD at the time of examination (Table 3 ). The lowest cathepsin K levels were evident in the leflunomide group, but no significant difference between these groups could be demonstrated. Discussion Bone resorption and formation is a well-balanced system and is mediated by osteoclasts. Cathepsin K is essential for bone resorption, which depends on the production of cathepsin K by osteoclasts and its secretion into the extracellular department. This leads to a degradation of the organic matrix between the osteoclasts and the bone surface [ 22 ]. In vivo the activation of cathepsin K occurs intracellularly, before secretion into the resorbing lacunae and the onset of bone resorption, whereby local factors may regulate the processing of procathepsin K to mature cathepsin K [ 23 ]. In accordance with this, synovial fibroblasts are also involved in joint destruction and in the pathogenesis of RA. Hou and colleagues found that cathepsin K has a potent aggrecan-degrading activity, whereby the aggrecan cleavage products increase the collagenolytic effects of this protease on collagen type I and type II. They were able to show that cathepsin K is also a critical protease in cartilage degradation by synovial fibroblasts [ 24 ]. Increased expression of cathepsin K around lymphocytic infiltrates in synovial tissue seems to facilitate the movement of mononuclear cells through the perivascular matrix [ 25 ] Proinflammatory cytokines such as IL-1β and tumour necrosis factor alpha influence the expression of cathepsin K. Its overexpression in the rheumatoid synovium, induced by IL-1β and tumour necrosis factor alpha due to the increase of cathepsin K-expressing cells, proves this protease to be a valuable tool for bone research, and cathepsin K also may become a new and highly specific biomarker for RA [ 26 ]. Votta and colleagues demonstrated high levels of cathepsin K expression in osteoclasts at sites of extensive bone loss. According to this, they developed a peptide aldehyde inhibitor of cathepsin K that inhibits osteoclast-mediated bone resorption in foetal rat long bone organ cultures and even in a human osteoclast-mediated assay in vitro . This inhibitor leads to a significantly reduced bone loss [ 27 ]. Furthermore, structure activity studies on a series of reversible ketoamide-based inhibitors of cathepsin K have led to the identification of potent and selective inhibitors [ 28 ]. Wittrant and colleagues demonstrated osteoprotegerin to be an inhibitor of cathepsin K. Osteoprotegerin is an osteoblast-secreted decoy receptor that inhibits osteoclast differentiation and activation. Human osteoprotegerin inhibits cathepsin K and tartrate-resistant acid phosphatase, both osteoclast markers, but stimulates the expression of tissue inhibitor of metalloproteinases-1 [ 29 ]. These results are a further step in the development of new therapies for the prevention of bone destruction. In the synovium of RA, the cathepsin K protein was localised in synovial fibroblasts, stromal multinucleated giant cells and CD68 + macrophage-like synoviocytes. Highly interesting is the expression of cathepsin K by fibroblasts and giant cells at sites of cartilage erosions. This was two to five times higher compared with osteoarthritic synovium. In normal synovium, cathepsin K expression was not increased and was restricted to fibroblast like cells [ 26 , 30 - 32 ]. The overexpression of cathepsin K in RA synovia proves that this protease is responsible for the degradation of articular tissue in rheumatoid joints and in normal synovial tissue. To our knowledge, no study has previously investigated the serum levels of cathepsin K in RA. Our results demonstrate that cathepsin K is elevated in the serum of patients with RA compared with that of a healthy control group (Table 4 ). The upregulation of cathepsin K and the correlation with the Larsen score as a parameter for radiological changes (Table 5 ) mirrors the destruction of bone structures in inflammatory diseases like RA. The measurement of cathepsin K seems an inexpensive tool that is independent of CRP and shows only a weak correlation with the erythrocyte sedimentation rate. Further studies should investigate whether elevated cathepsin K levels precede osseous destruction or whether they occur as result of them. In the first case, determination of cathepsin K could be an important additional tool to decide on aggressive forms of disease-modifying antirheumatic therapies. Conclusion This is the first study that demonstrates increased cathepsin K levels in the serum of patients with RA. As could be shown in the synovia of RA, the elevated serum levels of this protease are significantly correlated with the joint destruction, which in this study was assessed by the Larsen score. Cathepsin K seems to be a valuable parameter for the assessment of bone metabolism in patients with established RA and its measurement will probably contribute to developing targeted therapies for the prevention of further bone destruction. However, more studies need to be performed to verify the presence of cathepsin K in patients with early RA and its value as a prognostic factor for bone destruction in RA Abbreviations CRP = C-reactive protein; DMARD = disease-modifying antirheumatic drug; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; NF = nuclear factor; RA = rheumatoid arthritis. Competing interests Dr G Hawa and Prof. W Woloszczuk are members of BIOMEDICA who developed the Cathepinsin K kit, but they did not receive any financial benefits. Authors' contributions MS is the corresponding author, and GH and GK are coauthors of the manuscript. GH and WW developed the cathepsin K ELISA kit. AK performed the statistical analysis.

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1064889

MMP-3 expression and release by rheumatoid arthritis fibroblast-like synoviocytes induced with a bacterial ligand of integrin α5β1

Fibroblast-like synoviocytes (FLSs) play a major role in the pathogenesis of rheumatoid arthritis (RA) by secreting effector molecules that promote inflammation and joint destruction. How these cells become and remain activated is still elusive. Both genetic and environmental factors probably play a role in transforming FLSs into inflammatory matrix-degrading cells. As bacterial products have been detected in the joint and shown to trigger joint inflammation, this study was undertaken to investigate whether a bacterial ligand of integrin α5β1, protein I/II, could contribute to the aggressive behavior of RA FLSs. Protein I/II is a pathogen-associated molecular pattern (PAMP) isolated from oral streptococci that have been identified in the joints of RA patients. The response of RA and osteoarthritis FLSs to protein I/II was analyzed using human cancer cDNA expression arrays. RT-PCR and pro-MMP-3 (pro-matrix metalloproteinase) assays were then performed to confirm the up-regulation of gene expression. Protein I/II modulated about 6% of all profiled genes. Three of these, those encoding IL-6, leukemia inhibitory factor, and MMP-3, showed a high expression level in all RA FLSs tested, whereas the expression of genes encoding other members of the cytokine or MMP-family was not affected. Furthermore, the up-regulation of MMP-3 gene expression was followed by an increase of pro-MMP-3 release. The expression of interferon regulatory factor 1 and fibroblast growth factor-5 was also up-regulated, although the expression levels were lower. Only one gene, that for insulin-like growth factor binding protein-4, was down-regulated in all RA FLSs. In contrast, in osteoarthritis FLSs only one gene, that for IL-6, was modulated. These results suggest that a bacterial ligand of integrin α5β1 may contribute to the aggressive behavior of RA FLSs by inducing the release of pro-inflammatory cytokines and a cartilage-degrading enzyme, such as IL-6 and MMP-3, respectively.

Introduction Fibroblast-like synoviocytes (FLSs) appear to play a major role in the pathogenesis of rheumatoid arthritis (RA). These cells are characterized by pannus formation, cartilage invasion, and secretion of effector molecules, including cytokines and chemokines, that act on various cells to promote inflammation [ 1 - 3 ]. Recent experiments have shown that although FLSs were able to secrete large amounts of IL-6 and IL-8, they failed to release significant amounts of TNF-α, IL-1, IL-15, and IL-18 [ 4 , 5 ]. They showed a dissociated pattern of cytokine mRNA and protein expression, suggesting the existence of post-transcriptional regulation. FLSs are also the principal promoters of joint destruction [ 3 ], either through the release of proteolytic enzymes such as matrix metalloproteinases (MMPs), or indirectly through the stimulation of osteoclastogenesis. FLSs are the source of a broad range of MMPs, including MMP-1, MMP-13, and MMP-3; the last of these degrades different types of collagen and proteoglycans and activates other MMPs such as MMP-2 and MMP-9. How these cells become and remain activated is still not known. Numerous factors may account for the permanent changes observed in FLS functions. To date it is very difficult to define whether FLSs are changed in response to genetic modifications, such as mutations or microsatellite instability, or in response to environmental factors. Both factors probably play a role in transforming FLSs into invading, inflammatory, matrix-degrading cells. Exposure to an inflammatory environment may even result in DNA damage [ 6 ]. On the other hand, genetic modifications may contribute to inflammation: it has been shown that a decrease of the expression of p21, an inhibitor of the cyclin-dependent kinases which is regulated by p53, activates activating protein-1, leading to enhanced cytokine and MMP synthesis in RA FLSs [ 7 , 8 ]. Regarding environmental factors, there has been considerable interest in a possible role of innate immunity in the initiation and perpetuation of inflammation during RA. Bacterial products – also called pathogen-associated molecular patterns (PAMPs) – such as lipopolysaccharide (LPS), peptidoglycan, and bacterial DNA have been detected in the joint [ 9 , 10 ] and were shown to trigger joint inflammation: in fact, oral administration of LPS exacerbates collagen-induced arthritis in mice, and intra-articular injection of CpG oligonucleotides and of peptidoglycan leads to transient arthritis in mice [ 11 - 13 ]. As DNA and rRNA from a wide variety of bacterial species have been identified in the joints of RA patients [ 9 , 14 , 15 ], it has been suggested that joint inflammation could be triggered by PAMPs common to various microorganisms interacting with cellular pattern-recognition receptors (PRRs). FLSs express a large number of PRRs, such as Toll-like receptor (TLR)-2, TLR-4, and TLR-9 [ 16 ], as well as numerous integrins. Their interaction with a variety of PAMPs may contribute to the aggressive phenotype of RA FLSs. We reported previously that interaction of protein I/II, a cell wall component of oral streptococci, with FLSs triggers, through integrin α5β1, the production and release of inflammatory mediators such as IL-6 and IL-8 but not of TNF-α, IL-1, or IL-18 [ 17 - 19 ]. This cytokine synthesis involves extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinases, as well as activating protein-1-binding activity and nuclear translocation of nuclear factor κB [ 20 ]. Oral streptococci have been identified in the joints of RA patients [ 15 ] and have been shown to exacerbate collagen-induced arthritis in mice [ 21 ]. This study was undertaken to investigate whether a PAMP such as protein I/II could contribute to the aggressive behaviour of RA FLSs. Using cDNA array analysis, we show that this cell wall component triggers the synthesis of MMP-3 by FLSs and may therefore contribute to joint destruction. Furthermore, the genes modulated by protein I/II are mainly involved in cell signaling, protein turnover, and cellular communication, suggesting that protein I/II may contribute to the aggressive behavior of FLSs. Materials and methods Reagents Cell-culture media (RPMI 1640 and M199), FCS, penicillin, streptomycin, amphotericin B, Taq DNA polymerase, dNTPs, and primers were from Invitrogen (Cergy-Pontoise, France). Cell-culture media never had an endotoxin level above 0.04 ng/mL, as tested by the Limulus chromogenic assay. LPS from Escherichia coli O55:B5, polymyxin B, and type XI collagenase were obtained from Sigma (Saint Quentin Fallavier, France). The First Strand cDNA synthesis kits and 32 P dATP were from Amersham Pharmacia Biotech (Saclay, France). A Nucleospin RNA II extraction kit was from Macherey-Nagel (Souffelweyersheim, France). Atlas human cancer cDNA expression arrays were from Clontech (Ozyme, Saint Quentin Yvelines, France). BINDAZYME™ ProMMP-3 enzyme immunoassay kit was from The Binding Site (Saint Egreve, France). The SYBR ® Green PCR master mix was from Applied Biosystems (Courtaboeuf, France). Buffers were prepared with apyrogenic water obtained from Braun Medical (Boulogne, France). Cell culture Human FLSs were isolated from RA synovial tissues from three patients at the time of knee joint arthroscopic synovectomy, as described previously [ 22 ]. The diagnoses conformed to the revised criteria of the American College of Rheumatology [ 23 ]. Human FLSs were also isolated from osteoarthritis (OA) synovial tissues from three patients who were having joint replacements. FLS cultures were performed as previously described [ 20 ]. Briefly, tissues were minced, digested with 1 mg/mL collagenase in serum-free RPMI 1640 for 3 hours at 37°C, centrifuged (130 g for 10 minutes at 4°C) and resuspended in M199-RPMI 1640 (1:1) containing 2 mm l-glutamine, penicillin (100 IU/mL), streptomycin (100 μg/mL), amphotericin B (0.25 μg/mL), and 20% heat-inactivated FCS (complete medium). After overnight culture, nonadherent cells were removed and adherent cells were cultured in complete medium. At confluence, cells were trypsinized and passaged in 75-cm 2 culture flasks in complete medium containing 10% heat-inactivated FCS. Experiments were performed between the third and the ninth passages, during which time cultures were a homogeneous population of fibroblastic cells, negative for CD16 as determined by FACS analysis. Before activation experiments, cells were deprived of serum for 24 hours, and then the appropriate stimuli diluted in serum-free RPMI 1640 with antibiotics were added. To eliminate the possibility that the observed effects were due to LPS contamination, all the experiments were performed in the presence of polymyxin B (2 μg/ml). Cell numbers and cell viability were assessed using the MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test) as described elsewhere [ 24 ]. Purification of protein I/II Recombinant protein I/II of Streptococcus mutans OMZ 175 was purified from pHBsr-1 transformed E. coli cell extract by gel filtration and immunoaffinity chromatography as described elsewhere [ 25 ]. The purity of the protein was checked by SDS–PAGE after staining with Coomassie blue. Protein I/II migrated as a single band having an apparent molecular weight of 195 kDa. Stimulation of cells for total RNA extraction FLSs (3 ×10 6 cells) were stimulated with 600 μL of serum-free RPMI 1640 containing protein I/II (125 pm final concentration). After a 4-hour incubation period, cells were centrifuged (130 g for 10 min at 4°C), and total RNA was extracted from cell pellets using the Nucleospin RNA II extraction kit in accordance with the manufacturer's instructions. Stimulation of cells for pro-MMP-3 assay FLSs (5 ×10 3 cells) were grown to confluence in 96-well plates (7–10 days) and then stimulated with 200 μL of serum-free RPMI 1640 containing protein I/II (125 pm final concentration). After an 18-hour incubation period, a heterologous two-site sandwich ELISA was used to estimate pro-MMP-3 release in the culture supernatants. Gene expression profile analysis and quantification The gene expression profile was examined using Atlas human cancer cDNA expression arrays, consisting of nylon membranes spotted in duplicate with cDNA fragments of 588 known genes. These genes are classified into several functional groups, such as oncogenes and tumor suppressors, growth factors and receptors, and regulators of cell adhesion, angiogenesis, and cell cycle (listed at ). Total RNA (2 μg) was converted into 32 P-labeled first-strand cDNA following the protocol provided by the manufacturer. Briefly, total RNA was reverse-transcribed using MMLV (Moloney murine leukemia virus) reverse transcriptase and 32 P-labeled dATP in a PCR thermal cycler set at 50°C for 25 min. Labeled cDNA probes were then purified from unincorporated 32 P-labeled nucleotides and small (<0.1 kb) cDNA fragments using column chromatography. Probes (2–10 × 10 6 cpm) were freshly applied to cDNA array membranes to hybridize overnight at 68°C in continuously agitated roller bottles. After hybridization, membranes were washed four times, for 30 min each, at 68°C with 2 × SSC (standard saline citrate), 1% SDS, and once with 0.1 × SSC, 0.5% SDS, followed by one wash with 2 × SSC at room temperature. Array membranes were wrapped in plastic and exposed to a phosphor screen for 1–5 days, depending on the radiation intensity of the bound fragments. After image acquisition on a Storm phosphor imager (ImageQuant, Molecular Dynamics, Sunnyvale, CA, USA), spots were quantified using the AtlasImage 2.0 Software (Clontech), developed specifically for analysis of the Atlas cDNA expression arrays. All spots were individually checked by hand to ensure accuracy of the detection method. A dot was considered to be positive if it was well located and at least three times the local background level. Spots of poor quality were not included. For each patient, the relative expression level of each gene between protein-I/II-stimulated and control FLSs was evaluated and standardized based on expression levels of housekeeping genes included on each array. RT-PCR reactions Total RNA (2 μg) isolated from FLSs was reverse-transcribed using the First Strand cDNA Synthesis Kit in accordance with the manufacturer's instructions. Total RNA (8 μL) was mixed with 5 μl of the bulk reaction mix, 1 μl of DTT (200 mm), and 1 μl of the Not I-d(T) 18 bifunctional primer (0.2 μg/ml). The reaction was carried out for 1 hour at 37°C. Real-time PCR was performed in 96-well plates in a total volume of 25 μl using the SYBR ® Green PCR master mix (containing SYBRGreen dye, AmpliTAq Gold ® DNA Polymerase, dNTPs with dUTP, passive reference and optimized buffer components) and gene-specific primers (250 nm): MMP-3 1) 5' GCA GTT TGC TCA GCC TAT CC 3' and 2) 5' GAG TGT CGG AGT CCA GCT TC 3' [ 26 ]; GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 1) 5' AGC AAT GCC TCC TGC ACC ACC AAC 3' and 2) 5' CCG GAG GGG CCA TCC ACA GTC T 3' [ 27 ]. After incubation at 50°C for 10 min and at 95°C for 10 min, samples were subjected to 40 rounds of amplification for 15s at 95°C, 15s at 56°C, and 40s at 72°C using the AbiPrism 7700 Sequence Detection System (Applied Biosystems). Amplification products were detected as an increased fluorescent signal of SYBR ® Green during the amplification cycles. Results were obtained using SDS Software (Applied Biosystems, Foster City, CA, USA) and evaluated using Excel (Microsoft). Primer efficiency was calculated for MMP-3 and GAPDH using the standard curve method in accordance with the supplier's recommendations. As the MMP-3 and GAPDH amplifications were about equally efficient, the relative expression levels of the MMP-3 gene were evaluated using the 2 -ΔΔCT method as described by Applied Biosystems. Additionally, to confirm the amplification specificity of each gene product, the PCR products were subjected to a melting-curve analysis. Pro-MMP-3 assay Pro-MMP-3 levels in cell-culture supernatants were determined using the BINDAZYME™ pro-MMP-3 Enzyme Immunoassay Kit in accordance with the manufacturer's instructions. Briefly, duplicate samples were added to wells coated with anti-pro-MMP-3 and were then incubated with anti-pro-MMP-3 peroxidase conjugate; the peroxidase substrate (TMB) was then added. After the reaction had been stopped by the addition of phosphoric acid (3 m), the optical density of each well was read at 450 nm. Pro-MMP-3 levels were calculated using a standard curve. Statistical analysis Values are represented as means ± standard error of the mean. The significance of the results was analyzed using Student's two-tailed t -test. Values of P < 0.05 were considered significant. Results Gene expression in RA and OA FLSs after a stimulation with protein I/II In this study we used a gene array technique to further investigate the cellular response of FLSs to protein I/II. As we were particularly interested in genes that could contribute to the aggressive behavior of RA FLSs, we chose Atlas human cancer cDNA expression arrays containing genes involved in cell-cycle regulation, cellular adhesion, and inflammation. In parallel to RA FLSs, we used OA FLSs in order to study the protein-I/II-induced response of FLSs isolated from a noninflammatory arthropathy. After a 4-hour incubation in the presence or absence of 125 pm protein I/II, the total RNA from FLSs of three RA and three OA patients was extracted, and radiolabeled cDNA probes were subsequently hybridized to the cDNA arrays. Figure 1 shows the expression patterns of FLSs isolated from one RA and one OA patient in nonstimulated controls after 4 hours (Fig. 1a,1c ) and after stimulation with protein I/II for 4 hours (Fig. 1b,1d ). The products of some differentially expressed genes (e.g. the gene for IL-6) are easily observed. The relative expression level of each gene in protein-I/II-stimulated FLSs versus control FLSs was evaluated with the AtlasImage 2.0 Software. When a cut-off of a threefold change in mRNA expression was used, 28 genes were up-regulated and 5 genes were down-regulated in a least one of the three RA FLSs tested (Tables 1 and 2 ). These modulated genes account for about 6% of all profiled genes. Among the genes up-regulated by protein I/II in all three RA FLSs, two genes encoding proinflammatory cytokines, IL-6 and leukemia inhibitory factor, showed a strong expression level. The expression of other cytokine genes included on the array, such as TNF-α or IL-1, was not affected by protein I/II stimulation (IL-8 was not represented on the array). Protein I/II also strongly up-regulated the expression of MMP-3 (stromelysin 1), whereas the expression of genes encoding other members of the MMP family was not modified. Furthermore, a transcription factor, interferon regulatory factor 1 (IRF-1), as well as a growth factor, fibroblast growth factor-5 (FGF-5), were up-regulated by protein I/II in the three RA FLSs, although the expression levels were lower. Besides this group of genes which were strongly up-regulated in all RA FLSs, the expression of other genes was more heterogeneous and varied between the different RA FLS cultures. Among these genes, mainly involved in protein turnover and in cell signaling and communication, two genes, encoding CDC27Hs protein and interferon γ antagonist, were strongly up-regulated and the expression of three more genes (those encoding integrin αE, vimentin, and transmembrane protein SEX) was less intensely up-regulated in two of the three RA FLSs. Interestingly, the expression of a few genes was down-regulated by protein I/II and only one gene was down-regulated in all three RA FLSs tested, namely, that for insulin-like growth factor binding protein-4. This member of the family of insulin-like growth factor binding proteins, which act as carriers and regulators of insulin-like growth factor, is believed to play an important role in maintaining the equilibrium between synthesis and degradation of tissue matrix molecules. In OA FLSs, the number of genes modulated within 4 hours by protein I/II was much lower than in RA FLSs: protein I/II up-regulated the expression of only one gene, namely IL-6, in all OA FLSs tested. Other genes were up-regulated but with a high variability among FLSs cultures (Table 3 ). A very interesting finding was that one of the three OA FLS cultures (from patient 3) showed a gene expression pattern that was very close to the patterns obtained with protein-I/II-stimulated RA FLSs. Clinical investigations revealed that this patient had a secondary arthropathy of the knee occurring during a long remission phase of RA. This patient was therefore excluded from further experiments. The up-regulation of IL-6 gene expression found in both RA and OA FLSs in response to protein I/II has been previously shown to be followed by an increase of IL-6 release from these cells (MB Zeisel and colleagues, unpublished data) [ 20 ]. Since we were primarily interested in genes that could contribute to the aggressive, invasive behavior of RA FLSs, the marked up-regulation of the MMP-3 gene, which plays a major role in cartilage degradation, prompted us to further study its expression in FLSs stimulated with protein I/II. Confirmation of the differential expression of MMP-3 by RT-PCR analysis In order to confirm the up-regulation of the expression of MMP-3 gene in RA FLSs by protein I/II, we performed real-time PCR analysis for MMP-3. Total RNA was extracted from FLSs isolated from three RA FLSs stimulated with protein I/II or left untreated, reverse-transcribed, and then amplified by real-time PCR. MMP-3 mRNA showed a mean 36-fold up-regulation after protein I/II stimulation of RA FLSs in comparison with control cells, confirming the results obtained with the cDNA array experiments. Confirmation of the differential expression of MMP-3 by pro-MMP-3 assay To determine whether the up-regulation of the expression of MMP-3 gene by protein I/II stimulation in RA FLSs resulted in changes in gene translation and thereby contributed to an increase in the release of pro-MMP-3, we next measured the level of pro-MMP-3 in culture supernatants from control and RA FLSs that had been stimulated with protein I/II for 18 hours. As MMP-3 is first synthesized as a proenzyme (pro-MMP-3), which is subsequently cleaved to generate active MMP-3, we chose to assay pro-MMP-3 levels, as the activation process might not occur in our in vitro cultures. Figure 2 shows that control RA FLSs basally released pro-MMP-3 (1.4 ± 0.4 ng/mL) and that stimulation with protein I/II induced an increase in pro-MMP-3 secretion (3.1 ± 0.4 ng/mL, P < 0.01). Control OA FLSs basally released pro-MMP-3, but protein I/II stimulation did not significantly increase pro-MMP-3 secretion from these cells (Fig. 2 ). To confirm the activation of FLSs, we assayed IL-6 and IL-8 levels in the same supernatants used for pro-MMP-3 assay, and found that protein I/II induced a significant increase in the release of these cytokines from both RA and OA FLSs (data not shown). Discussion In this study, we analyzed gene expression changes in RA FLSs after stimulation with protein I/II, a ligand of integrin α5β1, a PRR highly expressed on FLSs. We chose to study the early induction of genes, within 4 hours of protein I/II stimulation, in order to analyze the direct effects of protein I/II, but we cannot rule out that some genes can be modulated in an autocrine fashion by some FLS-secreted mediators. Our results showed that stimulation of FLSs through this PAMP/PRR pathway induced the expression of several genes that may be involved in inflammation, matrix degradation, and invasiveness. Protein I/II strongly up-regulated the expression of genes encoding IL-6 and leukemia inhibitory factor in RA FLSs. Although a great many interleukins (IL-1 to IL-17 except IL-8 and IL-16) as well as members of the TNF family were represented on the cDNA arrays, the expression of genes for only these two cytokines was modulated by protein I/II. IL-6 and leukemia inhibitory factor, which both belong to the same family of cytokines, are known to participate substantively in the pathogenesis of murine models of arthritis as well as RA. In fact, they contribute to cartilage degradation by inducing proteoglycan resorption and inhibiting proteoglycan synthesis [ 28 ]. Elevated expression of MMP-3 gene as well as pro-MMP-3 secretion in response to protein I/II were also found in RA FLSs, whereas the expression of other MMPs (MMP-1 to MMP-3 and MMP-7 to MMP-18) present on the arrays were not affected by protein I/II stimulation. FLSs have been reported to express different kinds of MMPs, but, as was found for cytokines, protein I/II seemed to up-regulate only a limited number of genes encoding matrix-degrading enzymes. Interestingly, the only MMP up-regulated by protein I/II is MMP-3, which is one of the major MMPs involved in cartilage destruction, and it has also been associated with an increased invasive potential of cells [ 29 ]. Kyburz and colleagues found similar cytokine and MMP expression profiles (i.e. IL-6, IL-8, and MMP-3) after stimulation of RA FLSs with the TLR-2 ligand peptidoglycan [ 16 ]. Peptidoglycan is one of the bacterial components that have been detected in the synovial cavity [ 9 ] and have been shown to induce an inflammatory response in the joint [ 13 ]. In contrast to our results, peptidoglycan not only induced the expression of MMP-3 but also seemed able to up-regulate the expression of other MMPs, such as MMP-1, MMP-9, and MMP-13 [ 16 ]. Furthermore, this bacterial component up-regulated the expression of several chemokine genes, including RANTES (regulated upon activation, normal T-cell expressed and secreted) and MCP-1 (monocyte chemoattractant protein-1) [ 30 ], but these genes were not represented on the arrays used in this study. These data indicate that various PRRs present on FLSs may contribute in different ways to the aggressive behavior of RA FLSs. In this study, we also evaluated how FLSs isolated from OA patients responded to protein I/II. In our cDNA analysis, protein I/II induced strong IL-6 production in both RA and OA FLSs. In contrast to RA FLSs, OA FLSs did not express leukemia inhibitory factor or MMP-3. Our results are in contrast with those of Pierer and colleagues, who did not find a significant difference between RA and OA FLSs regarding MMP-3 and chemokine expression after stimulation with peptidoglycan. However, they found a higher chemokine level in the synovial fluid of RA patients than in OA patients [ 30 ]. The differences we noted between the gene expression profiles of protein-I/II-stimulated RA and OA FLSs could be due to particular features of RA FLSs but are probably not due to the absence of integrin α5β1, which is expressed on both RA and OA FLSs in vitro (data not shown). Moreover, we have shown that protein I/II induced the expression of the transcription factor IRF-1, the growth factor FGF-5, and vimentin in RA but not OA FLSs. IRF-1 activates transcription from genes that play a role in inflammation, such as cyclooxygenase-2 and caspase 1, but also in apoptosis or cell growth inhibition [ 31 ]. In contrast to IRF-1, FGF-5 and vimentin are associated with increased cell growth, motility, and invasiveness [ 32 , 33 ]. Protein I/II seems thus able to act on FLSs apoptosis/proliferation by inducing different genes, although their effects may be opposite. These PAMP–PRR interactions are probably not sufficient to explain activation of FLSs in RA and it is evident that additional factors, such as genetic polymorphisms, play an important role, because bacterial cell wall components, such as peptidoglycan, have also been found in joint tissues from OA patients but do not induce the inflammatory response occurring in RA. However, bacterial components probably have an important role in perpetuating joint inflammation. In support of this hypothesis, Choe and colleagues showed that innate immune functions via TLR-4 may perpetuate inflammatory mechanisms and bypass the need for IL-1 in chronic joint inflammation. In fact, in the transgenic K/BXN mice, IL-1 plays a key role in joint swelling and destruction, but administration of the TLR-4 ligand LPS along with arthritogenic serum from K/BXN mice resulted in joint swelling and destruction in IL-1R-deficient mice but not in MyD88-deficient mice [ 34 ]. Further experiments will be necessary to define the mechanisms underlying this PAMP-specific response of RA FLSs. Conclusion Our results suggest that a bacterial ligand of integrin α5β1 may contribute to the aggressive behavior of RA FLSs by inducing the release of pro-inflammatory cytokines, such as IL-6, and a cartilage-degrading enzyme, MMP-3. Abbreviations ELISA = enzyme-linked immunosorbent assay; FACS = fluorescence-activated cell sorter; FCS = fetal calf serum; FGF = fibroblast growth factor; FLS = fibroblast-like synoviocyte; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; IL = interleukin; IRF = interferon regulatory factor; LPS = lipopolysaccharide; MMP = matrix metalloproteinase; OA = osteoarthritis; PAMP = pathogen-associated molecular pattern; PCR = polymerase chain reaction; PRR = pattern-recognition receptor; RA = rheumatoid arthritis; RT = reverse transcriptase; SSC = standard saline citrate; TLR = Toll-like receptor. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MBZ carried out the study and drafted the manuscript. VAD participated in the study. DW and JS coordinated the study. All authors read and approved the manuscript.

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1064890

Quantitative ultrasonic assessment for detecting microscopic cartilage damage in osteoarthritis

Osteoarthritis (OA) is one of the most prevalent chronic conditions. The histological cartilage changes in OA include surface erosion and irregularities, deep fissures, and alterations in the staining of the matrix. The reversibility of these chondral alterations is still under debate. It is expected that clinical and basic science studies will provide the clinician with new scientific information about the natural history and optimal treatment of OA at an early stage. However, a reliable method for detecting microscopic changes in early OA has not yet been established. We have developed a novel system for evaluating articular cartilage, in which the acoustic properties of the articular cartilage are measured by introducing an ultrasonic probe into the knee joint under arthroscopy. The purpose of this study was to assess microscopic cartilage damage in OA by using this cartilage evaluation system on collagenase-treated articular cartilage in vivo and in vitro . Ultrasonic echoes from articular cartilage were converted into a wavelet map by wavelet transformation. On the wavelet map, the maximum magnitude and echo duration were selected as quantitative indices. Using these indices, the articular cartilage was examined to elucidate the relationships of the ultrasonic analysis with biochemical, biomechanical and histological analyses. In the in vitro study, the maximum magnitude decreased as the duration of collagenase digestion increased. Correlations were observed between the maximum magnitude and the proteoglycan content from biochemical findings, and the maximum magnitude and the aggregate modulus from biomechanical findings. From the histological findings, matrix staining of the surface layer to a depth of 500 μm was closely related to the maximum magnitude. In the in vivo study, the maximum magnitude decreased with increasing duration of the collagenase injection. There was a significant correlation between the maximum magnitude and the aggregate modulus. The evaluation system therefore successfully detected microscopic changes in degenerated cartilage with the use of collagen-induced OA.

Introduction Osteoarthritis (OA), also referred to as degenerative joint disease, is one of the most prevalent chronic conditions. It consists of a general progressive loss of articular cartilage, remodeling and sclerosis of the subchondral bone, and the formation of subchondral bone cysts and marginal osteophytes. In particular, the degenerative processes of articular cartilage can be accelerated by a single traumatic event, multiple repetitive loads, or local chemical and mechanical factors [ 1 ]. The histological changes that occur in cartilage in OA are a striking feature of the disease. The earliest alterations include surface erosion and irregularities, deep fissures and alterations in the staining of the matrix. The reversibility of these chondral alterations is still under debate [ 2 ]. It is expected that clinical and basic science studies will provide the clinician with new scientific information about the natural history and optimal treatment of OA at an early stage. However, a reliable method for detecting microscopic changes in early OA has not yet been established. We previously developed a novel system for evaluating articular cartilage, in which the acoustic properties of articular cartilage are measured by introducing an ultrasonic probe into the knee joint under arthroscopy [ 3 , 4 ]. The analysis system is based on wavelet transformation of the reflex echogram from articular cartilage. In detail, reflex echograms from many articular cartilage samples were transformed into wavelet maps by wavelet transformation and examined in detail. The results revealed two quantitative parameters on the wavelet maps that could be used as indices for the quantitative assessment of articular cartilage, namely the maximum magnitude and the echo duration at the 95% interval of the maximum magnitude. Macroscopic articular cartilage degeneration would result in a decreased magnitude and prolonged echo duration, as indicated by the L-shaped distribution obtained with human cadaver cartilage. However, the point at which this system can detect microscopic changes in articular cartilage degeneration is unknown. If our evaluation system can detect microscopic changes in cartilage in vivo and in vitro , it may provide a means to solve the problem of whether or not microscopic damage in OA is reversible. Moreover, this system will provide new information about the natural history and treatment of OA. The purpose of this study was to investigate the clinical usefulness of our system for evaluating microscopic damage in OA. We therefore evaluated articular cartilage with no visible disruption in collagenase-induced experimental OA, using our system to assess the microscopic damage. The present study was also performed to investigate the correlation between ultrasonic examination and biomechanical or biochemical examination. The goal of our study was to further elucidate the processes of articular cartilage degeneration with the use of our ultrasonic evaluation system. Materials and methods In vitro study Pig osteochondral plugs (diameter 5 mm; n = 77) were prepared for this study. The pig cartilage was delivered intact within 6 hours of slaughter, and the knee joints were stored at less than -30°C until use. During the preparation, the knee joints were first thawed in saline at 20°C and the joint cartilage was then exposed. Osteochondral plugs were excised from a flat area of the cartilage with a metal punch. The osteochondral samples were subsequently digested in PBS (Invitrogen Corporation, Carlsbad, CA, USA) containing 30 U/ml collagenase type ΙΙ (Worthington Biochemical Corporation, Lakewood, NJ, USA) at 37°C for 1, 2, 4, 8, 16 and 24 hours. Cartilage samples in PBS alone at 37°C were used as controls. After digestion, all the samples in each group ( n = 11) were examined by ultrasonic evaluation. Four samples in each group were prepared for mechanical testing by cartilage indentation. Four samples of each group were used for biochemical examination and were separated from the bone with the use of an autopsy saw. Three samples in each group were prepared for histological analysis. Ultrasonic analysis Our evaluation method was described in detail in a previous manuscript [ 3 ], and is illustrated in Fig. 1 . In brief, during arthroscopic examination, ultrasonic evaluation was performed by using an ultrasonic probe with a transducer fixed to the tip. The transducer (Panametrics Japan Co. Ltd., Tokyo, Japan) was small (diameter 3 mm; thickness 3 mm) and used a flat ultrasonic wave (center frequency 10 MHz). Ultrasonic echoes from the cartilage surface were converted into a wavelet map by wavelet transformation. The wavelet transformation ( W ( a , b )) of the reflex echogram ( f ( t )) is expressed by where Ψ( t ) is the mother wavelet function. For the mother wavelet function, Gabor's function was selected. The right side of Fig. 1 shows a typical ultrasonic echogram (upper) and wavelet map (lower) of an intact articular cartilage surface in vitro . The wavelet map shows a two-dimensional map whose x -axis and y -axis represent time and frequency, respectively, and the magnitude is indicated by the gray scale. As quantitative indices we used the maximum magnitude and echo duration, which was defined as the length of time that included 95% of the echo signal. These indices were calculated automatically by a computer. Articular cartilage was evaluated in vivo and in vitro with these two indices. Biochemical analysis The cartilage samples were freeze-dried overnight after measuring the wet weight. The dry weight of the samples was then measured, and the amount of water was calculated. The water content of the cartilage was determined as a percentage by using the following equation: 100 × (wet weight - dry weight)/wet weight. The samples were digested with papain (Sigma Chemical Co., St Louis, MO, USA) (40 μg/ml in 20 mM ammonium acetate, 1 mM EDTA, 2 mM dithiothreitol) for 48 hours at 65°C and then stored at -20°C until analysis. Aliquots of the digests were assayed separately for the proteoglycan and collagen contents. The proteoglycan content was estimated by quantifying the amount of sulfated glycosaminoglycans with the use of a dimethylmethylene blue dye binding assay (Polyscience Inc., Washington, PA, USA) and spectrophotometry (wavelength 525 nm). A standard curve for the analysis was generated with bovine trachea chondroitin sulfate A (Sigma). The collagen content was estimated by determination of the hydroxyproline content. Aliquots of the papain digest were hydrolyzed at 110°C in 6 M HCl for 18 hours. The hydroxyproline content of the resulting hydrolyzate was determined by the chloramine-T/Ehrlich reagent assay and spectrophotometry (wavelength 561 nm). A standard curve for this analysis was generated with L-hydroxyproline (Sigma). Biomechanical analysis A custom-made indentation testing device was used for mechanical testing to determine the creep and recovery behavior of the osteochondral samples. The samples were mounted on stainless steel plates with cyanoacrylate cement such that the rigid porous indenter tip was perpendicular to the test site on the cartilage surface. The porous indenter was made of titanium alloy particles (Ti-6Al-4V; diameter 75–180 μm). The porous permeable indenter tip (diameter 1.5 mm) was ultrasonically cleaned before testing to ensure ease of fluid flow from the specimen into the tip. The displacement of the indenter was measured using a laser measurement sensor (LB040/LB-1000; Keyence Corporation, Osaka, Japan). After equilibration under a tare load (0.0098 N), the test load (0.0098 N) was applied and the osteochondral specimen was allowed to creep to equilibrium. Equilibrium was determined as being when no further variations occurred in the observed creep value for 20 min. After creep equilibrium had been achieved, the test site was unloaded and the recovery was observed. The cartilage thickness was then measured at an exact location and orientation site with a penetrating steel needle probe. The aggregate modulus, H a , was determined from the equilibrium stress–strain data as described by Mow and colleagues [ 5 , 6 ]. Histological analysis The cartilage samples were fixed in 10% formalin, decalcified in EDTA and then embedded in paraffin. Sagittal sections of 5 μm thickness were prepared from the center of the samples and stained with Safranin-O. In vivo study The experimental OA model used in this study was created by intra-articular injection of collagenase into rabbit knee joints as reported by Kikuchi and colleagues [ 7 ]. Collagenase type ΙΙ (Worthington Biochemical Corporation) was dissolved in saline (530 U/ml), filtered with a 0.22 μm pore-size membrane, and used for the intra-articular injection. Japanese white adult rabbits (male, weight 3.0–5.5 kg; n = 24) were anesthetized with a mixture of ketamine (50 mg/ml) and xylazine (20 mg/ml) at a ratio of 2:1, by means of a dose of 1 ml/kg body weight injected intramuscularly into the gluteal muscle. After both knee joints had been shaved and sterilized, 0.5 ml of collagenase solution was injected intra-articularly into the right knee joint and/or saline was injected into the left knee joint as a control. The injection was performed twice, on days 1 and 4 of the experiment. The rabbits were returned to their cages and allowed to move freely without joint immobilization. For each experiment, four rabbits were killed at 0, 1, 4, 8, 12 and 16 weeks after the start of the experiment with an overdose of phenobarbital sodium salt, although two rabbits were discounted from the study because of a bacterial infection and a patellar dislocation, respectively. All the remaining knee joints were opened and the cartilage surfaces were observed macroscopically and photographed. The knee joint was dissected free from all soft tissues and the tibia was removed. The distal femur was cut proximally to the patellofemoral joint and cartilage samples were taken. Ultrasonic and biomechanical analyses were performed on the medial femoral condyle. For histological analysis, the lateral femoral condyles of the cartilage samples were fixed in 10% formalin, decalcified in EDTA and then embedded in paraffin. Sagittal sections of 5 μm thickness were prepared from the center of the samples and stained with Safranin-O. This study was approved by the Nara Medical University Ethics Committee. Statistical analysis All data in this study are reported as means ± SD. The changes in the maximum magnitude, echo duration, water content, chondroitin sulfate content, hydroxyproline content and aggregate modulus with respect to the collagenase treatment duration were analyzed by one-way analysis of variance. Pearson correlations were performed to determine the associations between the ultrasonic data and the biochemical or biomechanical data. The significance level was set at P < 0.05. Results In vitro study Ultrasonic measurement The maximum magnitude decreased as the duration of collagenase digestion increased. There was a rapid decrease in the maximum magnitude after 8 hours of digestion in comparison with the control, and then a gradual decrease from 8 to 24 hours (Fig. 2a ). There was no significant change in echo duration over the time course of digestion (Fig. 2b ). Biochemical measurement The water content gradually increased over the time course of collagenase digestion (Fig. 3a ). At the same time, the chondroitin sulfate content decreased rapidly with increasing duration of digestion. There was a rapid decrease in the chondroitin sulfate content after 8 hours of digestion and then a gradual decrease from 8 to 24 hours (Fig. 3b ). There was a significant correlation between maximum magnitude and chondroitin sulfate content ( R 2 = 0.6164, P < 0.01) (Fig. 3c ). There was very little change in hydroxyproline content during collagenase digestion (Fig. 3d ). There was no significant correlation between maximum magnitude and hydroxyproline content ( R 2 = 0.069, P = 0.176) (Fig. 3e ). Biomechanical measurement The aggregate modulus rapidly decreased during the first 4 hours of collagenase digestion, but there was no subsequent change from 4 to 24 hours (Fig. 4a ). There was a significant correlation between maximum magnitude and aggregate modulus ( R 2 = 0.739, P < 0.01) (Fig. 4b ). Histological findings Representative sections of collagenase-digested cartilage stained with Safranin-O are shown in Fig. 5 . In control cartilage, the Safranin-O staining of the extracellular matrix appeared almost homogeneous. After 1 hour of digestion, the superficial layer showed slight changes in the Safranin-O staining. After 8 hours of digestion, the surface layer to a depth of 500 μm was not stained with Safranin-O. Over the course of degeneration time, Safranin-O staining became less intense in the deeper layers. In vivo study Macroscopic and histological findings Figure 6 shows the macroscopic and histological findings of the collagenase-injected articular cartilage. Macroscopically, cartilage surface changes were not detected on either femoral condyle of the rabbits. Histologically, chondrocyte cluster formation was seen and the surface layer was not stained with Safranin-O at 4 weeks after injection. Several fissures were observed in the surface area at 8 weeks after injection. Ultrasonic measurement The maximum magnitude decreased with increasing time after collagenase injection. There was a rapid decrease in the maximum magnitude at 4 weeks after injection, in comparison with control samples (Fig. 7a ). However, there was no significant decrease in echo duration after injection (Fig. 7b ). Biomechanical measurement In the same manner as for the in vitro study, the relationship between the maximum magnitude and the aggregate modulus was investigated: there was a significant correlation ( R 2 = 0.5173, P < 0.05) (Fig. 8 ). Discussion The results of this study indicate that ultrasonic examination is promising as a minimally invasive method of evaluating microscopic damage in OA at an early stage. To evaluate microscopic damage to articular cartilage, reflex echoes from the cartilage were transformed into a wavelet map, and the echo duration and maximum magnitude were calculated and used as quantitative indices of cartilage degeneration. According to this study, the maximum magnitude was shown to reflect the proteoglycan content from biochemical analysis, the aggregate modulus from biomechanical analysis and the decrease in Safranin-O staining of the cartilage surface from histological analysis. There are numerous clinical methods of grading the degenerative changes and injuries to articular cartilage at the time of surgery or arthroscopy with direct observation of the cartilage surface [ 8 - 10 ]. The overall observation from macroscopic findings and probing is that cartilage lesions vary in location, depth, size and shape. In addition, it is well established that probing cannot evaluate the cartilage condition quantitatively. As a quantitative method that could replace probing, attempts have been made to evaluate cartilage using magnetic resonance imaging, but such in situ evaluation has been performed only in experimental trials [ 11 - 13 ]. Cartilage biopsy and histological examination have been performed to evaluate articular cartilage clinically. However, it is still difficult to measure the degree of cartilage degeneration in a non-destructive manner. Therefore, further developments in diagnostic techniques are required for in situ evaluation. Several different approaches have been investigated to improve the techniques for diagnosing the condition of cartilage, including optical coherence tomography [ 14 ], electromechanical evaluation [ 15 ], mechanical indentation [ 16 ], ultrasonic evaluation [ 17 , 18 ] and ultrasonic indentation [ 19 - 21 ]. Most of these approaches are still under development and only a few devices have been used successfully for cartilage evaluation during clinical investigations. Ultrasonic indentation methods are capable of determining the cartilage thickness and deformation, and can therefore be used to determine the Young's modulus of articular cartilage. In a clinical context, Lyyra and colleagues [ 19 ] reported the efficacy of an ultrasonic indentation instrument under arthroscopic control for the quantification of cartilage stiffness, as evaluated with three human cadaver knees. This might prove to be suitable for clinical use, but the rod of the instrument (5 mm in diameter) is too thick to evaluate the cartilage in all regions of knee joints or the cartilage in ankle and wrist joints [ 21 ]. In contrast, our ultrasonic probe is so small (4 mm wide and 2.5 mm thick) that we can evaluate living human joint cartilage under arthroscopy. Moreover, we have reported clinically relevant data obtained from living human cartilage in situ [ 4 ]. Ultrasonic measurement under arthroscopy has three merits in comparison with arthroscopic indentation. The first is that the possibility of tissue damage caused by the measurement device can be completely excluded owing to the non-contact measurement. The second is that the evaluation system can predict the histological findings of cartilage on the basis of studies in experimental animal models [ 22 , 23 ]: hyaline cartilage has a higher maximum magnitude than fibrous tissue, whereas imperfectly regenerated cartilage has a lower maximum magnitude, even when only fibrous tissue and fibrocartilage are present in the superficial layer of the repaired tissue. The third is that the ultrasonic probe used in the evaluation is so small that it should be useful not only for articular cartilage in the knee joint but also for that in the wrist and ankle joints under arthroscopy. Before this investigation, the maximum magnitude and echo duration were used as quantitative indices of degenerated cartilage, but it was not known what the indices were closely related to [ 3 ]. However, this study using a collagenase-induced OA model clarified the significance of the maximum magnitude. From an acoustic point of view, the maximum magnitude is a modification of the echo reflection from the cartilage surface, and hence differences in the surface reflection indicate significant alterations in the acoustic impedance among degenerated cartilage samples. From the histological findings, the matrix staining of the surface layer to a depth of 500 μm was closely related to the maximum magnitude. From a biochemical point of view, the proteoglycan content was more related to the maximum magnitude than the type ΙΙ collagen content. The collagen content showed little change after collagenase digestion in this study, although the collagen meshwork is widely known to be the main reflector of ultrasound and the source of ultrasound backscatter [ 24 - 26 ]. However, the apparent inconsistency between these observations and our results would be due to differences between the reflex echoes from flat ultrasound and focal ultrasound. From a biomechanical point of view, the maximum magnitude was related to the aggregate modulus from the mechanical properties of the articular cartilage. Therefore, the maximum magnitude reveals microstructural changes in degenerated cartilage and can provide diagnostically important information about the degenerated cartilage. In this study, the echo duration showed no change over the time course of collagenase digestion. From the histological findings, the cartilage surface was smooth after collagenase digestion in the in vitro study and had several fissures only at 8 weeks after the collagenase injection. According to the previous human cadaver study, the echo duration becomes longer with macroscopic roughening of the cartilage surface due to wear [ 3 ]. Moreover, Myers and colleagues showed that the width of the echo band can be related to the depth of fibrillation in the macroscopic degenerative cartilage surface [ 27 ]. The echo duration is therefore closely related to the macroscopic fibrillation of articular cartilage. There are three limitations to this study. First, the cartilage samples in this study were not human OA cartilage but collagenase-treated articular cartilage. However, OA-like changes were observed in the experimental animals after induction by intra-articular injection of collagenase, and enzyme-induced OA models are also used to investigate the pathogenesis of OA. Second, our evaluation system could not detect any microscopic roughness of the articular cartilage by using the index of echo duration. To detect this histological change, high-frequency ultrasound might be required. Finally, we did not detect the progression of cartilage degeneration in living humans. However, we have reported relevant clinical acoustic data from human cartilage in situ under arthroscopy. Further studies are therefore needed to determine whether this evaluation system will be beneficial for studying the pathogenesis of OA. Conclusion Ultrasonic evaluation using a wavelet map can support the evaluation of microscopic damage of articular cartilage in OA. The evaluation system is suitable for clinical use under arthroscopy. This evaluation successfully predicted the histological findings of degenerated cartilage with the use of a collagen-induced OA model. We believe that our findings offer the potential for standardized evaluation as an adjunct to further research in this field, which will lead to a reliable method for the quantification of articular cartilage treatments. Abbreviations OA = osteoarthritis. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KH conceived the study, participated in its design and performed all the experiments. KI and YM performed biomechanical studies. YT participated in the design of the study and participated in the in vivo study. All authors read and approved the final manuscript.

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1064891

Tumor necrosis factor alpha and epidermal growth factor act additively to inhibit matrix gene expression by chondrocyte

The failure of chondrocytes to replace the lost extracellular matrix contributes to the progression of degenerative disorders of cartilage. Inflammatory mediators present in the joint regulate the breakdown of the established matrix and the synthesis of new extracellular matrix molecules. In the present study, we investigated the effects of tumor necrosis factor alpha (TNF-α) and epidermal growth factor (EGF) on chondrocyte morphology and matrix gene expression. Chondrocytes were isolated from distal femoral condyles of neonatal rats. Cells in primary culture displayed a cobblestone appearance. EGF, but not TNF-α, increased the number of cells exhibiting an elongated morphology. TNF-α potentiated the effect of EGF on chondrocyte morphology. Individually, TNF-α and EGF diminished levels of aggrecan and type II collagen mRNA. In combination, the effects of TNF-α and EGF were additive, indicating the involvement of discrete signaling pathways. Cell viability was not compromised by TNF-α or by EGF, alone or in combination. EGF alone did not activate NF-κB or alter NF-κB activation by TNF-α. Pharmacologic studies indicated that the effects of TNF-α and EGF alone or in combination were independent of protein kinase C signaling, but were dependent on MEK1/2 activity. Finally, we analyzed the involvement of Sox-9 using a reporter construct of the 48 base pair minimal enhancer of type II collagen. TNF-α attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-α or basal activity. TNF-α and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function. These findings provide critical insights into the control of chondrocytes through the integration of multiple extracellular signals.

Introduction The role of epidermal growth factor (EGF) in the development of articular cartilage and the pathogenesis of arthritis is poorly understood. During development, EGF produced by the apical ectodermal ridge promotes the outgrowth of the limb bud mesoderm; however, migration away from the apical ectodermal ridge and downregulation of EGF expression in the mesodermal cells is necessary for differentiation of this cell population into chondrocytes [ 1 ]. We previously found that EGF encourages expansion of early committed chondrocytes but prevents the expression of link protein and aggrecan [ 2 ], two extracellular matrix components that are necessary for proper cartilage organization [ 3 ]. Proteoglycan accumulation is inhibited following treatment of mature articular chondrocytes with EGF in a monolayer or an organ culture [ 4 , 5 ]. We recently demonstrated an increase in proton efflux from chondrocytes treated with EGF resulting in localized acidification of the microenvironment that may contribute to altering both responsiveness of chondrocytes to extracellular stimuli and the activity of matrix metalloproteinases [ 6 ]. EGF is detectable in the synovial fluid of rheumatoid arthritis patients and influences the growth of synovial cells [ 7 ]. However, the effects on cartilage of EGF, alone or in conjunction with other mediators associated with inflammation, are poorly characterized. Among the inflammatory mediators associated with joint diseases, tumor necrosis factor alpha (TNF-α) is well established as a key mediator in the progression of cartilage degeneration. High levels of TNF-α are detected in the synovial lining of rheumatic joints and in chondrocytes of osteoarthritic joints [ 8 ]. TNF-α promotes further expression of cytokines and chemokines by synovial cells and chondrocytes, thereby sustaining a renewal of local inflammatory mediators (reviewed in [ 9 , 10 ]). The presence of TNF-α correlates with a general loss of cartilage matrix molecules, such as type II collagen and aggrecan, due to increased production of matrix metalloproteinases and a reduction in synthesis of matrix molecules [ 11 ]. We recently demonstrated that activation of the NF-κB and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways contributes to the TNF-α-mediated reduction of transcription of the type II collagen and link protein genes, as well as to a reduction in the steady-state mRNA levels of these key extracellular matrix components [ 12 ]. In rheumatic joints, elevated levels of EGF in the synovial fluid contribute to hyperplasia of the synovial lining, where synovial cells display increased expression of the EGF receptor ErbB-2 (also known as c-neu or HER2) [ 7 , 13 , 14 ] and amplify IL-1-mediated release of prostaglandin E 2 from synovial cells [ 15 ]. However, the combined effects of EGF and TNF-α have not been investigated previously. The objective of the present study was to determine whether EGF potentiates the response of chondrocytes to TNF-α. We investigated changes in chondrocyte morphology and function. The expression of type II collagen that is responsible for the structural integrity of articular cartilage and aggrecan that imparts resilience to the tissue were used as measures of chondrocyte function. Co-administration of TNF-α and EGF in the present study resulted in a marked increase in the proportion of elongated cells and an additive decrease in matrix gene expression. These changes in morphology and gene expression were found to be controlled in part by the MAPK pathway. Furthermore, EGF exerts its effects on matrix gene expression through a pathway independent of Sox-9. Materials and methods Primary cell culture Articular chondrocytes were isolated from the distal femoral condyles of 1-day-old Sprague–Dawley rats (Charles River, St Hyacinthe, QC, Canada) as previously described [ 12 ]. The Animal Use Subcommittee of the University of Western Ontario Council on Animal Care approved the use of rats for these studies. Cells were plated at a density of 4.25 × 10 4 cells/cm 2 on tissue culture-treated plates (Falcon; BD Biosciences, Mississauga, ON, Canada) and cultured in RPMI 1640 media supplemented with 5% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin and 10 mM HEPES (Invitrogen Life Technologies Inc., Burlington, ON, Canada). Culture media was replaced every 3 days. Culture medium was replaced with serum-free medium 16–20 hours prior to experiments. Primary chondrocyte cultures were treated with TNF-α (30 ng/ml; Sigma Aldrich, Oakville, ON, Canada), with EGF (10 ng/ml; Sigma Aldrich) or with vehicle (phosphate-buffered saline + 0.01% bovine albumin; Roche Diagnostics, Laval, QC, Canada) in serum-free medium. These concentrations were previously found to elicit maximal responses from these cells [ 6 , 12 ]. For analysis of signaling pathways, cells were treated prior to addition of TNF-α or EGF with pharmacologic inhibitors including 2-[1-(3-dimethylaminopropyl)-1 H -indol-3-yl]-3-(1 H -indol-3-yl)-maleimide (10 μM bisindolylmaleimide [BIS] I, protein kinase C [PKC] inhibitor), or 2,3- bis (1 H -indol-3-yl)- N -methylmaleimide (10 μM BIS V, inactive analog of BIS I), 1,4-diamino-2,3-dicyano-1,4- bis (2-aminophenylthio)-butadiene (10 μM U0126, mitogen-activated protein kinase kinase 1 and 2 [MEK1/2] inhibitor; Promega, Madison, WI, USA), and 1,4-diamino-2,3-dicyano-1,4- bis (methylthio)-butadiene (10 μM U0124, inactive analog of U0126). BIS I was used at a concentration that was greater than 500 times the inhibitory concentration 50% for conventional PKCs and twice the inhibitory concentration 50% for PKCζ. U0126 was used at a concentration previously found to be effective for inhibiting the phosphorylation of ERK1/2 [ 12 ]. The pharmacologic agents were obtained from EMD Biosciences (Calbiochem, La Jolla, CA, USA) unless otherwise stated. Imaging Digital images of confluent monolayers were obtained using a Sony Power HAD 3CCD mounted onto a Nikon TMS inverted phase-contrast microscope (20 × objective magnification) (Nikon Canada Inc., Mississauga, ON, Canada). Images were acquired with NorthernEclipse V.5 software (Empix, Mississauga, ON, Canada). For the present study, an elongated cell was defined as having a predominant axis length exceeding three times the maximum width of the cell. The number of elongated cells per field of view (1.376 mm 2 ) was counted and averaged. RNA extraction and northern blot analysis Total RNA was collected from cells using the acid–guanidium–phenol–chloroform extraction method (Trizol; Invitrogen Life Technologies Inc.), according to the manufacturer's instructions. RNA was quantified by ultraviolet spectrophotometry. Total RNA (10 μg) was resolved on a 1.1% agarose gel containing formaldehyde. Equivalent loading of samples was verified by ethidium bromide staining before RNA was transferred to Nytran membranes (Schleicher & Schuell, Keene, NH, USA). RNA was fixed to the Nytran membrane by incubation at 80°C for 2.5 hours under vacuum. cDNA probes corresponding to the mouse C-propeptide of type II collagen (pKN225) [ 16 ], to 18S rRNA (DECAtemplate 18S mouse; Ambion, Austin, TX, USA), and to the C-terminus of rat aggrecan [ 17 , 18 ] were labeled with [α 32 P]dCTP (3000 Ci/mmol; Perkin Elmer, Woodbridge, ON, Canada) by a random-primed oligonucleotide method (Prime-a-gene labeling kit; Promega). Membranes were hybridized with cDNA probes and processed as described previously [ 19 ]. Preparation of cell extracts and immunoblotting Cell extracts were prepared as described previously [ 12 ]. Equivalent amounts of protein (15–30 μg) were resolved by electrophoresis on 7.5% polyacrylamide-SDS gels. Protein was transferred to nitrocellulose membrane (Schleicher & Schuell) by electroblotting. Transfer and equivalent loading was verified by subsequent staining with Ponceau Red (3-hydroxy-4-(2-sulfo-4-[4-sulfophenylazo]-phenylazo)-2,7-napthalenedisulfonic acid) [ 20 ]. Immunoblotting was performed by blocking the membrane for 1 hour with 5% non-fat milk (Carnation, North York, ON, Canada)/TBS 0.5% Tween. Membranes were incubated with antibodies for poly(ADP ribose) polymerase (PARP) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-specific ERK1/2 (Anti-active MAPK; Promega) or ERK1 and ERK2 (Santa Cruz Biotechnology) according to the manufacturer's instructions. Target signals were detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology Inc., Rockford, IL, USA) and exposure to Hyperfilm ECL (Amersham Biosciences, Baie d'Urfé, QC, Canada). Apoptosis analysis Cells were seeded on Permanox chamber slides (Nalge Nunc, Naperville, IL, USA) at a density of 550 cells/mm 2 . Following treatment with factors, slides were fixed with 4% formalin solution. Apoptosis was assessed by the terminal deoxynucleotidyltransferase end-labeling with fluorescein-dUTP (TUNEL) method (Roche Diagnostics) as described in the manufacturer's instructions. Positive controls were treated for 10 min with DNAse I (Roche Diagnostics) to induce DNA breaks. Fluorescein activity was imaged by laser scanning confocal microscopy (LSM 510 Meta; Carl Zeiss Microscopy, Jena, Germany). MTT assay for cell viability Cell viability was analyzed using the Cell Proliferation Kit I (MTT; Roche Diagnostics) following the manufacturer's instructions. Cells were seeded on 96-well plates at 400 cells/mm 2 , were cultured for 5 days and were then treated with TNF-α, or with EGF, or with TNF-α + EGF for an additional 24 hours. The colorimetric reaction was read on a μQuant spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA) at 550 nm and 690 nm. The reading at 690 nm was used as a reference wavelength to calculate a corrected absorbance (A 550 – A 690 ). Transfections and luciferase reporter analysis Chondrocytes were transfected with reporter constructs for NF-κB (Clontech, Palo Alto, CA, USA) or the type II collagen enhancer region (pGl3 4 × 48; a kind gift from Dr TM Underhill, The University of British Columbia, Vancouver, BC, Canada) [ 21 ]. Briefly, per transfection reaction, 0.1 μg reporter DNA and 2 ng PRL-SV40, a constitutively expressed renilla luciferase plasmid for monitoring transfection efficiency, were incubated with Fugene 6 transfection reagent (Roche Diagnostics). The mixture was added to a well of a 48-well plate and overlayed with 3.5 × 10 4 cells in serum-free culture medium. After 5 hours, medium containing serum was added to the wells. The following day, cells were treated with TNF-α (30 ng/ml), with EGF (10 ng/ml), with a combination of both or with vehicle in serum-free medium for 24 hours. The cells were lysed with 1 × Reporter Lysis Buffer (Promega) and luciferase activity quantified using the Dual Luciferase Assay System (Promega). Nuclear extract preparation and electrophoretic mobility shift assays Isolation of nuclear extracts and the electrophoretic mobility shift assay were performed as previously described [ 12 , 22 ]. The double-stranded oligonucleotide containing the κB recognition sequence was purchased from Santa Cruz Biotechnology. Densitometry and statistical analysis All data are representative of at least three independent experiments. Bands appearing on exposed film were analyzed using Kodak Digital Science software (Eastman Kodak, Rochester, NY, USA). Relative expression levels of type II collagen mRNA and aggrecan mRNA were standardized to the expression levels of 18S rRNA. One-way analysis of variance or repeated-measures analysis of variance followed by Tukey–Kramer post-test comparisons was performed to determine the statistical significance of differences among means (GraphPad Prism version 3.00; GraphPad Software, San Diego, CA, USA). Results Effects of TNF-α and EGF on chondrocyte morphology The cellular morphology reflects the differentiation status of cells such as chondrocytes. For example, a change from a rounded to a more elongated morphology in response to EGF by CFK2 chondrocytic cells is associated with a diminished onset of expression of aggrecan and link protein gene [ 2 ]. To determine whether the morphology of primary chondrocytes expressing the matrix was affected by TNF-α or EGF, live cultures were examined by phase-contrast microscopy (Fig. 1a ) and the number of elongated cells per field was quantified (Fig. 1b ). Previous studies established concentrations for TNF-α (30 ng/ml) [ 12 ] and EGF (10 ng/ml) [ 6 ] for maximal activation of signaling pathways in primary chondrocytes. Following a 24-hour treatment with vehicle (control) or TNF-α, the monolayers exhibited a 'cobblestone' appearance. In contrast, treatment with EGF promoted cell elongation, a change that was significantly potentiated by the presence of TNF-α. The distribution and arrangement of actin filaments were analyzed by phalloidin labeling. An increase in stress fibers was observed in elongated cells; however, the density of cells and prevalence of filamentous actin throughout the monolayer precluded any further quantitative analysis (data not shown). Effects of TNF-α and EGF on levels of aggrecan and type II collagen mRNA We previously demonstrated that TNF-α reduces transcriptional expression of type II collagen and link protein genes [ 12 ]. In the present study, we characterized the effect of TNF-α on aggrecan mRNA levels and determined whether EGF altered type II collagen and aggrecan mRNA levels in the presence or absence of TNF-α. Cultures were treated with TNF-α or EGF individually or in combination (TNF-α + EGF) and the levels of aggrecan and type II collagen mRNA were analyzed (Fig. 2 ). Following 24 hours of treatment with TNF-α, levels of aggrecan and type II collagen mRNA were decreased by 42 ± 4% and 39 ± 2%, respectively. EGF alone decreased levels of aggrecan and type II collagen mRNA by 44 ± 5% and 42 ± 4%, respectively. Treatment of chondrocytes with TNF-α + EGF resulted in additive losses of aggrecan and type II collagen mRNA (93 ± 2% and 79 ± 4%, respectively). Treatment with TNF-α for 4 hours prior to the addition of EGF for the remainder of the 24 hours resulted in comparable decreases in levels of aggrecan and type II collagen mRNA (89 ± 2% and 81 ± 7%, respectively; data not shown). The combination of TNF-α and EGF therefore produces an additive decrease in both aggrecan and type II collagen mRNA levels, suggestive of discrete signals regulating mRNA expression by each factor. TNF-α and EGF do not alter the extent of apoptosis in the chondrocyte culture Cultures treated with TNF-α, with EGF or with TNF-α + EGF were assessed for evidence of apoptosis using an early marker, PARP (Fig. 3a ). PARP is a 116 kDa protein involved in DNA repair [ 23 ] that is cleaved as part of the caspase cascade initiated in cells undergoing apoptosis. Cell extracts were immunoblotted for the presence of intact and cleaved forms of PARP. Neither loss of intact PARP nor the appearance of cleaved moieties (85 kDa) was detected following 24 hours of treatment with TNF-α, with EGF or with TNF-α + EGF. Interestingly, TNF-α + EGF increased the amount of PARP present in the chondrocytes. To confirm the lack of apoptosis in factor-treated cultures, the presence of DNA strand breaks was evaluated by in situ labeling (TUNEL) (Fig. 3b ). TUNEL labeling was not detected following any of the treatments. Cell viability was also assessed using the MTT assay (Fig. 4 ). TNF-α did not significantly alter cell viability after 24 hours. EGF caused an increase in metabolism of the tetrazolium salt at 24 hours that was not, however, changed significantly by co-addition of TNF-α, probably reflecting an increase in chondrocyte number. These results suggest that reduction in aggrecan and type II collagen mRNA levels induced by TNF-α and EGF are not correlated with initiation of programmed cell death (Fig. 3 ) or a decrease in cell number (Fig. 4 ). EGF does not alter NF-κB activation by TNF-α Several signaling pathways known to mediate the effects of TNF-α and EGF were next investigated. We previously demonstrated that primary articular chondrocytes treated with TNF-α exhibit sustained activation of NF-κB at 24 hours, and that NF-κB partially mediated the reduction in type II collagen mRNA induced by TNF-α [ 12 ]. To assess whether changes in NF-κB activity contribute to the observed decrease in aggrecan and type II collagen mRNA, chondrocytes were transfected with a κB-driven reporter to detect functional activation of NF-κB (Fig. 5a ). As expected, TNF-α significantly increased reporter levels. In contrast, EGF did not activate NF-κB or alter activation of NF-κB by TNF-α. Furthermore, sustained NF-κB activation induced by TNF-α was unchanged by EGF as determined by the electrophoretic mobility shift assay (Fig. 5b ). The heightened decrease in aggrecan and type II collagen mRNA induced by TNF-α + EGF was therefore not the result of altered NF-κB activation. Inhibition of PKC does not prevent reduction in levels of aggrecan or type II collagen mRNA by TNF-α and EGF TNF-α and EGF have been found to activate PKC in other cell types. The role of PKC signaling in the reduction of aggrecan and type II collagen mRNA by TNF-α and EGF was examined using a pharmacologic inhibitor of PKC (Fig. 6 ). Cultures were pretreated with the PKC inhibitor BIS I at a concentration known to inhibit activation of several PKC isoforms, specifically PKCα, PKCβ I , PKCβ II , PKCγ, PKCδ, PKCε, and PKCζ [ 24 , 25 ], or with BIS V, an inactive analog of BIS I. TNF-α and/or EGF were added and the mRNA levels were analyzed by northern blot. Pretreatment with either BIS I or BIS V did not prevent the reduction in levels of aggrecan and type II collagen mRNA by TNF-α, by EGF or by TNF-α + EGF. Activation of PKC thus does not appear to be involved in the regulation of matrix gene expression by TNF-α and EGF. Neither BIS I nor BIS V treatment alone significantly altered the levels of aggrecan and type II collagen mRNA. Changes in cell morphology induced by EGF or the combination of TNF-α and EGF are suppressed by inhibition of MAPK EGF is a well-characterized activator of the MAPK/ERK pathway [ 26 ]. We investigated whether the changes observed in cell morphology were dependent on the MAPK/ERK pathway. Chondrocytes were treated with the selective inhibitor of MEK1/2 activation, U0126, at a concentration previously found to inhibit ERK1/2 phosphorylation in these cells [ 12 ], or the inactive analog U0124 followed by TNF-α and/or EGF or vehicle. After 24 hours, the chondrocytes treated with U0124 or with U0126 followed by treatment with either vehicle or TNF-α exhibited similar morphology (Fig. 7 a) to that observed in the absence of pharmacological agents (Fig. 1 ). The number of elongated cells per field was also counted (Fig. 7b ). The number of elongated cells induced by EGF and by TNF-α + EGF was markedly reduced following pretreatment with U0126. Cultures treated with U0124 followed by EGF or by TNF-α + EGF exhibited changes in the number of elongated cells comparable with vehicle-pretreated cultures. Changes in morphology in response to EGF and to TNF-α + EGF are thus dependent on a MEK1/2-regulated process. Inhibition of the MAPK pathway prevents TNF-α and EGF-mediated loss of aggrecan and type II collagen mRNA We previously demonstrated that activation of the MAPK signaling cascade contributed to a reduction in type II collagen mRNA levels [ 12 ]. The involvement of the MAPK/ERK pathway in the reduction in aggrecan and type II collagen mRNA levels by EGF and TNF-α was investigated. Cells were pretreated with U0124 or U0126 followed by the addition of TNF-α and/or EGF or vehicle for 24 hours (Fig. 8 ). Cultures treated with the inactive inhibitor exhibited no change in either the basal levels of mRNA (data not shown) or the extent of reduction in aggrecan or type II collagen mRNA levels from that of untreated cultures (Fig. 2 ). U0126 prevented the losses mediated by the individual factors and partially protected against the effect of TNF-α and EGF in combination. To determine the MAPK responsiveness of chondrocytes to the combination of these factors, the phosphorylation of ERK1/2 was assessed. Cell extracts were collected from cultures treated for 4 hours with vehicle or with TNF-α prior to the addition of EGF, and were immunoblotted with antibody specific for phosphorylated forms of ERK1/2 (Fig. 9 ). In a previous study [ 12 ], we demonstrated phosphorylation of ERK1/2 within 15 min of the addition of TNF-α. In the present study, we found that phosphorylation of ERK1/2 returned to control levels after 4 hours of treatment with TNF-α. Phosphorylation of both ERK1 and ERK2 by EGF was apparent after 5 min and had not diminished by 30 min in the vehicle-treated cells. As both simultaneous and sequential addition of TNF-α and EGF produced comparable reductions in matrix gene mRNA levels, the MAPK response to EGF was assessed in the presence or absence of a 4-hour TNF-α pretreatment. Cultures that received TNF-α pretreatment followed by EGF had levels of ERK1/2 phosphorylation comparable with those cultures treated with EGF alone. An increase in the level of phosphorylation therefore did not contribute to the greater loss of matrix gene mRNA expression. Effects of EGF on type II collagen mRNA are independent of the Sox-9 response region of the type II collagen enhancer Expression of both type II collagen and aggrecan genes is regulated by a transcriptional complex containing members of the Sox family, namely Sox-5, Sox-6, and Sox-9 [ 27 , 28 ]. A Sox-9 regulatory element resides in the type II collagen enhancer. To determine whether the reduction in type II collagen mRNA levels by EGF involves the minimal enhancer region, chondrocytes were transfected with a reporter construct for this 48 base pair region [ 21 ] and were treated with TNF-α and/or EGF or with vehicle (Fig. 10 ). As previously demonstrated [ 12 ], TNF-α markedly reduced activity at this regulatory region consistent with impairment of Sox-9 binding or activity. In contrast, EGF did not alter the activity of the enhancer region and the effect of TNF-α + EGF was not significantly different from that of TNF-α alone. These results indicate that the EGF-mediated reduction in levels of type II collagen mRNA is independent of the minimal enhancer regulatory region and, therefore, probably independent of Sox-9 regulation. Discussion The morphology of cells is regulated by extracellular signals including soluble mediators and a matrix. Moreover, a relationship exists between the morphology and the state of differentiation. In the present study we investigated the effects of TNF-α, a factor that did not alter cell morphology, and the effects of EGF, a factor that induced a change in cell morphology. It is well established that removal of chondrocytes from their environment rich in extracellular matrix to a two-dimensional culture causes a change in morphology from rounded/cuboidal to more flattened and spread cells [ 29 ]. These morphological changes are accompanied by changes in the organization of the actin cytoskeleton [ 30 ]. Coincident with the change in chondrocyte shape is a loss of expression of phenotypic markers such as type II collagen and aggrecan [ 29 , 31 , 32 ], a phenomenon referred to as dedifferentiation. In addition, non-matrix factors can influence the organization of the actin cytoskeleton and can have profound effects on differentiation of chondrocytes. For example, bone morphogenetic protein-7 and IL-1 promote and restrict chondrogenesis, respectively, through changes in the distribution of focal adhesion proteins that are essential components of the cytoskeletal complexes. Their induction or their repression, respectively, of type II collagen gene expression involves altering the organization of the actin cytoskeleton [ 33 ]. In the present study, however, while only EGF induced a notable change in cell morphology, both TNF-α and EGF brought about comparable reductions in the mRNA levels of cartilage matrix genes. Morphological changes may thus be linked to expression of a differentiated phenotype for some inflammatory mediators. Cell survival is essential for ensuring ongoing homeostatic maintenance of cartilage and for bringing about repair to damaged cartilage. Maintaining integrity of the nuclear material is critical, and the repair of damaged DNA is dependent on PARP. When a cell initiates apoptosis, PARP is targeted by caspase 3 and caspase 7, and is cleaved, rendering the enzyme inactive (properties of PARP are reviewed in [ 34 , 35 ]). Furthermore, in a caspase-independent manner, overactivation of PARP can lead to cell death through the release of apoptosis-inducing factor [ 35 ]. In the present study, apoptosis was not initiated by TNF-α and/or EGF as there was no cleavage of PARP and no evidence of DNA fragmentation (TUNEL staining). TNF-α and EGF separately had no effect on levels of PARP; however, when TNF-α and EGF were combined, increased levels of PARP were found. It is not clear whether the increase in PARP is due to increased de novo synthesis or to prevention of turnover. A similar phenomenon has been observed in retinal tissue following ischemia-reperfusion injury [ 36 ], another situation in which multiple inflammatory mediators are present. The upregulation of PARP by the combination of TNF-α and EGF suggests a protective response by chondrocytes as a certain cellular threshold for tolerance is exceeded. Furthermore, PARP can mediate transcriptional suppression through direct interaction with promoter DNA or modification of regulatory transcription factors such as NF-κB. Further investigation would be needed to determine whether PARP is involved in the reduced mRNA levels of type II collagen and aggrecan. TNF-α and EGF activate several intracellular signaling pathways through their respective receptors or via cross-talk of pathway components. The concentrations of factors used in this study are sufficient to elicit maximal responses in these chondrocytes [ 6 , 12 ]. The additive nature of the decrease in aggrecan and type II collagen mRNA suggests the involvement of at least two signaling pathways activated by TNF-α and EGF. We have previously shown that TNF-α does not activate p38 in this system [ 12 ]. Furthermore, NF-κB activity has been implicated in mediating the effects of TNF-α and IL-1β on the expression of type II collagen [ 12 , 37 ]. Disruption of the actin cytoskeleton with cytochalasin D or with latrunculin B results in an increase in NF-κB activation [ 38 ]. Although inducing a change in morphology, EGF did not alter the activity of NF-B, either basal or that induced by TNF-α. Similarly, PKC is typically activated in response to TNF-α or EGF and can mediate an activation of MAPK signaling [ 39 , 40 ]. In the present study, however, inhibition of several isoforms of PKC did not alter the observed losses in aggrecan and type II collagen mRNA. The pharmacological inhibitor of MEK1/2 suppressed mRNA loss and changes in cell morphology. The MAPK/ERK pathway is thereby at least partially involved in regulating the aggrecan and type II collagen genes and in remodeling of the cytoskeleton in response to factors present during inflammation. The MAPK/ERK pathway plays an important role in directing alterations of the cytoskeleton. For example, constitutively active MAPK induces morphological changes in fibroblasts, coinciding with disruption of stress fibers and disappearance of focal adhesions [ 41 ]. The MEK/ERK pathway is crucial in the control of hepatocyte cell morphology and cell cycle in response to EGF [ 42 ]. In chondrocytes, the MAPK/ERK pathway may have dual function in controlling the alteration in gene expression during cartilage degeneration and cytoskeletal remodeling. Induction of dedifferentiation may be a consequence of proliferation induced by growth factors, a process involving MAPK that may shift the balance away from differentiated phenotype towards amplification of the population. When both TNF-α and EGF are present, inhibition of MEK1/2 failed to completely prevent a reduction in mRNA levels of matrix components. The level of ERK1/2 phosphorylation induced by EGF was not altered in the presence of TNF-α, suggesting that MEK1/2 activity was also not altered and could be fully inhibited by the concentration of U0126 used. Taken together, these data suggest that although blockade of MEK1/2 can prevent the loss of aggrecan and type II collagen mRNA by TNF-α and EGF individually, additional signals beyond the MAPK pathway are probably involved when the factors are combined. The intracellular signals that control matrix gene expression elicit their effects through regulation of gene transcription or through post-transcriptional modification and turnover of gene products (i.e. stability of mRNA). A key molecule involved in the transcriptional regulation of both type II collagen and aggrecan is Sox-9 [ 28 , 43 , 44 ]. Sox-9 acts by binding to enhancer regions of the type II collagen gene and to regulatory regions of the aggrecan gene to drive promoter activity [ 43 ]. Although the exact mechanism of loss of aggrecan mRNA in response to TNF-α and EGF remains unclear, the lack of change in activity at the type II collagen enhancer in response to EGF suggests that changes in Sox-9 activity do not mediate the EGF effects. Furthermore, these results together with the fact that the MAPK/ERK pathway was activated by EGF in this system suggest that this region is independent of MAPK/ERK activity. There are, however, alternate sites within these genes that may govern expression, such as SP1/SP3, C-Krox, and Stat1 [ 45 - 48 ]. In addition, activation of signaling pathways can increase synthesis of proteins responsible for the breakdown of existing mRNA, thereby increasing mRNA turnover. In this regard, we previously demonstrated that TNF-α reduces levels of type II collagen mRNA by approximately 40% when transcription was fully inhibited pharmacologically [ 12 ]. The additional loss of mRNA species following the treatment with both EGF and TNF-α in the present study suggests that intracellular signals target regulatory elements external to the enhancer-like sequence or stability of the mRNA. Conclusion In this study, changes in chondrocyte phenotype and function were determined following treatment with TNF-α and EGF, mediators that contribute to sustaining the inflammatory processes associated with arthritis. The effects of this combination of factors have not been explored previously in cartilage or in other tissues. The expression of matrix genes critical for maintaining structural and functional integrity of cartilage was downregulated additively by TNF-α and EGF through mechanisms that involved at least two signals convergent on matrix gene regulation. Multiple inflammatory mediators can therefore profoundly reduce chondrocyte function and contribute to the progression of cartilage degeneration through several distinct signaling events. Abbreviations BIS = bisindolylmaleimide; EGF = epidermal growth factor; ERK = extracellular signal-regulated kinase; IL = interleukin; MAPK = mitogen-activated protein kinase; MEK1/2 = mitogen-activated protein kinase kinase 1 and 2; NF = nuclear factor; PARP = poly(ADP ribose) polymerase; PKC = protein kinase C; TNF-α = tumor necrosis factor alpha; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling; U0124 = 1,4-diamino-2,3-dicyano-1,4- bis (methylthio) butadiene; U0126 = 1,4-diamino-2,3-dicyano-1,4- bis [2-aminophenylthio] butadiene. Competing interests The author(s) declare that they have no competing interests. Authors' contributions ARK, an MSc candidate, participated in the design of the study, performed all the experiments and analysis, prepared the figures, and contributed to the writing of the manuscript. SMB conceived of the study, participated in its design and analysis, and prepared and revised the manuscript. Both authors read and approved the final manuscript.

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1064892

Circulating tumour necrosis factor-α bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response

Our objective was to clarify the heterogeneity in response to infliximab treatment in rheumatoid arthritis (RA); to this end, a bioassay was designed to explore the contribution of circulating tumour necrosis factor (TNF)-α bioactivity and its possible link to response. The bioassay is based on the induction of IL-6 and osteoprotegerin (OPG) production by synoviocytes in response to TNF-α. RA synoviocytes were cultured with TNF-α (5 ng/ml) and 42 RA plasma samples collected just before starting therapy. Levels of IL-6 and OPG were measured in supernatants. In 20 of the patients, plasma samples collected before and 4 hours after the first and the ninth infusions were tested in the same way. Plasma concentrations of TNF-α and p55 and p75 soluble receptors were measured using ELISA. TNF-α induced IL-6 and OPG production by synoviocytes, which was further increased with patient plasma dilutions and inhibited by infliximab. With plasma samples obtained before the first infusion, the IL-6-induced production was greater in patients with a good clinical response than in the poor responders (44.4 ± 23.3 ng/ml versus 27.4 ± 20.9 ng/ml; P = 0.05). This high circulating TNF-α bioactivity was strongly inhibited with the first infliximab infusion. The difference between IL-6 levels induced with plasma samples obtained before and 4 hours after the first infusion was greater in patients with a good clinical response (40.0 ± 23.7 ng/ml versus 3.4 ± 10.0 ng/ml; P = 0.001). Similar findings were obtained for OPG production (7.0 ± 6.2 ng/ml versus 0.0 ± 3.0 ng/ml; P < 0.05). Levels of circulating TNF-α bioactivity were predictive of clinical response to TNF-α inhibition, confirming a key role for TNF-α in these RA patients.

Introduction Rheumatoid arthritis (RA) is a chronic disease characterized by synovial inflammation that leads to progressive joint damage. Knowledge concerning the role played by cytokines in mediating cell–cell interactions in rheumatoid synovium has led to the rational development of treatment with anticytokine agents. Among these proinflammatory cytokines, tumour necrosis factor (TNF)-α has emerged as a major therapeutic target, based on clinical studies with biological inhibitors such as monoclonal antibodies and soluble receptors. In large proportions of patients, TNF-α inhibitors strongly reduced symptoms of synovitis, biological markers of inflammation and bone destruction [ 1 - 4 ]. However, the improvement varied between patients. In an attempt to explain these differences between patients, we explored whether heterogeneity exists in the contribution of circulating TNF-α bioactivity, with the hypothesis that patients with higher levels of bioactive TNF-α would be more sensitive to the systemic administration of a specific inhibitor. Such circulating TNF-α activity would reflect local joint production. The goal of the present study was to evaluate circulating TNF-α bioactivity in RA patients before infliximab treatment and to assess its acute modulation by infliximab. Indeed, the remaining TNF-α activity would represent the difference between total TNF-α and its fraction bound to specific and nonspecific inhibitors. Therefore, a bioassay was developed using the properties of synoviocytes to produce IL-6 and osteoprotegerin (OPG) in response to TNF-α [ 5 , 6 ]. Finally, we looked for a possible link between changes in OPG and IL-6 levels and the rate of clinical improvement during infliximab treatment. Methods Patients Forty-two patients with RA (35 women and 7 men, median age 46.8 years [range 20–67 years], disease duration 9.0 years [range 1–31 years]), diagnosed according to the revised criteria of the American College of Rheumatology (ACR) [ 7 ], were enrolled. Rheumatoid factor was present in 31 of the patients. All received infliximab according to the ATTRACT (Anti-TNF Trial in RA with Concomitant Therapy) protocol at 3 mg/kg every 8 weeks, combined with methotrexate [ 8 ]. The following indices were measured: tender joint count, swollen joint count, patient's assessment of pain, patient's global assessment of disease activity, physician's global assessment of disease activity, the Disability Index of the Health Assessment Questionnaire, serum levels of C-reactive protein and erythrocyte sedimentation rate. ACR response was recorded at 54 weeks [ 9 ]. RA patients were divided into two groups: good responders, with an ACR response equal to or greater than 50 ( n = 24); and poor responders, with an ACR response equal to or less than 20 ( n = 18). EDTA-treated venous blood was collected before infliximab therapy in all patients ( n = 42). In 20 patients, blood samples were collected during infliximab treatment before and 4 hours after the first and ninth infusions. Plasma samples obtained by centrifugation were stored at -20°C and thawed before use. The main characteristics of the patients are summarized in Table 1 . Bioassay for circulating TNF-α bioactivity A functional assay for TNF-α activity was designed using the ability of RA synoviocytes to produce IL-6 and OPG in response to TNF-α [ 5 ]. To isolate synoviocytes, RA synovial tissues were finely minced and digested with 4 mg/ml collagenase (Worthington, Freehold, NJ, USA) in phosphate-buffered saline (Life Technologies, Grand Island, NY, USA). These synovium pieces were obtained from RA patients undergoing joint replacement. Synoviocytes were used at passages four to eight. RA synoviocytes (10 4 cells/well) were cultured in 96-well plates (Falcon, Lincoln Park, NJ, USA) in a final volume of 200 μl in minimum essential medium (Life Technologies) supplemented with 10% heat-inactivated foetal calf serum (Life Technologies), 25 000 UI penicillin, 25 000 μg streptomycin and 250 μg fungizone. TNF-α (0–10 ng/ml) was added to RA synoviocytes with or without infliximab (10 μg/ml). Then, TNF-α (5 ng/ml) was combined with 20 μl plasma per well in order to increase the sensitivity of the synoviocyte response. Plasma samples were collected just before the first infusion. In addition, RA synoviocytes were stimulated with plasma samples collected before and 4 hours after the first and the ninth infusions from 20 RA patients. TNF-α (5 ng/ml), with or without infliximab (10 μg/ml), was preincubated with these four plasma samples for 1 hour before being added to the culture. IL-6 and OPG production were measured by ELISA in 48-hour supernatants, as previously described [ 5 , 6 ]. At baseline, in the 20 patients with a 1-year follow up, plasma concentrations of TNF-α p55 and p75 soluble receptors were measured using commercial ELISA kits (Biosource, Camarillo, CA, USA), in accordance with the manufacturer's instructions. Statistical analysis Statistical analysis was performed using the Statview software (Abacus Concept Inc., Cary, NC, USA). Means were compared using a nonparametric test. Spearman's correlation was used to determine a relationship between the changes in IL-6 and OPG levels and those of TNF-α or TNF soluble receptors detected by ELISA. χ 2 test was performed to detect differences between different subsets. Results Principle of the bioassay The principle of the bioassay, as shown in Fig. 1 , is based on the ability of TNF-α to induce IL-6 and OPG production by RA synoviocytes. With TNF-α concentrations ranging from 0.1 to 100 ng/ml, IL-6 production by synoviocytes increased in a dose-dependent manner. Addition of infliximab at 10 μg/ml completely inhibited the effect of TNF-α at 1 ng/ml, and reduced that of TNF-α at 10 ng/ml by 74% (32.9 ng/ml without versus 8.5 ng/ml with infliximab; Fig. 1a ). Similar studies were performed for OPG production. As for IL-6, OPG production by synoviocytes increased in a dose-dependent manner in response to TNF-α (Fig. 1b ). Maximal concentrations of IL-6 and OPG were in the same range up to 35 ng/ml. With regard to IL-6, addition of infliximab inhibited OPG production induced by TNF-α at 10 ng/ml. Addition of plasma further increased the effect of exogenous TNF-α. With RA plasma concentrations ranging from 0% to 20% used alone ( n = 4), IL-6 production by synoviocytes increased in a dose-dependent manner (Fig. 2a ). This effect was further increased when exogenous TNF-α at 5 ng/ml was added. The greatest effect was observed with 10% plasma. An inhibitory effect was often observed with concentrations of plasma at 20%. In further experiments, 5 ng/ml TNF-α and 10% plasma concentration were used. With RA plasma samples collected before infliximab therapy used alone, IL-6 production was 6.5 ± 4.8 ng/ml ( n = 20). The contribution of TNF bioactivity is shown in Fig. 2b . Combination of 10% concentration of RA plasma with TNF-α at 5 ng/ml increased IL-6 production to 43.5 ng/ml. This effect was inhibited by infliximab (6.5 ng/ml), demonstrating the specificity for TNF-α. Effect of patient plasma samples collected just before the first infliximab infusion Samples were obtained from 42 patients before the first infliximab infusion. The levels of IL-6 produced by stimulation with 10% plasma and TNF-α (5 ng/ml) are shown in Fig. 3 , stratified by ACR response observed at week 54. Levels were higher for the good responders (ACR response ≥ 50) than for poor responders (ACR ≤ 20; 44.4 ± 23.3 ng/ml versus 27.4 ± 20.9 ng/ml; P = 0.05). Circulating TNF-α bioactivity modulation by the first infliximab infusion For 20 of the patients, samples were collected before and after the first and the ninth infusions. There was no significant clinical difference between the 20 patients and the rest of the 42 patient cohort (Table 1 ). The high IL-6-inducing activity found in samples before treatment was strongly reduced in samples obtained 4 hours after the first infusion, following TNF-α /anti-TNF-α complex formation induced by the infliximab infusion (Fig. 4a,4b ). This reduction demonstrates the contribution of TNF-α to the activity following in vivo administration of the TNF-α inhibitor. This pattern of reduction was associated with a very good clinical response at 54 weeks for all patients (ACR 50; n = 11) except one (ACR 20). Conversely, no modulation in circulating bioactivity was observed in patients with a low level of TNF-α bioactivity (Fig. 4c ). This pattern was associated with a poor clinical response (ACR <20) except in one patient (ACR 50). The difference between the IL-6 levels induced with TNF-α at 5 ng/ml and plasma obtained before and 4 hours after the first infusion correlated with good clinical response (40.0 ± 23.7 ng/ml versus 3.4 ± 10.0 ng/ml; P = 0.001; Fig. 5a ). Similarly, OPG levels were measured in the same supernatants. With regard to IL-6, the difference between the levels of OPG with plasma samples obtained before and 4 hours after the first infliximab infusion correlated with good clinical response (7.0 ± 6.2 ng/ml versus 0.0 ± 3.0 ng/ml; P < 0.05; Fig. 5b ). A positive correlation was observed between changes in IL-6 and OPG production ( n = 20; r = 0.843; P < 0.001). Circulating TNF-α bioactivity modulation by the ninth infusion Patients with high circulating bioactivity at the first infusion could be separated into two subsets. For the first group, before the ninth infusion, no circulating bioactivity was detected (Fig. 4a ). This pattern was associated with very good clinical response at 54 weeks in all patients (ACR 50; n = 6). In the second group, circulating bioactivity was still present before the ninth infusion but remained sensitive to further infliximab administration (Fig. 4b ). With this pattern, five out of six patients had an ACR 50 response but one had a poor response (ACR ≤ 20). No modulation in circulating bioactivity was observed in patients with low circulating TNF-α bioactivity before infliximab therapy ( n = 8; Fig. 4c ). A link was observed between these patterns and the clinical response (χ 2 = 16.6; P < 0.001). Various clinical and biological parameters were analyzed according to these patterns. Before treatment, no difference was observed between joint counts, erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor positivity, or Disease Activity Score 28 (5.4 ± 0.9 in good responders versus 5.2 ± 1.4 in poor responders; P = 0.6). Absence of correlation between circulating TNF-α bioactivity and ELISA levels of TNF-α or soluble receptors Levels of TNF-α and of soluble receptors (p55 and p75) were measured in plasma samples obtained at baseline from 20 RA patients. The mean TNF-α level was 144.87 ± 130.36 pg/ml in good responders and 153.75 ± 132.93 pg/ml in poor responders ( P = 0.97). Similar results were observed with p55 (2534 ± 1074 pg/ml versus 2436 ± 953 pg/ml; P = 0.57) and p75 (3054.8 ± 673.8 pg/ml versus 2332.2 ± 921.3 pg/ml; P = 0.84) soluble receptors in good responders versus poor responders. No correlation was observed between circulating TNF-α bioactivity and TNF-α or soluble receptors, or the difference between TNF-α and p55 and p75 levels. Similarly, no negative correlation was observed between levels of circulating TNF-α bioactivity and of p55 or p75 soluble receptors. Discussion Prediction of the response of RA to treatment remains a hot topic. There is no evidence that simple determination of plasma TNF-α levels by ELISA allows such prediction for treatment with TNF-α inhibitors. However, it still makes sense that patients producing high levels of TNF-α will be more sensitive to TNF-α inhibition. A system was established to evaluate the circulating TNF-α-related bioactivity in plasma. Exogenous TNF-α alone stimulates IL-6 production and this effect can be abrogated by first incubating TNF-α with infliximab before exposure to the synovial cells. When plasma is combined, cells respond to free TNF-α and not to inactive TNF-α bound to specific soluble receptors and to other less specific binding sites on proteins. This bioassay was based on the IL-6 production induced by the combination of TNF-α and plasma. Addition of exogenous TNF-α was used to detect the presence of circulating inhibitors. Such inhibitors in plasma are probably involved in the lower effect of 20% plasma concentration as compared with the effect seen with a 10% concentration (Fig. 2a ). When the system was applied to explain part of the heterogeneity in treatment response, a third of the patients showed strong and prolonged inhibition in circulating TNF-α bioactivity, suggesting the critical contribution of systemic TNF-α in these patients. In another third of the patients circulating TNF-α bioactivity was inhibited by infliximab infusion for a short time, because bioactive TNF-α reappeared but was again inhibited by the next infliximab infusion. This profile suggested partial inhibition, although the clinical benefit was still very significant. In two thirds of the samples, the bioassay measured a strong inhibition of circulating TNF-α-related activity during the first infusion. The link between strong anti-TNF-α activity induced by the first infusion and the good clinical response confirms the key role played by TNF-α in approximately two thirds of the RA patients. This result is in accord with results from the ATTRACT study [ 8 ]. In contrast, in the last third of the patients the assay suggested no contribution or a reduced contribution of TNF-α bioactivity either before or after the first infusion of infliximab. All plasma samples from these patients inhibited the IL-6 production usually induced by TNF-α. This profile suggests that these patients may have a high level of innate neutralizing TNF-α activity and/or no circulating active TNF-α. Patients with this pattern exhibited a poor clinical response, suggesting that their RA was not much driven by TNF-α activity alone but probably by other cytokines or mechanisms. The heterogeneity in these patterns may be explained by higher disease activity for responders than nonresponders, but no difference was observed in clinical and biological parameters before treatment. One way to view the difference among patients with high circulating TNF-α bioactivity initially is the link observed between higher trough concentrations of infliximab in RA patient serum and good response and a reduced progression of radiographic joint damage [ 10 ]. This latter study suggested that RA patients with a poor clinical response tend to eliminate infliximab more rapidly from their circulation. RA synoviocytes produce higher levels of OPG than do peripheral blood mononuclear cells or synovial fluid mononuclear cells, but they do not produce soluble receptor activator of nuclear factor-κB ligand [ 5 ]. Accordingly, we focused on OPG, which is produced in response to TNF-α. Similar results were observed for IL-6 and OPG production, although changes in IL-6 levels appeared more sensitive. This is related to the very low levels of IL-6 produced by resting synoviocytes. Accordingly, the predictive value of changes in IL-6 was better than that of OPG. Our findings extended previous studies indicating a correction in high OPG serum levels in RA patients treated with infliximab [ 5 ]. No correlation was observed between circulating TNF-α bioactivity and its protein concentration in plasma measured by ELISA. Circulating TNF-α bioactivity levels could not be calculated as free protein TNF-α taking into account the levels of TNF-α and soluble receptors (p55 and p75) measured by ELISA. This discrepancy further indicates the usefulness of a bioassay when function is the key. Such complexity has been observed when trying to explain loss of infliximab response by the induction of anti-mouse antibodies [ 11 ]. Once again, only the demonstration of inhibitory activity in vivo would allow such a conclusion to be drawn. Conclusion In conclusion, this bioassay was able to predict correctly the clinical response in 69% of cases (29/42). Taking into account the effect of the first infusion increased the value to 90%. However, a simplified assay would need to be designed for routine application. Abbreviations ACR = American College of Rheumatology; ELISA = enzyme-linked immunosorbent assay; IL = interleukin; OPG = osteoprotegerin; RA = rheumatoid arthritis; TNF = tumour necrosis factor. Competing interests The author(s) declare that they have no competing interests. Authors' contributions HM conducted the experiments and wrote the paper. WM took OPG measurements. PM directed the research.

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1064893

Local expression of matrix metalloproteinases, cathepsins, and their inhibitors during the development of murine antigen-induced arthritis

Cartilage and bone degradation, observed in human rheumatoid arthritis (RA), are caused by aberrant expression of proteinases, resulting in an imbalance of these degrading enzymes and their inhibitors. However, the role of the individual proteinases in the pathogenesis of degradation is not yet completely understood. Murine antigen-induced arthritis (AIA) is a well-established animal model of RA. We investigated the time profiles of expression of matrix metalloproteinase (MMP), cathepsins, tissue inhibitors of matrix metalloproteinases (TIMP) and cystatins in AIA. For primary screening, we revealed the expression profile with Affymetrix oligonucleotide chips. Real-time polymerase chain reaction (PCR) analyses were performed for the validation of array results, for tests of more RNA samples and for the completion of the time profile. For the analyses at the protein level, we used an MMP fluorescence activity assay and zymography. By a combination of oligonucleotide chips, real-time PCR and zymography, we showed differential expressions of several MMPs, cathepsins and proteinase inhibitors in the course of AIA. The strongest dysregulation was observed on days 1 and 3 in the acute phase. Proteoglycan loss analysed by safranin O staining was also strongest on days 1 and 3. Expression of most of the proteinases followed the expression of pro-inflammatory cytokines. TIMP-3 showed an expression profile similar to that of anti-inflammatory interleukin-4. The present study indicates that MMPs and cathepsins are important in AIA and contribute to the degradation of cartilage and bone.

Introduction Rheumatoid arthritis (RA) is a chronic destructive autoimmune disease characterized by the inflammation and progressive destruction of distal joints. The initial histological features of RA are characterized by synovial lining hyperplasia, excessive angiogenesis and the accumulation of polymorphonuclear and mononuclear cells in the synovium [ 1 , 2 ]. The etiology of RA is still unknown, but the degradation of cartilage and bone observed in RA is caused by an increased expression of proteinases, resulting in an imbalance of these degrading enzymes and their inhibitors [ 3 , 4 ]. Proteinases have a pivotal function in endogenous angiogenesis, antigen presentation and pathological remodeling of cartilage and bone [ 5 - 7 ]. For an understanding of the pathogenesis of RA, it is important to investigate the time profiles of expression of proteinases and proteinase inhibitors during the development of arthritis and their relationship to cytokine expression. It has been suggested that the immune hyper-responsiveness in RA tissues is triggered by an unknown joint-specific antigen. Antigen-induced arthritis (AIA) in mice is an experimental model for RA, in which arthritis is induced by systemic immunization with the antigen methylated bovine serum albumin (mBSA) in complete Freund's adjuvant, followed by a single intra-articular injection of the antigen into the knee joint cavity [ 8 ]. The development of chronic arthritis is visible for several weeks. The advantage of AIA over other experimental arthritis models consists in the exactly defined stages of the development of arthritis elicited by antigen injection into the knee joint cavity. After this initiation of AIA, it is possible to distinguish between the acute stage from day 0 to day 7, characterized by inflammatory processes, and the subsequent chronic stage with pannus formation and joint destruction. As in RA, breakdown of articular cartilage and bone in AIA results from the overexpression of proteinases and deregulation of the balance between proteinases and their inhibitors. We investigated the expression patterns of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), cathepsins and cystatins by Affymetrix oligonucleotide microarray technology in combination with real-time polymerase chain reaction (PCR), to identify the mediators that were differentially expressed in murine AIA. We were able to follow the expression in murine knee joints from the induction of arthritis to the development of the chronic phase. Additionally, we studied the expression profiles of different cytokines on mRNA and protein level. To complete our oligonucleotide chip and PCR results, we investigated MMP expression and activity by fluorescence assays and zymography. Materials and methods Animals Female C57Bl/6 mice (age 7–9 weeks) were obtained from the Animal Research Facility of Friedrich Schiller University, Jena, Germany, and Charles River Laboratories, Sulzfeld, Germany, respectively. They were kept under standard conditions in a 12 hours:12 hours light:dark cycle and fed with standard pellets (Altromin no. 1326, Lage, Germany) and water ad libitum . All animal studies were approved by the governmental committee for animal protection. Immunization and arthritis induction Mice were immunized on days – 21 and – 14 by subcutaneous injections of 100 μg of mBSA (Sigma, Deisenhofen, Germany) in 50 μl of saline solution, emulsified in 50 μl of complete Freund's adjuvant (Sigma), adjusted to a concentration of 2 mg/ml heat-killed Mycobacterium tuberculosis strain H37RA (Difco, Detroit, MI, USA). In addition, intraperitoneal injections of 2 × 10 9 heat-killed Bordetella pertussis bacteria (Chiron Behring, Marburg, Germany) were performed on the same days. Arthritis was induced on day 0 by injection of 100μg of mBSA in 25 μl of saline solution into the right knee joint cavity. Joint swelling For clinical monitoring of AIA development, the joint diameters were analyzed before (nonarthritic mice, immunized with mBSA: day 0) and at various times after AIA induction (days 1, 3, 7, 14 and 21). The joint swelling was measured with an Oditest vernier caliper (Kroeplin Längenmesstechnik, Schluechtern, Germany). Joint swelling was expressed as the difference in diameter (mm) between the right knee joint on days 1, 3, 7, 14 and 21, and the same knee joint on day 0 before arthritis induction. For measurement of joint swelling, the mice were anesthetized by ether inhalation. Preparation of total RNA The expression of mRNA for proteinases and cytokines was analyzed for each individual animal. Arthritic and control mice were anesthetized with ether and killed by cervical dislocation. Then right knee joints (where arthritis was induced) were dissected and skinned. The muscle tissue was removed and the bony parts of knee joints were prepared, including the joint capsules with synovial tissue. The RNA in the knee joint was stabilized in RNAlater (Qiagen, Hilden, Germany), in accordance with the instructions in the manual. After incubation of the joints for 12 hours at 4°C in RNAlater, the samples were transferred to -80°C. The joints were mechanically disrupted by milling with a dismembrator U (Braun AG, Meiningen, Germany) for 15 s at 2000 Hz, followed by cooling the vessel in liquid nitrogen for 1 min. This procedure was repeated six or seven times. The tissue powder was rapidly transferred into 2 ml of cold TRIzol (Invitrogen, Carlsbad, CA, USA), immediately followed by dispersion for 1 min with a Polytron 1200 CL (Kinematica AG, Littau/Luzern, Switzerland). After homogenization and mechanical disruption, the RNA was extracted with TRIzol in accordance with the manual. Microarray data analysis The MG_U74Av2 oligonucleotide chip (array A; Affymetrix, Santa Clara, CA, USA), representing all functionally characterized sequences (about 6000 genes) in the Mouse UniGene database (Build 74) and additionally about 6000 expressed sequence tag clusters, was used to analyze the gene expression in murine arthritic knee joints during AIA. We hybridized six chips with samples from two individual animals from each investigated time point (two at day 0 [control], two at day 3 [acute phase] and two at day 14 [chronic phase]). RNA labelling, hybridization and scanning of gene chips were performed in accordance with the supplier's instruction. Expression levels were calculated with the commercially available software MAS 5.0 provided by Affymetrix. Normalization of the signal was based on the expression of the housekeeping gene β-actin. Expression in the acute and chronic inflamed knee joints was compared with expression in the knee joints of control animals at day 0. DNase treatment and complementary DNA (cDNA) synthesis for real-time PCR The DNase treatment was performed by DNA Free™ kit (Ambion, Woodward, Austin, TX, USA) in accordance with the manufacturer's instructions. Total RNA (5 μg) was digested with 1 μl of DNase I (1 U/μl) and 2 μl of 10 × DNase I buffer in a volume of 20 μl. Supernatant (15 μl), containing the DNase-treated RNA, was transferred into a fresh 0.5 ml PCR tube. The RNA was denatured by incubation at 65°C for 15 min. After incubation on ice for 5 min, the reverse transcriptase (RT) reaction was completed by adding 35 μl of RT reaction mix, containing 1 μl of Superscript RT (200 U/μl; Invitrogen), 10μl of 5 × RT buffer (Invitrogen), 5 μl of 0.1 M dithiothreitol (Invitrogen), 5 μl of dNTPs (10 mM; Promega, Mannheim, Germany), 2 μl of poly(T) primer (T 30 VN; 50μmol), and 12 μl of distilled water. Reverse transcription was performed at 42°C for 1 hour, after which the cDNA was precipitated with ethanol. The precipitated and air-dried cDNA was resuspended in 50 μl of distilled water. Real-time PCR analyses The oligonucleotide microarray data of interesting proteinases that could have a pivotal role in AIA were independently validated by real-time PCR. Cytokine expression at the mRNA level was also determined with this technique. Semiquantification of proteinase and cytokine expression by real-time PCR was performed with the Rotorgene 2000 (LTF Labortechnik, Wasserburg/Bodensee, Germany). The β-actin gene served as an endogenous control to normalize the differences in the amount of cDNA in each sample. SYBR Green I dye (Sigma) was supplied at 10,000 × concentration in dimethylsulfoxide. The enzyme Hot Star Taq (Qiagen) with the supplied reaction buffer was used for the PCR reaction. The reaction was performed in a volume of 25 μl, consisting of 2.5 μl of 10 × PCR buffer, 2.0–2.5 μl of MgCl 2 (25 mM), 0.5 μl of dNTPs (10 mM each; Promega), 0.5 μl of Hot Star Taq (5 U/μl), 0.5 μl of primer mix (20 μM each) and 2.5 μl of 1 × SYBR Green I solution. A standard curve was prepared by serial dilution of plasmid DNA (Vector pCR ® 2.1-TOPO ® ; Invitrogen), containing the cDNA of the analyzed gene. The cloned fragment was identical in sequence and length with the PCR product. All samples that had to be compared for expression differences were run in the same assay as duplicates together with the standards. After completion of PCR amplification, data were analyzed with Rotorgene Software version 4.4. Data were initially expressed as a threshold cycle and are expressed as fold increases in gene expression in mice on day 0 compared with the expression on the other days investigated. The mean value of day 0 was set at 100%. After amplification was complete, the PCR products were analyzed by agarose gel electrophoresis. The primers used and the resulting PCR product sizes are given in Table 1 . Preparation of joint extracts Arthritic and control mice were anesthetized with ether and killed by cervical dislocation. Knee joints were dissected, skinned and snap-frozen in propane/liquid nitrogen, and stored at -80°C until further analysis. Joint extract was obtained as described by Smith-Oliver and colleagues [ 9 ]. The frozen joints were ground under liquid nitrogen with mortar and pestle. The powdered tissue was transferred to a glass homogenizer, and exactly 2 ml of sterile saline solution was added. The powder suspension was homogenized by hand for 2 min and centrifuged for 20 min at 1500 × g and 4°C. The supernatant was spun again for 10 min at 3000 × g , and the resulting supernatant was aliquoted and frozen at -70°C. Protein concentration was determined by bicinchoninic acid assay (Pierce, Rockford, IL, USA). Cytokine analysis in joint extracts Concentrations of cytokines in joint extracts were determined by sandwich enzyme-linked immunosorbent assay (ELISA) in accordance with standard procedures, with the following antibody pairs: MP5-20F3 and MP5-32C11 (interleukin-6 [IL-6]; BD Biosciences, Palo Alto, CA, USA), R4-6A2 and XMG1.2 (interferon-γ [IFN-γ]; BD Biosciences), G281-2626 and MP6-XT3 (tumor necrosis factor-α [TNF-α]; BD Biosciences), MAB401 and BAF401 (IL-1β; R&D Systems, Minneapolis, MN, USA), BVD4-1D11 and BVD6-24G2 (IL-4; BD Biosciences). The second antibody of each antibody pair was biotinylated. Bound antibodies were detected with streptavidin-coupled peroxidase using o -phenylene diamine with 0.05% H 2 O 2 as substrate. Optical density was measured at 492 nm in a microtiter plate-reader, model 16 598 (SLT Lab instruments, Crailsheim, Germany). MMP analysis in joint extracts by zymography For analysis of proteolytic capacity, joint extracts were diluted to a final protein concentration of 1 mg/ml, mixed with sample buffer containing sodium dodecyl sulfate (SDS), glycerol and bromophenol blue. Equal amounts of each sample were separated on an SDS-polyacrylamide gel (7.5%) containing 0.8 mg/ml gelatin (Merck, Darmstadt, Germany). After SDS-polyacrylamide gel electrophoresis, the gels were washed twice with 2.5% Triton X-100 for 30 min to remove SDS, then twice with distilled water and were finally equilibrated with incubation buffer (100 mM Tris/HCl, 30 mM CaCl 2 , 0.01% NaN 3 ). The gel was then incubated in incubation buffer for 20 hours at 37°C. Staining of protein was performed with Coomassie Blue solution (10 ml of acetic acid, 40 ml of distilled water, 50 ml of methanol, 0.25% Coomassie Blue G250 [SERVA, Heidelberg, Germany]) for 40 min. Destaining was performed in methanol/acetic acid/distilled water (25:7:68, by vol.). After staining, white bands on blue gels indicate enzyme species. We used human pro-MMP-2 and pro-MMP-9 as controls (Novus Molecular Inc., San Diego, CA, USA) Determination of MMP activity in joint extract The synthetic peptide Mca-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH 2 (no. M-1895; Bachem, Heidelberg, Germany; Mca stands for (7-methoxycoumarin-4-yl)acetic acid, Dap for L-2,3-diaminopropionic acid, and Dnp for 2,4-dinitrophenyl) was used to quantify the activity of MMPs in joint extracts. This substrate can be cleaved by different MMPs (MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-12, MMP-13, MMP-14, MMP-17, MMP-25 and MMP-26). This fluorogenic peptide is a very sensitive substrate for continuous assays and for the in situ determination of matrix metalloproteinase activity. Cleavage at the Gly-Leu bond separates the highly fluorescent Mca group from the efficient Dnp quencher, resulting in an increase in fluorescence intensity. Joint extracts were diluted with phosphate-buffered saline to give a final protein concentration of 1 mg/ml, then incubated with 5 μM substrate at 37°C in incubation buffer (100 mM Tris-HCl, 30 mM CaCl 2 , pH 7.6) for 3 hours. Active and free (not inhibited by TIMPs) MMPs cleaved the quenched substrate, which led to an increase in fluorescence at 390 nm (λ ex = 330 nm). The fluorescence was serially measured in black microtiter plates (Greiner, Solingen, Germany) with a fluorescence reader (FLUOstar Galaxy; BMG, B&L Systems, AS Maarssen, Netherlands). To estimate the total MMP activity, latent MMPs were activated by incubation with 2 mM aminophenylmercuric acetate (APMA; Sigma) for 15 min before the addition of substrate. The subsequent fluorescence signal after activation with APMA reflects the sum of the pro-forms of MMPs and the active forms of MMPs, which are not inhibited by TIMPs. Histological evaluation of paraffin sections Mice were killed on days 0, 1, 3, 7, 14 and 21 (two animals per group) and the knees were dissected and fixed in 4% phosphate-buffered paraformaldehyde. Fixed joints were decalcified in 20% EDTA (Sigma), dehydrated and embedded in paraffin wax. Frontal sections (2 μm) of the whole knee joint were stained with safranin O (counterstained with hematoxylin) for microscopic examination. Proteoglycans of cartilage are stained red. Safranin O staining was used to reflect cartilage proteoglycan depletion during the development of experimental arthritis. Statistics Analyses were performed with the statistical software SPSS/Win version 10.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed with the Mann–Whitney U -test. After induction of arthritis, each group of animals was compared with the control day 0 group and the control normal group, respectively. For each test, P ≤ 0.05 was considered to be statistically significant. Correlations according to Pearson and Spearman's rho were also made with SPSS/Win version 10.0. Results Joint swelling Joint swelling reached its maximum on day 1 of arthritis in the acute phase, and gradually declined until day 7 in the beginning chronic phase of AIA (Fig. 1 ). Affymetrix chip and real-time PCR analyses Primarily, we screened the expression patterns during the course of AIA by MG_U74Av2 Affymetrix oligonucleotide chips (array A) to investigate the role of proteinases and their inhibitors in the development of arthritis. We compared the expression at the mRNA level in arthritic knee joints of two animals on day 3 and two animals on day 14 with the expression in knee joints of two control mice on day 0. By oligonucleotide chip technology we obtained a first impression of which MMPs, TIMPs, cathepsins and cystatins were strongly and differentially expressed in murine knee joints (Table 2 ). We identified 5 MMPs (MMP-3, MMP-8, MMP-9, MMP-13 and MMP-14), 3 TIMPs (TIMP-1, TIMP-2 and TIMP-3), 11 cathepsins (cathepsins B, C, D, E, F, G, H, K, L, S and Z) and 3 cystatins (cystatins B, F and C) that were highly expressed at the mRNA level in murine joints during AIA and resulted in a present call as a detection signal analyzed by MAS 5.0 (Table 2 ). Many proteinases and inhibitors were very weakly expressed and resulted in an absent call. Among the strongly expressed proteinases and inhibitors, some MMPs, cathepsins, TIMPs and cystatins showed interesting expression patterns, which seemed to be connected with arthritis development. We used real-time PCR to validate the expression of these molecules by an independent method. We confirmed the oligonucleotide chip data for most of the proteinases investigated (except cathepsin L; Fig. 2 ). It was possible to investigate additional time points of AIA and to study the expression behavior more exactly by this second molecular biological method. Of all the proteinases investigated by chip analysis and real-time PCR, the matrix metalloproteinase MMP-3 was heavily induced in the acute phase of AIA and showed the most impressive increase of expression on days 1 and 3 (Fig. 2a ). Its expression level decreased in the chronic phase in comparison with the acute phase but remained significantly elevated in chronically inflamed knee joints (days 14 and 21). MMP-13 was non-significantly overexpressed on day 1 compared with day 0. No significant changes in expression of MMP-13 mRNA were found for days 3, 7, 14 and 21. Although oligonucleotide chip technology showed MMP-12 (macrophage elastase) to be very weakly expressed (absent call; Table 2 ), we investigated its expression by real-time PCR, because macrophages are important in RA. MMP-12 was very strongly and significantly overexpressed in the course of AIA after arthritis induction (Fig. 2a ). The significantly increased expression of MMP-12 was observed until day 21 in the chronic phase. Gelatinase B (MMP-9) showed little change in expression at the mRNA level (Fig. 2a ). The transcription was weakly but not significantly downregulated in the acute phase at the mRNA level. MMP-2 (absent call on the array) was significantly downregulated at the mRNA level on day 1 in comparison with day 0 (Fig. 2a ), reaching the day 0 level again on day 3 and increasing until day 21 of the chronic phase. MMP-2 was non-significantly overexpressed at the mRNA level on day 21 in comparison with day 0. Next we investigated in more detail the differential expression of strongly expressed cathepsins by real-time PCR. After an analysis of the array data we found some candidate genes that might have a pivotal function in AIA. We analyzed cathepsins B, H, K, L and S more thoroughly by real-time PCR. The cathepsins B and H were non-significantly overexpressed in the acute phase. The mRNAs for cathepsins L and S were significantly elevated on day 3 in murine knee joints, where arthritis was induced (Fig. 2b ). In contrast to MMP-3 overexpression with the highest peak on day 1, the overexpression of cathepsins was strongest on day 3. Because cathepsin K was strongly expressed in murine knee joints and has an essential role in joint destruction, we investigated its expression pattern by real-time PCR (Fig. 2b ) and found a significant downregulation at the mRNA level on day 1 but no alteration of expression at the mRNA level on the other days in the course of AIA. Not only were MMPs and cathepsins differentially expressed after arthritis induction, but the expression of their inhibitors, the TIMPs and cystatins, was also changed in the course of AIA, as analyzed by Affymetrix oligonucleotide chips (Table 2 ). We performed real-time PCR for TIMP-1, TIMP-3 and cystatin B. TIMP-1 was the most strongly overexpressed (Fig. 3a ). TIMP-3 showed a totally different expression profile from that of TIMP-1 (Fig. 3a ): its expression was downregulated on day 1, increased from day 1 to day 7 and returned on day 14 to the level of control mice at day 0. The expression of cystatin B was increased about twofold on day 3 of the acute stage (Fig. 3a ). Like the expression of cathepsins, the expression of cystatin B was strongest on day 3 (Fig. 3a ). MMP measurement by fluorescence activity assay and zymography Fluorescence assay data of MMP activity in arthritic knee joints showed elevated latent MMP concentrations in the acute phase of AIA, in comparison with healthy control mice (Fig. 4a ). However, active MMP molecules, not bound by inhibitor molecules, were not significantly elevated in arthritic knee joints. Zymography of total protein knee joint extracts was performed for the different time points of AIA (Fig. 5 ). By gelatin zymography, bands could be seen at about 105 kDa (MMP-9) and 66 kDa (MMP-2). Protein solution from arthritic knee joints showed elevated gelatinolytic activity. In contrast to mRNA studies, zymography showed an increased expression of MMP-9 (105 kDa) in the course of AIA in comparison with day 0 (Fig. 5 ). Cytokine expression during AIA Because the production and secretion of proteinases and their inhibitors are regulated by cytokines, we investigated the expression of different cytokines at the mRNA and protein levels. Several cytokines were differentially expressed at the mRNA level. We showed an altered expression for the pro-inflammatory cytokines IFN-γ, IL-6, TNF-α, IL-1β and IL-17 (Fig. 2c ). IFN-γ was significantly elevated at the mRNA level on day 1 in the acute phase of inflammation. On day 7 it was significantly decreased at the mRNA level in comparison with day 0. IL-1β was non-significantly increased on day 1. IL-6 was significantly elevated at the mRNA level from day 1 to day 3, compared with day 0 mice at the mRNA level. Expression of IL-17 was non-significantly elevated at the mRNA level in the arthritic knee joint on days 1 and 3 of the acute phase. TNF-α expression was not significantly elevated at the mRNA level on day 1 of the acute phase or on day 21 of the chronic phase. The anti-inflammatory cytokine IL-4 was non-significantly decreased on day 1 at the mRNA level (Fig. 3b ). Afterwards the mRNA expression of IL-4 increased until day 7 and returned to the starting level until day 14 of the chronic phase. The expression patterns of these cytokines (exception IL-17) were also confirmed at the protein level by ELISA in murine knee joints after the induction of arthritis (Fig. 6 ). IFN-γ was significantly elevated at the protein level on days 1 and 3 of the acute phase. IL-1β was significantly elevated at the protein level from the induction of arthritis during the acute phase (days 1 and 3) until the beginning of the chronic phase of inflammation (day 7). IL-6 was also significantly increased in joint extracts on days 1 and 3. TNF-α expression was upregulated at the protein level on days 1 and 3 in the acute phase. In contrast to the mRNA level, no second increase in TNF-α expression was found. The anti-inflammatory cytokine IL-4 was downregulated on days 1, 3 and 7 at the protein level. Correlations between cytokine and proteinase expression The expression of TIMP-3 in knee joints was significantly correlated with IL-4 expression (correlation according to Spearman's rho, P ≤ 0.01; Fig. 3b ). At the protein level, total MMP activity was significantly correlated with IL-1β protein concentration in knee joints (correlation according to Pearson P ≤ 0.005) (Fig. 4b ). Safranin O staining Safranin O staining reflects the proteoglycan content of cartilage. Proteoglycan loss is an early marker of cartilage destruction. The staining intensity of proteoglycans was very weak during the acute stage of AIA on days 1 and 3, corresponding to the days of highest proteinase expression, in comparison with control joints on day 0 (Fig. 7 ). Discussion The aim of the present study was to investigate the contribution of various proteinases to bone and cartilage degradation during the development of AIA, an experimental model of human RA. MMPs are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in RA [ 10 , 11 ]. However, the role of individual MMPs in the pathogenesis of arthritis must be determined to identify specific targets for joint protective therapies. MMP-3 (stromelysin-1) was the proteinase that showed the highest differences in expression during the course of AIA. Our observation concurs with that of van Meurs and colleagues [ 12 ] who noted a fast upregulation of mRNA for stromelysin during acute flare-up of murine AIA. The overproduction of MMP-3 has a role not only in the direct lysis of collagen but also in the activation of other proteinases [ 13 ]. MMP-13 (collagenase 3) showed a non-significantly increased expression on day 1 of the acute phase of AIA. The significance of these results must be shown by further experimental analyses. However, Wernicke and colleagues [ 14 ] demonstrated that MMP-13 mRNA was induced at sites of cartilage erosion in synovial fibroblasts co-implanted with normal cartilage in non-obese diabetic/severe combined immunodeficient mice. In contrast to MMP-13, the macrophage elastase (MMP-12) was abundantly increased at all investigated time points in AIA. Janusz and colleagues [ 15 ] showed that murine MMP-12 degrades cartilage proteoglycan with an efficiency about equal to human MMP-12 and matrilysin (MMP-7) and twice that of stromelysin-1 (MMP-3). The gelatinases (MMP-2/gelatinase A and MMP-9/gelatinase B) are overproduced in joints of patients with RA [ 16 - 18 ]. Because of their degradative effects on the extracellular matrix, the family of gelatinases has been believed to be important in progression and cartilage degradation in this disease, although their precise roles still are to be defined. Itoh and colleagues [ 19 ] showed in the model of antibody-induced arthritis that MMP-9 knockout mice displayed milder arthritis than their wild-type littermates. Surprisingly, they found that MMP-2 knockout mice display a more severe arthritis than wild-type mice. These results indicate a suppressive role of MMP-2 and a pivotal role of MMP-9 in the development of inflammatory joint disease. In contrast to most of the investigated proteinases in our study, MMP-9 showed a decreased, nearly unaffected, expression at the mRNA level. For pro-MMP-9, we detected by zymography an increased protein content in knee joints during the development of experimental arthritis. The contradiction between high MMP-9 expression at the protein level and a decreased mRNA level in our experiments can be explained by the paracrine mechanism postulated by Dreier and colleagues [ 20 ]. MMP-2 showed a significantly decreased expression on day 1. A similar regulation mechanism for MMP-2 expression as for MMP-9 has not yet been described. Usually, metalloproteinases have been favored as potential matrix-degrading enzymes over cysteine proteinases because of an activity optimum at neutral pH. However, an increasing number of expression analyses of synovial tissues and fluids from patients with RA [ 21 - 27 ], and studies with models of experimental arthritis [ 28 , 29 ], have shown the pivotal role of cathepsins in arthritis development. Extracellular cathepsin S shows an elastin-degrading and proteoglycan-degrading activity at neutral pH [ 30 - 32 ]. Cathepsin B, secreted by invading tumor cells, can degrade collagen and elastin [ 33 ]. The role of cathepsin B, for instance in activation of pro-MMP-3 and its regulation in arthritis, was recently reviewed by Yan and Sloane [ 34 ]. However, the expression and function of most of the cathepsins in RA are unknown. Our results support the hypothesis that cathepsins participate in matrix-degrading processes. We detected strong increases in cathepsin S and L mRNA in the acute phase of AIA. Cathepsin B and H were non-significantly elevated on day 3 after the induction of experimental arthritis. Current interest is focused on the role of cathepsin K in RA. Besides its expression in osteoclasts and related chondroclasts as well as in multinucleated giant cells, cathepsin K has also been described in mononuclear cells that might act as precursor cells for osteoclasts, in macrophages and in various epithelial cells. In situ hybridization experiments have demonstrated cathepsin K expression in synoviocytes of patients with RA at sites of synovial destruction [ 35 , 36 ]. The overexpression of cathepsin K as shown in RA and collagen-induced arthritis of mice could not be confirmed in AIA at the mRNA level. We found a significant decrease in the expression of cathepsin K on day 1, when IFN-γ was expressed at its highest level. A feedback mechanism, as described for MMP-9, can be excluded, because Kamolmatyakul and colleagues [ 37 ] demonstrated that IFN-γ simultaneously downregulates cathepsin K expression in osteoclasts at the protein and mRNA levels. The cytokines, especially the pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-1, IL-6 and IL-17, also have a pivotal role in the pathology of RA. They are found in large quantities in RA synovium and synovial fluid [ 38 - 42 ]. Furthermore, cytokine knockout and transgenic mice showed a changed susceptibility for arthritis in animal models for RA [ 43 - 46 ]. A variety of cytokines including IL-1, TNF-α, and IL-17 increase the production and secretion of MMPs and cathepsins in fibroblasts [ 21 , 47 - 49 ]. In our studies, the local induction of AIA led to an impressive increase in IFN-γ, TNF-α, IL-1β and IL-6 in the joints. The latent MMP concentration at the protein level significantly correlated with the IL-1β concentration in joint extracts. We obtained very precise expression profiles for the cytokines and proteinases under investigation by using real-time PCR. At the transcriptional level, the increase in many MMPs and cathepsins is correlated with the highest expression levels of pro-inflammatory cytokines on days 1 and 3. The downregulation of cathepsin K on day 1 could be another example of the fact that the time course of cytokine expression determines the time course of proteinase and inhibitor expression. In contrast to pro-inflammatory cytokines, IL-4 is one of the major cytokines that are able to protect against severe cartilage destruction during experimental arthritis. IL-4 treatment can inhibit IL-1 mRNA levels in the synovium [ 50 , 51 ]. IL-4 overexpression in the arthritic knee joint strongly and locally inhibited the mRNA expression of MMP-3 in mice with collagen-induced arthritis [ 50 ]. In addition, Borghaei and colleagues [ 52 ] have shown that IL-4 suppresses the IL-1-induced transcription of collagenase 1 (MMP-1) and stromelysin-1 (MMP-3) in human synovial fibroblasts. Van Lent and colleagues [ 53 ] reported that IL-4 blocks the activation step for latent MMPs within the cartilage layer. In the course of AIA we have seen a downregulation of IL-4 on day 1 at the mRNA level, after which IL-4 mRNA expression increased until day 7. Together with the fact that IFN-γ is significantly downregulated on day 7, this might be the reason for the decreased expression of MMP-3 on day 7 at the mRNA level in our study. The biological activity of the proteinases can be regulated transcriptionally and post-transcriptionally by different TIMPs and by activation steps by the proteolytic cleavage of latent forms. Van der Laan and colleagues [ 54 ] demonstrated that the degradation and invasion of cartilage by rheumatoid synovial fibroblasts can be inhibited by gene transfer of TIMP-1 and TIMP-3. We have demonstrated in our analyses that TIMP-1 and TIMP-3 were differentially expressed in AIA. On account of the high similarity of expression profiles, TIMP-3 transcription seems to be directly or indirectly associated with IL-4 expression. Currently, there is no experimental evidence that IL-4 upregulated the TIMP-3 mRNA expression, but a similar mechanism to the upregulation of TIMP-2 by IL-4 in dermal fibroblasts described by Ihn and colleagues [ 55 ] cannot be excluded. TIMP-1 shows an expression profile very similar to that of MMP-3, being highly overexpressed during AIA. The overexpression of TIMP-1 at the mRNA level in our analysis is smaller and appears later than MMP-3 overexpression in arthritic knee joints. Hegemann and colleagues [ 56 ] demonstrated in canine rheumatoid arthritis that the amount of TIMP-1 was not sufficient to block the increased MMP-3 activity. We showed also in murine AIA, by safranin O staining of articular cartilage, that the balance between proteinase and inhibitor expression is disturbed and results in cartilage depletion. Conclusion This study was designed to detect proteinases and proteinase inhibitors that contribute to pathogenic processes in the development of experimental arthritis. We have been able to show that several MMPs, cathepsins and proteinase inhibitors are differentially expressed during the course of AIA. MMP-3, with the highest expression differences, seemed to have the major role in AIA development. We were able to show a correlation between, on the one hand, proteinase activity and proteinase inhibitor expression at the mRNA level and, on the other, cytokine expression. The mRNA data were manifested at the protein level by zymography and activity assays. Safranin O staining showed that the balance between proteinase and inhibitor expression is disturbed. Abbreviations AIA = antigen-induced arthritis; APMA = aminophenylmercuric acetate; ELISA = enzyme-linked immunosorbent assay; IFN = interferon; IL = interleukin; mBSA = methylated bovine serum albumin; MMP = matrix metalloproteinases; PCR = polymerase chain reaction; RA = rheumatoid arthritis; RT = reverse transcriptase; SDS = sodium dodecyl sulfate; TIMP = tissue inhibitor of matrix metalloproteinases; TNF = tumor necrosis factor. Competing interests The author(s) declare that they have no competing interests. Authors' contributions US and NS performed the Affymetrix chip analysis. US, NS and CP analysed the MMP, cathepsin and cytokine expression by real-time PCR. MH performed the zymography, the MMP activity assay and the cytokine ELISAs. US wrote the manuscript. BW and RB supervised the project and gave helpful comments about the manuscript. All authors read and approved the final manuscript.

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1064895

Increased interleukin-17 production via a phosphoinositide 3-kinase/Akt and nuclear factor κB-dependent pathway in patients with rheumatoid arthritis

Inflammatory mediators have been recognized as being important in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-17 is an important regulator of immune and inflammatory responses, including the induction of proinflammatory cytokines and osteoclastic bone resorption. Evidence for the expression and proinflammatory activity of IL-17 has been demonstrated in RA synovium and in animal models of RA. Although some cytokines (IL-15 and IL-23) have been reported to regulate IL-17 production, the intracellular signaling pathways that regulate IL-17 production remain unknown. In the present study, we investigated the role of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the regulation of IL-17 production in RA. Peripheral blood mononuclear cells (PBMC) from patients with RA ( n = 24) were separated, then stimulated with various agents including anti-CD3, anti-CD28, phytohemagglutinin (PHA) and several inflammatory cytokines and chemokines. IL-17 levels were determined by sandwich enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction. The production of IL-17 was significantly increased in cells treated with anti-CD3 antibody with or without anti-CD28 and PHA ( P < 0.05). Among tested cytokines and chemokines, IL-15, monocyte chemoattractant protein-1 and IL-6 upregulated IL-17 production ( P < 0.05), whereas tumor necrosis factor-α, IL-1β, IL-18 or transforming growth factor-β did not. IL-17 was also detected in the PBMC of patients with osteoarthritis, but their expression levels were much lower than those of RA PBMC. Anti-CD3 antibody activated the PI3K/Akt pathway; activation of this pathway resulted in a pronounced augmentation of nuclear factor κB (NF-κB) DNA-binding activity. IL-17 production by activated RA PBMC is completely or partly blocked in the presence of the NF-κB inhibitor pyrrolidine dithiocarbamate and the PI3K/Akt inhibitor wortmannin and LY294002, respectively. However, inhibition of activator protein-1 and extracellular signal-regulated kinase 1/2 did not affect IL-17 production. These results suggest that signal transduction pathways dependent on PI3K/Akt and NF-κB are involved in the overproduction of the key inflammatory cytokine IL-17 in RA.

Introduction Rheumatoid arthritis (RA) is characterized by infiltrations of macrophages and T cells into the joint, and synovial hyperplasia. Proinflammatory cytokines released from these cells are known to be important in the destruction of joints in RA [ 1 ]. The favorable clinical benefits obtained with inhibitors of tumor necrosis factor (TNF)-α) and interleukin (IL)-1 suggest that the blockade of key inflammatory cytokines has been the important issue in the development of new therapeutic applications [ 2 ]. A little over a decade ago, the primacy of T cells in the pathogenesis of autoimmune disease such as RA was undisputed because they are the largest cell population infiltrating the synovium. However, a series of studies demonstrated paucity of T cell-derived cytokines such as IL-2 and interferon-γ in the joints of RA, whereas macrophage and fibroblast cytokines including IL-1, IL-6, IL-15, IL-18 and TNF-α were abundant in rheumatoid synovium. This paradox has questioned the role of T cells in the pathogenesis of RA [ 3 ]. Because we have already demonstrated the enhanced proliferation of antigen specific T cells, especially to type II collagen, and the skewing of T helper type 1 (Th1) cytokines in RA [ 4 ], the role of T cells needs to be elucidated in different aspects. IL-17 is one of the inflammatory cytokines secreted mainly by activated T cells, which can induce IL-6 and IL-8 by fibroblasts [ 5 ]. This cytokine is of interest for two major reasons: first, similarly to TNF-α and IL-1, IL-17 has proinflammatory properties; second, it is produced by T cells [ 6 ]. Recent observations demonstrated that IL-17 can also activate osteoclastic bone resorption by the induction of RANKL (receptor activator of nuclear factor κB [NF-κB] ligand), which is involved in bony erosion in RA [ 7 ]. It also stimulates the production of IL-6 and leukemia inhibitory factor by synoviocytes, and of prostaglandin E 2 and nitric oxide by chondrocytes, and has the ability to differentiate and activate the dendritic cells [ 8 - 10 ]. Levels of IL-17 in synovial fluids were significantly higher in patients with RA than in patients with osteoarthritis (OA), and it was produced by CD4 + T cells in the synovium [ 11 , 12 ]. IL-15, secreted from activated macrophages, has been reported to be an important trigger of IL-17 production in RA peripheral blood mononuclear cells (PBMC) by cyclosporine and steroid sensitive pathways [ 13 ]. Recently, Happel and colleagues also showed that IL-23 could be an efficient trigger of IL-17 production from both CD4 + and CD8 + T cells [ 14 ]. Although the contribution of IL-17 in joint inflammation in RA has been documented in earlier studies [ 12 , 15 , 16 ], the intracellular signal transduction pathway for IL-17 production remains uncertain. In the present study we used various stimuli to investigate IL-17 production in PBMC of patients with RA and its signaling transduction pathway. We found that the intracellular signaling pathway involving phosphoinositide 3-kinase (PI3K)/Akt and NF-κB might be involved in the overproduction of the key inflammatory cytokine IL-17 in RA. These results might provide new insights into the pathogenesis of RA and future directions for new therapeutic strategies in RA. Materials and methods Patients Informed consent was obtained from 24 patients (5 men and 19 women) with RA who fulfilled the 1987 revised criteria of the American College of Rheumatology (formerly the American Rheumatism Association) [ 17 ]. The age of the patients with RA was 50 ± 8 (mean ± SEM) years (range 23–71 years). All medications were stopped 48 hours before entry to the study. Comparisons were made with 14 patients with OA (3 men and 11 women) and with 14 healthy controls (3 men and 11 women) who had no rheumatic diseases. The mean ages of the patients with OA and the healthy controls were 50 ± 8 years (range 34–68 years) and 30 ± 6 years (range 24–57 years). Informed consent was obtained, and the protocol was approved by the Catholic University of Korea Human Research Ethics Committee. Reagents Recombinant IL-17, IL-18, IL-15, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, IL-6 and IL-8 were purchased from R & D systems (Minneapolis, MN, USA). Recombinant transforming growth factor (TGF)-β was purchased from Peprotech (London, UK). Recombinant TNF-α and IL-1 were purchased from Endogen Inc. (Cambridge, MA, USA). Cyclosporin A was provided by Sandos Ltd. (Basel, Switzerland). Phytohemagglutinin (PHA), pyrrolidine dithiocarbamate (PDTC), rapamycin, dexamethasone and curcumin were all obtained from the Sigma Chemical Co. (St Louis, MA, USA). Anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody were obtained from BD Biosciences (San Diego, CA, USA). LY294002, SB203580, FK506, wortmannin and PD98059 were obtained from Calbiochem (Schwalbach, Germany). Production of IL-17 by T cell receptor activation, cytokines or chemokines PBMC were prepared from heparinized blood by Ficoll-Hypaque (SG1077) density-gradient centrifugation. Cell cultures were performed as described previously [ 18 ]. In brief, the cell suspensions were adjusted to a concentration of 10 6 /ml in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine. Cell suspension (1 ml) was dispensed into 24-well multi-well plates (Nunc, Roskilde, Denmark), and incubated for 24 hours at 37°C in 5% CO 2 . Subsequently, various concentrations of cyclosporin A (10–500 ng/ml) were added to the medium and cells were incubated for 24 hours. To each well was added FK506, rapamycin, curcumin, PDTC, LY294002, SB203580, PD98059, dexamethasone or wortmannin. After incubation for 24 hours (unless otherwise stated), cell-free media were collected and stored at -20°C until assayed. All cultures were set up in triplicate, and results are expressed as means ± SEM. CD4 + T-cell isolation by MACS Anti-CD4 microbeads were used essentially as recommended by the manufacturer (Miltenyi) [ 19 ]. PBMC were resuspended in 80 μl of FBS staining buffer. Anti-CD4 microbeads (20 μl) were added and incubated for 15 min at 6–12°C. Saturating amounts of fluorochrome-conjugated antibodies were added for a further 10 min. Cells were diluted in 2.5 ml of FBS staining buffer, pelleted, resuspended in 500 μl and magnetically separated, usually on an AutoMACS magnet fitted with a MACS MS column. Flow-through and two 1 ml washes were collected as the negative fraction. Enriched cells were collected in two 0.5 ml aliquots from the column after removal from the magnet. Alternatively, cells stained with anti-CD4–phycoerythrin were washed, magnetically labeled with anti-phycoerythrin microbeads (20 μl added to 80 μl of cell suspension; 15 min, 6–12°C), and magnetically separated as described above. The purity of cells was assessed by flow cytometric analysis of stained cells on a FACS Vantage sorter. Most (more than 97%) of the isolated cells had the CD4 T cell marker. Enzyme-linked immunosorbent assay of IL-17 IL-17 in culture supernatants was measured by sandwich enzyme-linked immunosorbent assay as described previously [ 20 ]. In brief, a 96-well plate (Nunc) was coated with 4 μg/ml monoclonal antibodies against IL-17 (R & D Systems) at 4°C overnight. After blocking with phosphate-buffered saline/1% bovine serum albumin (BSA)/0.05% Tween 20 for 2 hours at room temperature (22–25°C), test samples and the standard recombinant IL-17 (R & D Systems) were added to the 96-well plate and incubated at room temperature for 2 hours. Plates were washed four times with phosphate-buffered saline/Tween 20, and then incubated with 500 ng/ml biotinylated mouse monoclonal antibodies against IL-17 (R & D Systems) for 2 hours at room temperature. After washing, streptavidin–alkaline phosphate–horseradish peroxidase conjugate (Sigma) was incubated for 2 hours, then washed again and incubated with 1 mg/ml p -nitrophenyl phosphate (Sigma) dissolved in diethanolamine (Sigma) to develop the color reaction. The reaction was stopped by the addition of 1 M NaOH and the optical density of each well was read at 405 nm. The lower limit of IL-17 detection was 10 pg/ml. Recombinant human IL-17 diluted in culture medium was used as a calibration standard, ranging from 10 to 2000 pg/ml. A standard curve was drawn by plotting optical density against the log of the concentration of recombinant cytokines, and used for determination of IL-17 in test samples. Quantification of IL-17 mRNA by semiquantitative reverse transcription–polymerase chain reaction PBMC were incubated with various concentrations of anti-CD3 in the presence or absence of inhibitors (LY294002, PDTC). After 16 hours of incubation, mRNA was extracted with RNAzol B (Biotex Laboratories, Houston, TX, USA) in accordance with the manufacturer's instructions. Reverse transcription of 2 μg of total mRNA was performed at 42°C using the Superscript™ reverse transcription system (Takara, Shiga, Japan). PCR amplification of cDNA aliquots was performed by adding 2.5 mM dNTPs, 2.5 U of Taq DNA polymerase (Takara) and 0.25 μM of sense and antisense primers. The reaction was performed in PCR buffer (1.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl, pH 8.3) in a total volume of 25 μl. The following sense and antisense primers for each molecules were used: IL-17 sense, 5'-ATG ACT CCT GGG AAG ACC TCA TTG-3'; IL-17 antisense, 5'-TTA GGC CAC ATG GTG GAC AAT CGG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense, 5'-CGA TGC TGG GCG TGA GTA C-3'; GAPDH antisense, 5'-CGT TCA GCT CAG GGA TGA CC-3'. Reactions were processed in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA) through cycles for 30 s of denaturation at 94°C, 1 min of annealing at 56°C for GAPDH and IL-17, followed by 1 min of elongation at 72°C. PCR rounds were repeated for 25 cycles each for both GAPDH and IL-17; this was determined as falling within the exponential phase of amplification for each molecule. The level of mRNA expression was presented as a ratio of IL-17 PCR product over GAPDH product. Western blot analysis of Akt, phosphorylated Akt and IκB-α PBMC were incubated with anti-CD3 (10 μg/ml) in the presence or absence of LY294002 (20 μM). After incubation for 1 hour, whole cell lysates were prepared from about 10 7 cells by homogenization in the lysis buffer, and centrifuged at 14,000 r.p.m. (19,000 g ) for 15 min. Protein concentrations in the supernatants were determined with the Bradford method (Bio-Rad, Hercules, CA, USA). Protein samples were separated by 10% SDS–PAGE and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western hybridization, membrane was preincubated with 0.1% skimmed milk in TBS-T buffer (0.1% Tween 20 in Tris-buffered saline) at room temperature for 2 hours, then primary antibodies against Akt, phosphorylated Akt and IκB-α (Cell Signaling Technology Inc., Beverly, MA, USA), diluted 1:1000 in 5% BSA/TBS-T, were added and incubated overnight at 4°C. After washing four times with TBS-T, horseradish peroxidase-conjugated secondary antibodies were added and allowed to incubate for 1 hour at room temperature. After TBS-T washing, hybridized bands were detected with the enhanced chemiluminescence (ECL) detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia). Gel mobility-shift assay of NF-κB binding site Nuclear proteins were extracted from about 5 × 10 6 PBMC. Oligonucleotide probes encompassing the NF-κB binding site of the human IL-17 promoter (5'-ATG ACC TGG AAA TAC CCA AAA TTC-3') were generated by 5'-end labeling of the sense strand with [γ- 32 P]dATP (Amersham Pharmacia) and T4 polynucleotide kinase (TaKaRa). Unincorporated nucleotides were removed by NucTrap probe purification columns (Stratagene, La Jolla, CA, USA). Nuclear extracts (2 μg of protein) were incubated with radiolabeled DNA probes (10 ng; 100,000 c.p.m.) for 30 min at room temperature in 20 μl of binding buffer consisting of 20 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 5% glycerol, 1 mg/ml BSA, 0.2% Nonidet P40 and 50 ng/μl poly(dI-dC). Samples were subjected to electrophoresis on nondenaturing 5% polyacrylamide gels in 0.5 × Tris-borate-EDTA buffer (pH 8.0) at 100 V. Gels were dried under vacuum and exposed to Kodak X-OMAT film at -70°C with intensifying screens. Rabbit polyclonal antibodies against NF-κB subunits p50, p65 and c-Rel were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell viability (Trypan blue dye exclusion assay) For cell viability assays, the trypan blue dye exclusion method was used to evaluate the potential of direct cytotoxic effect of inhibitors on cells. After incubation for 24 hours, the cells were harvested and the percentage cell viability was calculated with the formula 100 × (number of viable cells/number of both viable and dead cells) [ 21 ]. Statistical analysis Data are expressed as means ± SEM. Statistical analysis was performed with Student's t -test for matched pairs. P values less than 0.05 were considered significant. Results IL-17 production in PBMC from patients with RA, patients with OA and normal individuals PBMC were separated and cultured with PHA (5 μg/ml) from patients with RA, patients with OA, and age-matched normal controls; IL-17 levels were then determined in the culture supernatants (Fig. 1 ). Although the amounts of basal IL-17 secretion were not different between RA, OA and normal controls (62 ± 31, 43 ± 19 and 43 ± 10 pg/ml, respectively), the IL-17 production stimulated by PHA was significantly higher in RA PBMC than in those from OA and controls (768 ± 295 versus 463 ± 211 pg/ml [ P < 0.05] and 241 ± 29 pg/ml [ P < 0.001]). Increased IL-17 production in PBMC of patients with RA by anti-CD3 and/or anti-CD28, and PHA Because IL-17 was already known from earlier reports to be produced mainly by activated T cells, we investigated the effect of different concentrations of anti-CD3 (1, 5 and 10 μg/ml) as a T cell activation, which showed a dose-dependent increase in IL-17 levels (data not shown). On the basis of this, we chose 10 μg/ml as a stimulation concentration for anti-CD3. As shown in Table 1 , anti-CD3 significantly upregulated IL-17 production up to 3.7-fold, and the combination of anti-CD28 and anti-CD3 produced more IL-17 (approximately 1.3-1.5-fold) than anti-CD3 alone. Furthermore, when incubated with T cell mitogens such as PHA, increased IL-17 production was more pronounced than with anti-CD3 and anti-CD28 (588 ± 85 versus 211 ± 1 pg/ml; P < 0.05). Regulation of IL-17 production in RA PBMC by inflammatory cytokines and chemokines Because RA PBMC include several cell types in addition to T cells, some inflammatory cytokines released from macrophages and other lymphocytes might have affected the production of IL-17 from T cells. To evaluate the effects of inflammatory cytokines released by activated PBMC, we tested the effects of several cytokines and chemokines on IL-17 production. We detected an increase in IL-17 level after stimulation with IL-15 (10 ng/ml), whereas with IL-1β (10 ng/ml), TNF-α (10 ng/ml), IL-18 (10 ng/ml) or TGF-β (10 ng/ml) the levels in IL-17 were unchanged (Fig. 2a ). When treated with MCP-1 (10 ng/ml) or IL-6 (10 ng/ml), significant upregulations of IL-17 proteins were observed (62 ± 42 and 50 ± 10 versus 31 ± 11 pg/ml, respectively; P < 0.05), whereas none was observed with IL-8 (10 ng/ml), MIP-1α (10 ng/ml) or MIP-1β (10 ng/ml) (Fig. 2b ). Inhibition of IL-17 production by signal transduction inhibitors and anti-rheumatic drugs Having observed the increased IL-17 production in RA PBMC, it was important to know which signal transduction pathways were involved. As illustrated in Fig. 3 , an significant decrease in anti-CD3-induced IL-17 production was observed when co-incubated with NF-κB inhibitor, PDTC and dexamethasone in comparison with anti-CD3 alone (38 ± 5 and 54 ± 11 versus 98 ± 19 pg/ml, respectively; P < 0.05). LY294002 and wortmannin, as an inhibitor of PI3K, also markedly inhibited the anti-CD3-induced IL-17 production in RA PBMC (98 ± 19 versus 38 ± 10 pg/ml [ P < 0.005] and 48 ± 4 pg/ml [ P < 0.05], respectively). The calcineurin inhibitors cyclosporin A and FK506 also downregulated the IL-17 secretion as well as the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 did, whereas rapamycin and PD98059 had no effect on IL-17 levels (Fig. 3 ). To evaluate the possibility of non-specific inhibition by the drug at high concentrations, we observed the dose response of PDTC and LY294002 for the inhibition of IL-17 production in PBMC. There were dose-dependent inhibitions of IL-17 production with chemical inhibitors (Fig. 4a ). The other inhibitors in addition to PDTC and LY294002 showed the same pattern of inhibition. Cytotoxic effects on PBMC by the chemical inhibitors at experimental concentrations were not observed (Fig. 4b ). IL-17 mRNA expression in RA PBMC To see whether enhanced IL-17 production could be regulated at a transcriptional level, semi-quantatitive reverse transcription–polymerase chain reaction was performed. We observed a dose-dependent increase in IL-17 mRNA transcripts after stimulation with anti-CD3; this was inhibited by the PI3K inhibitor LY294002 and by the NF-κB inhibitor PDTC (Fig. 5 ). Activation of PI3K/Akt signal transduction pathway on IL-17 production by anti-CD3 To determine downstream effector molecules of the PI3K pathway, we evaluated the activation of Akt by western blotting. As shown in Fig. 6 , at 10 min of incubation with anti-CD3 (10 μg/ml) or LY294002 (20 μM), no difference in the amounts of phosphorylated Akt was observed. However, after 30 min of incubation, phosphorylated Akt increased (lane 2), and the effect of inhibition by LY294002 (lane 3) reached a peak at 60 min, lasting to 120–240 min. In contrast, non-phosphorylated Akt and β-actin remained unchanged regardless of incubation time. PHA, concanavalin A and IL-15 also demonstrated the same effect on phosphorylated Akt as shown with anti-CD3, which was an inhibition by wortmannin and PDTC as well as by LY294002 (data not shown). Activation of the NF-κB and activator protein-1 (AP-1) pathway in the IL-17 promoter region To investigate further the intracellular signaling pathway activated by anti-CD3 plus anti-CD28, concanavalin A, PHA and IL-15, and responsible for inducing IL-17 expression, we performed an electrophoretic mobility-shift assay (EMSA) of NF-κB recognition sites in the promoters of IL-17. As shown in Fig. 7a , nuclear extracts from RA PBMC stimulated with anti-CD3 plus anti-CD28 (lane 2) demonstrated increased binding of NF-κB to IL-17 promoters in comparison with that of controls (lane 1). A supershift assay demonstrated shifted bands in p65 and p50 (lanes 3 and 4) not in c-Rel (lane 5). In normal PBMC the same pattern was observed, but the degree of NF-κB activation by anti-CD3 plus anti-CD28 was less intense than that in RA PBMC (Fig. 7b ). To confirm the link between PI3K activity and NF-κB, we performed EMSA to determine the NF-κB binding activity after treatment with both LY294002 and PDTC. Both agents block NF-κB DNA-binding activity in the IL-17 promoter (Fig. 7c ). Western blotting for IκB-α showed inhibition of degradation of IκB-α by LY294002 and PDTC at the same time (Fig. 7c ). In contrast, the AP-1 pathway was not activated by stimulation with anti-CD3 plus anti-CD28 (data not shown), demonstrating that NF-κB is the main intracellular signaling pathway in IL-17 production by activated PBMC from patients with RA. Discussion IL-17 was first described as a T cell product with proinflammatory properties [ 5 , 22 ]. RA is characterized by hyperplasia of synovial lining cells and an intense infiltration by mononuclear cells [ 23 ]. Proinflammatory cytokines such as IL-1 and TNF-α are abundant in rheumatoid synovium, whereas the T cell-derived cytokines, especially IL-4 and interferon-γ, have often proved difficult to detect in RA synovium [ 24 ]. Although T cells may have a role in the augmentation of rheumatoid synovial inflammation, the lack of T cell-derived cytokines has limited its importance. In this respect, IL-17 is appealing because it has been described as a T cell-derived cytokine with proinflammatory properties. In our studies, we tried to evaluate how IL-17 production is regulated in RA PBMC, and which signaling pathway it used. Levels of IL-17 were found to be higher in RA synovial fluid than in OA synovial fluid [ 15 ]. However, there are few data available on the agents that stimulate IL-17 production in RA, although the highest level of IL-17 production can be achieved by anti-CD3/anti-CD28 stimulation in healthy individuals [ 25 ]. In our experiments, PHA as mitogens, as well as anti-CD3/anti-CD28 for signaling through the T cell receptor, increased IL-17 production from RA PBMC in a dose-dependent manner. We found, by a cell proliferation assay (data not shown), that this upregulation of IL-17 might be due to increased cellular activity rather than to cellular proliferation. IL-17 is produced mainly by activated CD4 + T cells, especially for Th1/Th0 cells, not the Th2 phenotype [ 26 ]. However, it can also be produced by CD8 + T cells via an IL-23 triggering mechanism in Gram-negative pulmonary infection [ 14 ]. In addition, IL-17 production was significantly augmented by T cells recognizing type II collagen in a collagen-induced arthritis model [ 27 ]. A complex interaction between cells in inflamed RA joints might produce a variety of proinflammatory cytokines and chemokines, which also activate other cells in the joints. For example, IL-17 stimulates rheumatoid synoviocytes to secrete several cytokines such as IL-6, IL-8 and tumor necrosis factor-stimulated gene 6 as well as prostaglandin E 2 in vitro [ 12 , 28 , 29 ]. There are as yet few data available on the agents that stimulate IL-17 production in RA, although some cytokines (IL-15 and IL-23) have been known to regulate IL-17 production [ 13 , 14 ]. We therefore investigated the in vitro production of IL-17 in RA PBMC responding to a variety of cytokines/chemokines and mitogens as well as T cell receptor (TCR) ligation using anti-CD3/anti-CD28. Our studies demonstrated that IL-15 and MCP-1 as well as TCR ligation significantly increased the production of IL-17 in RA PBMC. Adding IL-15 or MCP-1 to TCR ligation augmented IL-17 production more markedly. In contrast, IL-1 and TNF-α, which are known to have proinflammatory properties and to be increased in RA joints, did not affect IL-17 production. Our data were consistent with a recent report that IL-15 triggered in vitro IL-17 production in PBMC, but TNF-α did not do so [ 13 ]. Although there were no data that MCP-1 directly induces T cell activation, it might exert effects indirectly on T cells through the activation of monocytes/macrophages in PBMC cultures. As reported for normal individuals [ 25 ], T cell activation through anti-CD3/anti-CD28 also increases IL-17 induction in RA PBMC. Although the signaling pathway for the induction of cytokines/chemokines by IL-17 has been documented widely [ 8 , 30 , 31 ], no data have been available on how IL-17 production can be regulated by certain signaling pathways. By using signal transduction inhibitors, we therefore examined which signaling pathway was mainly involved in the induction of IL-17 in RA PBMC. We identified that anti-CD3-induced IL-17 production in RA PBMC was significantly hampered by the PI3K inhibitor LY294002 and the NF-κB inhibitor PDTC to comparable levels of basal production without stimulation. We also found that anti-CD3-induced IL-17 production was downregulated by the addition of SB203580, a p38 MAPK inhibitor. It is interesting that a series of evidence supports crosstalk between NF-κB and p38. In myocytes, IκB kinase-β is activated by p38 [ 32 ], and the activated p38 can stimulate NF-κB by a mechanism involving histone acetylase p300/CREB-binding protein [ 33 ]. Our results revealed that p38 MAPK activation was not affected by LY294002, whereas NF-κB binding activity was decreased by LY294002, which provided the evidence for a p38 MAPK pathway independent of PI3K activation. The direct relationship between p38 and NF-κB for IL-17 production needs to be studied in future experiments. The search for a downstream pathway of PI3K seemed to have a maximal response of Akt activation at 1 hour and a gradual loss of activity at 2 hours. The fact that Akt is phosphorylated upon anti-CD3 stimulation suggests the possible involvement of PI3K in the induction of IL-17 in RA. In view of the fact that NF-κB was also activated by anti-CD3/anti-CD28, IL-15 or mitogens in our experiments, it is most likely that the NF-κB pathway is also actively involved in the induction of IL-17 in RA PBMC. In contrast, the AP-1 signal transduction pathway, another important signaling pathway for cytokines/chemokines, was not activated in our experiments (data not shown). Although PI3K and its downstream kinase Akt in association with NF-κB have been reported to deliver activating signals in many cell types, the data on the signal inducing IL-17 are lacking. Our data clearly demonstrated that PI3K/Akt and resultant NF-κB activation could be an important arbitrator of the upregulation of IL-17 in RA, on the basis of our experiments showing simultaneous blocking of NF-κB binding activity in the IL-17 promoter by PDTC and LY294002. Considering its proinflammatory activities and successful induction of anti-IL-17 for ameliorating arthritis in animal models [ 2 , 6 , 34 - 36 ], understanding the IL-17 signaling pathway is an important element of developing new targeted therapies in RA. Conclusions We have detected a more pronounced production of IL-17 from RA PBMC in response to IL-15 and MCP-1 as well as stimulation by anti-CD3/anti-CD28. We have also shown that upregulation of IL-17 by activated T cells in patients with RA could be the result of activation via the PI3K/Akt pathway with resultant NF-κB activation. Our data provide insights into cellular mechanisms of the regulation of IL-17 production in RA, and highlight the role of T cells, which has hitherto been neglected in RA pathogenesis. Together with recent data on the successful introduction of anti-IL-17 in RA, our results have added information for the future molecular targeting of new therapeutic applications in RA. Abbreviations AP-1, activator protein-1; BSA = bovine serum albumin; EMSA = electrophoretic mobility-shift assay; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; IL = interleukin; MAPK = mitogen-activated protein kinase; MCP-1 = monocyte chemoattractant protein-1; MIP = macrophage inflammatory protein; NF-κB = nuclear factor κB; OA = osteoarthritis; PBMC = peripheral blood mononuclear cells; PDTC = pyrrolidine dithiocarbamate; PHA = phytohemagglutinin; PI3K = phosphoinositide 3-kinase; RA = rheumatoid arthritis; TGF = transforming growth factor; Th = T helper; TNF = tumor necrosis factor. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KWK performed the cellular immune response studies and participated in the immunoassays. MLC participated in the design of the study and performed the statistical analysis. MKP participated in the isolation of the cells. CHY drafted the manuscript. SHP participated in the molecular biology and in the PCR. SHL conceived the study, participated in its design and coordination and helped to draft the manuscript. HYK helped to draft the manuscript. All authors read and approved the final manuscript.

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1064896

Early and stable upregulation of collagen type II, collagen type I and YKL40 expression levels in cartilage during early experimental osteoarthritis occurs independent of joint location and histological grading

While morphologic and biochemical aspects of degenerative joint disease (osteoarthritis [OA]) have been elucidated by numerous studies, the molecular mechanisms underlying the progressive loss of articular cartilage during OA development remain largely unknown. The main focus of the present study was to gain more insight into molecular changes during the very early stages of mechanically induced cartilage degeneration and to relate molecular alterations to histological changes at distinct localizations of the joint. Studies on human articular cartilage are hampered by the difficulty of obtaining normal tissue and early-stage OA tissue, and they allow no progressive follow-up. An experimental OA model in dogs with a slow natural history of OA (Pond–Nuki model) was therefore chosen. Anterior cruciate ligament transection (ACLT) was performed on 24 skeletally mature dogs to induce joint instability resulting in OA. Samples were taken from different joint areas after 6, 12, 24 and 48 weeks, and gene expression levels of common cartilage molecules were quantified in relation to the histological grading (modified Mankin score) of adjacent tissue. Histological changes reflected early progressive degenerative OA. Soon after ACLT, chondrocytes responded to the altered mechanical conditions by significant and stable elevation of collagen type II, collagen type I and YKL40 expression, which persisted throughout the study. In contrast to the mild to moderate histological alterations, these molecular changes were not progressive and were independent of the joint localization (tibia, femur, lateral, medial) and the extent of matrix degeneration. MMP13 remained unaltered until 24 weeks, and aggrecan and tenascinC remained unaltered until 48 weeks after ACLT. These findings indicate that elevated collagen type II, collagen type I and YKL40 mRNA expression levels are early and sensitive measures of ACLT-induced joint instability independent of a certain grade of morphological cartilage degeneration. A second phase of molecular changes in OA may begin around 48 weeks after ACLT with altered expression of further genes, such as MMP13, aggrecan and tenascin. Molecular changes observed in the present study suggest that dog cartilage responds to degenerative conditions by regulating the same genes in a similar direction as that observed for chondrocytes in late human OA.

Introduction Osteoarthritis (OA) is a disease with a high prevalence, and it occupies a very important place in orthopedic surgery. It is characterized by progressive degeneration of articular cartilage and damage to subchondral bone. While macroscopic, histological and biochemical features of OA have been extensively studied [ 1 - 4 ], the molecular changes in chondrocyte metabolism underlying the pathophysiological process of cartilage degeneration remain largely unknown. Studies on human articular cartilage are hampered by the difficulty of obtaining normal tissue and early-stage OA tissue. For this reason a number of animal models have been developed in which cartilage degeneration is induced by causing permanent joint instability [ 5 - 7 ]. In the Pond–Nuki model in dogs, anterior cruciate ligament transection (ACLT) leads to joint laxity and altered mechanical loading in the knee joint, resulting in cartilage degeneration over time. This model has the advantage of a fairly slow natural history of OA, since full-thickness loss of articular cartilage does not develop until about 4–5 years after ACLT. The resulting degenerative changes in cartilage and synovial tissue thus closely resemble those in natural canine OA and human OA [ 1 , 4 , 6 , 8 ]. Recent studies have examined the gene expression levels of collagen type (col) II, aggrecan and small proteoglycans in dogs [ 9 - 11 ] and of several cartilage-related and OA-related molecules in rabbits [ 12 - 14 ] over time after ACLT. However, most of these studies used insensitive methods of RNA detection (northern blot) [ 9 , 11 ], or the methods were only semiquantitative [ 13 , 14 ] or failed to relate specific gene expression to the basic expression level of housekeeping genes [ 9 , 10 ]. This may be one reason why contradictory results have been obtained; for example in rabbits, in which no alteration of col II gene expression [ 12 , 13 ] or upregulation at region-specific sites [ 14 ] is reported. Other matrix molecules, such as aggrecan and fibromodulin, seem to be altered only at isolated time points [ 13 , 14 ]. Col II expression in dogs was reported to be higher in ACLT-treated knees than in control knees. These changes in col II expression progressed in one study [ 9 ] and decreased in another one [ 10 ], while the elevation of aggrecan was stable. The histological appearance of OA cartilage makes it obvious that the severity of changes may vary with the location in the joint. It has been demonstrated in the rabbit knee joint that there are differences in RNA levels between different cartilage regions, which emphasizes that it may be risky to pool samples from distinct regions of the knee for molecular analysis [ 15 ]. Nonetheless, the difficulty of extracting sufficient RNA from a limited quantity of cartilage has hampered attempts to find direct correlations between the histological appearance of the cartilage and the metabolism of the chondrocytes in this region. For this reason, histological information has either not been considered at all [ 9 , 10 ] or has been derived from separate animals [ 12 , 14 ] in earlier gene expression studies, although region-specific histological progression of cartilage degeneration was reported in the rabbit ACLT model [ 14 ]. The aim of the present study was to perform a highly sensitive and quantitative molecular analysis of the response of articular cartilage to increased joint instability and altered mechanical loading, and thus to gain more insight into the very early stages of mechanically induced cartilage degeneration. The major objective was to focus on local aspects of gene expression with reference to the histological grading of cartilage degeneration. By improving RNA yields from small cartilage samples and selecting highly sensitive methods for quantitative gene expression analysis, we studied alterations in chondrocyte metabolism side by side with the histological appearance of cartilage degeneration in adjacent tissue. The Pond–Nuki model was chosen because of its close similarity to human OA, and the gene expression levels of six OA-related molecules were followed longitudinally over 48 weeks. Methods Animals Twenty-four skeletally mature beagle dogs were each assigned randomly to one of four experimental groups. The animals' ages ranged from 1 to 2 years (average, 17 months) and the animal body mass was 15–22 kg (average, 19 kg). The dogs were cared for according to the guidelines of the local Council of Animal Care. ACLT was performed on each dog's left knee as described elsewhere [ 7 ], with the right knee serving as the control. After 3 days in individual indoor kennels the animals were allowed to move about freely in groups in outdoor pens. The dogs showed no abnormalities in posture and movement before surgery and at euthanasia. Dogs in the four groups were euthanized after 6, 12, 24 and 48 weeks, respectively. Preparation of specimens Samples were taken within 2 hours after euthanasia. A full-thickness sample about 5 × 2 mm 2 in area, including cartilage and bone, was excised with a hammer and chisel from the lateral and the medial femoral condyle and from the lateral and the medial tibial plateau. For molecular analysis, articular cartilage was shaved off the articular surface 2–4 mm around the site of the histological samples and was immersed in liquid nitrogen. Peripheral areas of cartilage were not included. Histology Samples were fixed in 4% formalin, embedded in paraffin, cut into 3-μm-thick slices and were stained with Safranin O. Specimens were analyzed for the degree of histological change using the Mankin score [ 16 ] modified as previously published [ 17 ]. All sections were graded by three independent observers blinded to the group, and median scores were determined for statistical analysis. Gene expression analysis After measurement of the frozen tissue mass, cartilage samples were pulverized in a freezer mill (Dismembrator S; Braun Biotech, Melsungen, Germany). Messenger RNA was extracted from the powder using oligo(dT)-coated beads (Dynabeads; Dynal, Oslo, Norway) according to the manufacturer's instructions, and was quantified and tested for quality by measurement of the optical density at 280 and 260 nm in a NanoDrop ND100 photometer (Kisker, Steinfurt, Germany). First-strand cDNA was generated using reverse transcriptase (SuperScript II; Invitrogen Life Technologies, Karlsruhe, Germany) and oligo(dT) primers. The cDNA was further purified using a commercially available kit (PCR purification kit; Qiagen, Hilden, Germany). In a pilot study, tissue requirements had been minimized to confine the analysis to the close proximity around the histological tissue sample. About 50 mg tissue was sufficient for quantitative analysis of up to 10 genes. Canine-specific PCR primers for GAPDH, col I, col II, aggrecan and MMP13 were designed on the basis of gene bank information. For tenascinC, YKL39 and YKL40 degenerated primers were applied on canine chondrocyte cDNA to obtain specific DNA fragments. Amplified fragments were purified and sequenced, and specific primers were designed based on this sequence: GAPDH, 5'-GATTGTCAGCAATGCCTCCT-3' and 5'-GTGGAAGCAGGGATGAT-GTT-3' ; col I A1, 5'-GAGAAAGAGGCTTCCCTGGT-3' and 5'-AGGAGAACCATCTCGTCCAG-3' ; col II A1, 5'-TGAATGGAAGAGCGGAGACT-3' and 5'-CCACCATTGAT-GGTTTCTCC-3' ; YKL40, 5'-TCTGTTGGAGGATGGAGCTT-3' and 5'-CAGCCTTCATTTCCTTGACC-3' ; MMP13, 5'-CAGAGCGCTACCTGAAATCC-3' and 5'-CATTGTACTCGCCCACATCA-3' ; aggrecan, 5'-ACCCCT-GAGGAACAGGAGTT-3' and 5'-GTGCCAGATCATCACCACAC-3'; tenascinC, 5'-AGGGGGTCTTCGACAGTTTT-3' and 5'-CATGGCTGTTGTTGCTATGG-3'. Quantitative reverse transcriptase-PCR was performed in a LightCycler (Roche Diagnostics, Mannheim, Germany) with optimized parameters according to the Operator's Manual (version 3.5; Roche). Melting curves were checked for correctness and the size of the fragments was verified on agarose gels. In order to obtain a GAPDH standard curve, dilutions of GAPDH cDNA in a range from 3 × 10 -6 to 3 ng were subjected to LightCycler analysis. The absolute amount of GAPDH mRNA contained in each cartilage sample was obtained after LightCycler analysis by deduction from the GAPDH standard curve. A GAPDH standard was included in every LightCycler run, and the expression of each gene was normalized to the mRNA level of the housekeeping gene GAPDH in the corresponding sample (set as 100% GAPDH). Statistics The mean and standard deviation of each variable were computed. The median and interquartile range were also calculated. A two-way analysis of variance was used to control for the side factor and for the time factor. The side factor was analyzed as the paired measure. Post-hoc tests were also performed to compare time points. Significant changes were depicted. Post-hoc test results were calculated (Scheffé tests) and a two-tailed P ≤ 0.05 was considered significant. An explorative Mann–Whitney U-test and the Wilcoxon test were chosen to evaluate differences between two groups without alpha adjustment. Data analysis was performed with SPSS for Windows 11.0.1 (SPSS Inc., Chicago, IL, USA). Results The age and body mass of the animals were similar in all experimental groups. All but one of the dogs, which was in the 24-week group, were male. One animal had to be excluded after euthanasia following severe injury, so that in the 12-week group only five animals were evaluated instead of six animals. There were no surgical complications, and none of the dogs showed clinical signs of OA, such as altered posture or motion. The animals did not show partially or totally restricted use of the ACLT-treated extremity. When opened, the joints were found to show no macroscopic signs of inflammation or cartilage degeneration at 6 and 12 weeks. In those joints opened 24 weeks after ATLC surgery incipient cartilage discoloration and softening were seen, which were slightly more frequent and pronounced at 48 weeks. Most dogs had an increased volume of synovial fluid in treated knees at the two later time points. Histological examination Histological features observed in cartilage from ACLT-treated knees and from control knees were similar to those described previously [ 3 , 4 , 18 - 20 ]. We found slight changes of the cartilage surface and chondrocytes at 6 weeks after surgery but such changes appeared also in part of the control samples. Decreased Safranin O staining and loss of zonal structure were noticeable in some specimens 12 weeks after ACLT, and were more pronounced in joints examined 48 weeks after surgery. Chondrocyte clustering and fissures never extending beyond the transitional zone were observed at 24 and 48 weeks, which never extended beyond the transitional zone. Variations in histopathological scores were evident within each group, indicating variations in the progression of osteoarthritic changes over time (Fig. 1 ). Median modified Mankin scores at 48 weeks (12.4) were significantly higher than those at 6 weeks (7.4) ( P = 0.036), confirming progressive degeneration of cartilage in ACLT-treated knees. There was no obvious difference between the lateral and the medial compartments of the tibia or of the femur (48 weeks) and no progressive changes were obvious in the control knees over time. In summary, the ACLT-induced changes were progressive and consistent with early stages of OA. Gene expression analysis The concentration of mRNA per sample was determined and used to calculate the absolute amount of mRNA per milligram of tissue wet weight. The mRNA content was similar in all study groups, providing no evidence for major differences in RNA extraction efficiency, tissue water content or overall transcriptional activity of cells between groups. The absolute amount of GAPDH mRNA per total mRNA was determined for each sample by LightCycler analysis using a GAPDH standard curve. A considerable variability of GAPDH per microgram of mRNA was noted that, according to values of the control side (right knee) and the ACLT side (left knee), primarily reflected differences between individuals. Absolute amounts of GAPDH levels per microgram of mRNA determined in all samples were compared by variance analysis comprising the factors time, side and treatment. Post-hoc tests were also performed to analyze differences between all groups. Statistical analysis revealed no significant differences in GAPDH levels between any of the study groups (controls, ACLT, locations, time). Analysis of tenascinC and the chitinase-like molecules YKL39 and YKL40 was included in the present study because they have been related to human OA [ 21 - 23 ]. Since canine DNA sequence information on tenascinC, YKL39 and YKL40 was not available from public databases, degenerated primers were designed on the basis of multiple sequence alignment. Resulting PCR fragments for tenascinC (923 bp) and for YKL40 (665 bp) were sequenced, and they showed 90% and 83% nucleotide identity with the corresponding human cDNA sequence, respectively. The deduced protein sequence of the canine YKL40 fragment had 83% identical and 91% similar amino acids to human YKL40, and was only 48% identical to human YKL39. In spite of intense primer design and PCR analysis, it was not possible to obtain any fragments for YKL39 from dog cartilage. The dog genome database [ 24 ] also contained no sequence information with high homology to human YKL39. Interestingly, in spite of eightfold sequence coverage of the mouse genome, no YKL39 sequence is available from mouse databases and there is no orthologous gene at the locus corresponding to the one where human YKL39 is located. This suggested that mouse and dog lack the YKL39 gene, while YKL40 is present in the human, mouse, and dog. Early upregulation of col II, YKL40 and col I Quantitative reverse transcriptase-PCR analysis of cartilage from the tibial plateau revealed significant upregulation of YKL40 and col II expression at all time points in ACLT-treated knees compared with the untreated joints. The median elevation was twofold to sevenfold for col II, and was threefold to 17-fold for YKL40 (Fig. 2a,2b ). Gene expression of col I was significantly elevated at 12, 24 and 48 weeks after surgery in cartilage from the tibial plateau (Fig. 2c ). Higher expression in OA samples was also evident at 6 weeks, but owing to a large standard deviation the difference did not reach statistical significance. There was no significant increase or decrease over time in the levels of col I or col II or of YKL40 in osteoarthritic cartilage. Femoral condyle samples were analyzed at 48 weeks after surgery. In keeping with our observations in the tibial plateau, expression of col II and of YKL40 was significantly higher in osteoarthritic cartilage than in control cartilage, while col I was highly variable (Table 1 ). Aggrecan, MMP13 and tenascinC expression While mRNA levels for aggrecan tended to be lower in OA samples than in control samples, tenascinC and MMP13 levels tended to be higher (Fig. 3 ). Overall, for all time points, osteoarthritic and control knees did not differ significantly in aggrecan, MMP13 or tenascinC expression. At 24 and 48 weeks, however, MMP13 was significantly higher in ACLT-treated knees than in normal knees (Fig. 3a ). Aggrecan mRNA levels showed relatively wide variation between samples taken from animals in the same study group, and median values tended to be slightly lower in osteoarthritic samples than in control samples. At 48 weeks after ACLT surgery the difference was twofold, and it reached statistical significance ( P = 0.009) (Fig. 3c ). In addition, elevated levels of tenascinC were seen in OA samples at 48 weeks after surgery (Fig. 3b ). mRNA levels of aggrecan, tenascinC and MMP13 in the femur samples (48 weeks) were unchanged in OA samples compared with controls (Table 1 ). Region-specific differences in gene expression Significantly higher expression of col II, YKL40 and col I was evident in osteoarthritic samples than in normal cartilage samples from both the lateral and the medial tibial plateau (Fig. 4a,4b,4c ) at all time points studied. The results for joints examined at all time points were pooled since no time effects were evident for col II, col I and YKL40. On average, the levels of col II in osteoarthritic joints were fourfold those in normal joints in the lateral compartment ( P < 0.001) and were sevenfold those in normal joints in the medial compartment ( P <0.001). YKL40 was elevated to 6.5-fold normal values ( P < 0.001) in the lateral compartment and to eightfold in the medial compartment ( P < 0.001). Expression of col I in the lateral compartment of ACLT-treated knees was fourfold that in control joints ( P = 0.004); levels in the medial compartment were 22-fold normal ( P < 0.001). The baseline expression of several genes tended to be higher in the lateral compartment than in the medial tibial compartment, reaching significance for col II expression (3.5-fold, P = 0.045) and for col I expression (ninefold, P < 0.001) (Fig. 4a,4c ). A similar tendency was seen for basic YKL40 and aggrecan expression, but not for MMP13 and tenascinC expression. In spite of region-specific baseline expression differences, robust generalized molecular effects were seen after ACLT surgery in canine knee cartilage, indicating a consistent regulation of the chondrocytes' response to an altered mechanical loading. No correlation between molecular changes and histological scoring In order to correlate a particular histological outcome with gene expression independently of the study group or the location, samples adjacent to sections with extreme signs of chondrocyte cloning and with the highest ( n = 8) or the lowest ( n = 7) modified Mankin score in the ACLT-treated group were selected. The corresponding samples were compared with seven normal control samples (modified Mankin score 0–2). Comparison of extreme samples preselected for the highest histological scores (Fig. 5a ) and the lowest histological scores (Fig. 5b ) in the ACLT group will increase the statistical power to detect differences that would correlate with histological scoring. Nevertheless, we did not obtain molecular differences between these two ACLT groups, although both kept statistically significant molecular differences to the contralateral control group (Fig. 5c ). Even samples with the lowest modified Mankin score in the ACLT group had already elevated col II ( P = 0.004), col I ( P = 0.007) and YKL-40 ( P = 0.052) expression levels. Upregulation of col II, col I and YKL-40 was thus a very sensitive measure of cartilage degeneration that did not progress any further during the moderate advancement of cartilage degeneration examined in the present study. Discussion Although OA has been well studied, many of the basic molecular mechanisms underlying its development are still unknown. The use of an animal model opens up the possibility of studying the early stages of disease progression and the regional pattern of matrix degradation by comparing diseased and healthy joints in the same individual. The results presented in this report demonstrate that ACLT leads to early and robust upregulation of the extracellular matrix molecule col II and of YKL40 in knee cartilage, which is independent of the time lapse since surgery, of the particular joint region studied and of the structural appearance of the extracellular matrix in adjacent histological sections. In addition, some evidence for a later change of gene expression of MMP13, aggrecan and tenascinC was obtained, which differed significantly from that on the control side by 24 and/or 48 weeks after surgery. Our data might be interpreted as indicative of an early phase of OA development characterized by upregulation of col II, col I and YKL40, which is followed by a second phase of OA progression characterized by further upregulation of MMP13 and tenascinC. From a clinical point of view, the possibility of differentiating successive stages of OA progression by means of early and late disease markers is quite an attractive prospect. According to our data, col II and YKL40 are good candidates for use as robust, early and sensitive markers of joint instability. Although tenascinC and MMP13 may appear on a list of potential marker genes characterizing a second phase of OA, their expression will deserve further attention since no difference between ACLT samples with the lowest and the highest modified Mankin grades is yet obvious for these molecules. Such a distinction would be expected since cartilage degeneration progresses with time. Since the latest time point studied in our dog model is about 2–3 years before full-thickness loss of articular cartilage can be anticipated, the median and late alterations of gene expression cannot be expected to have occurred. Longer studies will be required to decide whether regulation of tenascinC and MMP13 indicate a further stage of molecular alterations in the Pond–Nuki model of OA development. One of the major objectives of this project was to focus on local aspects of gene expression with reference to the histological grading of cartilage degeneration. Most strikingly, upregulation of col II, col I and YKL40 was pronounced in ACLT-treated knees even if the histological grading of adjacent tissue was only weak above control level and there was no progression with OA development (Fig. 5 ). Differing levels of severity or 'doses' of injury have been suggested by others as a possible reason for regional changes in mRNA expression [ 9 ]. Our study, however, lends little credence to this suggestion [ 10 , 13 ]. Knee instability is very likely to increase inappropriate mechanical loading in many parts of the joint and, in keeping with this, col II and YKL40 expression rose in all locations after ACLT in our study. Surprisingly, we detected significant differences in col II and col I expression between the lateral and the medial tibial plateau already in normal control knees, while levels of the housekeeping gene GAPDH did not differ significantly among the compartments at any time point of the study. Given that mechanical forces are modulators of chondrocyte metabolism, even physiologic loading differences may influence basal expression levels of sensitive genes and be the explanation for this effect. This is in line with reports of upregulation of col II and col I synthesis in chondrocytes in response to cyclic loading in vitro [ 25 , 26 ]. Elevated col II and aggrecan mRNA levels were reported previously in early-stage and median-stage canine OA after ACLT [ 9 , 10 ]. In contrast to our data, however, either a progressive [ 9 ] or a declining [ 10 ] elevation of col II RNA was observed over time in those studies. Beside the fact that semiquantitative methods have been used in these previous studies, the normalization of gene expression data will strongly influence the results [ 27 ]. While others decided to express changes in mRNA expression on a per cell basis by referring data to the DNA content of the tissue, we referred specific mRNA expression levels to expression of the housekeeping gene GAPDH. The strength of our method is that we can detect a specific regulation of cartilage-relevant molecules beyond generalized effects that may occur after ACLT, such as the overall activation of chondrocyte metabolism. Its weakness is that there is a risk that the housekeeping gene itself may be regulated by ACLT. Among a selection of housekeeping genes, GAPDH correlated best with total RNA content per milligram of tissue in dog cartilage [ 28 ]. Thorough statistical analysis of our data provided no evidence for either a regulation of total mRNA per milligram of tissue or a regulation of GAPDH in response to ACLT at any time point of the study. Given that the very weak trend to higher GAPDH levels after ACLT would become significant when data are referred on a per cell basis, the upregulation of col II, col I, YKL40, MMP13 and tenascinC per cell would be even more pronounced. Elevated release of proteoglycan and col II protein degradation products into body fluids of ACLT-treated dogs [ 29 ] and of patients with advanced knee OA [ 30 , 31 ] indicate that loss of such molecules from the cartilage matrix makes a major contribution to the development of OA. Chondrocytes may sense this loss and respond to it with upregulation of col II mRNA levels, but they did not adapt mRNA levels for the aggrecan core protein accordingly in the present study. Although there is evidence for enhanced sulfate incorporation into proteoglycans of ACLT-treated cartilage versus normal dog cartilage [ 32 - 34 ], the identity of these molecules remained unknown. Enhanced mRNA levels for small proteoglycans like biglycan, decorin and fibromodulin after ACLT [ 11 ] may explain such observations, but due to the small sample size they unfortunately could not be included in the present study. Human chondrocytes secrete two distinct chitinase-like molecules, called YKL39 and YKL40. While YKL40 has been linked with tissue remodeling, with joint injury and with in situ inflammatory macrophages [ 35 - 40 ], clinical correlates of YKL39 expression remained unknown. Enhanced expression of YKL39, but not of YKL40, was demonstrated in severe human OA cartilage [ 41 , 42 ]. However, in spite of reasonable effort, we have not been able to detect YKL39 in canine chondrocytes and databases. The upregulation both of YKL40 in early stages of dog OA and of YKL39 in late-stage human OA suggests, however, that chitinase-like molecules may have some function in cartilage remodeling and are potentially interesting marker molecules for osteoarthritic joint disease. Human late-stage osteoarthritic cartilage from joint replacement surgery subjected to cDNA array analysis showed significantly elevated levels of col II, col I, chitinase precursor, tenascin and several matrix metalloproteinases, including MMP13, while aggrecan levels remain unaltered [ 43 ]. Taking into account the high donor-dependent variability in human samples, the lower sensitivity of the cDNA array technique than of quantitative PCR and the different time frames studied, the overlap between molecular alterations in natural human OA and experimental canine OA is considerable. This indicates that chondrocytes in dog cartilage respond to degenerative conditions by regulating the same genes as chondrocytes in human OA, and in a similar direction. Conclusion In conclusion, upregulation of col II, col I and YKL40 was a very sensitive and robust response to the altered mechanical situation after ACLT surgery, which occurred quite independent of joint location and a certain grade of morphological cartilage degeneration. Levels did not progress any further during the moderate advancement of cartilage degeneration examined in the present study, and more progressed stages of cartilage degeneration may therefore rather be characterized by regulation of additional cartilage-relevant molecules like MMP13 and tenascinC. We interpret the molecular alterations in natural human OA and experimental canine OA as considerable and we suggest that the Pond–Nuki model may be a suitable experimental model to unravel further basic anabolic and catabolic molecular mechanisms of relevance for human disease development. Abbreviations ACLT = anterior cruciate ligament transection; bp = base pair; col = collagen type; GAPDH = glyceraldehydes-3-phosphate dehydrogenase; OA = osteoarthritis; PCR = polymerase chain reaction. Competing interests The author(s) declare that they have no competing interests. Authors' contributions HL participated in the design of the study, coordinated and assisted surgery, carried out the molecular analysis, evaluated histology and drafted the manuscript. WW participated in the design of the study, performed surgery and evaluated histological samples. MI assisted with surgery and evaluated histological samples. ES participated in the design and evaluation of molecular analysis, and contributed to the manuscript. WR conceived of the study, participated in its design, and contributed to histological and molecular data analysis and to the manuscript. All authors read and approved the manuscript.

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1064897

JNK1 is not essential for TNF-mediated joint disease

Tumour necrosis factor (TNF) signalling molecules are considered as promising therapeutic targets of antirheumatic therapy. Among them, mitogen-activated protein kinases are thought to be of central importance. Herein, we investigate the role in vivo of TNF-α signalling through c-Jun N-terminal kinase (JNK)1 in destructive arthritis. Human TNF transgenic (hTNFtg) mice, which develop inflammatory arthritis, were intercrossed with JNK1-deficient ( JNK1 -/- ) mice. Animals ( n = 35) of all four genotypes (wild-type, JNK1 -/- , hTNFtg, JNK1 -/- hTNFtg) were assessed for clinical and histological signs of arthritis. Clinical features of arthritis (swelling and decreased grip strength) developed equally in hTNFtg and JNK1 -/- hTNFtg mice. Histological analyses revealed no differences in the quantity of synovial inflammation and bone erosions or in the cellular composition of the synovial infiltrate. Bone destruction and osteoclast formation were observed to a similar degree in hTNFtg and JNK1 -/- hTNFtg animals. Moreover, cartilage damage, as indicated by proteoglycan loss in the articular cartilage, was comparable in the two strains. Intact phosphorylation of JNK and c-Jun as well as expression of JNK2 in the synovial tissue of JNK1 -/- hTNFtg mice suggests that signalling through JNK2 may compensate for the deficiency in JNK1. Thus, JNK1 activation does not seem to be essential for TNF-mediated arthritis.

Introduction Proinflammatory cytokines bind to their receptors on the plasma membrane and transmit the stimulatory effects to the nucleus via intracellular signalling molecules. Therefore, these cytokines are considered as promising therapeutic targets. Drugs specifically inhibiting such proteins are usually small molecules and are thought to open a new frontier in antirheumatic therapy along with newly arisen cytokine-blocking strategies. Among the many downstream molecules of cytokine signalling, mitogen-activated protein kinases (MAPKs) are of central importance in shuttling the signal of proinflammatory cytokines, such as IL-1 or tumour necrosis factor (TNF)-α, to their respective target tissues [ 1 , 2 ]. Cellular activation by TNF-α is a critical step in chronic synovial inflammation and progressive joint destruction. This is supported by the overwhelming effects of TNF blockade, which has revolutionized the therapy of rheumatoid arthritis (RA). It inhibits both inflammation and destruction of joints, in a majority of patients suffering from RA [ 3 - 5 ]. The hypothesis is supported by animal models in which specific overexpression of TNF-α is sufficient to cause chronic destructive arthritis [ 6 ]. Increased levels of this cytokine in the synovial fluid and tissue of RA patients have also been reported [ 7 - 9 ]).). To design therapeutic tools that not only interfere with TNF signalling but also effectively block TNF-mediated inflammatory responses, it is essential to identify the major signalling targets of TNF in inflammatory joint disease. In fact, TNF-α signalling is a complex process, involving not only MAPKs but also other pathways including nuclear factor κB and the caspase cascade [ 10 , 11 ]. MAPKs are thought to be of central importance for mediating the proinflammatory effects of TNF-α. Interestingly, all three MAPK families – p38 protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) – are activated in the synovial membrane of RA patients, and TNF-α has the potential to signal through all of them [ 12 ]. Therefore, each of these different MAPKs is a possible therapeutic target. We investigated the role of JNK1 in TNF-mediated inflammatory joint disease. Our findings show that the JNK1 signal pathway is not essential for the development of arthritis and joint destruction. Materials and methods Animals The heterozygous human TNF transgenic (hTNFtg) mouse (strain: tg197; background: C57/BL6) has been described previously [ 6 ]. As reported elsewhere, mice of this strain develop destructive arthritis resembling RA within 4–6 weeks of birth [ 6 , 13 ]. JNK1-deficient ( JNK1 -/- ) mice were generated as previously described [ 14 ]. The hTNFtg and JNK1 -/- strains were intercrossed to obtain double mutant animals. F 2 generations were used and all data were generated from littermates. A total of 35 mice (wt, n = 7; hTNFtg, n = 13; JNK1 -/- , n = 6; and JNK1 -/- hTNFtg, n = 9) of six different breedings were studied. This study was approved by the local ethical committee pf the Medical University of Vienna. Clinical assessment Arthritis was evaluated in a blinded manner as described in earlier reports [ 13 ]. Assessments were started when the mice were 5 weeks old and were repeated weekly. In brief, joint swelling was assessed using a clinical score graded from 0 to 3 (0, no swelling; 1, mild swelling of toes and ankle; 2, moderate swelling of toes and ankle; 3, severe swelling of toes and ankle). In addition, the grip strength of each paw was analysed on a wire 3 mm in diameter, using a score from 0 to -4 (0, normal grip strength; -1, mildly reduced grip strength; -2, moderately reduced grip strength; -3, severely reduced grip strength; -4, no grip strength at all). After cervical dislocation, the blood was withdrawn by heart puncture and the paws of all animals were dissected and preserved for histological analysis. The last evaluation was performed 10 weeks after birth. Histological sections and histochemistry A total of 26 mice (wt, n = 7; hTNFtg, n = 6; JNK1 -/- , n = 6; and JNK1 -/- hTNFtg, n = 7) were assessed histologically. Hind and front paws and right knee joints were fixed in 4.0% formalin overnight and then were decalcified in a 14% EDTA/ammonium hydroxide buffer at pH 7.2 (Sigma-Aldrich, St Louis, MO, USA) at 4°C until the bones were pliable. Serial paraffin sections (2 μm) were stained with H&E, or with toluidine blue for tartrate-resistant acid phosphatase (TRAP) activity. TRAP staining was performed as previously described [ 13 ]. For immunohistochemistry, deparaffinized, ethanol-dehydrated tissue sections were boiled for 2 min in 10 mM sodium citrate buffer (pH 6.0) using a 700-W microwave oven. Tissue sections were cooled to room temperature and then rinsed using detergent solution: 0.5% Tween in phosphate-buffered saline (PBS). For quantification of inflammation, areas of H-&-E-stained sections were measured (5 sections/animal). The total area of inflammation for each single animal was calculated by evaluating all digital, carpal, and tarsal joints and the right knee joint. The same H-&-E-stained sections were analysed as described above for quantification of erosions. The number of osteoclasts was counted as described above analysing TRAP-stained serial sections. Cartilage breakdown (i.e., proteoglycan loss and matrix dissolution) was measured from toluidine-blue-stained serial sections by assessing cartilage according to the method of Joosten and colleagues [ 15 ]. Total and destained cartilage areas were measured and percentages of destained areas indicating low proteoglycan content were ascertained. For immunohistochemistry, dewaxed, ethanol-dehydrated tissue sections were boiled for 2 min in 10 mM sodium citrate buffer (pH 6.0) using a 700-W microwave oven, then allowed to cool to room temperature and rinsed in detergent solution (0.5% Tween in PBS) for 10 min. Tissue sections were blocked for 20 min in PBS containing 20% rabbit serum and were then incubated for 1 hour at room temperature with the following antibodies (Abs): rat monoclonal antimacrophage (F4/80) Ab (Serotec Inc, Oxford, UK); diluted 1:80), rat monoclonal anti-CD3 Ab (Novocastra, Newcastle, UK; diluted 1:100), rat monoclonal anti-CD45R/B220 Ab (Pharmingen International, Oxford, UK) and rat monoclonal antineutrophil Ab (clone7/4, Serotec), mouse monoclonal antiphosphorylated-JNK Ab (clone G7, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-JNK2 Ab (clone D2, Santa Cruz), and mouse monoclonal phospho-specific anti-c-Jun Ab (clone KM-1, Santa Cruz). The sections were rinsed, and then endogenous peroxidase was blocked with 0.3% hydrogen peroxide in Tris-buffered saline (10 mM Tris/HCl, 140 mM NaCl, pH 7.4) for 10 min. This was followed by 30 min of incubation with a biotinylated antirat IgG secondary Ab (Vector, Burlingame, CA, USA). Then, sections were incubated with the appropriate VECTASTAIN@ABC reagent (Vector) for another 30 min using 3,3'-diaminobenzidine (Sigma). Statistical analysis Data are shown as means ± standard deviation. Group mean values were compared using a two-tailed Student's t test. Results TNF-induced clinical signs of arthritis develop independently of JNK1 To study the role in vivo of JNK1 activation in TNF-mediated arthritis, we intercrossed hTNFtg with JNK1 -/- mice. The offspring of all four genotypes (wt, hTNFtg, JNK1 -/- , and JNK1 -/- hTNFtg) were born at Mendelian frequency and were viable. To evaluate arthritis, we assessed joint swelling and grip strength weekly in all four genotypes. Neither wt nor JNK1 -/- mice developed any signs of paw swelling, and both maintained normal grip strength (data not shown). In contrast, the hTNFtg animals developed joint swelling at 6 weeks of age, which increased to a maximum at week 10 ( P < 0.05 in comparison with wt and JNK1 -/- ). In the JNK1 -/- hTNFtg group, joint swelling started at age 7 weeks and increased significantly thereafter ( P < 0.05 in comparison with wt and JNK1 -/- ). There was no significant difference between the hTNFtg and JNK1 -/- hTNFtg mice at any time of the analysis (Fig. 1a ). In addition, grip strength significantly decreased in both hTNFtg and JNK1 -/- hTNFtg strains, but no significant difference between the two genotypes was found (Fig. 1b ). Thus, overexpression of TNF-α induces clinical signs of arthritis also in the absence of JNK1. Similar degrees of synovial inflammation and cellular composition of synovial inflammatory tissue in hTNFtg and JNK1 -/- hTNFtg mice We next more closely evaluated arthritis by quantitative and qualitative histological analysis of inflammatory tissue (Fig. 2 ). Animals of both control groups, wt and JNK1 -/- , did not show any sign of joint inflammation or destruction. In contrast, hTNFtg mice not only developed intense inflammation but also showed multiple bone erosions. Comparable destructive changes were observed in joints from JNK1 -/- hTNFtg mice. Quantitative analysis of the area of synovial inflammation revealed no significant differences between hTNFtg and JNK1 -/- hTNFtg mice (Fig. 3a ). Similarly, quantification of erosive changes was comparable in these two genotypes (Fig. 3b ). Furthermore, immunohistochemical analysis revealed similar distributions of T cells, B cells, granulocytes, and macrophages within the synovial membranes of the two genotypes (Table 1 ). Synovial osteoclast formation is not affected by the absence of JNK1 Since JNK1 is involved in osteoclast differentiation, we wanted to know whether the absence of JNK1 influences TNF-driven osteoclast formation in the inflamed joints. Staining for the osteoclast-specific enzyme TRAP revealed numerous osteoclasts within arthritic bone erosions in both hTNFtg and JNK1 -/- hTNFtg mice (Fig. 4a ). Quantification of osteoclasts revealed no significant difference between the two groups (Fig. 4b ), reflecting that the absence of JNK1 does not alter the progression of joint destruction in conditions of TNF overexpression. Proteoglycan content is reduced in cartilage of hTNFtg and JNK1 -/- /hTNFtg mice Lastly, we addressed whether the absence of JNK1 influences cartilage damage in TNF-α-induced arthritis. Quantitative assessment of proteoglycan loss by toluidine blue staining showed a marked reduction of proteoglycan content in both hTNFtg and JNK1 -/- hTNFtg mice (Fig. 5a ). But again, no significant difference in the percentage of the affected cartilage surface was detected between hTNFtg and JNK1 -/- hTNFtg mice. In contrast, wt and JNK1 -/- animals revealed virtually no alterations of articular cartilage (Fig. 5b ). This suggests that TNF-α induces degradation of cartilage independently of JNK1. Lack of JNK1 decreases expression of phosphorylated JNK but does not affect activation of c-Jun Surprisingly, JNK1 -/- hTNFtg mice developed destructive arthritis comparable to that of hTNFtg animals. Therefore, we assessed JNK signalling in animals of both genotypes. Histological staining of synovial membrane from JNK1 -/- hTNFtg mice revealed significantly fewer cells expressing phosphorylated JNK than in hTNFtg animals (Fig. 6a ; P < 0.05). The expression within the synovial membrane of JNK2 (Fig. 6b ) or phosphorylated c-Jun (Fig. 6c ) was similar in both groups that developed arthritis. Discussion In RA, cytokine-mediated cell activation leads to chronic synovial inflammation as well as bone and cartilage destruction. Evidence from basic and clinical research has established TNF-α as one of the major players in the pathogenesis of RA. The effects of TNF-α are mediated via membrane receptors which themselves activate intracellular messenger cascades, such as MAPK. JNKs are especially important, because of their ability to phosphorylate the activator protein (AP)-1 component c-Jun, making them critical regulators of transcription [ 16 ]. The JNK proteins include three different isoforms, of which JNK1 and (with even higher affinity) JNK2 phosphorylate c-Jun [ 17 ]. JNK2 is preferentially bound to c-Jun in unstimulated cells, whereas JNK1 becomes the major c-Jun-interacting kinase after cell stimulation [ 18 ]. Notably, JNK1 appears to be a key regulator of the differentiation of type 1 T helper cells in mice [ 19 ]. Like other MAPK pathways, JNK signalling is activated in the synovial tissue of RA patients [ 12 , 20 - 22 ]. Studies with cultured synovial fibroblast-like cells from RA patients have established proinflammatory cytokines, such as TNF-α and IL-1, as important activators of JNK signalling, and JNK2 is the dominant isoform in synovial cells [ 12 , 20 - 22 ]. In these cells, TNF-induced phosphorylation of JNK leads to the phosphorylation of c-Jun and finally activation of the transcription factor complex AP-1 [ 20 ]. Loss of either JNK1 or JNK2 suppresses AP-1 [ 21 ]. The JNK pathway is therefore involved in the regulation of genes coding for collagenases, chemoattractants such as macrophage chemoattractant protein-1, or the adhesion molecule E-selectin [ 23 , 24 ]. These data indicate that JNK1 is dispensable in TNF-mediated joint disease. Of interest, arthritis in hTNFtg mice is directly induced by overexpression of TNF, a potent trigger of the JNK pathway. Thus, it is surprising that even if disease is caused by the overexpression of a trigger of JNK-signalling, the absence of JNK1 is not essential for the development of the disease. However, the hTNFtg animal model has its limits, since it bypasses the autoimmune inductive phase, which is seen in other arthritis models such as collagen-induced arthritis or adjuvant arthritis, as well as in human disease. Thus, although these findings are relevant for TNF-mediated inflammation and connective tissue destruction, their implication for human RA should be seen with caution. Interestingly, the role of JNK has been investigated in autoimmune-based models of experimental arthritis in two outstanding studies. Treatment of rat adjuvant arthritis with SP600125, an inhibitor that affects kinase activity of both JNK1 and JNK2, was followed by a modest decrease of inflammation and paw swelling and an impressive inhibition of radiographic damage [ 21 ]. The influence of selective deletion of JNK2 was investigated in collagen-induced arthritis in mice. The severity of arthritis was even slightly increased, and histological evaluation showed a degree of synovial inflammation comparable to that in wt mice [ 25 ]. These latter findings suggest that selective inhibition of JNK2 is not effective to block destructive arthritis and raises the question whether JNK1 is a more promising target or effective inhibition of arthritis depends on the nonselective inhibition of JNK1 and JNK2. Our data extend this knowledge: the selective inhibition of JNK1 is not effective to block inflammation in TNF-driven arthritis. In fact, we show that even in the complete absence of JNK1, phosphorylation of JNK, although to a reduced amount, still occurs via engagement of JNK2. The latter is expressed in the synovial inflammatory tissue and its activation by TNF is sufficient to induce c-Jun transcription factor. Taken together, these findings from various experimental models of arthritis suggest that only the inhibition of both JNK isoforms, JNK1 and JNK2, may be a feasible approach to achieve a major blockade of synovitis and thus achieve therapeutic efficacy. Development of local bone erosions in the joints affected by chronic arthritis depends on the presence of osteoclasts [ 13 , 26 ]. Interestingly, JNK1 plays an important role in osteoclastogenesis. Cells derived from JNK1 -/- mice revealed an impaired differentiation of osteoclasts in vitro [ 27 ]. Thus, targeting of JNK1 could have been considered as a feasible approach to protect from inflammatory joint damage. Surprisingly, however, the degree of bone erosions as well as numbers of osteoclasts was not affected by the lack of JNK1, indicating that TNF-driven osteoclastogensis is independent of JNK1 in vivo . In fact, matrix metalloproteinases substantially contribute to irreversible degradation of collagen. The IL-1-dependent induction of matrix metalloproteinases in chondrocytes is regulated through complex pathways including MAPKs, AP-1, and nuclear factor κB transcription factors [ 28 ]. Interestingly, lack of JNK2 led to a modest but significant reduction of cartilage damage in collagen-induced arthritis [ 25 ]. Therefore, we hypothesized that a reduction of cartilage damage could be expected after knocking out JNK1 in hTNFtg mice. However, much as with synovial inflammation and bone loss, no significant protection of articular cartilage has been observed in JNK1 -/- hTNFtg, suggesting that JNK1 does not seem to be important in TNF-mediated cartilage destruction. Conclusion Taken together, these findings show that JNK1 is not essential for TNF-mediated joint disease. Specific inhibitors of JNK1 in mice probably work in conditions that are not primarily dependent on TNF-α. Moreover, an effective inhibition of synovitis and joint destruction may necessitate a combined blockade of the JNK isoforms or even additional MAPKs. Abbreviations Ab = antibody; AP-1 = activator protein-1; H&E = hematoxylin and eosin; hTNFtg = human tumour necrosis factor transgenic; IL-1 = interleukin-1; JNK = c-Jun N-terminal kinase; MAPK = mitogen-activated protein kinase; PBS = phosphate-buffered saline; RA = rheumatoid arthritis; TNF = tumour necrosis factor; TRAP = tartrate-resistant acid phosphatase; wt = wild-type. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MK carried out histological analyses and drafted the manuscript. SH, EW, and GS conceived of the study and participated in its design and coordination. KR carried out histological and statistical analyses. RR participated in breeding of mice. JD participated in the design of the study and breeding of mice. GSt coordinated immunohistological analysis. JS participated in the design of the study. All authors read and approved the final manuscript.

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1065018

Effects of positive end-expiratory pressure on gastric mucosal perfusion in acute respiratory distress syndrome

Introduction Positive end-expiratory pressure (PEEP) improves oxygenation and can prevent ventilator-induced lung injury in patients with acute respiratory distress syndrome (ARDS). Nevertheless, PEEP can also induce detrimental effects by its influence on the cardiovascular system. The purpose of this study was to assess the effects of PEEP on gastric mucosal perfusion while applying a protective ventilatory strategy in patients with ARDS. Methods Eight patients were included. A pressure–volume curve was traced and ideal PEEP, defined as lower inflection point + 2 cmH 2 O, was determined. Gastric tonometry was measured continuously (Tonocap). After baseline measurements, 10, 15 and 20 cmH 2 O PEEP and ideal PEEP were applied for 30 min each. By the end of each period, hemodynamic, CO 2 gap (gastric minus arterial partial pressures), and ventilatory measurements were performed. Results PEEP had no effect on CO 2 gap (median [range], baseline: 19 [2–30] mmHg; PEEP 10: 19 [0–40] mmHg; PEEP 15: 18 [0–39] mmHg; PEEP 20: 17 [4–39] mmHg; ideal PEEP: 19 [9–39] mmHg; P = 0.18). Cardiac index also remained unchanged (baseline: 4.6 [2.5–6.3] l min -1 m -2 ; PEEP 10: 4.5 [2.5–6.9] l min -1 m -2 ; PEEP 15: 4.3 [2–6.8] l min -1 m -2 ; PEEP 20: 4.7 [2.4–6.2] l min -1 m -2 ; ideal PEEP: 5.1 [2.1–6.3] l min -1 m -2 ; P = 0.08). One patient did not complete the protocol because of hypotension. Conclusion PEEP of 10–20 cmH 2 O does not affect gastric mucosal perfusion and is hemodynamically well tolerated in most patients with ARDS, including those receiving adrenergic drugs.

Introduction Recent studies have shown that lung protective strategies using low tidal volumes and high levels of positive end-expiratory pressure (PEEP) reduce mortality and are becoming standard practice in patients with acute respiratory distress syndrome (ARDS) [ 1 , 2 ]. Although PEEP improves arterial oxygenation, it can adversely affect systemic hemodynamics, reducing venous return and cardiac output. These effects are proportional to the PEEP level. Regional perfusion can also be affected by PEEP, independently of cardiac output changes. The splanchnic perfusion is particularly sensitive, and any reduction can compromise its barrier function, promote bacterial translocation, and contribute to the development of multiple organ failure [ 3 ]. In experimental models, PEEP has markedly decreased mesenteric and portal blood flow, despite only moderate reductions in cardiac output [ 4 - 8 ]. Similar results have been reported in patients without lung injury [ 9 , 10 ]. These effects are usually dose related, becoming more pronounced with PEEP levels around 20 cmH 2 O. Kiefer reported that PEEP did not significantly alter splanchnic blood flow in six patients with acute lung injury [ 11 ]. Nevertheless, caution should be exercised in extending these results to clinical practice, because only hemodynamically stable patients without adrenergic drugs were studied, and PEEP levels never exceeded 14 cmH 2 O [ 12 ]. Our aim was to evaluate the effects of PEEP levels up to 20 cmH 2 O on gastric mucosal perfusion and systemic hemodynamics in mechanically ventilated patients with ARDS under hemodynamic support. Methods Patients The study was approved by the Ethics Committee of the Medicine Faculty and was performed in the Surgical Intensive Care Unit of the Catholic University Hospital of Chile. Adult mechanically ventilated patients were considered eligible for the study if they met the following criteria for ARDS during the 24 hours that preceded the study: acute onset of respiratory failure; diffuse bilateral infiltrates in the chest radiograph; a ratio of partial pressure of O 2 (PaO 2 ) to fraction of inspired oxygen (FiO 2 ) of less than 200 mmHg; and a pulmonary arterial occlusion pressure less than 18 mmHg and no cardiac failure. Hemodynamic monitoring included an arterial line and a pulmonary artery catheter (Baxter Edwards Critical-Care, Irvine, CA). Patients could be under vasopressor or inotropic support, but had to be hemodynamically stable for at least 3 hours before starting the protocol. Patients were excluded if they had any of the following conditions: pregnancy, pre-existing respiratory dysfunction, cardiac index of less than 2.5 l min -1 m -2 , or were receiving enteral nutrition. Interventions A nasogastric tonometer (TRIP ® Tonometry Catheter 14F, with biofilter connector for TONOCAP™ Monitor) was inserted into the stomach and connected to air automated tonometry (TONOCAP™ Monitor; Datex-Engstrom, Helsinki, Finland). All patients were sedated with midazolam and morphine, and paralyzed with rocuronium. Neuromuscular relaxation was measured by a TOF watch ® device. An intravenous 20 mg dose of famotidine was administered before starting the protocol. Patients were connected to volume-controlled mechanical ventilation (Servo 900 C; Siemens, Solna, Sweden). A pressure–volume curve was obtained for each patient by the airway occlusion technique [ 13 ], and ideal PEEP was defined as the lower inflection point + 2 cmH 2 O, or 12 cmH 2 O if no lower inflection point was found. PEEP levels of 10, 15, 20 cmH 2 O, and ideal PEEP, with tidal volumes of 8 ml kg -1 , were applied in four consecutive 30 min periods, respectively. Respiratory rate was modified to maintain end tidal CO 2 within ± 10 mmHg of basal. All patients were receiving a constant infusion of 6% hetastarch before the beginning of the study (40–80 ml h -1 ). Cardiac output was optimized before and during the trial by determining the respiratory variation of systolic arterial pressure [ 14 ]. Whenever the variation was more than 10% a 100 ml bolus of 6% hetastarch was infused and the volume status was reassessed. No change in adrenergic support was allowed during the protocol. If hypotension (mean arterial pressure < 65 mmHg) persisted for more than 1 min, the protocol was stopped. Measurements At baseline, and at the end of each period, hemodynamic, ventilatory and tonometric measurements were performed, and arterial blood samples withdrawn. Hemodynamic records included mean arterial pressure, heart rate, cardiac output, pulmonary artery occlusion pressure, central venous pressure and left ventricular stroke work index. Cardiac output was measured by thermodilution as the average of three values obtained after injections of 10 ml of 5% dextrose in water at room temperature. Mean airway pressure, oxygenation index and PEEP levels were registered. Oxygenation index was calculated as mean airway pressure × FiO 2 × 100/PaO 2 . The CO 2 gap (gastric partial pressure of CO 2 [pCO 2 ] minus arterial pCO 2 ) was calculated by comparing simultaneous measurements of tonometric gastric mucosal pCO 2 and arterial pCO 2 . Statistical analysis Results are presented as median and range. The software Statview 5.0 was used to perform the statistical analysis. Nonparametric tests were used because of the small sample size. Data were analyzed with a Friedman test followed by a Wilcoxon signed-rank test if necessary. Results were considered statistically significant at P < 0.05. Results Eight patients with ARDS were enrolled. They had a median (range) age of 63.5 years (23–86), and an Acute Physiology and Chronic Health Evaluation II score of 14 (7–20) at admission to the intensive care unit. On the day of the study they had a median Sepsis-related Organ Failure Assessment (SOFA) [ 15 ] score of 10 (7–13). All patients fulfilled criteria for ARDS, as defined by the inclusion criteria, during the 24 hours before the study and they had been on mechanical ventilation for 32 (12–72) hours. They were being ventilated with a median PEEP level of 9 (4–12) cmH 2 O, they had a PaO 2 /FiO 2 ratio of 235 (144–388) mmHg and their respiratory system compliance was 45 (27–60) ml per cmH 2 O. Seven patients had sepsis (two pneumonia and five extrapulmonary sepsis), and one a severe thoracic trauma. Of the septic patients, six were in septic shock. Characteristics of individual patients are shown in Table 1 . Table 1 Baseline characteristics of the patients Patient Age (years) Sex Diagnosis APACHE II SOFA PaO 2 /FiO 2 (mmHg) pH Bicarbonate (mEq/L) PEEP (cmH 2 O) Crs (ml/cmH 2 O) LIP (cmH 2 O) Vasopressors/inotropes a Outcome (S/NS) 1 55 M Hepatic lobectomy 14 13 144 7.38 25.4 10 51 10 NA 0.08 S Dbt 3.3 2 23 F Peritonitis 20 10 388 7.36 23.5 8 32 10 NA 0.12 S 3 32 M Mucormycosis and sepsis 7 7 282 7.42 21.5 6 60 6 NA 0.09 S 4 68 F Acute pancreatitis 9 13 208 7.38 20.4 10 40 NL NA 0.2 NS 5 59 F Pneumonia and sepsis 16 8 197 7.28 25.5 10 55 NL NA 0.03; Dp 6.8; Dbt 3.4 S 6 68 M Thoracic trauma 14 10 289 7.36 21.6 4 37 13 NA 0.05 S 7 72 M Sepsis 17 9 263 7.25 13.8 4 50 8 Dbt 5.4 S 8 86 M Pneumonia and sepsis 14 12 150 7.37 20.3 12 27 13 NA 0.02 NS APACHE, Acute Physiology and Chronic Health Evaluation; Crs, Respiratory system compliance; Dbt, dobutamine; Dp, dopamine; LIP, lower inflection point; NE, norepinephrine (noradrenaline); NL, no LIP found; NS, not significant; PEEP, positive end-expiratory pressure; S, significant; SOFA, Sepsis-related Organ Failure Assessment. a Doses are in μg kg -1 min -1 . No changes in cardiac index or in CO 2 gap were found at any of the study periods (Table 2 ). Oxygenation index, mean arterial pressure, pulmonary mean arterial pressure, pulmonary artery occlusion pressure, central venous pressure and left ventricular stroke work index also remained stable through the study. Only mean airway pressure and PaO 2 /FiO 2 ratio differed between periods, as expected. Five patients required a 100 ml bolus of hetastarch during the trial; in no patient was it necessary to repeat it. Individual changes in CO 2 gap and cardiac index are presented in Figs 1 and 2 , respectively. At baseline three patients had already a CO 2 gap of more than 20 mmHg. After starting the protocol with 10 cmH 2 O PEEP, patient 6, who was previously being ventilated with 4 cmH 2 O PEEP, had a further increase in CO 2 gap. When PEEP was increased from 10 to 15 cmH 2 O, six patients decreased their CO 2 gap and two increased it. When PEEP was increased from 15 to 20 cmH 2 O, three patients increased their CO 2 gap, three decreased it and in one patient it remained unchanged. Patient 4 did not complete the protocol because of moderate hypotension (mean arterial pressure 60 mmHg) when PEEP was increased to 20 cmH 2 O. This patient recovered after an increased dose of norepinephrine (noradrenaline) and a return of PEEP to baseline levels. Table 2 Respiratory, hemodynamic and tonometric measurements Parameter Baseline ( n = 8) PEEP 10 ( n = 8) PEEP 15 ( n = 8) PEEP 20 ( n = 7) Ideal PEEP ( n = 7) P PEEP (cmH 2 O) 9 (4–12) 10 15 20 12 (8–15) Mean airway pressure (cmH 2 O) 13.2 (8–18.7) 14 (12–17) 19 (17–22.2) 24 (22–26.4) 16.2 (11.5–22.2) 0.0001 a OI (cmH 2 O per mmHg) 5.3 (2.9–12.4) 7 (3–14.5) 6.7 (4.1–12.3) 7 (5–12.3) 6.6 (2.9–12.3) 0.3 PaO 2 /FiO 2 (mmHg) 235 (144–388) 210 (117–402) 285 (154–412) 333 (196–440) 243 (164–467) 0.0009 b PaCO 2 (mmHg) 36 (31–54) 41 (28–63) 42 (31–66) 45 (32–60) 43 (28–52) 0.08 Cardiac index (l min -1 m -2 ) 4.6 (2.5–6.3) 4.5 (2.5–6.9) 4.3 (2–6.8) 4.7 (2.4–6.2) 5.1 (2.1–6.3) 0.08 LVSWI (g m m -2 ) 45 (22–71) 43 (22–60) 40 (14–60) 36 (15–58) 42 (14–66) 0.13 MAP (mmHg) 79 (74–103) 81 (69–99) 74 (69–97) 74 (66–93) 73 (69–96) 0.24 PAOP (mmHg) 16 (10–19) 17 (8–22) 17 (11–23) 18 (12–26) 14 (11–23) 0.22 CVP (mmHg) 14 (9–17) 15 (7–19) 15 (9–24) 15 (10–19) 12 (8–18) 0.27 CO 2 gap (mmHg) 19 (2–30) 19 (0–40) 18 (0–39) 17 (4–39) 19 (9–39) 0.18 Results are presented as median (range). CVP, central venous pressure; CO 2 gap, arterial partial pressure of CO 2 [pCO 2 ] minus gastric pCO 2 ; FiO 2 , fraction of inspired oxygen; LVSWI, left ventricular stroke work index; MAP, mean arterial pressure; OI, oxygenation index, defined as mean airway pressure × FiO 2 × 100/arterial pCO 2 ; PaO 2 , partial pressure of O 2 ; PaCO 2 , partial pressure of CO 2 ; PAOP, pulmonary arterial occlusion pressure; PEEP, positive end-expiratory pressure. a P < 0.05 for all comparisons except baseline versus PEEP 10 and PEEP 10 versus ideal PEEP. b P < 0.05 for all comparisons except baseline versus PEEP 10, baseline versus PEEP 15, baseline versus ideal PEEP, and PEEP 15 versus ideal PEEP. Six of the eight patients studied survived (75%). The median length of stay in the intensive care unit was 17 (10–34) days and the median duration of mechanical ventilation was 9 (5–34) days. Figure 1 Individual changes in CO 2 gap (gastric pCO 2 minus arterial pCO 2 ) with different positive end-expiratory pressure levels. Figure 2 Individual changes in cardiac index with different positive end-expiratory pressure levels. Discussion Our results show that high PEEP levels (up to 20 cmH 2 O) do not compromise gastric mucosal perfusion, as assessed by tonometry, and do not affect systemic hemodynamics in most patients with ARDS. This is consistent with the findings of two other studies on the effects of PEEP on splanchnic perfusion in patients with ARDS. Nevertheless, in contrast with our study, neither of those studies included patients in septic shock or under adrenergic support [ 11 , 16 ]. Shock and cardiovascular dysfunction are frequently associated with ARDS. This is an important issue, because hemodynamic safety concerns could preclude the use of high or optimal PEEP levels in that setting, even if necessary. A major finding of our study is that PEEP levels up to 20 cmH 2 O can be well tolerated, even in patients with ARDS and septic shock. Nevertheless, our trial was relatively short and we cannot exclude the possibility that keeping high PEEP levels for a longer period might result in increased fluid requirements, which could be deleterious in the longer term. Experimental and clinical research has demonstrated that in mechanically ventilated subjects without lung injury, PEEP decreases venous return and, secondarily, cardiac output [ 17 - 19 ]. In addition, Trager and colleagues showed that, in patients with acute respiratory failure associated with septic shock, high PEEP levels induced a decrease in cardiac output [ 20 ]. In contrast, we found no decrease in cardiac output in our patients tested with increasing PEEP levels when fluid administration was optimized according to respiratory variation in systolic arterial pressure. A similar result was reported by Kiefer and colleagues and by Akinci and colleagues [ 11 , 16 ]. Possible explanations for these contradictory results are a higher rate of fluid administration and the use of lower tidal volumes in the latter studies. Although we did not determine the upper inflection point of the pressure–volume curve, we think that by keeping tidal volume at 8 ml kg -1 any overdistension of the lungs was minimized. Lung volumes are a critical component of the hemodynamic effects of ventilation [ 21 ]. Thus, it seems that it is possible to preserve cardiac output in patients with ARDS, despite the use of high PEEP levels, by optimizing fluid administration and limiting tidal volumes. Gastric mucosal perfusion, as assessed by CO 2 gap, also remained unchanged during the PEEP trial. This is consistent with the results reported by Kiefer and Akinci in similar studies. In all these studies cardiac output remained unchanged [ 11 , 16 ]. In contrast, Trager reported, in a series of septic shock patients with acute respiratory failure, that an increase in PEEP from 5 to 15 cmH 2 O induced a decrease in cardiac output associated to a decrease in hepatic vein O 2 saturation and in hepatic glucose production [ 20 ]. It therefore seems that by avoiding decreases in cardiac output, splanchnic perfusion can be preserved in the majority of the patients. In spite of the fact that no significant changes in CO 2 gap or cardiac index were found during the protocol, when looking at the individual data certain patients evidenced an adverse effect when their PEEP level was increased. Patient 4, who had an extrapulmonary ARDS, presented hypotension when 20 cmH 2 O PEEP was applied. In this case, no simultaneous records of cardiac output or CO 2 gap could be made for safety reasons (we immediately proceeded to decrease PEEP level). Patient 6, who had a pulmonary ARDS and who before starting the study had a 30 mmHg CO 2 gap while being ventilated with 4 cmH 2 O PEEP, presented a further deterioration in CO 2 gap after starting the protocol with 10 cmH 2 O PEEP, which was not associated with a decrease in cardiac output. Thereafter, the CO 2 gap remained unchanged despite increasing PEEP up to 20 cmH 2 O. These events suggest that not all patients with ARDS can tolerate high PEEP levels; if required, careful hemodynamic monitoring including assessment of regional perfusion should be applied. One major limitation of our study is the small number of patients studied. Thus, a type II error cannot be excluded. We did not perform any a priori power analysis because we had no estimation of the possible magnitude of the effects that PEEP could have on gastric tonometry. Another limitation is the rather moderate severity of ARDS in our study. Although all patients fulfilled criteria for ARDS during the 24 hours that preceded the study, at inclusion their PaO 2 /FiO 2 ratio and their respiratory system compliance were only moderately decreased. Two recent papers provide an explanation for this observation [ 22 , 23 ]. They show in patients diagnosed with ARDS that after a few hours of treatment with PEEP or a high FiO 2 , more than half of the patients present a PaO 2 /FiO 2 ratio of more than 200 mmHg. In addition, respiratory system compliance increased by more than 10 ml per cmH 2 O after 6 hours of treatment with PEEP [ 23 ]. At inclusion our patients had already been ventilated with a median PEEP level of 9 cmH 2 O for more than 12 hours, which could have explained the rather improved respiratory performance at baseline. In any event, this improvement demonstrated a less severe ARDS. It is possible that more severely compromised patients might present a lower tolerance to high PEEP levels. Other limitation is that tonometry was the sole method used to assess gastric mucosal perfusion. Nevertheless, Elizalde and colleagues showed that gastric mucosal blood flow, measured by laser Doppler flowmetry and by reflectance spectrophotometry, is well correlated with gastric intramucosal acidosis in mechanically ventilated patients [ 24 ]. Conclusions Our study supports the findings of previous studies suggesting that high PEEP levels do not affect splanchnic perfusion and are hemodynamically well tolerated in most patients with ARDS. Furthermore, our study shows that gastric mucosal perfusion can be well preserved while high PEEP levels are applied even in patients presenting cardiovascular dysfunction and receiving adrenergic support, which is a frequent occurrence in critical care. Nevertheless, two of the eight patients studied exhibited adverse effects during the PEEP trial, which highlights the importance of a close monitoring of systemic and regional perfusion while applying high PEEP levels to patients with ARDS. Future studies should assess the effects of PEEP on splanchnic perfusion in a longer term. Key messages • High PEEP levels do not affect gastric mucosal perfusion and are hemodinamically well tolerated in most patients with ARDS Competing interests None declared. Abbreviations ARDS = acute respiratory distress syndrome; CO 2 gap = gastric pCO 2 minus arterial pCO 2 ; FiO 2 = fraction of inspired oxygen; PaO 2 = partial pressure of O 2 ; pCO 2 = partial pressure of CO 2 ; PEEP = positive end-expiratory pressure.

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1065019

Comparison of two percutaneous tracheostomy techniques, guide wire dilating forceps and Ciaglia Blue Rhino: a sequential cohort study

Introduction To evaluate and compare the peri-operative and postoperative complications of the two most frequently used percutaneous tracheostomy techniques, namely guide wire dilating forceps (GWDF) and Ciaglia Blue Rhino (CBR). Methods A sequential cohort study with comparison of short-term and long-term peri-operative and postoperative complications was performed in the intensive care unit of the University Medical Centre in Nijmegen, The Netherlands. In the period 1997–2000, 171 patients underwent a tracheostomy with the GWDF technique and, in the period 2000–2003, a further 171 patients with the CBR technique. All complications were prospectively registered on a standard form. Results There was no significant difference in major complications, either peri-operative or postoperative. We found a significant difference in minor peri-operative complications ( P < 0.01) and minor late complications ( P < 0.05). Conclusion Despite a difference in minor complications between GWDF and CBR, both techniques seem equally reliable.

Introduction Tracheostomy is usually performed in patients who need prolonged mechanical ventilation, frequent suctioning of bronchopulmonary toilet or have obstruction of the upper airway. The percutaneous tracheostomy is a minimally invasive, effective and reliable procedure and has become the alternative to surgical tracheostomy [ 1 ]. Almost all percutaneous procedures in The Netherlands are performed with one of the three following techniques: guide wire dilating forceps (GWDF) tracheostomy, Ciaglia Blue Rhino (CBR) tracheostomy, and sequential dilation tracheostomy (classic Ciaglia) [ 2 ]. We have extensive experience with the first two techniques [ 3 , 4 ]. This study is a sequel to our previous reports. Several studies have compared different percutaneous techniques [ 5 - 12 ], but because CBR is relatively new, a comparison with GWDF has been made only twice in two small prospective cohorts [ 5 , 12 ]. The strength of the present study is the large group of patients, so the incidence of relevant complications is more meaningful. The aim of this study was to compare GWDF and CBR. The study not only focuses on the immediate peri-operative complications but also describes the long-term sequelae of both techniques. Methods This is a retrospective analysis of all patients who underwent percutaneous tracheostomy in the University Medical Centre Nijmegen between March 1997 and April 2003. We compared the two historic data sets that we have published previously [ 3 , 4 ], but we specifically focused on the precise definition of early complications and long-term sequelae. Between March 1997 and February 2000 we performed percutaneous tracheostomy on 171 patients, using the GWDF technique. Between March 2000 and April 2003 we performed percutaneous tracheostomy on a further 171 patients, using the CBR technique. Indications, contra-indications and technique for percutaneous tracheostomy are standardised [ 3 , 4 ]. Patients or family gave informed consent before the procedure. Ethical approval from the institution's medical ethical committee was not obtained because the standard of care was provided and no other experimental treatments were introduced. Published data cannot be reduced to a single recognisable patient. All data were recorded prospectively on pre-designed forms. 'Procedure time' was defined as the time from incision to successful placement of the cannula. A 'peri-operative complication' was defined as a complication related to the procedure and occurring during or within 24 hours of the procedure. Postoperative complications were divided into 'complications while cannulated' and 'late complications'. A 'complication while cannulated' was defined as a complication occurring in the period between 24 hours after the procedure until removal of the cannula. A 'late complication' was defined as a complication occurring after removal of the cannula up to a follow-up of 3 years. Complications were divided into minor and major (see Tables 1 , 2 , 3 ). Moreover, complications were classified as procedure-specific and procedure-non-specific. Hypotension was defined as a systolic blood pressure of less than 90 mmHg. Hypoxaemia was defined as an arterial oxygen saturation of less than 90%. It was considered minor when lasting less than 5 min, and major when lasting 5 min or longer. Information regarding late complications was obtained by structured interviews with patients who were decannulated successfully. Patients or close relatives were asked about voice changes, dyspnoea, stridor, pain, and cosmetic problems. Patients were also asked to grade specific problems as absent, minor or major. Table 1 Peri-operative complications Complication GWDF ( n = 171) CBR ( n = 171) P Conversion to surgical tracheostomy No. % No. % GWDF ( n = 6) CBR ( n = 2) No complications 128 74.9 100 58.5 <0.01 Minor complications Procedure-specific Bleeding (local pressure) 11 6.4 24 14.0 0.04 Difficult dilation 0 23 13.5 <0.01 Difficult procedure 6 3.5 7 4.1 NS Subcutaneous emphysema 2 1.2 2 1.2 NS Cannula insertion difficult 0 3 1.8 NS Air leakage cuff 0 2 1.2 NS Procedure-non-specific Puncture endotracheal tube 9 5.3 8 4.7 NS Puncture posterior tracheal wall 4 2.3 2 1.2 NS Accidental detubation 1 0.6 3 1.8 NS Hypotension 1 0.6 2 1.2 NS Total 34 19.9 75 43.9 <0.01 Major complications Procedure-specific Bleeding (exploration) 6 3.5 4 2.3 NS 2 Bleeding (life-threatening) 1 0.6 1 0.6 NS Fausse route 2 1.2 1 0.6 NS 1 Oesophageal perforation 1 0.6 0 NS 1 Cannula insertion impossible 3 1.8 0 NS 3 Pneumothorax 0 3 1.8 NS 1 Total 13 7.6 9 5.3 NS a Some patients had more than one complication. CBR, Ciaglia Blue Rhino; GWDF, guide wire dilating forceps; NS, not significant. Table 2 Complications while cannulated Complication GWDF CBR P No. % No. % Surgical tracheostomy 6 2 Lost to follow-up 1 0 Available for analysis 164 169 No complications 139 84.8 138 81.7 NS Minor complications Bleeding (local pressure) 15 9.1 14 8.3 NS Infection 4 2.4 6 3.6 NS Granulation tissue around stoma 1 0.6 1 0.6 NS Pain from stoma 1 0.6 0 NS Tracheal oedema 0 1 0.6 NS Subcutaneous emphysema 0 1 0.6 NS Dyspnoea 0 1 0.6 NS Total 21 12.8 24 14.2 NS Major complications Bleeding (exploration) 0 2 1.2 NS Bleeding (life-threatening) 0 0 NS Stridor (with empty cuff) 2 1.2 0 NS Cardiopulmonary resuscitation 1 0.6 0 NS Cannula obstruction 1 0.6 3 1.8 NS Hypoxaemia 0 2 1.2 NS Total 4 2.4 7 4.1 NS CBR, Ciaglia Blue Rhino; GWDF, guide wire dilating forceps; NS, not significant. All data were analysed with Statistical Product and Service Solutions (SPSS) version 11.0. All variables were checked for normal distribution. Data are given as means ± SD or medians. Continuous variables were compared with Student's t -test or the Mann–Whitney test as appropriate. Bonferroni's correction for multiple comparisons was used. Categorisable variables were compared with the χ 2 test. A cut-off level of P < 0.05 was accepted as statistically significant. Table 3 Late Complications Complication GWDF CBR P No. % No. % Surgical tracheostomy 6 2 Lost to follow up 5 6 Still cannulated 0 3 Deceased 53 60 Available for analysis 107 100 No complications 86 80.2 73 73.0 NS Minor Complications Voice 9 8.5 22 22.0 <0.01 Cosmetic problems 10 9.4 2 2.0 0.04 Pain 0 2 2.0 NS Total minor complications 19 17.9 26 26.0 NS Major complications Stridor 2 1.9 1 1.0 NS CBR, Ciaglia Blue Rhino; GWDF, guide wire dilating forceps; NS, not significant. Results Demographic data are shown in Table 4 . The procedure was successful in 165 of 171 patients (96.5%) in the GWDF group and in 169 of 171 patients (98.8%) in the CBR group. Most tracheostomies were performed by an intensivist or a fellow (under supervision). More procedures were performed by a fellow in the CBR group than in the GWDF group (51 versus 27, respectively; P < 0.01). Table 4 Demographic data Parameter GWDF ( n = 171) CBR ( n = 171) P Mean SD Median Mean SD Median Age (years) 57.5 18.2 62 57.5 18.4 62 NS Male/Female 99/72 114/157 NS Endotracheal intubation (days) 16.9 12.2 14 20.3 12.3 18 0.03 Procedure time (min) 9.1 8.3 5.0 10.8 10.5 7.0 NS Cannulation time (days) 38.4 63.4 24 29.6 39.8 18 NS Time in ICU (days) 39.4 29.8 33 44.1 38.3 34 NS CBR, Ciaglia Blue Rhino; GWDF, guide wire dilating forceps; ICU, intensive care unit; NS, not significant. Peri-operative complications Peri-operative complications are described in Table 1 . In total, there were 47 peri-operative complications in 43 patients in the GWDF group, and 84 peri-operative complications in 71 patients in the CBR group ( P < 0.05). This difference is explained by a greater number of difficult dilations ( P < 0.01) and minor bleedings with the CBR technique. After the introduction of a Crile's forceps for blunt dissection of the pretracheal tissues preceding CBR, the procedure became much easier. In the GWDF group, 13 patients (7.6%) had a major complication, compared with 9 patients (5.3%) in the CBR group. All these major peri-operative complications were procedure-specific. One life-threatening bleeding in the GWDF group led to severe hypoxia at the end of the procedure. After removal of the cannula, large blood clots were suctioned from the trachea. There was no significant difference in the number of patients in whom conversion to a surgical tracheostomy was necessary. In the GWDF group, six patients underwent conversion to a surgical tracheostomy: one patient had a major venous bleeding after dilation of the trachea and the cannula could not be inserted. In another patient, arterial blood was aspirated and the procedure was terminated. In two patients, the trachea was difficult to locate, resulting in hypoxaemia and hypercapnia. In one patient the guide wire was placed correctly but the cannula perforated the posterior tracheal wall and entered the oesophagus. Surgical exploration confirmed rupture of the oesophagus, and the tracheo-oesophageal wall was immediately repaired. The post-operative course was uneventful. In the last patient the distance between skin and trachea was too large for the insertion of a cannula. In the CBR group two patients underwent surgical tracheostomy: in one patient the trachea was difficult to locate, and the cannula was placed pretracheally as a result of guide wire kinking. Another patient developed major bleeding and tension pneumothorax several hours after the procedure. After immediate drainage with a chest tube, surgical exploration showed that the tracheostomy tube had perforated the cricothyroid membrane. No deaths were seen after either procedure. Complications while cannulated In total, 164 GWDF and 169 CBR patients were analysed for complications while cannulated (Table 2 ). Four major complications (2.4%) occurred in the GWDF group, and seven major complications (4.1%) in the CBR group. One patient in the GWDF group had an obstruction of the cannula by a mucous plug, leading to a cardiorespiratory arrest. Another patient sustained a cardiorespiratory arrest shortly after decannulation, possibly due to aspiration. Both patients were resuscitated successfully. Three patients in the CBR group had an obstruction of the cannula: one of them died on his first day on the ward, possibly owing to an obstructive blood clot in the cannula. The second patient had a mucous plug causing severe hypoxaemia. He received a minitracheotomy through the old tracheostomy opening. The third patient with an obstructed cannula was found in bed on the ward, having a respiratory arrest. The inner cannula, which was obstructed by a blood clot, was removed. The patient recovered uneventfully. Late complications Of 164 patients in the GWDF group, 53 (32.3%) died with the cannula in place or within 1 week after decannulation, and five patients were lost to follow-up. One hundred and seven GWDF patients (62.6%) were decannulated successfully and analysed for late complications (Table 3 ). Of 169 CBR patients, 60 (35.5%) died with the cannula in place or within 1 week of decannulation, six patients were lost to follow-up, and three patients had the cannula still in situ . Finally, 100 CBR patients (58.5%) were analysed for late complications. There was no significant difference between both groups with regard to total late complications. All patients with voice problems were given the opportunity to consult an ENT specialist. None of these had an objective laryngeal abnormality explaining their voice problems. Patients with cosmetic problems relating to the tracheostomy scar were offered specialist consultation. Six GWDF patients underwent scar revision. Three patients developed a severe stridor after decannulation. In the GWDF group, an 83-year-old woman had tracheal stenosis and was treated with an endotracheal stent, and an 80-year-old woman was treated with laser for a granuloma just above the tracheostomy opening. In the CBR group, an 18-year-old man suffered from severe tracheal stenosis. He had a tracheal stent placed initially, but because of recurrence of the stenosis, a tracheal resection was necessary. The patient recovered uneventfully. Discussion In this study we have compared two different techniques of percutaneous tracheostomy, GWDF and CBR. Both techniques are frequently used in The Netherlands and are replacing the surgical technique [ 2 ]. This study showed no significant differences in clinically relevant complications between the two techniques. This is in agreement with two other studies comparing these techniques [ 5 , 12 ]. Although the total number of complications in the two groups in the study of Ambesh and colleagues was not significantly different, the authors noticed an increased rate of minor peri-operative bleeding in the GDWF group [ 5 ]. This was balanced by an increase in the number of patients with one or more tracheal ring fractures in the CBR group (30%). The increase in major peri-operative bleeding with the GDWF technique might be explained by the poorly controllable dilation with the forceps [ 9 ]. Although the study of Añón and colleagues did not find any significant differences, in three of 26 patients in the GWDF group there was an inability to insert the cannula [ 12 ]. Several other studies comparing sequential dilation (classic Ciaglia) and CBR [ 6 , 8 ], and comparing sequential dilation and GWDF [ 7 , 9 - 11 ], have been described in the literature. Van Heurn and colleagues concluded that sequential dilation and GWDF are both reliable but that sequential dilation has fewer early complications than GWDF [ 7 ]. Nates and colleagues also preferred sequential dilation to the GWDF technique, because of fewer surgical complications, less peri-operative and postoperative bleeding, and easier use [ 9 ]. Añón and colleagues found a comparable complication rate, but the procedural time of the GWDF method was significantly shorter [ 10 ]. Unfortunately, comparing these studies is difficult because complications were not defined uniformly. In our study, a major complication while cannulated was obstruction of the cannula, which occurred in four patients. These figures correspond to the prevalence of cannula obstruction in the literature (0.3–3.5%) [ 13 - 15 ]. Strict adherence to nursing protocols and a low threshold for cleaning the inner cannula should be the standard of care in the intensive care unit. An outreach team from the intensive care unit should visit patients, discharged to the general ward with a cannula in place, on a daily basis. There are only few data available concerning late complications of percutaneous tracheostomy. Unfortunately, many confounders might be present, such as the disease process itself, the duration of endotracheal intubation, and other treatments in the intensive care unit (such as sedation or physical therapy). Moreover, both patients and caregivers often interpret late complications subjectively. The total number of late complications in our study was not significantly different between the two groups. Subjective voice changes and hoarseness were more frequent in the CBR group ( P < 0.01). An explanation might be the longer mean endotracheal intubation time, because this is possibly the most important cause of voice problems. With sequential dilation tracheostomy, the incidence of voice problems ranges between 0% and 21% [ 16 - 22 ]. More patients in the GWDF group complained of cosmetic problems. Only a few studies have mentioned cosmetic complaints, but differences of opinion between patient and caregiver are frequent [ 23 ]. In each group in our study, one patient developed a critical symptomatic tracheal stenosis. More patients might have had an asymptomatic tracheal stenosis, but because no additional diagnostic tests such as computed tomography or magnetic resonance imaging scans were performed, the actual incidence is unknown. Several studies have incriminated the GWDF technique as a cause of tracheal stenosis, but no studies with the CBR have been described. The incidence varied from 0% to 63% [ 18 , 23 - 27 ]. Most of these tracheal stenoses were asymptomatic. Several factors might decrease the strength of our conclusions. First, the study used historical data sets with a sequential design; a time bias is therefore possible. As experience with percutaneous tracheostomy increases, the number of complications will decrease, even if another technique is used, although in our study this might well have been balanced by the fact that over time more fellows performed the procedure. Second, scoring of the peri-operative complications by different physicians might be variable because of different interpretations. Despite these shortcomings, we conclude from our study that, although the CBR technique has more minor peri-operative complications, the two techniques are comparable. More prospective, randomised studies are required to compare these different tracheostomy techniques adequately. We are currently conducting a prospective, randomised study in which we compare GWDF and CBR tracheostomies; we are specifically looking for the occurrence of precisely defined early and late complications. The occurrence of tracheal stenosis will be analysed using the forced oscillation technique and magnetic resonance imaging. Key messages • GWDF and CBR tracheostomy seem equally reliable. • Major peri-operative complications occur in 5.3–7.6% of patients. • Late complications are rare Competing interests None declared. Abbreviations CBR = Ciaglia Blue Rhino; GWDF = guide wire dilating forceps.

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1065024

Early tracheostomy in intensive care trauma patients improves resource utilization: a cohort study and literature review

Introduction Despite the integral role played by tracheostomy in the management of trauma patients admitted to intensive care units (ICUs), its timing remains subject to considerable practice variation. The purpose of this study is to examine the impact of early tracheostomy on the duration of mechanical ventilation, ICU length of stay, and outcomes in trauma ICU patients. Methods The following data were obtained from a prospective ICU database containing information on all trauma patients who received tracheostomy over a 5-year period: demographics, Acute Physiology and Chronic Health Evaluation II score, Simplified Acute Physiology Score II, Glasgow Coma Scale score, Injury Severity Score, type of injuries, ICU and hospital outcomes, ICU and hospital length of stay (LOS), and the type of tracheostomy procedure (percutaneous versus surgical). Tracheostomy was considered early if it was performed by day 7 of mechanical ventilation. We compared the duration of mechanical ventilation, ICU LOS and outcome between early and late tracheostomy patients. Multivariate analysis was performed to assess the impact of tracheostomy timing on ICU stay. Results Of 653 trauma ICU patients, 136 (21%) required tracheostomies, 29 of whom were early and 107 were late. Age, sex, Acute Physiology and Chronic Health Evaluation II score, Simplified Acute Physiology Score II and Injury Severity Score were not different between the two groups. Patients with early tracheostomy were more likely to have maxillofacial injuries and to have lower Glasgow Coma Scale score. Duration of mechanical ventilation was significantly shorter with early tracheostomy (mean ± standard error: 9.6 ± 1.2 days versus 18.7 ± 1.3 days; P < 0.0001). Similarly, ICU LOS was significantly shorter (10.9 ± 1.2 days versus 21.0 ± 1.3 days; P < 0.0001). Following tracheostomy, patients were discharged from the ICU after comparable periods in both groups (4.9 ± 1.2 days versus 4.9 ± 1.1 days; not significant). ICU and hospital mortality rates were similar. Using multivariate analysis, late tracheostomy was an independent predictor of prolonged ICU stay (>14 days). Conclusion Early tracheostomy in trauma ICU patients is associated with shorter duration of mechanical ventilation and ICU LOS, without affecting ICU or hospital outcome. Adopting a standardized strategy of early tracheostomy in appropriately selected patients may help in reducing unnecessary resource utilization.

Introduction Patients with multiple trauma often require mechanical ventilation for prolonged periods because of their inability to protect their airways, persistence of excessive secretions, and inadequacy of spontaneous ventilation [ 1 ]. Tracheostomy plays an integral role in the airway management of such patients, but its timing remains subject to considerable practice variation [ 2 ]. The decision to proceed to tracheostomy is often made only if the patient could not be extubated within 10–14 days or more [ 3 ]. In 1989, the American College of Chest Physicians Consensus Statement on Artificial Airways in Patients Receiving Mechanical Ventilation considered translaryngeal intubation to be the preferred technique for patients requiring up to 10 days of mechanical ventilation [ 4 ]. For those with anticipated need for artificial airway for more than 21 days, tracheostomy was recommended. For all other patients, the decision regarding the timing of tracheostomy was left to daily assessment and physician preference. Such practice was based on earlier reports showing high tracheal stenosis rates with tracheostomy as compared with endotracheal intubation [ 5 , 6 ]. For example, one study reported in 1981 [ 6 ] found an incidence of tracheal stenosis after tracheostomy of 65%, as compared with 19% after endotracheal intubation. The authors of that study concluded that tracheostomy for patients requiring an artificial airway for periods as long as 3 weeks could not be recommended. However, the incidence of tracheal stenosis has decreased substantially with recognition of its aetiology and improvements in tracheostomy materials, design and management [ 7 ], particularly with the use of high-volume, low-pressure cuffs. Also, the complications associated with prolonged endotracheal intubation are increasingly being recognized, including injury to the larynx and trachea, and patient discomfort. In addition, endotracheal intubation often requires the administration of systemic sedation, with attendant complications. Finally, the incidence of ventilator-associated pneumonia is related directly to the duration of mechanical ventilation [ 8 ] – a complication that carries significant morbidity and mortality [ 9 ]. One of the under-appreciated consequences of delaying tracheostomy is prolonged mechanical ventilation and intensive care unit (ICU) stay. Notably, the large body of literature addressing local complications of tracheostomy contrasts with the paucity of reports on the advantages of this procedure, especially its impact on resource utilization. This contrast may have encouraged practitioners to consider alternatives to tracheostomy. The aim of the present study is to examine the impact of early tracheostomy on resource utilization in ICU trauma patients. This examination is followed by a review of the existing literature in this area. Methods Settings The study was performed at a major tertiary care trauma centre in Riyadh, Saudi Arabia. The 600-bed hospital has a 21-bed medical/surgical ICU staffed by full-time, on-site intensivists 24 hours a day and 7 days a week. Our department has nine consultant intensivists, all of whom are certified in critical care. The hospital has a designated trauma service, including a consultant surgeon, available 24 hours a day. Medical care in the ICU is provided by the ICU team, with the trauma team being responsible for surgical aspects of care. Ventilatory management, and decisions regarding extubation or tracheostomy and discharge from the ICU are made primarily by the ICU team. All percutaneous tracheostomies are performed at the bedside by the ICU team. Data collection We have maintained a prospective database including all consecutive ICU patients admitted since March 1999. For the present study we extracted data on all consecutive patients admitted to the ICU over a 5-year period (March 1999 to February 2004) with new trauma and who underwent tracheostomy during their ICU stay. We excluded patients with history of previous trauma but admitted to the ICU for other reasons, readmissions to the ICU and trauma referrals from other hospitals. Data were collected on demographics and admission severity of illness, estimated using the Acute Physiology and Chronic Health Evaluation (APACHE) II [ 10 ], Simplified Acute Physiology Score II [ 11 ], postresuscitation Glasgow Coma Score (GCS) and Injury Severity Score (ISS) [ 12 , 13 ]. We documented the presence of injuries to brain, maxillofacial bones, chest, abdominal organs, spinal cord and pelvis/lower extremities. We documented whether an extubation trial was given before tracheostomy. The type of tracheostomy procedure (surgical versus percutaneous) was recorded. The number of days from initiation of ventilation to tracheostomy, from admission to tracheostomy, from tracheostomy to weaning, from tracheostomy to discharge from ICU, the duration of mechanical ventilation, ICU length of stay (LOS) and hospital LOS were all calculated. All these durations were calculated as the number of calendar days, with the day of admission being considered day 0. ICU and hospital mortality rates were documented. We stratified patients into two groups: the early tracheostomy group, in which tracheostomy was performed within the first 7 days of initiation of mechanical ventilation; and the late tracheostomy group, in which tracheostomy was performed after 7 days. Prolonged ICU stay was defined as ICU stay in excess of 14 days. Statistical analysis Minitab for Windows, release 12.1 (Minitab Inc., State College, PA, USA), was used for statistical analysis. Continuous variables are expressed as means ± standard error of the mean, and were compared using t-tests. Medians and interquartile ranges are also given. Categorical variables are expressed as absolute and relative frequencies, and were compared using χ 2 tests. Linear correlation was performed to test for associations between the duration from initiation of mechanical ventilation to tracheostomy and ICU LOS. To assess further the impact of delayed tracheostomy on ICU LOS, univariate and multivariate analyses were performed to examine whether delayed tracheostomy is an independent predictor of prolonged ICU stay. Results of prediction are expressed as odds ratios (ORs) and 95% confidence intervals (CIs). P ≤ 0.05 were considered statistically significant. Results Baseline patient characteristics Table 1 summarizes the patients' characteristics at baseline. During the period of study there were 653 trauma admissions to the ICU. The number of patients who required tracheostomy was 136 (21%); 29 patients had tracheostomy within 7 days of mechanical ventilation and the remaining 107 underwent tracheostomy after 7 days. Comparison of demographic data between the two groups revealed no significant differences with regard to age, sex, APACHE II score, Simplified Acute Physiology Score II or ISS. GCS was slightly lower in the early tracheostomy group (5.2 ± 0.5 versus 6.5 ± 0.4; P = 0.04). There was no significant difference in the presence of head, chest, abdominal, or pelvic injuries between the groups. Maxillofacial injuries were more common in patients who received early tracheostomy (34% versus 16%; P = 0.03) whereas spinal cord injuries were less common (3% versus 16%; P = 0.08). The proportions of percutaneous and surgical tracheostomies were not different between the early and late groups. Table 1 Baseline patient characteristics Tracheostomy ≤ 7 days Tracheostomy >7 days P Number 29 107 Age (years) 33 ± 3 31 ± 1 0.5 Male sex (%) 26 (90%) 98 (92%) 0.75 APACHE II score 20 ± 1 19 ± 1 0.35 SAPS II score 42 ± 2 39 ± 1 0.36 ISS score 33 ± 2 34 ± 1 0.79 GCS score 5.2 ± 0.5 6.5 ± 0.4 0.04 Type of injury ( n [%]) Head 20 (69%) 66 (62%) 0.47 Maxillofacial 10 (34%) 17 (16%) 0.03 Chest 11 (38%) 51 (48%) 0.35 Abdomen 3 (10%) 14 (13%) 0.69 Spinal cord 1 (3%) 17 (16%) 0.08 Pelvic/lower extremities 10 (34%) 40 (37%) 0.77 Percutaneous tracheostomy ( n [%]) 21 (72%) 75 (70%) 0.81 Values are expressed as mean ± standard error of the mean, where appropriate. APACHE, Acute Physiology and Chornic Health Evaluation; GCS, Glasgow Coma Scale; ISS, Injury Severity Score; SAPS, Simplified Acute Physiology Score. Tracheostomy timing and main outcomes Table 2 shows tracheostomy timing data and main outcomes. Extubation trials were performed in 22% of patients with late tracheostomy as compared with 3% of those with early tracheostomy ( P = 0.019). After placement of the tracheostomy, both groups were weaned off mechanical ventilation and discharged from the ICU after similar periods. Early tracheostomy was associated with a significantly shorter duration of mechanical ventilation (9.6 ± 1.2 days versus 18.7 ± 1.3 days; P < 0.0001) and shorter ICU LOS (10.9 ± 1.2 days versus 21.0 ± 1.3 days; P < 0.0001). Hospital LOS, ICU mortality and hospital mortality were not different between the two groups. Table 2 Main findings Tracheostomy ≤7 days Tracheostomy >7 days P Ventilation days before tracheostomy 4.6 ± 0.5 (6, 2.5–7) 13.9 ± 0.5 (13, 10–16) <0.0001 Days from ICU admission to tracheostomy 4.6 ± 0.5 (6, 2.5–7) 14.1 ± 0.5 (13, 11–17) <0.0001 Number (%) of patients with extubation trials 1 (3%) 24 (22%) 0.019 Days from tracheostomy to weaning 4.9 ± 1.2 (2, 1–7) 4.9 ± 1.1 (1, 1–4) 1.0 Days from tracheostomy to ICU discharge 6.3 ± 1.3 (4, 2–8.5) 6.9 ± 1.1 (3, 2–7) 0.72 Total duration of mechanical ventilation (days) 9.6 ± 1.2 (8, 6–13) 18.7 ± 1.3 (15, 12–20) <0.0001 ICU LOS (days) 10.9 ± 1.2 (10, 7–14) 21.0 ± 1.3 (17, 14–23) <0.0001 Hospital LOS (days) 101 ± 19 (68, 33–139) 105 ± 7 (83, 54–136) 0.84 ICU mortality ( n [%]) 1 (3%) 1 (1%) NS Hospital mortality ( n [%]) 5 (17%) 15 (14%) 0.66 Values are expressed as mean ± standard error of the mean (median, interquartile range), where appropriate. ICU, intensive care unit; LOS, length of stay. Figure 1 shows the distribution of patients by timing of tracheostomy and the mean ICU LOS for patients, stratified by timing of tracheostomy. There was a direct correlation between the timing of tracheostomy and mean ICU LOS (r = 0.91; P < 0.001). Figures 2 and 3 show Kaplan–Meier curves of the duration of mechanical ventilation and ICU LOS in the two groups. Similarly, both the duration of mechanical ventilation and ICU LOS were significantly shorter in the early tracheostomy group (log rank P value < 0.001 for both). Figure 1 Distribution of patients by timing of tracheostomy and corresponding intensive care unit (ICU) length of stay (LOS). There was a direct correlation between timing of tracheostomy and mean ICU LOS (r = 0.91; P < 0.001). Figure 2 Kaplan–Meier curves of the duration of mechanical ventilation in early and late tracheostomy groups. Early tracheostomy was associated with a significantly shorter duration of mechanical ventilation. Figure 3 Kaplan–Meier curves of intensive care unit (ICU) length of stay (LOS) in early and late tracheostomy groups. Early tracheostomy was associated with a significantly shorter ICU LOS. Using univariate analysis the following factors were found to be associated with prolonged ICU stay (>14 days): late tracheostomy (OR 7.7, 95% CI 3.0–19.9; P < 0.001), spinal cord injury (OR 6.1, 95% CI 1.3–27.7; P = 0.019) and extubation trials (OR 3.1, 95% CI 1.1–8.7; P = 0.037). The presence of head injury was a significant negative predictor of prolonged ICU stay (OR 0.5, 95% CI 0.2–1; P = 0.047), as was the presence of maxillofacial bone injuries (OR 0.4, 95% CI 0.2–1.01; P = 0.052). APACHE II score, ISS and GCS score exhibited no significant association with prolonged ICU stay. Using multivariate analysis, late tracheostomy (OR 6.9, 95% CI 2.6–18.1; P < 0.001) and, to a much lesser extent, spinal cord injury (OR 4.7, 95% CI 0.99–22.6; P = 0.052) emerged as independent predictors of prolonged ICU stay. Discussion In our study we found that early tracheostomy in trauma ICU patients was associated with a significant reduction in the duration of mechanical ventilation and ICU LOS without affecting patient outcome. Weaning patients from mechanical ventilation and discharge occurred shortly and in similar periods after tracheostomy in both groups, suggesting that tracheostomy was a critical factor in weaning and discharge. We also found that late tracheostomy was an independent predictor of prolonged ICU stay. The study also showed that tracheostomy was more likely to be performed early in patients with maxillofacial fractures, reflecting the need for this procedure for airway management. In patients with spinal cord injury tracheostomy was more likely to be performed late because many of these patients had to undergo surgical spinal fixation before tracheostomy. In such cases, the surgeons preferred to wait until the surgical wound in anterior spinal fusion was healed before performing the tracheostomy. Patients with early tracheostomy had lower GCS, reflecting the common practice of performing tracheostomies earlier in patients with low GCS while delaying tracheostomy in patients with higher GCS in case extubation becomes possible. The very low mortality seen in the patients we studied may be explained by selection of proper candidates for tracheostomy, excluding those patients who were unlikely to survive. Hospital LOS in these patients was prolonged, reflecting their severe injuries that required lengthy rehabilitation periods. The very limited rehabilitation facilities meant that the patients had to undergo rehabilitation while they were hospitalized, prolonging further the hospital LOS. Table 3 summarizes studies that examined the impact of early tracheostomy on resource utilization [ 2 , 3 , 14 - 18 ]. All of these studies, except one [ 2 ], found reduction in the duration of mechanical ventilation, ICU LOS and/or hospital LOS. Some of these studies found reduction in ventilator-associated pneumonia or colonization incidence. Some of the studies [ 3 , 14 - 16 , 18 ] were retrospective, and all found a positive impact of early tracheostomy on duration of mechanical ventilation, ICU LOS, hospital LOS, or pneumonia rates. The study by Rodriguez and coworkers [ 17 ] was a prospective randomized trial in which patients were assigned to early tracheostomy (≤7 days) if they were admitted on an odd day and to late tracheostomy if admitted on an even day. That study found a reduction in duration of mechanical ventilation, ICU LOS and hospital LOS. Sugerman and coworkers [ 2 ] conducted a 'multicenter' randomized trial in five centres involving patients with head trauma, nonhead trauma and no trauma. Those investigators randomized patients on days 3–5 to receive tracheostomy or to continue with translaryngeal intubation. A second randomization for patients who remained intubated was performed on days 10–14. Those authors found no differences in ICU LOS or frequency of pneumonia between early and late tracheostomy. However, the study had several limitations. Out of the five participating centres, only one completed the study. Out of 157 eligible patients, only 112 completed the study because of physician bias and incomplete information. Only 14 patients entered the second randomization. That report illustrates the difficulty in performing studies that challenge widely accepted beliefs. Reviewing these studies also illustrates the lack of consensus regarding the definition of early tracheostomy, with different cutoff points used ranging between 3 and 14 days. Table 3 Literature review Ref. Type of study Number of patients Reason for admission Timing of tracheostomy Main outcomes [3] Retrospective 101 Blunt multiple trauma Early tracheostomy ≤4 days Late Tracheostomy >4 days ↓Duration of MV, ↓incidence of nosocomial pneumonia [14] Retrospective 31 Head trauma Early tracheostomy ≥7 days Late tracheostomy >7 days ↓Duration of MV, ↓hospital LOS, ↓ICU LOS [15] Retrospective 118 Multiple trauma Early tracheostomy ≤3 days Intermediate tracheostomy 4–7 days Late tracheostomy >7 days ↓Incidence of pneumonia [18] Retrospective 157 Blunt trauma Early tracheostomy ≤6 days Late tracheostomy >6 days ↓Duration of MV, ↓ICU LOS, ↓hospital LOS, ↓hospital charges [16] Retrospective 30 Neurosurgical (CVA, head injury, trauma, infection) Early tracheostomy ≤7 days Late tracheostomy >7 days ↓Duration of MV, ↓incidence of colonization, ↓faster recovery from pneumonia [17] Prospective randomized 106 Multiple trauma Early tracheostomy ≤7 days Late tracheostomy >7 days ↓Duration of MV, ↓ICU LOS, ↓hospital LOS, ↓pneumonia if tracheostomy was performed earlier than 3 days [2] a Prospective randomized multicentre 157 eligible patients Head-trauma, Nonhead trauma, no trauma First randomization: 3–5 days Second randomization: 10–14 No difference in ICU LOS, frequency of pneumonia, or death a Of five participating centres, only one completed the study; of 157 eligible patients, only 112 completed the study because of physician bias and incomplete information; and only 14 patients entered the second randomization. ICU, intensive care unit; LOS, length of stay; MV, mechanical ventilation. Strengths of our study include prospective data collection ensuring complete data and the relatively large number of patients. However, data extraction and analysis was retrospective. Because the database was not designed specifically to examine tracheostomy practices, certain issues were not documented, such as when the decision for tracheostomy was made and how different intensivists and surgeons varied in their timing of tracheostomy. In addition, the study was observational and was conducted from one centre. A large multicentre randomized controlled trial in which patients are randomized to early versus late tracheostomy would be the ideal way to test the impact of procedure timing on resource utilization. In summary, the present study, in addition to the existing literature, indicates that early tracheostomy is associated with reduced ICU LOS. Adopting a standardized strategy may help in improving resource utilization. In addition, there is an urgent need for a multicentre randomized controlled trial to assess the most appropriate timing for tracheostomy. Key messages • Early tracheostomy in trauma ICU patients was associated with shorter duration of mechanical ventilation and ICU LOS without affecting ICU or hospital outcomes. • There was a direct correlation between timing of tracheostomy and ICU LOS. • Using multivariate analysis, late tracheostomy emerged as an independent predictor of prolonged ICU LOS. Competing interests None declared. Abbreviations APACHE = Acute Physiology and Chronic Health Evaluation; CI = confidence interval; ICU = intensive care unit; ISS = Injury Severity Score; GCS = Glasgow Coma Score; LOS = length of stay; OR = odds ratio.

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1065025

Mild hypothermia after near drowning in twin toddlers

Introduction We report a case of twin toddlers who both suffered near drowning but with different post-trauma treatment and course, and different neurological outcomes. Methods and results Two twin toddlers (a boy and girl, aged 2 years and 3 months) suffered hypothermic near drowning with protracted cardiac arrest and aspiration. The girl was treated with mild hypothermia for 72 hours and developed acute respiratory dysfunction syndrome and sepsis. She recovered without neurological deficit. The boy's treatment was conducted under normothermia without further complications. He developed an apallic syndrome. Conclusion Although the twin toddlers experienced the same near drowning accident together, the outcomes with respect to neurological status and postinjury complications were completely different. One of the factors that possibly influenced the different postinjury course might have been prolonged mild hypothermia.

Introduction Of drowning and near drowning victims who are younger than 20 years, 63–68% are 0–5 years old [ 1 , 2 ]. Of submersion events in the age group 1–4 years, 56% occurred in artificial pools [ 3 ]. Death from drowning is the second leading cause of accidental death in children [ 4 ], and one-third of all survivors have neurological damage [ 4 ]. Hypothermia frequently accompanies submersion accidents, especially in children with a relatively large ratio of surface area to body mass [ 3 ]. Mild hypothermia (32–34°C) reduces oxygen consumption by 7% per 1°C decrease in temperature, and reduces cerebral blood flow and cerebral intracranial pressure [ 5 - 7 ]. Temperature under 28°C leads to cardiocirculatory depression and finally cardiac arrest [ 3 ]. Hypoxaemia and capillary leak develop due to apnoea, regardless of whether aspiration occurs [ 3 ]. The degree of cerebral protection that can be expected due to hypothermia depends, among other factors, on the amount of time that elapses before induction of mild hypothermia [ 1 , 3 , 6 ]. Induced mild hypothermia for cerebral protection after near drowning accidents has yielded controversial results in terms of mortality and neurological outcome [ 1 , 3 , 8 ]. However, induced mild hypothermia after cardiac arrest has led to improved neurological results, whereas life-threatening complications such as infections and resultant sepsis may counter these neurological benefits [ 9 ]. We report here a case of twins who both suffered near drowning, but with different post-trauma treatment and different neurological outcomes. Case report The twins (a girl and boy, aged 2 years and 3 months old) were found lifeless by their father in the neighbours' garden pond. It was early spring, and the toddlers had been unattended for at least 10 min. Bystander cardiopulmonary resuscitation (CPR) was performed. The emergency doctor could not palpate any pulse, the children were hypothermic, and the pupils were dilated and pupil reflexes absent. Both children had aspirated. Under CPR the children exhibited pulseless bradycardia on the electrocardiogram. The girl The girl was transported to a university hospital. Admission parameters are presented in Table 1 . After rewarming to 32°C and successful CPR, 180 min after admission to the hospital, haemodynamic stability was achieved with adrenaline (epinephrine) infusion and the child was admitted to the intensive care unit (ICU). The pupils were slightly dilated with reaction to light and the corneal reflex was absent. Cranial computed tomography (CT; Fig. 1 ), done 7 hours after admission, revealed cerebral oedema; this was regressive, as indicated by cranial CT obtained 3 days later. Mannitol therapy and prolonged mild hypothermia (32–34°C) were begun the day of the accident. Repeated fundoscopy did not show signs of papillary congestion. Intracranial pressure was not monitored. Under sedation with fentanyl and midazolam to a Ramsay level of 6 and controlled mild hyperventilation (arterial CO 2 tension 30–35 torr), mild hypothermia was continued and reduced gradually (0.5°C/8 hours). Seventy-two hours after the accident the child was normothermic without development of rebound hyperthermia. After rewarming the pupils were tight and reflexes present. Catecholamine therapy on admission to the ICU was switched to dobutamine and dopamine infusion. To achieve a mean arterial pressure greater than 70 mmHg, noradrenaline (norepinephrine) infusion had to be added. Under pressure controlled ventilation the oxygenation index improved initially and the inspiratory oxygen fraction could be reduced to 0.3 over the first 48 hours after the accident. However, 72 hours after the accident oxygenation deteriorated. The initial CT of the thorax had shown infiltrations in the basal dorsal thorax after aspiration (Fig. 2 ). The following chest X-ray films revealed increasing bilateral infiltrations of the lung (Fig. 3 ). After 3 days in the ICU, sepsis with multiple organ failure developed (acute respiratory dysfunction syndrome [ARDS] with an oxygenation index of 109 torr, circulatory failure requiring catecholamines, liver dysfunction with increased transferases and reduced prothrombin time, and disseminated intravascular coagulopathy). Substitution of blood products was necessary. Acute renal failure did not develop. Antibiotic treatment was started (ceftazidime for Pseudomonas aerguinosa in the tracheal aspirate, vancomycin for Enterococcus faecium at the central venous catheter tip). Under differentiated pressure controlled ventilation, oxygenation did not improve. Because it was unclear at this time whether extracorporeal membrane oxygenation would be required, 5 days after the accident the child was transferred by helicopter to another university hospital because of limited capacity at our hospital. Under high-frequency oscillatory ventilation and nitric oxide inhalation, oxygenation improved and extracorporeal membrane oxygenation was not necessary. Conventional pressure controlled ventilation could be restored 7 days after the accident, and at the same time the multiple organ failure improved. Sedation was reduced and the girl was extubated 11 days after the accident, with no neurological deficit. Twenty-three days after the accident she was transferred to the community hospital where her brother was initially hospitalized, and she was discharged 1 day later completely restored to health. The boy The brother was transported to a community hospital. Admission parameters are presented in Table 1 . Haemodynamic stability was achieved 150 min after admission to the hospital under dopamine and dobutamine therapy. The pupils were slightly dilated with reaction to light and the corneal reflex was present. He was rewarmed and normothermia was achieved 5 hours after admission. Continuous catecholamine therapy was stopped 4 days after the accident. The boy was sedated with fentanyl and midazolam, and ventilated to achieve normocapnia using a pressure-controlled mode. With improvement in oxygenation, he was extubated 6 days after the accident. The initial chest X-ray films showed bilateral infiltrations of the lung as a sign of aspiration pneumonia, which improved within the next few days. Liver and kidney function remained normal. After the end of sedation, an apallic syndrome with extension posturing developed. The initial cranial CT obtained 36 hours after admission was normal, and fundoscopy did not show signs of papillary congestion. A cranial CT obtained 32 days after the accident showed marked expansion of the internal and external cerebral fluid interspaces with marked cerebral atrophy. At discharge from hospital, 41 days after the accident, the little boy remained in an apallic state, with flexion and extension posturing. Discussion We present a case of twin toddlers with different neurological outcomes after near drowning with severe hypothermia and protracted cardiac arrest. Hypothermia at the scene has yielded controversial results with respect to cerebral protection. Factors such as time to achieve hypothermia (e.g. water temperature), the degree of hypothermia, the time of submersion, and other effects such as cardiocirculatory depression or arrest have various influences on the cerebral protection conferred [ 3 , 8 ]. Some of these factors are unclear in this case report. The two institutional approaches to management of the twins were optimal because both hospitals have paediatric departments with paediatric ICUs. In addition, the community hospital is a training hospital and part of the university hospital. Lavelle and Shaw [ 8 ] described three patients with body temperature under 28°C on arrival at the emergency department. All three patients had a good neurological outcome, but they fell into icy water. The use of prolonged or induced mild hypothermia for cerebral protection after near drowning has yielded controversial results [ 4 , 8 ]. Bohn and coworkers [ 6 ] reported on 40 children aged under 15 years who suffered severe near drowning accidents with submersion time longer than 5 min and need for CPR. Twenty-four children were treated with hypothermia (30–33°C) for 24–36 hours, and 14 survived but three of these children had permanent neurological damage. Sixteen children were kept normothermic, and 13 survived but four had permanent neurological damage. Nussbaum and Maggi [ 10 ] investigated 31 children aged under 6 years who had undergone near drowning and were in a flaccid state of coma. All children were treated with hypothermia (32–34°C) for 48 hours (half of them received additional barbiturate therapy). Twelve children recovered completely, 12 children had brain damage and seven died. Two recently published studies, conducted in patients who had suffered out-of-hospital cardiac arrest, compared induced mild hypothermia for 12–24 hours with normothermic management [ 7 , 9 ]; they found that a significantly greater percentage of patients in the groups treated with mild hypothermia had good neurological outcomes. In patients affected by brain injury with a Glasgow Coma Scale score from 3 to 8, induced mild hypothermia for 24–48 hours yielded controversial findings [ 11 , 12 ]. In these patients hypothermia on admission correlated with poor outcome, suggesting that spontaneous hypothermia may be a result of major brain injury [ 11 ]. In the present case report, hypothermia on the scene and on admission was probably the result of external factors such as water and air temperature and the children's age, suggesting cerebral protection from hypothermia. Up until the arrival of the twins at hospital, the treatment was identical. The boy was passively warmed to achieve normothermia, and the girl underwent prolonged (72 hours) mild hypothermia (32–34°C). The different neurological outcomes could have been influenced by these different treatments. However, some factors remain uncertain. For example, was the boy the first to go into the water, with resulting longer submersion and hypoxaemia times? How effective was bystander CPR in the two children? Was the time to achieve hypothermia the same in both children? Excluding bystander CPR, the remaining factors are considered strong predictors of outcome after near drowning [ 1 , 3 , 8 ]. The girl developed ARDS and septic shock, whereas the boy recovered from aspiration pneumonia without further complications. There is concern that prolonged mild hypothermia has adverse effects on cardiac and lung function, coagulation and the immune system [ 3 , 5 , 7 ]. In a series of 41 patients with submersion injury (temperature on admission >32°C, no induced mild hypothermia), 32% developed pneumonia and one person ARDS [ 8 ]. Significantly higher infection rates, predominantly pneumonia, were described in patients treated with induced mild hypothermia as compared with patients treated under normothermic conditions [ 5 , 13 , 14 ]. However, other investigations evaluating patients following out-of-hospital CPR and with brain damage did not identify any differences in the incidence of infection between normothermic and hypothermic groups treated. just for 12–24 hours [ 7 , 9 , 15 ]. It seems posssible that the duration of mild hypothermia has an impact in the incidence of infection and sepsis. Among the 41 normothermic patients described by Lavelle and Shaw [ 8 ], after submersion 14% developed sepsis. In experimental animal models it was shown that hypothermia under 29°C leads to a reduced neutrophil response to endotoxin [ 16 ]. Leukocytopenia has been described to be significantly more frequent in patients with induced mild hypothermia [ 13 , 14 ]. The girl was highly catecholamine dependent in the first 7 days after the accident. It has been reported that, in patients with mild hypothermia, significantly higher doses of catecholamines are required in comparison with normothermic patients after acute brain injury [ 11 ]. Vasopressor requirements have been described as having a significant impact on outcome [ 2 ]. The rate of other organ dysfunctions (liver, kidney) has also been found to be significantly higher in patients under induced mild hypothermia. The girl also developed transient liver dysfunction. Together with sepsis syndrome, coagulopathy developed. Disturbances of this system with resultant bleeding complications are known to occur during therapy with mild hypothermia [ 5 , 13 , 14 , 17 ]. Conclusion Although the twin toddlers experienced a near drowning accident together, the outcomes in terms of neurological status and postinjury complications were completely different. One of the factors that possibly influenced the different postinjury courses might have been prolonged mild hypothermia. Key messages • Two twin toddlers suffered hypothermic near drowning with protracted cardiac arrest and aspiration. • The girl was treated with mild hypothermia and developed acute respiratory dysfunction syndrome and sepsis, but recovered without neurological deficits. • The boy was treated under normothermic conditions and developed an apallic syndrome. • One of the factors that possibly influenced the different postinjury course might have been prolonged mild hypothermia. Competing interests None declared. Abbreviations ARDS = acute respiratory dysfunction syndrome; CPR = cardiopulmonary resuscitation; CT = computed tomography; ICU = intensive care unit.

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1065049

Combination of histopathological and electromyographic patterns can help to evaluate functional outcome of critical ill patients with neuromuscular weakness syndromes

Introduction The aim of the study was to describe patterns of neuromuscular weakness using a combination of electromyography and histology, and to evaluate functional outcome in patients following complicated cardiovascular surgery. Methods Fifteen adults requiring long-term mechanical ventilation (>15 days) following cardiovascular surgery associated with postoperative complications were prospectively included. Electrophysiological and histological analyses (muscle and nerve) were performed when failure to wean from mechanical ventilation associated with peripheral neuromuscular weakness was noticed. Functional disability was evaluated 12 months after surgery. Results Six patients had a predominantly axonal neuropathy, six presented with myopathy, and three patients had a combination of axonal neuropathy and myopathy. All of them presented with acute tetraparesis and failure to wean from mechanical ventilation. All of the study patients who received corticosteroids exhibited a myopathic pattern (with or without axonopathic changes) but never an axonopathic pattern only. Only two of the eight survivors at 12 months were not ambulatory. These two patients had no detectable compound muscle action potential on electrophysiological examination. Conclusion The combination of electromyographic evaluation and neuromuscular histological abnormalities could help to identify the type and severity of neuromuscular weakness, in turn helping to evaluate the patient's potential functional prognosis.

Introduction Critical illness polyneuropathy (CIP) and myopathy are neuromuscular disorders that occur in critically ill patients [ 1 , 2 ]. Clinical features often consist of difficulty in weaning from mechanical ventilation, tetraparesis and muscle wasting of the limbs, with tendon reflexes absent or markedly decreased. Regarding the causes of these disorders, it has been hypothesized that systemic inflammatory response syndrome (SIRS) and sepsis, with their impact on the body's defence system, may be involved [ 3 , 4 ]. Associations with drugs such as neuromuscular blocking agents, steroids and catecholamines have also been suggested [ 3 , 5 , 6 ]. In addition, other factors such as malnutrition, underlying disease, immobility and antibiotics have been considered [ 7 ]. Patients with weakness acquired in the intensive care unit (ICU) are often sedated and mechanically ventilated, and have unreliable sensory and motor examinations, and so diagnosis can be quite difficult because of the prolonged sedation. [ 8 ] Electromyography is useful for identifying and localizing a lesion to a particular component of the motor unit. Using electromyography, Bolton [ 9 ] and Zochodne [ 10 ] and their colleagues were the first to identify one of the major neuromuscular causes of neuromuscular weakness in acute ill patients, namely CIP [ 9 , 10 ]. However, use of electromyography and clinical examination without obtaining neuromuscular biopsy findings may lead to some patients being diagnosed as having CIP only [ 2 , 11 ]. Therefore, such examinations may distract attention from other neuromuscular disorders that occur in the critically ill, thus leading to identification of phenomena of neuromuscular junction blockade or critical illness myopathy, or a combined picture involving both syndromes. Because of this, and because of the lack of studies evaluating neuromuscular disorders in ICUs using a combination of histopathological (nerve and muscle) and electromyographic studies, we conducted the present study in which we monitored critically ill patients prospectively following complicated cardiovascular surgery. After inclusion in the study, serial clinical, neuromuscular biopsy and electromyographic analyses were systematically conducted. Using this method we were able to diagnose neuromuscular disorders; to identify the predominant neurogenic or myogenic pattern, or a combined picture involving both lesions; and to compare these findings with functional outcome. Methods Hospital This prospective study was carried out in the ICU of a teaching hospital with an 11-bed surgical unit. Annually, 650 critically ill patients are admitted following cardiac surgery using cardiopulmonary bypass. Patients From 1998 to 2002, 15 patients who had a complicated course (with one or more organ dysfunctions) following cardiovascular surgery were prospectively included after prolonged mechanical ventilation (>15 days) associated with tetraparesis and failure to wean. Peripheral electromyographic analysis and neuromuscular biopsy were performed in all of these patients to determine the cause of limb weakness and diaphragm dysfunction. The medical committee of the hospital approved the study, and informed consent was obtained from the relatives of the patients. Excluded were those patients who were suspected of having pre-existing polyneuropathy because they had a diagnosis of chronic diabetes mellitus, alcohol abuse, HIV infection, or end-stage renal disease (associated with chronic haemodialysis), or had used neurotoxic medication. Patients with acute or chronic spinal cord lesion, myasthenia gravis, or Guillain–Barré syndrome were also excluded. After the patients had been enrolled, clinical examination was performed daily during their stay in the ICU, and we assessed motor deficit, muscle wasting, sensory loss and tendon reflexes. In these patients undergoing cardiac surgery, at ICU admission the Euroscore was caculated to evaluate risk for postoperative mortality [ 12 ]. This prognostic scoring system was developed in Europe for use in patients undergoing cardiac surgery. The score is calculated by simple arithmetic (additive model) using risk factors that were found to be robust in predicting postoperative mortality, such as age, sex, emergency surgery, preoperative left ventricular dysfunction and type of surgery. Organ failure score and presence of SIRS were noted, according to criteria presented by Bone and coworkers [ 13 ], at admission and during the clinical course [ 7 ]. The diagnosis of SIRS required the presence of two or more of the following criteria [ 13 ]: body temperature >38°C or <36°C; heart rate >90 beats/min; tachypnoea >20 breaths/min; and hyperventilation, as indicated by an arterial carbon dioxide tension <32 mmHg, leucocyte count >12 g/l or <4 g/l, or presence of >10% immature neutrophils. Use and dosage of neuromuscular blocking agents and intravenous corticosteroids were registered daily. Electrophysiological monitoring and neuromuscular biopsies were performed as soon as the neuromuscular disorder was recognized after the end of prolonged sedation associated with mechanical ventilation (>15 days after the onset of mechanical ventilation). Failure to wean from mechanical ventilation was characterized by inability to extubate or inefficient spontaneous ventilation through tracheostomy. Peripheral weakness affected both proximal and distal muscle groups and was defined as failure to move against gravity. Electromyography Nerve conduction studies were performed with Nicolet Viking IV apparatus via percutaneous stimulation and surface recording. Quantitative concentric needle electromyography was performed in distal and proximal muscles of upper extremities (such as deltoid, muscle interosseus I, and muscle abductor pollicis brevis) and lower extremities (such as tibialis anterior and flexor hallucis brevis). Electroneurography included upper (median or ulnar) nerves and lower (peroneal or sural) nerves. In patients who could not exercise, repetitive stimulation at 20–30 Hz was also given. Diaphragmatic and phrenic nerve studies were not performed. Electromyography reports were analyzed by the same doctor and categorized as axonal polyneuropathy or myopathy, or a combination thereof. Patients were diagnosed as having axonal sensorimotor polyneuropathy if electrodiagnostic studies revealed very low amplitude or absent sensory responses and low motor amplitudes with normal or mildly reduced conduction velocities. Patients were diagnosed as having myopathy in the setting of low or normal motor amplitudes, with relatively normal sensory responses. Short duration of motor unit potentials with normal or early recruitment with or without fibrillation potentials, or fibrillation potentials and either no firing or polyphasic motor unit potentials of normal duration were also considered to reflect myopathy. Combined muscle and nerve biopsies Muscular biopsies were obtained in all 15 patients, from skeletal muscle specimens of the vastus lateralis or anterior tibialis muscles, under local anaesthesia (lidocaine 1% 100–150 mg) if necessary; these patients did not have thombocytopenia or coagulopathy. Muscle samples were first snap frozen in isopentane precooled in liquid nitrogen and stored at -80°C until examination. For routine histology, the samples were placed in formaldehyde fixative and paraffin embedded. For conventional transmission electron microscopy, specimens were fixed in 2.5% glutaralehyde in 0.1 mol/l phosphate-buffered saline, postfixed with 1% osmic acid and embedded in araldite. Semithin resin sections were stained using toluidine blue. Ultrathin sections were double stained with uranylacetate. Histoenzymology was performed in serial transverse cryostat sections (6 µm thick), stained using routine histochemical methods [ 14 ]. Sensitive nerve biopsies were obtained from sural, peroneal nerves or the sensory branch of the musculocutaneous nerve (in the distal third of the leg). Nerve samples were fixed in 2.5% glutaraldehyde in 0.1 mol/l phosphate-buffered saline, postfixed with 1% osmic acid and embedded in araldite. Semithin resin transverse and longitudinal sections were stained using haematein and eosin, solochrome blue and paraphenylene diamine. Ultrathin sections were double stained with uranylacetate and citrate. Histopathological reports and original slides were reviewed by two medical experts who were blinded to the electromyographic findings. Functional outcome Follow-up data were available in all 15 patients. The end-points were death or time to ambulation without assistance. The maximal duration of follow up was 12 months. Findings were not analyzed statistically because of the relatively small numbers included in the various groups. Results Clinical features A total of 25 patients, suffering in most cases from sepsis or SIRS following cardiac surgery, and who were undergoing long-term mechanical ventilation (>15 days), were enrolled. Ten patients were excluded because of previous alcoholic liver disease or end-stage renal failure ( n = 4) or a previous history of neuromuscular disease ( n = 6). The patients' median age was 53 years (range 33–82 years), and the median Euroscore was 7 (range 1–20). All patients presented with at least one episode of sepsis or a systemic inflammatory response. Multiorgan dysfunction was diagnosed in 10 patients, with a median Multiple Organ Dysfunction Score of 3 (range 1–4; Table 1 ). The most common organ system failure was cardiovascular, and more than 86% of patients fulfilled criteria for heart failure. Postoperative renal dysfunction requiring continuous venovenous haemofiltration was diagnosed in nine patients (60%). The severity of weakness and variability in reflex abnormality were noted. Patients were noted to be weak 15–40 days after ICU admission. Because patients received neuromuscular blocking agents and sedatives, the exact time of onset of weakness was usually not possible to determine. Of the patients studied, 50% presented with asymmetric tetraparesia, predominantly involving the legs. The other 50% had global tetraparesia with much reduced muscle tone (Table 1 ). Five patients were areflexic, seven had hyporeflexia and three had normal tendon reflexes. All patients grimaced in response to painful stimuli, but sensory testing was initially unreliable in most. One patient was transiently encephalopathic. Table 2 summarizes clinical diagnoses and medications affecting the neuromuscular system in these patients with neuromuscular weakness syndromes. Patients in all groups received muscle relaxants; only two were used in our unit during the study period – vecuronium and atracurium. The groups included patients who received large doses of muscle relaxants as well as patients who received none. Muscle relaxants were used in 66% of the patients included in the study; 20% of these received muscle relaxants for less than 24 hours. Six patients received intravenous corticoids; four of these patients had undergone transplants and the other two patients were asthmatic. Electrodiagnostic testing The spectrum of neuromuscular causes of weakness, along with electromyographic localization and neuromuscular biopsy findings, is summarized in Table 2 . The two most common causes of weakness in these patients were polyneuropathy or myopathy in isolation. Six patients presented with neuropathy, three of whom had sensory motor neuropathy characterized by reduced sensory and motor action potential amplitudes. Six other patients presented with acute myopathy, characterized by sensory and motor action potentials in the normal range or partially reduced. Three patients presented with CIP associated with myopathy. Motor unit potentials were usually reduced, with pathological fibrillation potentials associated. Muscle and nerve pathology A range of histopathological abnormalities was identified in the neuromuscular biopsies from the 15 patients (Table 2 ). The most common muscle abnormality was diffuse atrophy of fibre types I and II (14 out of 15 patients) associated with acute necrosis (12 out of 15 patients; Figs 1 and 2 ). There were abnormalities in the nerve biopsy from most patients (Fig. 3 ; except those with acute myopathy); these included axonal degeneration and demyelinating lesions. In some patients (patients 5, 7 and 8), analysis of neuromuscular biopsy revealed myopathic lesions associated with necrotic and atrophic fibres. This was associated with a huge reduction in sensory or motor action potential amplitude. Ten patients received neuromuscular blocking agents, five of whom exhibited an acute myopathy and the other five a peripheral neuropathy. However, two patients who did not receive any muscle relaxant developed a neuropathic pattern (with or without myopathic lesions). All patients who received corticosteroids exhibited a myopathic pattern (with or without axonopathic lesions) but never an exclusively axonopathic pattern. The overall mortality rate was 40%, with a further patient dying on the ward after ICU discharge, giving a hospital mortality of 46%. Among the eight survivors at 12 months, two patients were not ambulatory (patients 2 and 8; Table 3 ). Compound muscle action potentials (CMAPs) were undetectable in these two patients on electrophysiological examination (Table 2 ). Discussion Although acute neuromuscular weakness appears prevalent among patients on prolonged mechanical ventilation, few prospective studies have been reported that include both electrophysiological and histological patterns [ 5 , 15 , 16 ]. Furthermore, nerve biopsy findings are absent, even in recent prospective studies [ 5 ]. Our study is among the first to perform electromyography and obtain neuromuscular biopsies prospectively for all patients included. The neurophysiological abnormalities identified were of three types, namely CIP alone, acute myopathy and mixed neurogenic and myogenic disturbances, and they developed in a group of long-term mechanically ventilated patients who had undergone cardiovascular surgery. This type of severe and disturbing complication following cardiac surgery has been described previously [ 6 , 17 ], and led to an evaluation of hypothetical risk factors for such neurological disorders. It is also widely believed that the development of critical illness neuropathy is invariably associated with multiple organ failure, sepsis and SIRS [ 3 , 6 , 8 , 9 ]. Thus, CIP probably represents an organ failure caused by sepsis and SIRS, presumably as a result of the same basic mechanisms that lead to multiple organ dysunction, including inflammation, thrombosis, apoptosis and oxidant injury [ 18 ]. In the present study, however, peripheral neurological changes occurred in a few patients who did not fulfill accepted objective criteria for sepsis or single organ failure. This observation was previously reported in four series of patients with respiratory failure [ 7 , 15 , 19 , 20 ]. As might be expected, in the present study of long-term mechanically ventilated patients following cardiovascular surgery, sepsis and multiple organ failure were common but did not seem to be a prerequisite for the development of acute neuromuscular weakness, the cause of which remains unclear [ 18 ]. It was previously suggested that use of neuromuscular relaxants is associated with neuromuscular disorders in the ICU [ 21 - 24 ]. Possible mechanisms include persistent effects of these drugs or their active metabolites, pharmacological denervation hastening muscle atrophy, or association of these drugs with intravenous corticosteroids or aminoglycosides [ 22 , 24 ]. Vecuronium and its steroid components were also implicated as a cause of weakness [ 25 ]. In our study atracurium was also administered in patients with prolonged neuromuscular weakness, although it has no steroidal component and there is no accumulation of this molecule in the event of kidney or liver failure. However, we are unable to conclude that neuromuscular relaxants predispose to the development of neuromuscular disorders in general, or any type of neuromuscular disease in particular, because of the lack of a control group. Other observational studies failed to identify neuromuscular relaxants as possible additional risk factors [ 20 , 31 ]. The link between use of aminoglycosides and CIP was previously reported [ 26 , 27 ]. In the present study 66% of patients presenting with CIP received intravenous aminoglycosides, but only five patients out of 15 received intravenous aminoglycosides. Therefore, intravenous use of aminoglycosides may be another measure of severity of sepsis and multiple organ failure. Steroid administration appears to be associated with muscular lesions, regardless of association with neuropathy. All study patients who received high doses of corticosteroids (cumulative equivalent dose >1000 mg methylprednisolone) exhibited a myopathic pattern (with or without associated axonopathic lesion) but never an exclusive axonopathic pattern. This finding supports the deleterious effect of corticosteroids predominantly on the muscles, as suggested by several studies [ 5 , 28 - 32 ]. Electromyographic abnormalities and neuromuscular histology patterns were concordant in 13 patients out of 15. In the two discordant cases, electromyographic examination failed to show any myogenic component, whereas muscle histology suggested severe myopathy with necrotic fibres and vacuolization zones. This lack of complete agreement between neurophysiological testing and muscle histology has already been noted by Coakley and colleagues [ 20 ]. Some authors have also indicated that it could be difficult to differentiate myopathy from axonal motor neuropathy via electromyographic analysis alone, especially in unconscious patients [ 2 , 5 ]. Thus, in the absence of systematic muscle biopsy, some patients can be misdiagnosed with myopathy when motor axonopathy is present and, as shown in the present study, it is conceivable that some patients have both myopathy and neuropathy [ 4 ]. The combination of neurophysiological testing with neuromuscular histology could therefore help in the precise identification of the type and severity of neuromuscular weakness, and may lead to a better understanding of the causes and consequences of neuromuscular weakness [ 5 ]. There was a high mortality rate in patients with acute neuromuscular weakness. Patients who died did so as a result of their underlying diseases and not from neuromuscular affection. These findings are similar to those from previous studies that reported on neuromuscular abnormalities [ 4 , 33 ]. In fact, the mortality rate and functional prognosis were similar between survivors with acute myopathy and those with neuropathy. However, of the three patients with combined neurological and muscular lesions, two died and the third survived but with severe functional disability. Survivors from critical illness have sustained impairments in physical function and health status, even after 1 year of recovery [ 34 - 36 ]. In our study, of the eight survivors at 12 months only two were not ambulatory (patients 2 and 8). These were the only two survivors in whom CMAPs were undetectable on electrophysiological examination. This electrophysiological finding attests to the severity of the axonopathy and may be a predictor of prolonged functional disability. However, because of the relatively small number of patients in this group, additional studies are necessary to confirm this clinical finding. Certain limitations of present study are worthy of mention. This prospective study evaluated electrophysiological and histological neuromuscular patterns in 15 ICU patients with prolonged mechanical ventilation after a complicated course following cardiovascular surgery. However, because the lack of control group and the small numbers of patients included, we were not able to determine precisely the risk factors for each pathology. Furthermore, as in previous studies [ 20 , 37 ], we were unable to determine exactly the time of onset of weakness. Therefore, electrophysiological and histological analyses were not performed at the same time point for all patients. Conclusion Neurophysiological and neuromuscular histological abnormalities associated with acute neuromuscular weakness were identified in mechanically ventilated patients in the ICU who had undergone cardiovascular surgery. Such patients are assumed to present with reversible neurological damage, although in a proportion this damage could be irreversible. Among survivors the absence of CMAPs on electrophysiological examination could suggest prolonged functional disability. Because of the lack of sensitivity of clinical examination in such patients, combined electromyographic diagnosis and neuromuscular abnormalities on histology could help to identify the type and severity of neuromuscular weakness, and the functional prognosis. Key messages • In patients undergoing neuromuscular weakness syndrome following cardiovascular surgery, the combination of electromyographic evaluation and neuromuscular histological abnormalities could help identify type and severity of these neuromuscular weakness, in turn helping to evaluate more precisely the patient's functional prognosis. Competing interests None declared. Abbreviations CIP = critical illness polyneuropathy; CMAP = compound muscle action potential; ICU = intensive care unit; SIRS = systemic inflammatory response syndrome.

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1065052

Population-based epidemiology of intensive care: critical importance of ascertainment of residency status

Introduction Few studies evaluating the epidemiology of critical illness have used strict population-based designs that exclude subjects external to the base population. The objective of this study was to evaluate the potential effects of inclusion of nonresidents in population-based studies in intensive care. Methods A population-based cohort study including all adults admitted to Calgary Health Region (CHR) multidisciplinary and cardiovascular surgical intensive care units (ICUs) between 1 May 1999 and 30 April 2003 was conducted. A comparison of patients resident and nonresident in the base population was then performed. Results A total of 12,193 adult patients had at least one admission to an ICU; 7767 (63.7%) were CHR residents, for an incidence of 263.7 per 100,000 per year. Male CHR residents were at significant increased risk for ICU admission as compared with females (330.5 per 100,000 versus 198.2 per 100,000; relative risk, 1.67; 95% confidence interval, 1.59–1.74; P < 0.0001), as were CHR residents aged 65 years and older as compared with younger patients (1719.9 per 100,000 versus 238.7 per 100,000; relative risk, 7.21; 95% confidence interval, 6.95–7.47; P < 0.0001). The mortality rate was significantly lower among non-CHR residents (12.7%) as compared with CHR residents (20.0%; P < 0.0001). Logistic regression modeling identified CHR residency as an independent risk factor for death (odds ratio, 1.4; 95% confidence interval, 1.2–1.5; P < 0.0001). Conclusion This study provides information on the incidence of and demographic risk factors for admission to ICUs in a defined population. Inclusion of patients that are nonresident in base study populations may lead to gross errors in determination of the occurrence and outcomes of critical illness.

Introduction Knowledge of the occurrence of and determinants of critical illness is important for establishing its burden and the risk factors for acquisition to guide wise allocation of limited healthcare and research resources. Population-based cohort studies that strictly include all episodes of disease occurring in residents of a geographically defined region are commonly accepted as the optimal design for such purposes [ 1 - 3 ]. However, these designs have rarely been used in the critical care medical literature [ 4 - 6 ]. Studies attempting to evaluate the distribution and determinants of critical illness typically have been case series reported from academic tertiary care referral hospitals [ 7 - 9 ]. Multicentered studies that include intensive care units (ICUs) in different regions and/or countries have less commonly been performed [ 10 - 13 ]. A major limitation to these institution-based studies is that if the population at risk is unknown, then incidence rates may not be calculated. Furthermore, if these studies focus on tertiary care centers and fail to include critically ill patients admitted to ICUs in other hospitals, a biased assessment of disease occurrence and severity may occur [ 14 ]. This may still be problematic even if all ICUs in a defined geographic region are included if investigators do not exclude patients nonresident in that base population from analysis [ 15 ]. Although referral bias has been shown to be of major importance in a number of disease conditions [ 16 - 20 ], its importance in the ICU has only been systematically assessed in one study reported from a single medical ICU in a tertiary care university hospital [ 21 ]. The importance of excluding patients external to the base population in observational studies in the critically ill has not been well defined. Furthermore, few population-based studies have been conducted among the critically ill and none in the English-language literature have assessed the overall burden and risk factors associated with ICU admission. The objective of this study was to evaluate the impact of inclusion of nonresidents in population-based studies on the occurrence of, on the risk factors for, and on the outcomes of ICU admission. Materials and methods Study population The Calgary Health Region (CHR) administers all medical and surgical acute care to the residents of the cities of Calgary and Airdrie, and to approximately 20 nearby small towns, villages, and hamlets (2001 population, 958,610). In April 2003 the CHR was expanded to include the adjacent mountain parks and Wheatland regions [ 22 ]. All tertiary care services are provided by the CHR with the only exception being liver, heart, or lung transplantation, where patients are referred to the provincial program in Edmonton. The acute care institutions within the CHR also serve as referral centers for other communities in southern Alberta and the neighboring provinces of British Columbia and Saskatchewan. All adult ICUs within the CHR are closed units staffed by fully trained intensivists, and they are administered by the Department of Critical Care Medicine, University of Calgary and the CHR. These ICUs currently include a 14-bed cardiovascular surgery intensive care unit (CVICU) and a 22-bed multidisciplinary ICU that serves as the regional trauma and neurosurgical referral center at the Foothills Medical Centre, a 12-bed multidisciplinary ICU at the Peter Lougheed Centre that is also the vascular surgery referral center, and a 10-bed multidisciplinary ICU at the Rockyview General Hospital. All patients 18 years and older admitted to an adult multidisciplinary ICU or the CVICU in the CHR between 1 May 1999 and 30 April 2003 were included. Ethics approval was obtained from the Conjoint Health Research Ethics Board at the University of Calgary and the CHR. Protocol The study utilized a population-based surveillance cohort design with linkage of data collected from regional critical care and administrative databases. Demographic data, clinical data, basic laboratory data, and scoring data were obtained from all patients admitted to ICUs in the CHR in a consistent manner across all sites using the ICU Tracer database, as previously described [ 23 , 24 ]. Patients were classified as CHR residents or nonresidents using data from the CHR Data Warehouse (a regional administrative database), where regional residents are flagged if their home address is within the geographical boundaries of the CHR. Severity of illness at admission was assessed using the Acute Physiology and Chronic Health Evaluation (APACHE) II score, and the intensity of care was assessed using the Therapeutic Intervention Scoring System score [ 25 , 26 ]. Shock was defined as a mean arterial pressure < 60 mmHg on the first day of admission to the ICU or requirement for a vasopressor infusion. The diagnosis of systemic inflammatory response syndrome (SIRS) was based on a modification of consensus criteria and required at least two of the following; heart rate > 90/min, respiratory rate > 20/min, temperature < 36°C or > 38°C, or white blood cell count < 4 × 10 9 /l or > 12 × 10 9 /l [ 15 ]. A surgical patient was any patient recorded as having an operative diagnosis or any patient admitted from the trauma ward or directly from the operating room. Statistical analysis Analysis was performed using Stata version 8.0 (Stata Corp, College Station, TX, USA). With the exception of calculating SIRS criteria, where missing values were treated as normal, missing data were not replaced and a reduced number ( n ) reported where they ocurred. Only first ICU presentations were analyzed from patients with multiple ICU admissions. Normally or near-normally distributed variables were reported as means ± standard deviations and non-normally distributed variables were reported as medians with interquartile ranges (IQRs). Means were compared using the Student t test and medians were compared using the Mann–Whitney U test. Differences in proportions among categorical data were assessed using Fisher's exact test. Incidence rates were calculated using regional denominator data and compared as previously described [ 2 ]. Levels of significance were not a priori adjusted for multiple testing, and a two-sided P < 0.05 was considered significant for all comparisons. A multivariable logistic regression model was developed to assess independent risk factors for death. The initial model included clinically suspected variables and those identified as potentially important predictors, including CHR residency, multidisciplinary ICU admission as compared with CVICU admission, the presence of SIRS, shock, hypothermia, age, gender, surgical diagnosis, and APACHE II and Therapeutic Intervention Scoring System scores. Backward stepwise variable elimination was then performed to develop the final model. The final model discrimination was assessed using the area under the receiver operator curve and calibration using the Hosmer–Lemeshow goodness-of-fit test. Results During the 4-year study period 12,193 adult patients had a total of 13,638 admissions to CHR ICUs; 4509 were surgical admissions for less than 48 hours. Overall 7767 (63.7%) patients were classified as CHR residents, for an incidence of ICU admission of 263.7 per 100,000 per year. Both the quarterly and yearly numbers of admissions were stable over the study. More than one-third (4426) of patients were nonresident in the CHR (incidence not able to be calculated) and were primarily (3424 patients) from other health regions in Alberta, 705 patients were from British Columbia, 121 were from Saskatchewan, 135 were from other Canadian provinces and territories, and 41 were international residents. Among the four study ICUs there were 4715 admissions to the CVICU, 3584 to Foothills Medical Centre ICU, 2144 to Peter Lougheed Centre ICU, and 1750 to Rockyview General Hospital ICU, of which 2587 (54.9%), 2264 (63.2%), 1541 (71.9%), and 1375 (78.6%) were CHR residents, respectively. A significant proportional difference in admission rates for CHR and non-CHR residents was observed ( P < 0.001) between each of the ICUs. Demographic features The overall median age (IQR) was 64.6 years (50.6–74.0 years) and 7819 patients (64.1%) were male. Although the overall median age of CHR and non-CHR residents was not different, in the subgroup of patients aged 85 years and older patients were nearly twice as likely to be CHR residents (relative risk [RR], 1.80; 95% confidence interval [CI], 1.43–2.27; P < 0.0001). There was a gender difference associated with residency status as non-CHR residents were significantly more likely to be male as compared with CHR residents (67.9% versus 62.0%; P < 0.0001). Age-specific and gender-specific population incidence rates were established for the population-based cohort as shown in Fig. 1 . Males were at significant increased risk for ICU admission as compared with females (330.5 per 100,000 versus 198.2 per 100,000; RR, 1.67; 95% CI, 1.59–1.74; P < 0.0001), and this was consistent observed among all age groups (Fig. 1 ). Increasing risk was associated with incrementally advancing age up to the age of 85 years, where a decrease in incidence was then observed (Fig. 1 ). As compared with younger individuals, those aged 65 years and older were at substantially increased risk of admission to an ICU (1719.9 per 100,000 versus 238.7 per 100,000; RR, 7.21; 95% CI, 6.95–7.47; P < 0.0001). Clinical features Although the magnitudes of differences were small, a number of clinical features were significantly different among CHR and non-CHR residents, as presented in Table 1 . In general, non-CHR residents had more markers of increased severity as compared with CHR residents (Table 1 ). No difference was observed between CHR and non-CHR residents in the occurrence of SIRS, although overall 90% (11,020) of patients fulfilled criteria. Outcomes The overall medians of ICU length of stay and hospital length of stay were 1.9 (IQR, 1–3.9) and 11 (IQR, 6–21), respectively. No significant differences were observed between CHR and non-CHR residents with respect to length of stay. In total, 1443 (11.8%) patients died in the ICU and a further 667 died during that hospitalization, for an overall inhospital case fatality rate of 17.3%. There was a significant effect of CHR residency on case fatality; CHR residents were much more likely to die in the ICU (1016 [13.1%] versus 427 [9.6%]; RR, 1.36; 95% CI, 1.22–1.51; P < 0.0001) and in hospital (1547 [20%] versus 563 [12.7%]; RR, 1.57; 95% CI, 1.43–1.71; P < 0.0001) as compared with non-CHR resident patients. A multivariable logistic regression model ( n = 11,569) was developed that had good fit ( P = 0.4) and discrimination (area under receiver operator curve = 0.83). As presented in Table 2 , CHR residency status was independently associated with inhospital death. Discussion This study describes the occurrence of, the demographic risk factors for, and the outcome associated with ICU admission in a large nonselected North American population. Although it is notable that the annual incidence of ICU admission is reported, it is of greater interest that demographic risk groups in the population that were at increased risk for admission to an ICU were defined. Not surprisingly, older age and male gender were associated with an increased need for ICU admission. This may be at least partly due to a higher rate of comorbid conditions, such as smoking or alcohol use, or other high-risk behaviors or activities among males as compared with females [ 4 , 5 ]. The population-based cohort design is an excellent method for defining the actual magnitude of such risks [ 2 ]. However, detailed information on each of the patient's comorbidities was not available for all patients in this study, and as a result the risk factor analysis was limited to the evaluation of demographic features alone. The actual burden of disease requiring ICU admission in an entire population was established in a minimally biased fashion in this study. Such accurate information on the degree of human suffering and death related to critical illness is important to potentially support continued or increased funding of clinical ICUs and critical care medical research. This study demonstrates that inclusion of nonresidents of a base population may have a major impact on biasing the results of studies in the ICU. When nonresidents of the CHR were included in this study, the occurrence of ICU admission in the CHR was overestimated by more than 50%. This observation is consistent with previous studies in the CHR and elsewhere in noncritically ill specific populations [ 2 , 16 , 19 , 27 ]. On the other hand, it is highly unlikely that a significant number of CHR residents requiring ICU admission were missed in this study. This is because all multidisciplinary and cardiovascular surgical ICUs in the CHR were included in surveillance and that, with the exception of acute liver, heart, and lung transplantation, patients are rarely referred out of the CHR for provision of healthcare. Furthermore, the CHR is relatively geographically isolated, with the closest tertiary care center to the CHR in Edmonton approximately 300 km away. Therefore, with the exception of the small number of CHR residents who may have required ICU admission while traveling, it seems unlikely that a substantial number of CHR residents requiring ICU admission would have been lost to analysis in this study. A number of statistically significant differences in the clinical features between CHR and non-CHR residents (Table 1 ) were observed, and with the exception of fever these would remain significant even if a conservative correction for multiple statistical comparisons such as the Bonferroni method were used. However, although statistically significant, the magnitudes of these differences are small and may not be of practical clinical difference. On the other hand, there was a dramatic effect of residency status on the outcome of patients admitted to ICUs in the CHR. The observation of a lower mortality among non-CHR patients is in contrast to the recent hospital-based study reported by Rosenberg and colleagues, although the definition of 'referral' was different in their study [ 21 ]. The reason why non-CHR patients were at lower risk for inhospital mortality is unexplained by the present study data, especially given that they appeared in general to be somewhat sicker on average than CHR residents (Table 1 ). The possibility exists that non-CHR patients may have died after transfer back to their 'home' health region hospitals and have therefore not been captured in the study inhospital mortality. This would explain the apparent lower inhospital case fatality rate among non-CHR residents but is only speculation. Of note, there were no significant pair-wise interactions between residency status and each of the other variables in the multivariable model. This study demonstrates that if nonresidents of a base population are included in studies of patients admitted to ICUs, gross errors in the determination of occurrence and outcomes may occur. The results of this study raise concerns regarding the generalization of results obtained from hospital-based reviews or population-based studies where nonresidents are included. However, this may not always be of major practical significance depending on specific study objectives. For example, a hospital-based study defining the outcome of a certain patient population such as transplant patients may be generalizable to other transplant centers because transplant recipients are nearly always managed at academic tertiary care referral institutions [ 28 ]. Generalization of results to other populations may therefore not be necessary. Similarly, population-based studies that strictly exclude nonresidents may not always be necessary for providing important information to guide allocation of health resources at regional levels. For example, Manns and colleagues conducted an economic evaluation of activated protein C for severe sepsis using clinical information from such a 'population-based' cohort in the CHR [ 29 ]. Although they included non-CHR residents, their results should be widely generalizable to other centers in North America and worldwide because typically patients requiring this therapy are admitted in tertiary care ICUs that are composed of a substantial number of referral patients. It should be recognized, however, that although studies that suffer from such selection bias may provide useful clinical information, results should not be generalized to unlike patient cohorts, and rarely, if ever, to the population as a whole. There are some limitations to this study that merit discussion. First, the CHR may have a different socioeconomic and demographic profile as compared with other regions, and this may influence the validity of generalizing results to other populations. One advantage, however, is that since this study was population-based, age and gender standardization against a reference population may be performed to facilitate comparison among different regions. This has been demonstrated to be of significant value in other population-based studies conducted in the United States [ 3 ]. Second, although the data were collected in a uniform fashion at each of the regional ICUs and much of this was directly linked from bedside monitors, systematic manual auditing of the information was not performed. However, previous work has suggested a high degree of accuracy [ 24 ]. Third, the need for admission to an ICU in this study was determined by the attending intensivist and not on some predefined objective criteria. This may be important for generalization to other centers that use different criteria for ICU admission. For example, patients admitted to Canadian ICUs tend to be sicker than those admitted to American ICUs, although adjustment according to APACHE II scores is possible [ 30 ]. Fourth, we did not have adequate admission data to further define patients into more refined subgroups for analysis. Finally, it is possible that some case patients were missed by our study surveillance as a result of care external to the CHR. However, given the comprehensiveness of the critical care system in the CHR and its relative geographic isolation, this would be only expected to have a minor effect on the study findings. Conclusion This study demonstrates the adverse effect of inclusion of nonresident patients of the base population on the determination of occurrence and outcome in studies of patients admitted to ICUs. Further well-designed, population-based studies in other regions that exclude nonresidents of the base population are required to better define the distribution and determinants of ICU admission internationally. Key messages • This population-based cohort study included all adults admitted to CHR multidisciplinary and cardiovascular surgical ICUs during a 4-year period. The effect of inclusion of non-residents in the study was evaluated. • Failure to exclude non-residents would lead to an overestimation of the incidence of ICU admission by more than 50%. A number of clinical features were significantly different between resident and non-resident patients; most notably, the in-hospital mortality rate was much lower in the non-resident cohort. • This study supports that non-resident patients should be strictly excluded from population-based studies. Competing interests The authors declare that they have no competing intrests. Abbreviations APACHE = Acute Physiology and Chronic Health Evaluation; CHR = Calgary Health Region; CI = confidence interval; CVICU = cardiovascular surgery intensive care unit; ICU = intensive care unit; IQR = interquartile range; RR = relative risk; SIRS = systemic inflammatory response syndrome.

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1065053

Complex systems and the technology of variability analysis

Characteristic patterns of variation over time, namely rhythms, represent a defining feature of complex systems, one that is synonymous with life. Despite the intrinsic dynamic, interdependent and nonlinear relationships of their parts, complex biological systems exhibit robust systemic stability. Applied to critical care, it is the systemic properties of the host response to a physiological insult that manifest as health or illness and determine outcome in our patients. Variability analysis provides a novel technology with which to evaluate the overall properties of a complex system. This review highlights the means by which we scientifically measure variation, including analyses of overall variation (time domain analysis, frequency distribution, spectral power), frequency contribution (spectral analysis), scale invariant (fractal) behaviour (detrended fluctuation and power law analysis) and regularity (approximate and multiscale entropy). Each technique is presented with a definition, interpretation, clinical application, advantages, limitations and summary of its calculation. The ubiquitous association between altered variability and illness is highlighted, followed by an analysis of how variability analysis may significantly improve prognostication of severity of illness and guide therapeutic intervention in critically ill patients.

Introduction Biological systems are complex systems; specifically, they are systems that are spatially and temporally complex, built from a dynamic web of interconnected feedback loops marked by interdependence, pleiotropy and redundancy. Complex systems have properties that cannot wholly be understood by understanding the parts of the system [ 1 ]. The properties of the system are distinct from the properties of the parts, and they depend on the integrity of the whole; the systemic properties vanish when the system breaks apart, whereas the properties of the parts are maintained. Illness, which presents with varying severity, stability and duration, represents a systemic functional alteration in the human organism. Although illness may occasionally be due to a specific singular deficit (e.g. cystic fibrosis), this discussion relates to illnesses characterized by systemic changes that are secondary to multiple deficits, which differ from patient to patient, with varied temporal courses, diverse contributing events and heterogeneous genetic contributions. However, all factors contribute to a physiological alteration that is recognizable as a systemic illness. Multiple organ dysfunction syndrome represents the ultimate multisystem illness, really representing a common end-stage pathway of inflammation, infection, dysfunctional host response and organ failure in critically ill patients, and frequently leading to death [ 2 ]. Although multiple organ dysfunction syndrome provides a useful starting point for discussion regarding complex systems and variability analysis [ 3 ], the application of variability analysis to other disease states is readily apparent and exciting. Life is composed of and characterized by rhythms. Abnormal rhythms are associated with illness and can even be involved in its pathogenesis; they have been termed 'dynamical diseases' [ 4 ]. Measuring the absolute value of a clinical parameter such as heart rate yields highly significant, clinically useful information. However, evaluating heart rate variability (HRV) provides additionally useful clinical information, which is, in fact, more valuable than heart rate alone, particularly when heart rate is within normal limits. Indeed, as is demonstrated below, there is nothing 'static' about homeostasis. Akin to the concept of homeorrhesis (dynamic stability) introduced by CH Waddington, homeokinesis describes 'the ability of an organism functioning in a variable external environment to maintain a highly organized internal environment, fluctuating within acceptable limits by dissipating energy in a far from equilibrium state' [ 5 ]. Clinicians have long recognized that alterations in physiological rhythms are associated with disease. The human eye is an excellent pattern recognition device, which is capable of complex interpretation of ECGs and electroencephalograms (EEGs) [ 6 ], and physicians make use of this skill on a daily basis. However, more sophisticated analysis of variability provides a measure of the integrity of the underlying system that produces the dynamics. As the spatial and temporal organization of a complex system define its very nature, changes in the patterns of interconnection (connectivity) and patterns of variation over time (variability) contain valuable information about the state of the overall system, representing an important means with which to prognosticate and treat our patients [ 3 ]. As clinicians, our goal is to make use of this observation in order to improve patient care. This technology of variability analysis is particularly valuable in the intensive care unit (ICU), where patients are critically ill and numerous parameters are routinely measured continuously. The intensivist is poised to marshal the science of variability analysis, becoming a 'dynamicist' [ 6 ], to measure and characterize the variability of physiological signals in an attempt to understand the information locked in the 'homeokinetic code' [ 7 ], and thus contribute to a breakthrough in our ability to treat critically ill patients. The focus of this review and analysis is the measurement and characterization of variability, a science that has undergone considerable growth in the past two decades. The development of mathematical techniques with a theoretical basis in chaos theory and nonlinear dynamics has provided us with greater ability to discern meaningful distinctions between biological signals from clinically distinct groups of patients. The science of variability analysis has developed from a close collaboration between mathematicians, physicists and clinicians. As such, the techniques for measuring variability sometimes represent a bewildering morass of equations and terminology. Each technique represents a unique and distinct means of characterizing a series of data in time. The principal objectives of this review are as follows: to present a concise summary, including definition, interpretation, advantages, limitations and calculation of the principal techniques for performing variability analysis; to discuss the interpretation and application of this technology; and to propose how this information may improve patient care. Although the majority of the discussion relates to the analysis of HRV because is it readily and accurately measured on an ECG, the techniques are applicable to any biological time signal. Two tables are included to facilitate review of the techniques for characterizing variability (Table 1 ) and the evidence for altered variability in illness (Table 2 ). Science of variability analysis Sampling The analysis of patterns of change over time or variability is performed on a series of data collected continuously or semicontinuously over time. For example, a heart rate tracing may be converted to a time series of intervals between consecutive heart beats (measured as R–R' intervals on an ECG). The same may be done with inter-breath intervals, albeit not as easily. When there is no intrinsic rhythm such as a heart or respiratory rate, sampling a signal occurs in discrete time intervals (e.g. serum concentrations of a hormone measured every few minutes). In order to reconstruct the underlying signal without error, one must respect the Nyquist Theorem, which states that the sampling frequency must be at least twice the highest frequency of the signal being sampled. Stationarity Stationarity defines a limitation in techniques designed to characterize variability. It requires that statistical properties such as mean and standard deviation of the signal remain the same throughout the period of recording, regardless of measurement epoch. Stationarity does not preclude variability, but it provides boundaries for variability such that variability does not change with time or duration of measurement. If this requirement is not met, as is the case with most if not all biological signals when physiological and/or pathophysiological conditions change, then the impact of trends with change on the mean of the data set must be considered in the interpretation of the variability analysis. The relative importance of stationarity to individual techniques of variability analysis is addressed below. Artifact Variability analysis should be performed on data that are free from artifact, with a minimal noise:signal ratio. Noise is measurement error, or imprecision secondary to measurement technology. Often present in patient monitoring, artifact must be removed, often by visual inspection of the raw data. For example, in the evaluation of HRV the presence of premature atrial and/or ventricular beats require that the data be removed, and appropriate interpolation be performed without compromising the integrity of the variability analysis. Several techniques, such as a Poincaré Plot of the difference between consecutive data points, have been developed to facilitate automated identification and removal of artifact [ 8 - 10 ]. Different techniques are more or less sensitive to artifact, which again is addressed below. Standardized technique Various factors alter variability measurement. For example, standing or head-up tilt (increased sympathetic activity) and deep breathing (increased respiratory rate induced HRV) will alter HRV indices in healthy individuals. With deference to Heisenberg, experimental design should take into account that the process of measurement may alter the intrinsic variation. An important component of standardized technique is the duration of measurement for analysis. For example, indices of HRV may be calculated following a duration of 15 min or 24 hours. In general terms, it is inappropriate to compare variability analysis from widely disparate durations of measurements [ 11 ]. More specifically, the impact of duration of measurement varies in relation to individual analysis technique, and is discussed below. Time domain analysis Definition Time series analysis represents the simplest means of evaluating variability, identifying measures of variation over time such as standard deviation and range. For example, quantitative time series analysis is performed on heart rate by evaluating a series of intervals between consecutive normal sinus QRS complexes (normal–normal, or NN or RR' interval) on an ECG over time. In addition, a visual representation of data collected as a time series may be obtained by plotting a frequency distribution, plotting the number of occurrences of values in selected ranges of values or bins. Calculation Mathematically, standard deviation is equal to the square root of variance; and variance is equal to the sum of the squares of difference from the mean, divided by the number of degrees of freedom. Evaluating HRV, the standard deviation of a series of NN intervals (SDNN) represents a coarse quantification of overall variability. As a measure of global variation, standard deviation is altered by the duration of measurement; longer series will have greater SDNN. Thus, SDNN can be calculated for short periods between 30 s and 5 min and used as a measure of short-term variability, or calculated for long periods (24 hours) as a measure of long-term variation [ 12 ]. Because it is inappropriate to compare SDNNs from recordings of different duration, standardized duration of recording has also been suggested [ 11 ]. Various permutations of measurement of standard deviation, in an effort to isolate short-term, high frequency fluctuations from longer term variation, are possible. For example, SDANN (standard deviation of the average NN interval calculated over 5-min intervals within the entire period of recording) is a measure of longer term variation because the beat-to-beat variation is removed by the averaging process. In contrast, the following variables were devised as a measure of short-term variation: RMSSD (square root of the mean squared differences of consecutive NN intervals), NN50 (number of pairs of adjacent NN intervals differing by more than 50 ms), and pNN50 (proportion of NN intervals differing by more than 50 ms = NN50 divided by total number of NN intervals). These measures of high frequency variation are interrelated; however, RMSSD has been recommended because of superior statistical properties [ 11 ]. The conventional 50 ms used in the NN50 and pNN50 measurements represents an arbitrary cutoff, and is only one member of a general pNNx family of statistics; in fact, a threshold of 20 ms may demonstrate superior discrimination between physiological and pathological HRV [ 13 ]. In order to characterize a frequency distribution, it may be fitted to a normal distribution, or rather a log-normal distribution – one in which the log of the variable in question is normally distributed. The skewness or degree of symmetry may be calculated, with positive and negative values indicating distributions with a right-sided tail and a left-sided tail, respectively. Kurtosis may also be calculated to identify the peakedness of the distribution; positive kurtosis (leptokurtic) indicates a sharp peak with long tails, and negative kurtosis (platykurtic) indicates a flatter distribution. Interpretation and clinical application Time domain analysis involves the statistical evaluation of data expressed as a series in time. Clinical evaluation of time domain measures of HRV have been extensive, using overall standard deviation (SDNN) to measure global variation, standard deviation of 5-min averages (SDANN) to evaluate long-term variation, and the square root of mean squared differences of consecutive NN intervals (RMSSD) to measure short-term variation. An abridged review of an extensive literature suggests that diminished overall HRV measured with time domain analysis portends poorer prognosis and/or increased mortality risk in patients with coronary artery disease [ 14 , 15 ], dilated cardiomyopathy [ 16 ], congestive heart failure [ 17 , 18 ] and postinfarction patients [ 19 - 23 ], in addition to elderly patients [ 24 ]. Time domain HRV analysis has been used to compare ß-blocker therapies postinfarction [ 25 ], to evaluate percutaneous coronary interventions [ 26 , 27 ], to predict arrhythmias [ 28 ] and to select patients for specific antiarrhythmic therapies [ 29 ], which are a few examples of a vast body of literature that is well reviewed elsewhere [ 30 , 31 ]. Time series of parameters derived from biological systems are known to follow log-normal frequency distributions, and deviations from the log-normal distribution have been proposed to offer a means with which to characterize illness [ 32 ]. For example, in paediatric ICU patients with organ dysfunction, HRV evaluated using a frequency distribution (plotting frequency of occurrence of differences from the mean) revealed a reduction in HRV and a shift in the frequency distribution to the left with increasing organ failure; these changes improved in surviving patients and were refractory in nonsurvivors [ 33 ]. The authors utilized a technique that was initially described in the evaluation of airway impedance variability, demonstrating increased variability in asthma patients characterized by altered frequency distribution [ 5 ]. Advantages and limitations Statistical measures of variability are easy to compute and provide valuable prognostic information about patients. Frequency distributions also offer an accurate, visual representation of the data, although the analysis may be sensitive to the arbitrary number of bins chosen to represent the data. Time domain measures are susceptible to bias secondary to nonstationary signals. A potential confounding factor in characterizing variability with standard deviation is the increase in baseline heart rate that may accompany diminished HRV indices. The clinical significance of this distinction is unclear, because the prognostic significance of altered SDNN or SDANN remains clinically useful. A more condemning limitation of time domain measures is that they do not reliably distinguish between distinct biological signals. There are many potential examples of data series with identical means and standard deviations but with very different underlying rhythms [ 34 ]. Therefore, additional, more sophisticated methods of variability analysis are necessary to characterize and differentiate physiological signals. It is nonetheless encouraging that, using rather crude statistical measures of variability, it is possible to derive clinically useful information. Frequency domain analysis Definition Physiological data collected as a series in time, as with any time series, may be considered a sum of sinusoidal oscillations with distinct frequencies. Conversion from a time domain to frequency domain analysis is made possible with a mathematical transformation developed almost two centuries ago (1807) by the French mathematician Jean-Babtiste-Joseph Fourier (1768–1830). Other transforms exist (e.g. wavelet, Hilbert), but Fourier was first and his transformation is used most commonly. The amplitude of each sine and cosine wave determines its contribution to the biological signal; frequency domain analysis displays the contributions of each sine wave as a function of its frequency. Facilitated by computerized data harvest and computation, the result of converting data from time series to frequency analysis is termed spectral analysis because it provides an evaluation of the power (amplitude) of the contributing frequencies to the underlying signal. Calculation The clinician should note that the power spectrum is simply a different representation of the same time series data, and the transformation may be made from time to frequency and back again. It is not necessary for the clinician to know how to perform power spectral density analysis using the fast Fourier transformation because computers can do so quickly and reliably, calculating a weighted sum of sinusoidal waves, with different amplitudes and frequencies. This provides an analysis of the relative contributions of different frequencies to the overall variation in a particular data series. Interpretation of the analysis must factor in the assumptions inherent to this calculation, namely stationarity and periodicity. Note that the square of the contribution of each frequency is the power of that frequency to the total spectrum, and the total power of spectral analysis (area under the curve of the power spectrum) is equal to the variance described above (they are different representations of the same measure) [ 11 ]. The fast Fourier transform or analysis (see Appendix 1) represents a nonparametric calculation because it provides an evaluation of the contribution of all frequencies, not discrete or preselected frequencies. Interpretation and clinical application Spectral analysis of heart rate was first performed by Sayers [ 35 ]. It was subsequently used to document the contributions of the sympathetic, parasympathetic and renin–angiotensin systems to the heart rate power spectrum, which introduced frequency domain analysis as a sensitive, quantitative and noninvasive means for evaluating the integrity of cardiovascular control systems [ 36 ]. Spectral analysis has been utilized to evaluate and quantify cardiovascular and electroencephalographic variability in numerous disease states, and is perceived as an important tool in clinical medicine [ 37 ]. The power spectral density function or power spectrum provides a characteristic representation of the contributing frequencies to an underlying signal. By identifying and measuring the area of distinct peaks on the power spectrum, it is possible to derive quantitative connotation to facilitate comparison between individuals and groups. In 2–5 min recordings, spectral analysis reveals three principal peaks, identified by convention with the following ranges: very low frequency (VLF; frequency = 0.04 Hz [cycles/s], cycle length >25 s), low frequency (LF; frequency 0.04–0.15 Hz, cycle length >6 s) and high frequency (HF; frequency 0.15–0.4 Hz, cycle length 2.5–6 s). In 24 hour recordings VLF is further subdivided into VLF (frequency 0.003–0.04 Hz) and ultralow frequency (ULF; frequency = 0.003 Hz, cycle length >5 hours) [ 11 ]. Correlations between time and frequency measures have also been demonstrated, for example in healthy newborns [ 38 ] and in cardiac patients following myocardial infarction [ 39 ]. Numerous factors in health and disease have an impact on the amplitude and area of each peak (or frequency range) on the HRV power spectrum. Akselrod and coworkers [ 36 ] first demonstrated the contributions of sympathetic and parasympathetic nervous activity and the renin–angiotensin system to frequency specific alterations in the HRV power spectrum in dogs. Several authors have evaluated and reviewed the relationship between the autonomic nervous system and spectral analysis of HRV [ 40 - 44 ]. Although autonomic regulation is clearly a significant regulator of the HRV power spectrum, evidence demonstrates a lack of concordance with direct evaluation of sympathetic tone, for example in patients with heart failure [ 45 ], and reviews increasingly conclude that HRV is generated by multiple physiological factors, not just autonomic tone [ 46 , 47 ]. In interpreting the significance of the HRV power spectrum, investigators initially focused on peaks because of a presumed relationship with a single cardiovascular control mechanism leading to rhythmic oscillations; however, others documented nonrhythmic (no peak) fluctuations in both heart rate and blood pressure variability, indicating the need to analyze broadband power [ 48 ]. Thus, the calculation of HF, LF, VLF and ULF using the ranges listed above serve to facilitate data reporting and comparison, but they are nonetheless arbitrary ranges with diverse physiological input. A recent review of HRV [ 47 ] documented the evidence that ULF reflects changes secondary to the circadian rhythm, VLF is affected by temperature regulation and humoral systems, LF is sensitive to cardiac sympathetic and parasympathetic nerve activity, and HF is synchronized to respiratory rhythms, primarily related to vagal innervation. What does spectral analysis of HRV tell us about our patients? Despite nonspecific pathophysiological mechanisms, there is ample evidence that the frequency contributions to HRV are altered in illness states, and that the degree of alteration correlates with illness severity. It is illustrative that alterations in the spectral HRV analysis related to illness severity have been demonstrated from hypovolaemia [ 49 ] to heart failure [ 50 - 52 ], from hypertension [ 53 , 54 ] to coronary artery disease [ 55 , 56 ], and from angina [ 57 ] to myocardial infarction [ 58 ], in addition to chronic renal failure [ 59 ], autonomic neuropathy secondary to diabetes mellitus [ 60 ], depth of anaesthesia [ 61 ] and more. Spectral analysis of HRV has been applied in the ICU. For example, using spectral HRV and blood pressure variability analyses in consecutive patients admitted to an ICU, increasing total and LF HRV power were associated with recovery and survival, whereas progressive decreases in HRV were associated with deterioration and death [ 62 ]. In separate investigations involving patients in the emergency room [ 63 ] or admitted to an ICU after 48 hours [ 64 ], decreased total, LF and LF/HF HRV was not only present in patients with sepsis but also correlated with subsequent illness severity, organ dysfunction and mortality. Several reviews discuss the application of HRV spectral analysis to the critically ill patient [ 65 - 68 ]. Thus, alterations in spectral analysis correlate with severity of illness, a finding consistently reported in cardiac and noncardiac illness states, providing the clinician with a means with which to gauge prognosis and determine efficacy of intervention. Advantages and limitations In order to derive a valid and meaningful analysis using a fast Fourier transform and frequency domain analysis, the assumptions of stationarity and periodicity must be fulfilled. The signal must be periodic, namely it is a signal that is comprised of oscillations repeating in time, with positive and negative alterations [ 69 ]. In the interpretation of experimental data, periodic behaviour may or may not exist when evaluating alterations in spectral power in response to intervention. The assumption of stationarity may also be violated with prolonged signal recording. Changes in posture, level of activity and sleep patterns will alter the LF and HF components of spectral analysis [ 70 ]. Spectral analysis is more sensitive to the presence of artifact and/or ectopy than time domain statistical methods. In addition, given that different types of Holter monitors may yield altered LF signals [ 71 ], it is essential to ensure that the sampling frequency of the monitor used to read QRS complexes does not contribute to error in the variability analysis [ 11 , 72 ]. Thus, the performance and interpretation of spectral analysis must incorporate these limitations. Recommendations based upon the stationarity assumption include the following [ 11 ]: short-term and long-term spectral analyses must be distinguished; long-term spectral analyses are felt to represent averages of the alterations present in shorter term recordings and may hide information; traditional statistical tests should be used to test for stationarity when performing spectral analysis; and physiological mechanisms that are known to influence HRV throughout the period of recording must be controlled. Time spectrum analysis Another means to address the stationarity assumption inherent in the Fourier transform is to evaluate the power spectral density function for short periods of time when stationarity is assumed to be present, and subsequently follow the evolution of the power spectrum over time [ 73 ]. This combined time varying spectral analysis allows the continuous evaluation of change in variability over time. One can use sequential spectral approach [ 74 ], Wavelet analysis [ 75 ], the Wigner-Ville technique or Walsh transforms, all of which provide an analysis of frequency alteration over time, which is useful in clinical applications [ 37 ]. For example, time frequency analysis has demonstrated increased LF HRV power during waking hours (considered primarily a marker of sympathetic tone) and increased HF HRV during sleep (thought to be related to respiratory fluctuations secondary to vagal tone) [ 70 ]. The authors hypothesized that observations of increased cardiovascular events occurring during waking hours may be secondary to sudden increases in sympathetic activity. However, spectral analysis should not be the only form of variability analysis because there are patterns of variation that are present across the frequency spectrum, involving long-range organization and complexity. Power law Definition Power law behaviour describes the dynamics of widely disparate phenomena, from earthquakes, solar flares and stock market fluctuations to avalanches. These dynamics are thought to arise from the system itself; indeed, the theory of self-organized criticality has been suggested to represent a universal organizing principle in biology [ 76 ]. It is illustrative to discuss the frequency distribution of earthquakes. A plot of the log of the power of earthquakes (i.e. the Richter scale) against the log of the frequency of their occurrence reveals a straight line with negative slope of -1. Thus, the probability of an earthquake may be determined for a given magnitude, occurring in a given region over a period of time, providing a measure of earthquake risk. In areas of increased earthquake activity, the line is shifted to the right, but the straight line relationship (and the slope) remains unchanged. Thus, the vertical distance between the straight line log–log frequency distributions or the intercept provides a measure of the difference in probabilities of an earthquake of all magnitudes between the two regions. Power law behaviour in physics, ecology, evolution, epidemics and neurobiology has also been described and reviewed [ 77 ]. Power laws describe dynamics that have a similar pattern at different scales, namely they are 'scale invariant'. As we shall see, detrended fluctuation analysis (DFA) is also a technique that characterizes the pattern of variation across multiple scales of measurement. A power law describes a time series with many small variations, and fewer and fewer larger variations; and the pattern of variation is statistically similar regardless of the size of the variation. Magnifying or shrinking the scale of the signal reveals the same relationship that defines the dynamics of the signal, analogous to the self-similarity seen in a multitude of spatial structures found in biology [ 78 ]. This scale invariant self-similar nature is a property of fractals, which are geometric structures pioneered and investigated by Benoit Mandelbrot [ 79 ]. Akin to a coastline, fractals represent structures that have no fixed length; their length increases with increased precision (magnification) of measurement, a property that confers a noninteger dimension to all fractals. In the case of a coastline, the fractal dimension lies between 1 (a perfectly straight coastline) and 2 (an infinitely irregular coastline). With respect to time series, the pattern of variation appears the same at different scales (i.e. magnification of the pattern reveals the same pattern) [ 78 ]. This is often referred to as fractal scaling. Of principal interest to clinicians and scientists is that one can measure the long range correlations that are present in a series of data and, as we shall see, measure the alterations present in states of illness. Calculation As with frequency domain analysis (discussed above), the first step in the evaluation of the power law is the calculation of the power spectrum. This calculation, based on the fast Fourier transform (defined above), yields the frequency components of a series in time. By plotting a log–log representation of the power spectrum (log power versus log frequency), a straight line is obtained with a slope of approximately -1. As the frequency increases, the size of the variation drops by the same factor, and this patterns exists across many scales of frequency and variation, within a range consistent with system size and signal duration. Mathematically, power law behaviour is scale invariant; if a variable x is replaced by Ax', where A is a constant, then the fundamental power law relationship remains unaltered. A straight line is fitted using linear regression, and the slope and intercept are obtained (see Appendix 1). Interpretation and clinical implications Power law behaviour has been observed for numerous physiological parameters and, relevant to clinicians, a change in intercept and slope is both present and prognostic in illness. Power law behaviour describes fluctuations in heart rate (first noted by Kobayashi and Musha [ 80 ]), foetal respiratory rate in lambs [ 81 ], movement of cells [ 82 ] and more. Power laws in pulmonary physiology were recently reviewed [ 83 ], noting a link between fractal temporal structure and fractal spatial anatomy. Alterations in the heart rate power law relationship (decreased or more negative slope) are present with ageing in healthy humans [ 84 ] as well as in patients with coronary artery disease [ 85 ]. Illness also confers changes in heart rate power law relationship. In over 700 patients with a recent myocardial infarction, as compared with age-matched control individuals, a steeper (more negative slope) power law slope was the best predictor of mortality evaluated [ 86 ]. In a random sample of 347 healthy individuals aged 65 years or older, a steep slope in the power law regression line (ß < -1.5) was the best univariate predictor of all-cause mortality, with an odds ratio for mortality at 10 years of 7.9 (95% confidence interval 3.7–17.0; P < 0.0001) [ 87 ]. Furthermore, only power law slope and a history of congestive heart failure were multivariate predictors of mortality in this cohort. Thus, changes in both slope and intercept have been documented to provide prognostic information in diverse patient populations. Given that power law analysis is performed by plotting the log of spectral power versus the log of frequency using data derived from spectral analysis, what is the relationship between the two methods of characterizing variability? Although derived using the same data, the two methods assess different characteristics of signals. Spectral analysis measures the relative importance or contribution of specific frequencies to the underlying signal, whereas power law analysis attempts to determine the nature of correlations across the frequency spectrum. These analyses may have distinct and complementary clinical significance; for example, investigations of multiple HRV indices in patients following myocardial infarction [ 86 ] and in paediatric ICU patients [ 33 ] found that the slope of the power law had superior ability to predict mortality and organ failure, respectively, as compared with traditional spectral analysis. Limitation Because determining power law behaviour requires spectral analysis, namely the determination of the frequency components of the underlying signal, the technique becomes problematic when applied to nonstationary signals. This limitation makes it difficult to draw conclusions regarding the mechanisms that underlie the alteration in dynamics observed in different patient groups. In addition, because power law behaviour measures the correlation between a large range of frequencies, it requires prolonged recording to achieve statistical validity. Nonetheless, as with the time and frequency domain analysis, valid clinical distinctions based on power law analysis have been demonstrated. Specifically addressing the problem of nonstationarity, there is a problem in differentiating variations in a series of data that arise as an epiphenomenon of environmental stimuli (such as the effect of change in posture on heart rate dynamics) from variations that intrinsically arise from the dynamics of a complex nonlinear system [ 88 , 89 ]. Both lead to a nonstationary variations but nonetheless represent clinically distinct phenomena. The subsequent technique was developed to address this issue. Detrended fluctuation analysis Definition Introduced by Peng and coworkers [ 90 ], DFA was developed specifically to distinguish between intrinsic fluctuations generated by complex systems and those caused by external or environmental stimuli acting on the system [ 88 ]. Variations that arise because of extrinsic stimuli are presumed to cause a local effect, whereas variations due to the intrinsic dynamics of the system are presumed to exhibit long-range correlation. DFA is a second measure of scale invariant behaviour because it evaluates trends of all sizes, trends that exhibit fractal properties (similar patterns of variation across multiple time scales). A component of the DFA calculation involves the subtraction of local trends (more likely related to external stimuli) in order to address the correlations that are caused by nonstationarity, and to help quantify the character of long-range fractal correlation representing the intrinsic nature of the system. Calculation The calculation of DFA involves several steps (see Appendix 1). The analysis is performed on a time series, for example the intervals between consecutive heartbeats, with the total number of beats equal to N. First, the average value for all N values is calculated. Second, a new (integrated) series of data (also from 1 to N) is calculated by summing the differences between the average value and each individual value. This new series of values represents an evaluation of trends; for example, if the difference between individual NN intervals and the average NN interval remains positive (i.e. the interval between heartbeats is longer than the average interbeat interval), then the heartbeat is persistently slower than the mean, and the integrated series will increase. This trend series of data displays fractal, or scaling behaviour, and the following calculation is performed to quantify this behaviour. In this third step, the trend series is separated into equal boxes of length n, where n = N/(total number of boxes); and in each box the local trend is calculated (a linear representation of the trend function in that box using the least squares method). Fourth, the trend series is locally 'detrended' by subtracting the local trend in each box, and the root mean square of this integrated, detrended series is calculated, called F(n). Finally, it is possible to graph the relationship between F(n) and n. Scaling or fractal correlation is present if the data is linear on a graph of log F(n) versus log(n). The slope of the graph has been termed a, the scaling exponent. A single scaling exponent represents the limit as N and n approach infinity; however, applicable to real life data sets, the linear relationship between log F(n) and log n has been noted to be distinct for small n (n < 11) and large n (11 < n > 10,000), yielding two lines with two slopes, labelled the scaling exponents a 1 and a 2 , respectively. For a more detailed description, see Appendix 1; excellent descriptions of the calculation of DFA may be found elsewhere [ 34 , 88 ]. Interpretation and clinical applications DFA offers clinicians the advantage of a means to investigate long range correlations within a biological signal due to the intrinsic properties of the system producing the signal, rather than external stimuli unrelated to the 'health' of the system. In addition, the calculation is based on the entire data set and is 'scale free', offering greater potential to distinguish biological signals based on scale specific measures [ 91 ]. Theoretically, the scaling exponent will vary from 0.5 (random numbers) to 1.5 (random walk), but physiological signals yield scaling exponents close to 1. A scaling exponent greater than 1.0 indicates a loss in long range scaling behaviour and a pathological alteration in the underlying system [ 88 ]. The technique was initially applied to detect long range correlations in DNA sequences [ 90 ] but has been increasingly applied to biological time signals. As with other techniques of variability analysis, DFA has been used to evaluate cardiovascular variation. Elderly individuals [ 92 ], patients with heart disease [ 93 ] and asymptomatic relatives of patients with dilated cardiomyopathy who have enlarged left ventricles [ 94 ] all exhibit a loss of 'fractal scaling'. To date, a 1 has demonstrated greater clinical discrimination of distinct heart rate data sets, as compared with a 2 [ 88 , 94 ]. For example, a 1 provided the best means of distinguishing patients with stable angina from age-matched control individuals; however, the correlation did not extend to angiographical severity of coronary artery disease [ 95 ]. In a retrospective evaluation of 2 hour ambulatory ECG recordings in the Framingham Heart Study [ 96 ], DFA was found to carry additional prognostic information that was not provided by traditional time and frequency domain measures. In a retrospective comparison between 24 hour HRV analysis using several techniques in patients post-myocardial infarction with or without inducible ventricular tachyarrhythmia [ 97 ], a decrease in the scaling exponent a 1 was the strongest predictor of risk for ventricular arrhythmia. DFA was superior to spectral analysis in the analysis of HRV alteration in patients with sleep apnoea [ 98 ]. In a prospective, multicentre evaluation of HRV post-myocardial infarction, reduced short-term scaling exponent (a 1 < 0.65) was the single best predictor of subsequent mortality [ 99 ]. In patients who had undergone coronary artery bypass surgery, reduced short-term scaling exponent in the postoperative period was the best predictor of a longer ICU stay, as compared with other HRV measures [ 100 ]. Thus, alteration in DFA scaling exponent (both increased and decreased) of heart rate fluctuation provides additional diagnostic and prognostic information that appears independent of time and frequency domain analysis. In addition to cardiovascular variation, DFA has increasingly been applied to investigate other systems. Alterations in the scaling exponent of respiratory variation (inter-breath intervals) have been noted in elderly individuals [ 101 ]; and the finding of long-range correlations in breath–breath end-tidal carbon dioxide and oxygen fluctuations in healthy infants introduce novel avenues for investigation of respiratory illness [ 102 ]. Remarkably, the scaling properties of temperature measurements (every 10 min for 30 hours) are altered in association with ageing [ 103 ]. In addition, DFA provides meaningful information on EEG signals and has been utilized to distinguish normal individuals from stroke patients [ 104 , 105 ]. Advantages and limitations The principal advantage to DFA is the lack of confounding due to nonstationary data. DFA is readily calculated using a computer algorithm available through a cooperative academic internet resource, Physionet [ 106 ]. Although DFA represents a novel technological development in the science of variability analysis and has proven clinical significance, whether it offers information distinct from traditional spectral analysis is debated [ 107 ]. Data requirements are greater than with other techniques and have been suggested to include at least 8000 data points, as noted by empirical observations [ 88 ]. It is inappropriate to simply 'run' the DFA algorithm blindly on data sets; for example, a clear shift in the state of the cardiovascular system (e.g. spontaneous atrial fibrillation) would prohibit meaningful DFA interpretation. Finally, although appealing in order to simplify clinical comparison, the calculation of two scaling exponents (one for small and one for large n) represents a somewhat arbitrary manipulation of the results of the analysis. The assumption that the same scaling pattern is present throughout the signal remains flawed, and therefore techniques without this assumption are being developed and are referred to as multifractal analysis. Multifractal analysis DFA is a monofractal technique, in that the assumption is that the same scaling property is present throughout the entire signal. Multifractal techniques provide multiple, possibly infinite exponents, such that the analysis produces a spectrum rather than a discrete value. For example, wavelet analysis is a multifractal analysis technique similar to DFA, which is capable of distinguishing the heart rate dynamics of patients with congestive heart failure from healthy control individuals [ 34 ]; a full discussion of multifractality of biological signals can be found elsewhere [ 108 ]. A separate technique recently introduced by Echeverría and colleagues [ 109 ] utilizes an a–ß filter (a technique imported from real-time radar tracking technology) to characterize heart rate fluctuations. Those authors suggested that this representation provides a superior means of identifying clinically distinct signals, and in order to demonstrate this they evaluated both theoretically and experimentally derived data sets. It remains unclear whether the added complexity and theoretical advantages of these techniques will afford consistent clinically significant improvements in the ability to distinguish physiological from pathological rhythms. Entropy analysis Definition Entropy is a measure of disorder or randomness, as embodied in the Second Law of Thermodynamics, namely the entropy of a system tends toward a maximum. In other words, states tend to evolve from ordered statistically unlikely configurations to configurations that are less ordered and statistically more probable. For example, a smoke ring (ordered configuration) diffuses into the air (random configuration); the spontaneous reverse occurrence is statistically improbable to the point of impossibility. Entropy is the measure of disorder or randomness. Related to time series analysis, approximate entropy (ApEn) provides a measure of the degree of irregularity or randomness within a series of data. It is closely related to Kolmogorov entropy, which is a measure of the rate of generation of new information [ 110 ]. ApEn was pioneered by Pincus [ 111 ] as a measure of system complexity; smaller values indicate greater regularity, and greater values convey more disorder, randomness and system complexity. Calculation In order to measure the degree of regularity of a series of data (of length N), the data series is evaluated for patterns that recur. This is performed by evaluating data sequences of length m, and determining the likelihood that other runs in the data set of the same length m are similar (within a specified tolerance r); thus two parameters, m and r, must be fixed to calculate ApEn. Once the frequency of occurrence of repetitive runs is calculated, a measure of their prevalence (negative average natural logarithm of the conditional probability) is found. ApEn then measures the difference between the logarithmic frequencies of similar runs of length m and runs with the length m+1. Small values of ApEn indicate regularity, given that the prevalence of repetitive patterns of length m and m+1 do not differ significantly and their difference is small. A derivation is included in Appendix 1, and a more comprehensive description of ApEn may be found elsewhere [ 112 - 114 ]. Interpretation and clinical application ApEn is representative of the rate of generation of new information within a biological signal because it provides a measure of the degree of irregularity or disorder within the signal. As such, it has been used as a measure of the underlying 'complexity' of the system producing the dynamics [ 111 , 112 , 115 ]. The clinical value of a measure of 'complexity' is potentially enormous because complexity appears to be lost in the presence of illness [ 114 , 116 , 117 ] (discussed in greater detail below). As with other means of characterizing biological signals, ApEn has been most extensively studied in the evaluation of heart rate dynamics. Heart rate becomes more orderly with age and in men, exhibiting decreased ApEn [ 118 ]. Heart rate ApEn has demonstrated the capacity to predict atrial arrhythmias, including spontaneous [ 119 ] and postoperative atrial fibrillation after cardiac surgery [ 120 ], and to differentiate ventricular arrhythmias [ 121 ]. Heart rate ApEn is decreased in infants with aborted sudden infant death syndrome [ 122 ]; among adults, postoperative patients with ventricular dysfunction [ 123 ] and healthy individuals infused with endotoxin [ 124 ] exhibit reduced heart rate ApEn. Because ApEn may be applied to short, noisy data sets, it was applied to assess the variation of parameters in which frequent sampling is more difficult (e.g. a blood test is necessary) and a paucity of data exists. This was most apparent in the evaluation of endocrine variability, as demonstrated in the following investigations. By applying ApEn to measurements of growth hormone (GH) every 5 min for 24 hours in healthy control individuals and patients with acromegaly, reduced orderliness (i.e. increased ApEn) was observed in acromegaly [ 125 ]; and normalization of GH ApEn values was demonstrated after pituitary surgery for acromegaly [ 126 ]. Increased disorderliness has been observed in insulin secretion in healthy elderly individuals as compared with young control individuals (insulin measured every minute for 150 min) [ 127 ], and in first-degree relatives of patients with non-insulin-dependent diabetes mellitus (insulin measured every minute for about 75 min) [ 128 ]. ApEn of adrenocorticotrophic hormone, GH, prolactin and cortisol levels (sampled every 10 min for 24 hours) is altered in patients with Cushing's disease [ 129 , 130 ]. Finally, altered dynamics of parathyroid hormone pulsatile secretion has been demonstrated in osteoperosis and hyperparathyroidism [ 131 ]. ApEn has also been used to evaluate neurological, respiratory and, recently, temperature variability. ApEn offers a means of assessing the depth of anaesthesia [ 132 - 134 ], and ApEn of tidal volume respiratory rate has been evaluated in patients with respiratory failure weaning from mechanical ventilation [ 135 ]. Alterations in respiratory variability are present in psychiatric illness; for example, increased entropy of respiration has been observed in patients with panic disorder [ 136 ]. Comparing chest wall movement and EEG activity in healthy individuals, sleep (stage IV) produced more regular breathing and more regular EEG activity [ 137 ]. Finally, demonstrating the remarkable potential and novel applications of variability analysis, ApEn of temperature measurements (every 10 min for 30 hours) revealed increased regularity and decreased complexity associated with age [ 103 ]. Advantages and limitations ApEn statistics may be calculated for relatively short series of data, a principal advantage in their application to biological signals. Referring to both theoretical analysis and clinical applications, Pincus and Golberger [ 112 ] concluded that m = 2 and r = 10–25% of the standard deviation of all the N values, and an N value of 10 m , or preferably 30 m , will yield statistically reliable and reproducible results (i.e. 100–900 data points). Pincus [ 114 ] also reported that ApEn is applicable to any system with at least 50 data points. In contrast to time domain measures of variability, which are independent of the sequence of the data set, ApEn required an evaluation of vectors representing consecutive data points, and thus the order of the data is integral to the calculation of ApEn and must be preserved during data harvest. Significant noise or nonstationary data compromise meaningful interpretation of ApEn [ 113 ]; therefore, it should not be used as the only means to measure signal characteristics. Sample and multiscale entropy An inherent bias within the ApEn calculation exists because the algorithm counts similar sequences to a given sequence of length m, including counting the sequence itself (to avoid the natural logarithm of 0 within the calculations). As a result, ApEn can be sensitive to the size of the data set, giving inappropriately low values when the total number of data points is low; this, and a lack of consistency in differentiating signals when m and r are altered, have led to the development of a new family of statistics named sample entropy (SampEn), in which self-matches are excluded in the analysis [ 110 ]. SampEn has the advantage of being less dependent on the length of the data series in question, and has been applied to heart rate fluctuations in the paediatric ICU [ 138 ]. Finally, because both ApEn and SampEn are noted to evaluate differences between sequences of length m and m+1, they evaluate regularity on one scale only, the shortest one, and ignore other scales. Thus, given the temporal complexity of biological signals on multiple scales, a novel technique, multiscale entropy, was developed as a more robust measure of complexity [ 139 ]. Initial investigations of multiscale entropy have been promising [ 140 ], but comprehensive evaluation remains to be performed. Summary and discussion of variability techniques The preceding sections highlight the considerable range of techniques that have been developed to characterize biological signals. Each with distinct theoretical background and significance, they contribute complementary information regarding signal characteristics. Time domain measures of variation represent an evaluation of overall, short-term or long-term variation, and are clinically proven as a means of identifying clinically significant alterations in biological signals, in particular with cardiovascular variability. Frequency domain analysis also has prognostic value, and has been useful in demonstrating the importance of sympathovagal balance in regulating HF and LF cardiovascular oscillations. Power law analysis contributes an analysis of fractal, long range correlations, allowing distinction between physiological and pathological signals with the slope and intercept of the power law. DFA also represents a means of detecting long range correlations, and is less bound by the stationarity assumption inherent to the other techniques. By measuring the degree to which sequences of data repeat themselves within a signal, ApEn provides a measure of signal irregularity, related to the rate of production of new information. Although techniques have shown consistent prognostic capacity, prediction of mortality is not the sole virtue of HRV analysis; separate techniques also may clarify mechanisms of disease [ 141 ]. Attempts to characterize biological signals should incorporate the 'toolkit' of techniques discussed in this review as well as the publication of raw data and code to facilitate comparison and development of this still young, exciting science [ 117 ]. Interpretation and significance of altered variability Following this review of the technology of variability analysis, the meaning of altered variability in biological signals must be addressed. A synthesis of the multiple but consistent theories regarding the significance of altered variability is presented to assist in the clinical application of this novel technology. A leading investigator within this field, Goldberger [ 142 ] proposed that increased regularity of signals represents a 'decomplexification' of illness, citing numerous examples of illness states with increased regularity of rhythms. For example, Cheyne–Stokes respiration, Parkinsonian gait, loss of EEG variability, preterminal cardiac oscillations, neutrophil count in chronic myelogenous leukaemia and fever in Hodgkin's disease all exhibit periodic, more regular variation in the dynamics of disease states [ 142 ]. Given that scale invariance is believed to be a central organizing principle of physiological structure and function, breakdown in this scale invariant, fractal behaviour, leads to uncorrelated randomness or more predictable behaviour, both representing a pathological alteration to the underlying system [ 78 , 84 ]. Thus, health is characterized by 'organized variability' and disease is defined by decomplexification, increased regularity and reduction in variability. In contrast to the 'decomplexification' hypothesis, Vaillancourt and Newell [ 143 ] noted increased complexity and increased approximate entropy in several disease states, including acromegaly and Cushing's disease, and hypothesized that disease may manifest with increased or decreased complexity, depending on the underlying dimension of the intrinsic dynamic (e.g. oscillating versus fixed point). In a rebuttal, Goldberger [ 142 ] noted that increased complexity demonstrated by lower entropy (specifically ApEn) requires corroboration by other techniques, given potential problems with using ApEn as the only technique to assess variability. A rebuttal to the rebuttal (all published concurrently) [ 144 ] noted that others accept the fundamental premise that increased and decreased variability occur in disease. In addition to the discussion regarding complexity, increased short-term variation in airway calibre in patients with asthma is observed, and reproduced experimentally with activation of airway smooth muscle with inhaled methacholine [ 5 ]. Given that smooth muscle activation is associated with increased metabolic rate, energy dissipation and an increased likelihood of statistically unlikely airway configurations, Macklem's hypothesis states that asthma is a disease of higher energy dissipation, greater distance from thermodynamic equilibrium, lower entropy and greater variation [ 5 ]. This suggests that health is defined by a certain distance from thermodynamic equilibrium; too close (decreased variation, too little energy dissipation, low entropy) or too far (increased variation and energy dissipation, high entropy) both represent pathological alterations. The science of complex systems is intimately related to variability analysis. Taking a broad systems based interpretation, the human organism is a complex system or, more accurately, it is a complex system of complex systems. The host response to sepsis, shock, or trauma is an example of a biological complex system that is readily apparent to intensivists [ 3 ]. Every complex system has 'emergent' properties, which define its very nature and function, including the presence of health versus illness. Variability or patterns of change over time (in addition to connectivity or patterns of interconnection over space) represent technology with which to evaluate the emergent properties of a complex system, which may be physiological or pathological [ 3 ]. It is possible to conceive complex systemic host response in a phase space of variability parameters, in which health represents stable 'holes' in space, exhibiting marked systemic stability accompanied by specific patterns of variability (and connectivity). Illness represents an alteration from health, separate 'holes' with distinct patterns of variability. Often, it takes a major insult to change a stable healthy state to an illness state, which may have varying degrees of stability. It is within this complex systems conception of health and illness that the clinical utility of variability analysis may be appreciated. How can variability analysis improve outcome in the intensive care unit? What does variability analysis offer that conventional monitoring does not? What is the clinical utility of this technology? We propose that multi-system continuous variability analysis offers the intensivist a unique monitoring tool that is capable of improving prognostication and directing therapeutic intervention. Intuitively, there is additional information in this analysis. Variability analysis tracks specific patterns of change in individual parameters over time (akin to calculating the first derivative or velocity in calculus). Monitoring patterns of change in variability continuously over time offers an additional dimension of analysis (akin to a second derivative evaluation or acceleration). Just as monitoring individual system variability offers an evaluation of the underlying individual system producing those dynamics, evaluating multisystem variability provides an evaluation of the whole, namely the systemic host response. By using variability analysis at different time points or, more powerfully, continuously over time, it is theoretically possible to track the 'system state' over time. Then, by selecting patients according to pathological patterns of variability and pursuing interventions with a therapeutic response or physiological alteration in variability, we hypothesize that outcomes in critically ill patients may be improved. Why does this individualized variability directed therapy offer exciting clinical potential? First, as the host response is a complex system, response to intervention in individual patients is unpredictable, although response to an intervention may be statistically beneficial for a cohort of patients. Thus, only by evaluating the response to intervention in individual patients can it be ascertained that the intervention is beneficial in those patients. Interventions that have not proven beneficial for the 'average' patient may still be beneficial in selected individual patients, in whom pathological variability is both present and improved by therapy. In summary, continuous, individualized, variability directed, goal directed therapeutic intervention has numerous theoretical advantages over conventional epidemiological cohort analysis evaluating response to a single intervention given to a heterogeneous population of patients. This technology is well suited to the ICU, in which real-time, continuous, digital physiological data acquisition (including waveform analysis) has been demonstrated [ 145 - 147 ]. Unresolved questions include whether, how and when is it possible to convert pathological to physiological variability, to prod our patients from illness to health. Answering these questions will determine the impact variability analysis has on ICU patient outcome. Conclusion The science of analyzing biological signals has undergone tremendous growth over the past decade, with the development of advanced computational methods that characterize the variation, oscillation, complexity and regularity of signals. These methods were developed in response to theoretical limitations of the others; however, all appear to have clinical significance. There is no consensus that any single technique is the single best means of characterizing and differentiating biological signals; rather, investigators agree that multiple techniques should be performed simultaneously to facilitate comparison between methods, techniques and studies. Variability analysis represents a novel means to evaluate and treat individual patients, suggesting a shift from epidemiological analytical investigation to continuous individualized variability analysis. Existing literature documents the clinical value of measuring variability to provide diagnostic, prognostic and pathophysiological information; future research must utilize this technology to improve care and the outcomes of our patients. Key messages • A complex systems paradigm provides insights regarding research and treatment of critically ill patients. • Variability analysis is the science of measuring the degree and character of patterns of variation of a time-series of a biologic parameter, in order to evaluate the state of the underlying complex system responsible for the biologic signal. • Using techniques that measure overall variation, frequency contribution, scale-invariant variation and degree of disorder, altered variability in consistently present in illness states, and the degree of alteration provides a measure of prognosis. • Using continuous multiogan variability analysis (CMVA), we hypothesize that goal-directed variability-directed therapeutic intervention will improve outcome and reduce mortality in critically ill patients, a novel individualized systems approach that complements analytical basic science and epidemiologic population science. Appendix 1: techniques of variability analysis Variability analysis The description of means to characterize and differentiate biological signals, or sequences of data in time produced by biological systems, is referred to as 'variability analysis'. For example, a heart rate recording may be considered a series of intervals between consecutive heart beats, referred to as NN intervals (interval between consecutive normal sinus beats) or RR intervals (interval between consecutive R waves on an ECG). With the goal of providing a single means of characterizing a whole series of data, the following techniques were developed to perform variability analysis and applied to clinical data sets. Time domain analysis Considered the simplest means of measuring variability, time domain analysis involves performing a statistical analysis of data expressed as a sequence in time. For example, SDNN (the standard deviation of NN intervals) has been used as a measure of HRV; greater variation yeilds higher standard deviation. Standard deviation is the square root of the average of the squared differences from the mean. SDANN (standard deviation of the average NN interval calculated over 5-min intervals within the entire period of recording) is a measure of longer term variation because the averaging process removes beat-to-beat variations. In contrast, the following variables were devised as a measure of short-term variation: RMSSD (square root of the mean squared differences of consecutive NN intervals), NN50 (number of pairs of adjacent NN intervals differing by more than 50 ms), and pNN50 (proportion of NN intervals differing by more than 50 ms = NN50 divided by total number of NN intervals). Frequency domain analysis Physiological data collected as a series in time may be considered a sum of rhythmic oscillations with distinct frequencies. Conversion from time domain to frequency domain analysis is performed most commonly using the Fourier transform, which decomposes the signal into a series of sine and cosine waves with frequencies that are multiples of the fundamental frequency (reciprocal of the time length to the input data record); the fast Fourier transform is a discrete Fourier transform that reduces the number of computations. The result of the Fourier transform is a complex number (a number multiplied by the square root of -1) for each frequency, the square of which is considered the spectral power of that frequency. The whole process is called spectral analysis, because it provides an evaluation of the spectral power (amplitude) of the contributing frequencies of an underlying signal. Power law analysis Power law behaviour may be described by the following equation: F(x) = ax ß Where a and ß are constants. Taking the logarithm of both sides, a straight line (graph log f [x] versus log x) with slope ß and intercept log a is revealed: Log f(x) = log (ax ß ) = log a + log x ß = log a + ß log x Thus, power law behaviour is scale invariant; if a variable x is replaced by Ax', where A is a constant, then the fundamental power law relationship remains unaltered. If dynamics follow a power law, a log–log representation of the power spectrum (log power versus log frequency) reveals a straight line, always within a defined range consistent with the size and duration of the system. The straight line is fitted using linear regression, and the slope ß and intercept can readily be obtained. When ß = -1, the dynamics are described as 1/f noise. Power law behaviour describes the dynamics of widely disparate phenomena, including heart rate fluctuations, inter-breath intervals, earthquakes, solar flares, stock market fluctuations, and avalanches. Detrended fluctuation analysis Variations that arise because of extrinsic stimuli are presumed to cause a local effect, whereas variations due to the intrinsic dynamics of the system are presumed to exhibit long range correlation. DFA attempts to quantify the presence or absence of long range scale-invariant (fractal) correlation. The first step in the technique to calculate DFA is to map a biological signal, such as a series of heart beats, to an integrated series. The integrated series is calculated by the sum of the differences between individual inter-beat intervals represented as NN i and the average interbeat interval for the whole data set, equal to NN ave . y(k) = S i = 1 N (NN i - NN ave ) This series y(k) represents an evaluation of trends; for example, if the difference NN i - NN ave remains negative (heart beat is persistently faster than the mean), then y(k) increases as k increases. This trend function y(k) is then separated into equal boxes of length n, where n = N/(total number of boxes). In each box, the local trend y n (k) is calculated as a linear representation of the function y(k) in that box using the least squares method. Least squares analysis involves the principle of optimization of the estimate based on minimizing the sum of the squared differences from the values predicted by the model. The series y(k) is then 'detrended' by subtracting the local trend y n (k). The root mean square of this integrated and detrended series is represented by the following: F(n) = v (1/N S k = 1 N [y(k) 2 - y n (k) 2 ]) By performing this analysis for all values of n, it is possible to calculate the relationship between F(n) and n. Scaling or fractal correlation is present if the data is linear on a graph of log F(n) versus log(n). The slope of the graph has been termed a, the scaling exponent, which will vary from 0.5 (white noise or uncorrelated random data) to 1.5 (Brownian noise or integrated white noise or random walk). When a = 1, behaviour corresponds to the 1/f noise. As a increases above 1 to 1.5, behaviour is no longer determined by a power law. Because the linear relationship between log F(n) and log(n) appears to have two distinct linear segments, one for small (n < 11) and large n (n > 11), the slopes of both lines are calculated separately and termed a 1 and a 2 , respectively; repeatedly, a 1 has proven superior to a 2 in terms of prognostic ability. Approximate entropy ApEn is a measure of 'irregularity'; smaller values indicate a greater chance that a set of data will be followed by similar data (regularity), and a greater value for ApEn signifies a lesser chance of similar data being repeated (irregularity). To calculate ApEn of a series of data, the data series is evaluated for patterns that recur. This is performed by evaluating data sequences or runs of length m, and determining the likelihood that other runs of length m are similar, within a tolerance r. Thus, two parameters, m and r, must be fixed to calculate ApEn. Increased regularity is associated with illness. The following is a description of the calculation of ApEn. Given any sequence of data points u(i) from i = 1 to N, it is possible to define vector sequences x(i), which consist of length m and are made up of consecutive u(i), specifically defined by the following: x(i) = (u [i], u [i + 1], ... u [i + m - 1]) In order to estimate the frequency that vectors x(i) repeat themselves throughout the data set within a tolerance r, the distance d(x [i],x [j]) is defined as the maximum difference between the scalar components x(i) and x(j). Explicitly, two vectors x(i) and x(j) are 'similar' within the tolerance or filter r (i.e. d(x [i],x [j]) = r) if the difference between any two values for u(i) and u(j) within runs of length m are less than r (i.e. |u(i + k) - u(j+k)| = r for 0 = k = m). Subsequently, C i m (r) is defined as the frequency of occurrence of similar runs m within the tolerance r: C i m (r) = (number of j such that d(x [i],x [j]) = r)/(N - m - 1), where j = (N - m - 1) Taking the natural logarithm of C i m (r), F m (r) is defined as the average of ln C i m (r): F m (r) = S i ln C i m (r)/(N - m - 1), where S i is a sum from I = 1 to (N - m - 1) F m (r) is a measure of the prevalence of repetitive patterns of length m within the filter r. Finally, approximate entropy, or ApEn(m,r,N), is defined as the natural logarithm of the relative prevalence of repetitive patterns of length m as compared with those of length m + 1: ApEn(m,r,N) = F m (r) - F m+1 (r) Thus, ApEn(m,r,N) measures the logarithmic frequency that similar runs (within the filter r) of length m also remain similar when the length of the run is increased by 1. Thus, small values of ApEn indicate regularity, given that increasing run length m by 1 does not decrease the value of F m (r) significantly (i.e. regularity connotes that F m [r] ~ F m+1 [r]). ApEn(m,r,N) is expressed as a difference, but in essence it represents a ratio; note that F m (r) is a logarithm of the averaged C i m (r), and the ratio of logarithms is equivalent to their difference. Competing interests None declared. Abbreviations ApEn = approximate entropy; DFA = detrended fluctuation analysis; EEG = electroencephalogram; GH = growth hormone; HF = high frequency; HRV = heart rate variability; ICU = intensive care unit; LF = low frequency; NN50 = number of pairs of adjacent NN intervals differing by more than 50 ms; pNN50 = proportion of NN intervals differing by more than 50 ms; RMSSD = square root of the mean squared differences of consecutive NN intervals; SampEn = sample entropy; SDANN = standard deviation of the average NN interval calculated over 5 min intervals within the entire period of recording; SDNN = standard deviation of a series of NN intervals; ULF = ultralow frequency; VLF = very low frequency.

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1065055

Early management after self-poisoning with an organophosphorus or carbamate pesticide – a treatment protocol for junior doctors

Severe organophosphorus or carbamate pesticide poisoning is an important clinical problem in many countries of the world. Unfortunately, little clinical research has been performed and little evidence exists with which to determine best therapy. A cohort study of acute pesticide poisoned patients was established in Sri Lanka during 2002; so far, more than 2000 pesticide poisoned patients have been treated. A protocol for the early management of severely ill, unconscious organophosphorus/carbamate-poisoned patients was developed for use by newly qualified doctors. It concentrates on the early stabilisation of patients and the individualised administration of atropine. We present it here as a guide for junior doctors in rural parts of the developing world who see the majority of such patients and as a working model around which to base research to improve patient outcome. Improved management of pesticide poisoning will result in a reduced number of suicides globally.

Introduction Pesticide self-poisoning is a major clinical problem in many parts of the world [ 1 , 2 ], probably killing about 300,000 people every year [ 3 , 4 ]. Although most deaths occur in rural areas of the developing world [ 2 ], pesticide poisoning is also a problem in industrialized countries, where it may account for a significant proportion of the deaths from self-poisoning that do occur [ 5 , 6 ]. The case fatality for self-poisoning in the developing world is commonly 10–20%, but for particular pesticides it may be as high as 50–70% [ 2 ]. This contrasts with the less than 0.3% case fatality ratio normally found for self-poisoning from all causes in Western countries. The causes of the high case fatality are multifactorial but include the high toxicity of locally available poisons, difficulties in transporting patients across long distances to hospital, the paucity of health care workers compared with the large numbers of patients, and the lack of facilities, antidotes, and training for the management of pesticide-poisoned patients [ 2 , 4 ]. The problem is compounded by a lack of proven interventions with which to develop treatment protocols. In 2002 we set up a cohort study in the North Central Province of Sri Lanka that sought to follow 10,000 acutely self-poisoned patients prospectively. So far, over 6000 patients have been recruited, of whom more than 3000 have ingested pesticides. All patients are rapidly resuscitated on admission to hospital and stabilised according to a standard protocol. Basic pharmacology and animal work suggests that early antagonism of pesticide toxicity should be associated with better outcomes [ 7 , 8 ]. Although there are few studies on the subject, there is some evidence that patients in the developing world often die soon after admission ([ 9 ], and CGS Rao, unpublished data). The rapid and effective stabilisation and treatment of pesticide-poisoned patients on their admission should reduce the number of early deaths, improve the prognosis for surviving patients over the next few days, and reduce the number and severity of long-term sequelae. Organophosphorus and carbamate pesticide poisioning This paper presents the protocol that we use to treat organophosphorus (OP)-poisoned or carbamate-poisoned patients on admission, based on our clinical experience and the best available evidence (see Additional file 1 ). It focuses on intentional ingestion of pesticides because such patients are more often severely poisoned than those with accidental or occupational exposure. We have not used any of the published severity poisoning scales because none have been independently validated. More importantly, pesticide-poisoned patients are unstable and a mildly poisoned patient can rapidly become very ill. An initial severity score suggesting a mild poisoning might allow doctors to relax with unfortunate results, as recognised by the IPCS/EC/EAPCCT poison severity score, which is designed only to be used retrospectively [ 10 ]. Poisoning with other pesticides We concentrate here on OPs and carbamate pesticides because OPs in particular are responsible for most pesticide deaths across Asia [ 2 , 11 - 13 ]. In addition, careful administration of oxygen, atropine and mechanical ventilation offers the opportunity to make a significant difference in outcome. However, the protocol can be adapted for the resuscitation of patients poisoned with other pesticides. Readers are referred to textbooks of clinical toxicology for details of subsequent treatment. Initial assessment of the unconscious patient Initial assessment involves checking airway, breathing and circulation. As part of this process, provide high-flow oxygen if available and ensure a patent airway through the placement of a Guedel airway or access. Place the patient in the left lateral position, ideally in a head-down position, to reduce the risk of aspiration. Extension of the neck in this position helps to keep the airway patent. Watch out for convulsions and treat with intravascular (IV) diazepam immediately if they do occur. Record a baseline Glasgow Coma Score to help with subsequent monitoring of the patient's condition. If available, affix a pulse oximeter. Does the patient require atropine? Recognition of OP/carbamate poisoning Next, assess whether the patient requires atropine. Textbooks list many features of the cholinergic syndrome [ 14 , 15 ]. However, we use five in routine assessment: miosis, excessive sweating, poor air entry into the lungs due to bronchorrhoea and bronchospasm, bradycardia, and hypotension. Severely OP- or carbamate-poisoned patients are typically covered with sweat, and have small pinpoint pupils and laboured breathing (often with marked bronchorrhoea and wheeze). The presence of pinpoint pupils and excessive sweat suggests that the patient has taken an OP or carbamate and requires atropine. The heart rate may be slowed, but normal or even fast heart rates are common. If none of these signs are present, then the patient does not yet have clinical cholinergic poisoning and does not require atropine. However, it is possible that these signs will occur later, for example as a pro-poison (thion) OP is converted to the active oxon form, as a fat-soluble OP such as fenthion leaches out of fat stores into the blood, or if the patient has presented soon after the ingestion. Careful observation is required to look for the development of cholinergic signs. Loading with atropine and IV fluids Dose of atropine For an unconscious patient, give atropine 1.8–3 mg (three to five 0.6 mg vials) rapidly IV into a fast-flowing IV drip. Although it is preferable that oxygen is given early to all ill patients, do not delay giving atropine if oxygen is unavailable. Because atropine dries secretions and reduces bronchospasm, its administration will improve patient oxygenation. There is no good evidence that giving atropine to a cyanosed patient causes harm. Atropine takes only a few minutes to work. During the 5 min after atropine administration, record three other signs of cholinergic poisoning against which atropine dosing will be titrated (Table 1 ): (1) air entry into lungs; (2) blood pressure; (3) heart rate. There is no need to do this before atropine is given, because pinpoint pupils and sweating in a region where these pesticides are common are sufficient to indicate OP/carbamate poisoning and trigger the decision to give atropine. If the clinical presentation is not clear, administer atropine 0.6–1 mg. A marked increase in heart rate (more than 20–25 beats/min) and flushing of the skin suggest that the patient does not have significant cholinergic poisoning and further atropine is not required. Giving fluids While waiting for the atropine to have effect, ensure that the two IV drips have been set up (one for fluid and drugs, the other for atropine). Give 500–1000 ml (10–20 ml/kg) of normal saline over 10–20 min. Assess whether enough atropine has been given – is the patient atropinised? Three to five minutes after giving atropine, check the five markers of cholinergic poisoning (Table 2 ). Mark them on an OP/carbamate observation sheet (Table 1 ). A uniform improvement in most of the five parameters is required, not improvements in just one. However, the most important parameters are air entry on chest auscultation, heart rate, and blood pressure. Pupil dilatation is sometimes delayed. Because patients do not die from constricted pupils, and the other parameters may improve more rapidly, it is reasonable to wait for the pupils to dilate. Check frequently and carefully that the other parameters are improving. When all the parameters are satisfactory, the patient has received enough atropine and is 'atropinised'. Continuation of bolus atropine loading to reach atropinisation If after 3–5 min a consistent improvement across the five parameters has not occurred, then more atropine is required. Double the dose, and continue to double each time that there is no response [ 16 , 17 ] (Table 1 ). Do not simply repeat the initial dose of atropine. Some patients need tens or hundreds of mg of atropine, so repeating 3 mg doses will mean that it may take hours to give sufficient atropine [ 16 ]. Severely ill patients will be dead by this point – atropinise the patient as quickly as possible. Beware of pupils that do not dilate because pesticide has been splashed into them directly, and lung crepitations that are due to aspiration of the pesticide rather than the systemic effects of the pesticide. Generalised wheeze may be a better sign of under-atropinization in a patient who has aspirated pesticide. Atropine treatment after atropinization Once atropinised (with clear lungs, adequate heart rate [more than 80 beats/min] and blood pressure [more than 80 mmHg systolic with good urine output], dry skin, and pupils no longer pinpoint), set up an infusion using one of the two IV cannulae. This should keep the blood atropine concentration in the therapeutic range, reducing fluctuation compared with repeated bolus doses. In the infusion, try giving 10–20% of the total amount of atropine that was required to load the patient every hour. If very large doses (more than 30 mg) were initially required, then less can be used. Larger doses may be required if oximes are not available. It is rare that an infusion rate greater than 3–5 mg/hour is necessary. Such high rates require frequent review and reduction as necessary. Observation of the patient Review the patient and assess the five parameters every 15 min or so to see whether the atropine infusion rate is adequate. As atropinisation is lost, with for example recurrence of bronchospasm or bradycardia, give further boluses of atropine until they disappear, and increase the infusion rate (Table 1 ). Once the parameters have settled, see the patient at least hourly for the first 6 hours to check that the atropine infusion rate is sufficient and that there are no signs of atropine toxicity. As the required dose of atropine falls, observation for recurrence of cholinergic features can be done less often (every 2–3 hours). However, regular observation is still required to spot patients at risk of, and going into, respiratory failure. Atropine toxicity Excess atropine causes agitation, confusion, urinary retention, hyperthermia, bowel ileus and tachycardia [ 15 ]. During regular observation for signs of overtreatment, check for the features given in Table 3 . The presence of all three suggests that too much atropine is being given. Stop the atropine infusion. Check again after 30 min to see whether the features of toxicity have settled. If not, continue to review every 30 min or so. When they do settle, restart at 70–80% of the previous rate. The patient should then be seen frequently to ensure that the new infusion rate has reduced the signs of atropine toxicity without permitting the reappearance of cholinergic signs. Do not follow heart rate and pupil size because they can be fast or slow, and big or small, respectively, depending on the balance of nicotinic and muscarinic features. Tachycardia also occurs with rapid administration of oximes and with pneumonia, hypovolaemia, hypoxia, and alcohol withdrawal, and is not a contraindication to giving atropine. Catheterise unconscious patients soon after resuscitation is completed. Look for urinary retention in an agitated confused patient; agitation may settle after insertion of the catheter. Care of the airway If a pesticide-poisoned patient is unconscious, place an endotracheal (ET) tube at this point even if a Guedel airway is working well, to minimise the risk of aspiration and to facilitate respiratory care if there is deterioration. Use diazepam to keep the patient sedated and tolerant of the ET tube. Because patients are often unstable during the first 6–12 hours, it may be better to sedate the patients to keep their ET tube in position if they start to waken with the atropine and the first dose of oxime. Active cooling and sedation Hyperthermia is a serious complication in hot and humid wards. A febrile patient should receive the minimum amount of atropine needed to control muscarinic signs, sedation if there is excessive agitation and muscle activity, and active cooling. Lay a towel soaked with water over the patient's chest and place in a fan's airflow. Cold water soaked towels can also be placed at points of maximum heat loss (for example axillae, groins). Reduce agitation with diazepam 10 mg given by slow IV push, repeated as necessary in an adult, up to 30–40 mg per 24 hours. Tying a non-sedated agitated patient to the bed is associated with complications, including death. Such patients struggle against their bonds and generate excess body heat, which may result in hyperthermic cardiac arrest. Diazepam is preferred over haloperidol because large doses of haloperidol may be required in patients receiving atropine. Haloperidol is also non-sedating, associated with disturbances of central thermoregulation and prolongation of the QT interval, and pro-convulsant. Diazepam may also have other advantages because animal studies suggest that it reduces damage to the central nervous system [ 18 ] and diminishes central respiratory failure [ 8 ]. Confirmation of exposure to cholinergic compounds Confirmation of poisoning by anti-cholinesterase pesticides can be sought by measuring butyrylcholinesterase and/or red-cell acetylcholinesterase activity. However, such assays cannot be performed in the ward. Furthermore, emergency therapy should be determined by the patient's clinical features, not by knowledge of the ingested poison. Treatment of the resuscitated and stable patient – should gastric decontamination be performed? Consider the need for gastric decontamination once the patient has been stabilised. Do not perform gastric decontamination until the patient is stable and, if necessary, intubated. Ipecac is contraindicated in pesticide-poisoned patients. The effectiveness of both gastric lavage and activated charcoal is unknown. Gastric lavage Consider lavage only if a patient has taken a highly toxic pesticide and arrives at hospital within 1–2 hours. It can be given to calm patients who have given explicit consent to the procedure or to unconscious intubated patients. Its use in agitated non-compliant patients or un-intubated drowsy or unconscious patients risks major complications including death. Pass a nasogastric tube to decompress the stomach and to suck out its contents. If patients have been previously given forced emesis, their stomach may well be already filled with fluid. If a decision is made to give lavage, after aspirating the stomach contents give water or normal saline in lots of 300 ml through a nasogastric tube. Larger volumes of fluid may push the poison into the small bowel. There is no reason to use a large-bore oro-gastric lavage tube for liquid poisons unless food blocks the nasogastric tube. Take off 300 ml before giving a further two or three 300 ml aliquots, otherwise the stomach may become distended, allowing fluid to pass into the small bowel or causing the patient to vomit. Measure the amount of fluid taken off to ensure that fluid is not left in the stomach. Activated charcoal A dose of activated charcoal can be left in the stomach at the end of the lavage. There is currently no evidence that either single-dose or multiple-dose regimens of activated charcoal result in clinical benefit after pesticide poisoning. Oximes and other therapies The clinical benefit of oximes for OP pesticide poisoning is not clear, being limited by the type of OP, poison load, time to start of therapy, and dose of oxime [ 19 , 20 ]. Current World Health Organisation guidelines recommend giving a 30 mg/kg loading dose of pralidoxime over 10–20 min, followed by a continuous infusion of 8–10 mg/kg per hour until clinical recovery (for example 12–24 hours after atropine is no longer required or the patient is extubated) or 7 days, whichever is later [ 20 , 21 ]. Where obidoxime is available, a loading dose of 250 mg is followed by an infusion giving 750 mg every 24 hours [ 20 ]. Too rapid administration will result in vomiting, tachycardia and hypertension (especially diastolic hypertension). Oximes are not recommended for carbamate poisoning. The role of hydrocortisone and antibiotic treatment after aspiration is not known. Aspiration of pesticide and stomach contents initially causes a chemical pneumonitis and not pneumonia [ 22 ]. It is unknown whether pneumonitis benefits from steroids. Pneumonia is diagnosed if the fever persists for more than 48 hours or there is focal consolidation on X-ray. Earlier use of antibiotics risks antibiotic-associated diarrhoea. Alcohol co-ingestion requires assessment of blood sugar levels and vitamin B supplementation. Care after the first few hours General observation OP/carbamate-poisoned patients are unstable and require regular observation to pick up changes in their general condition and their atropine requirements. Consider repeated doses of diazepam to keep the patient calm and settled. If facilities permit, give patients a general anaesthetic, and intubate and mechanically ventilate them. This should reduce the number of deaths from respiratory complications. Observation for impending respiratory failure and recurring cholinergic crises Watch for early signs of intermediate syndrome in OP-poisoned patients. Weakness of neck flexion is common: the patient has difficulty lifting their head off the pillow; subsequent signs include the use of accessory muscles of respiration, nasal flaring, tachypnoea, sweating, cranial nerve palsies and proximal muscle weakness in the limbs with retained distal muscle strength. Not all patients with neck weakness will develop the full intermediate syndrome requiring intubation and ventilation, but such patients are at risk and should be seen regularly. Measure tidal or minute volume and blood gases, if available. A locally agreed value should act as a trigger for prophylactic sedation and intubation, followed as necessary by ventilation. Recurrence of toxicity, requiring atropine therapy, commonly occurs after poisoning with fat-soluble OPs, such as fenthion, that leak out of fat over days and even weeks. Recurring cholinergic crises may occur with little notice. Conclusions Medical management of severe cholinergic pesticide poisoning is difficult, with high mortality. Some patients will die no matter how well managed. However, careful resuscitation with appropriate use of antidotes, followed by good supportive care and observation, should minimise the number of deaths in the period after admission to hospital. Key messages • Initial treatment of OP/carbamate pesticide poisoned patients involves the standard ABC of resuscitation. • Since most deaths occur from respiratory failure, airway protection and ventilatory support is essential. • Atropine can be given in an individualised dosing regimen to stabilise the patient. • Careful observation probably saves many lives. • Decontamination should only be done after the patient is fully stabilised, and not directly on admission. Competing interests The authors declare that they have no competing interests. Abbreviations ET = endotracheal; IV = intravascular; OP = organophosphorus. Supplementary Material Additional file 1 Evidence for the protocol. Click here for file

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1065056

Effect of lung compliance and endotracheal tube leakage on measurement of tidal volume

Introduction The objective of this laboratory study was to measure the effect of decreased lung compliance and endotracheal tube (ETT) leakage on measured exhaled tidal volume at the airway and at the ventilator, in a research study with a test lung. Methods The subjects were infant, adult and pediatric test lungs. In the test lung model, lung compliances were set to normal and to levels seen in acute respiratory distress syndrome. Set tidal volume was 6 ml/kg across a range of simulated weights and ETT sizes. Data were recorded from both the ventilator light-emitting diode display and the CO 2 SMO Plus monitor display by a single observer. Effective tidal volume was calculated from a standard equation. Results In all test lung models, exhaled tidal volume measured at the airway decreased markedly with decreasing lung compliance, but measurement at the ventilator showed minimal change. In the absence of a simulated ETT leak, calculation of the effective tidal volume led to measurements very similar to exhaled tidal volume measured at the ETT. With a simulated ETT tube leak, the effective tidal volume markedly overestimated tidal volume measured at the airway. Conclusion Previous investigators have emphasized the need to measure tidal volume at the ETT for all children. When ETT leakage is minimal, it seems from our simulated lung models that calculation of effective tidal volume would give similar readings to tidal volume measured at the airway, even in small patients. Future studies of tidal volume measurement accuracy in mechanically ventilated children should control for the degree of ETT leakage.

Introduction Three investigators have reported that tidal volume ( V T ) in children is inaccurate when measured at the ventilator, even when effective V T is used [ 1 - 3 ]. Cannon and colleagues [ 1 ] studied 98 infants and children and found a significant discrepancy between expiratory V T measured at the ventilator and that measured with a pneumotachometer. Calculation of the effective V T did not alter this discrepancy. Castle and colleagues [ 2 ] studied 56 intubated children and found that exhaled V T displayed by the Servo 300 significantly overestimated V T measured at the airway by between 2% and 91%. After correcting for gas compression, effective V T overestimated true V T by as much as 29% in older children but underestimated the true V T by up to 64% in the smallest infants. Neve and colleagues [ 3 ] studied 27 infants and found that V T was overestimated by the ventilator in comparison with V T measured at the Y piece. None of these investigators controlled for endotracheal tube (ETT) leakage, which is more of a problem in children than in adults because of the use of uncuffed ETTs. Accurate measurement of V T is increasingly important because the Acute Respiratory Distress Syndrome (ARDS) Network investigators have shown that the use of a low effective V T leads to decreased mortality in their patient population [ 4 ]. The effective V T goal in their ventilator protocol was 6 ml/kg but could be reduced to as low as 4 ml/kg if the plateau pressure was above 30 cmH 2 O. At such low V T values, accurate measurement is imperative to prevent atelectasis and subsequent ineffective minute ventilation. Clinically, there are three methods to estimate delivered V T : first, direct measurement at the expiratory limb of the ventilator; second, direct measurement at the ETT with a pneumotachometer; and third, indirect calculation of effective V T by using set V T minus calculated compressible volume lost in the ventilator circuit [ 5 ]. The principle of Boyle's law (the volume of gas decreases as the absolute pressure exerted by the gas increases, and vice versa) is used to calculate the compressible volume in ventilator circuits. How effective V T compares with V T measured at the airway has not been rigorously tested. Using V T measured at the ETT as the gold standard, we used three test lung models in a controlled laboratory setting to evaluate the accuracy of ventilator measured V T and effective V T under conditions of poor lung compliance, with and without ETT leakage, across a range of simulated patient sizes. We proposed that the discrepancy between effective V T and V T measured at the ETT in children was due mainly to ETT leakage around uncuffed ETTs, and that in situations with minimal ETT leakage there would be minimal difference between the effective V T and V T measured at the airway. Materials and methods Experimental conditions A Servo 300 ventilator (Siemens-Elema, Solna, Sweden) in the SIMV volume control mode was used. A pressure differential pneumotachometer (CO 2 SMO Plus; Novametrix Medical Systems, Wallingford, CT) was used between the ventilator and ETT connection. The temperature of the humidifier was set at 37°C. A heated disposable respiratory circuit (Allegiance Healthcare Corporation, McGaw Park, IL) was used. We tested the compliance of the circuit to ensure that it was stable across a range of conditions. To do this, we first set the ventilator on the following: inspiratory time of 1.3 s, positive end-expiratory pressure (PEEP) of 0, respiratory set frequency of 6 breaths per minute, and a pause time of 15%. V T was increased by increments of 50 ml and the plateau pressure was recorded from the ventilator with the patient outlet occluded. No component other than the humidifier was added to the circuit [ 6 ]. A linear relationship was found, with no change of the circuit compliance at high airway pressure. In the pediatric and infant models, a valve distal to the ETT was used to adjust volume leaks of 0%, 10%, 20%, and 30%. A shown in Fig. 1 , a separate pneumotachometer (NVM-1; Thermo Respiratory Group, Palm Springs, CA) was used for independent measurement of the percentage of ETT leakage. The Servo 300 was used for all test conditions. To control for differences between the ventilators, we tested each set of experimental conditions on three different ventilators. The CO 2 SMO Plus respiratory mechanics monitor was used to measure the V T at the ETT. This monitor measures flow with a fixed-orifice differential pressure pneumotachometer located at the ETT. Respired gas flowing through the flow sensor produces a small pressure decrease across the two tubes connected to the sensor. This pressure decrease is transmitted through the tubing sensor to a differential pressure transducer inside the monitor and is correlated with flow according to a factory-stored calibration. The pressure transducer is automatically 'zeroed' to correct for changes in ambient temperature. Data are filtered and sampled at 100 Hz. The monitor continuously displays a range of ventilatory variables, including both V T and airway pressures. Three CO 2 SMO Plus sensors are available: neonatal, pediatric, and adult. The manufacturer recommends that the choice of sensor be based on various criteria: first, the diameter of the tracheal tube; second, the patient's age; third, the expected flow/volume range; and fourth, the acceptable levels of dead space and resistance. Table 1 lists the experimental conditions for all lung models. Before data collection, all ventilators, respiratory mechanics monitors, and tachometers used in this study were calibrated in accordance with the manufacturer's recommendation. To ensure that different ventilators and monitors did not influence the results, all data were repeated three times, each time with a different Servo 300 ventilator and a different CO 2 SMO Plus monitor. Adult lung model A TTL™ adult test lung (Vent Aid; Michigan Instruments Inc., Grand Rapids, MI) was used. This device has two separate lungs, each with a functional residual capacity (FRC) of 900 ml. The lung compliance can be adjusted by moving a spring up and down with a compliance ranging from 10 to 150 ml/cmH 2 O per lung. Each lung is tested before use to assess for leakage. Lung–thorax compliance levels were set at 10, 20, 40, 60, 100, and 150 ml/cmH 2 O. Pediatric lung model A TTL™ adult test single lung was used with the FRC adjusted to give 30 ml/kg by displacing the extra volume with water-filled bags. Lung–thorax compliance levels were set at 5, 10, 20, 40 and 60, ml/cmH 2 O. Infant lung model An infant lung simulator (D.B&M products, Redlands, CA) was used. The model has three different preset compliances of 1, 3, and 10 ml/cmH 2 O. Data recording Data were recorded from both the ventilator light-emitting diode display and the CO 2 SMO Plus monitor display by a single observer. Variables recorded were inspired V T , expired V T , peak inspiratory pressure (PIP), PEEP, and plateau pressure. Effective V T was calculated from the following equation [ 2 ]: set inspired V T - [circuit compliance × (PIP - PEEP)]. Analysis The major outcome variable was the calculated difference between the effective V T and the exhaled V T measured either at the ventilator or at the ETT in each experiment. For each set of test conditions (Table 1 ) we used the mean of the three replicate measurements and also give the highest and lowest values. V T was adjusted for the simulated weights and expressed as ml/kg. We determined a priori that the difference between the V T values would be considered excessive if it exceeded 10% of the 6 ml/kg goal (0.6 ml/kg). Results Test lung models As shown in Fig. 2 , for the adult, pediatric, and infant models with no ETT leak, the difference between V T measured at the ETT and at the ventilator increased with decreasing lung compliance. V T measured at the ventilator was always higher than that measured at the ETT. The ventilator measurement overestimated V T by more than 10% (0.6 ml/kg) as lung compliance dropped to moderately low values and the difference exceeded 20% (1.8 ml/kg) at the lowest lung compliances in each model. The standard deviation of the difference was 0–0.2 ml/kg for all sets of measurements. In all models, in the absence of ETT leakage the difference between effective V T and V T measured at the ETT was less than 10% across the range of lung compliances with a standard deviation of 0–0.2 ml/kg for all sets of measurements. As shown in Fig. 3 , however, the agreement between effective V T and V T measured at the ETT was poor when a 20% and 30% simulated ETT leak was added in the infant and pediatric test lung models. Under these conditions, the effective V T was at least 10% higher than that measured at the ETT for all simulated conditions, and the standard deviation was 0.1–0.4 ml/kg for all sets of measurements. Discussion Using well-controlled experimental conditions, we showed that in the absence of ETT leakage, effective V T approximated the V T measured at the ETT in the test lung even when lung compliance was poor. As expected, exhaled V T measured at the ventilator became increasingly inaccurate with poor lung compliance. In the presence of ETT leakage, effective V T overestimated the V T measured at the ETT by at least 0.6 ml/kg. It is clear that in the presence of ETT leakage, effective V T is inaccurate and V T is most accurately estimated at the airway. We used an in vitro model to manipulate experimental conditions while controlling for all other variables. Accurate measurement of V T is essential when a low- V T strategy is used to protect injured lungs as is recommended by the recent ARDS Network study [ 4 ]. In the adult lung model, we manipulated the compliance to simulate the lung compliance quartiles reported in the ARDSNet study [ 4 ]. Our findings have clinical implications. In agreement with other investigators [ 1 - 3 ], we found that unadjusted V T measured at the ventilator is highly inaccurate. We found this inaccuracy to increase markedly when lung compliance was abnormal. This means that dual-control automated ventilator modes (for example volume support or pressure-regulated volume control) that make adjustments based on V T measured at the ventilator might ineffectively ventilate patients with poor lung compliance. Automated ventilator modes should be used with care in critically ill children. We support the current recommendations of previous investigators [ 1 - 3 ] that V T should be measured at the ETT in critically ill children receiving mechanical ventilator support. These investigators emphasized the need to measure V T at the ETT for all children; they did not control for the presence of uncuffed ETTs in their studies or evaluate the effect of leakage. Significant loss of V T occurs when both ETT leakage and poor lung compliance are present. Although the V T measured at the ETT may underestimate the actual V T being delivered in this situation, it is still the best estimation of the tidal volume delivered to the lung. Use of cuffed ETTs to minimize ETT leakage may lead to more accurate measurement of V T when lung compliance is poor [ 7 ]. When ETT leakage is 20% or greater, Main and colleagues [ 8 ] reported inconsistent tidal volume delivery and gross overestimation of respiratory compliance and resistance in children. When ETT leakage is minimal, it seems from our simulated lung models that calculation of effective V T would give similar readings to V T measured at the airway, even in small patients. This could potentially negate the need for the addition of sensors at the airway and their associated increase in airway resistance for small ETTs [ 2 ]. Unfortunately, ETT leakage is dynamic and dependent on head position. Unless a simple, accurate and continuous means of measuring ETT leakage is available, it is safest to measure V T at the airway in all mechanically ventilated children. Future studies of V T measurement accuracy in mechanically ventilated children should control for the degree of ETT leakage. Key messages • Previous investigators have emphasized the need to measure tidal volume at the endotracheal tube for all mechanically ventilated children. • When endotracheal leakage is minimal, it would appear from this study using simulated lung models that calculation of effective tidal volume would give similar readings to tidal volume measured at the airway, even in small patients. • Future studies of tidal volume measurement accuracy in mechanically ventilated children should control for the degree of endotracheal tube leakage. Competing interests None declared. Abbreviations ARDS = acute respiratory distress syndrome; ETT = endotracheal tube; FRC = functional residual capacity; PEEP = positive end-expiratory pressure; PIP = peak inspiratory pressure; V T = tidal volume.

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1065057

The routine use of pediatric airway exchange catheter after extubation of adult patients who have undergone maxillofacial or major neck surgery: a clinical observational study

Introduction We conducted the present study to determine the usefulness of routinely inserting a pediatric airway exchange catheter (PAEC) before tracheal extubation of adult patients who had undergone maxillofacial or major neck surgery and have risk factors for difficult reintubation. Methods A prospective, observational and clinical study was performed in the 25-bed general intensive care unit of a university hospital. Thirty-six adult patients who underwent maxillofacial or major neck surgery and had risk factors for difficult reintubation were extubated after insertion of the PAEC. Results Four of 36 (11.1%) patients required emergency reintubation after 2, 4, 6 and 18 hours after tracheal extubation, respectively. Reintubation of these patients, which was thought to be nearly impossible by direct laryngoscopy, was easily achieved over the PAEC. Conclusion The PAEC can be a life-saving device during reintubation of patients with risk factors for difficult reintubation such as laryngeo-pharyngeal oedema due to surgical manipulation or airway obstruction resulting from haematoma and anatomic changes. We therefore suggest the routine use of the PAEC in patients undergoing major maxillofacial or major neck surgery.

Introduction Maxillofacial and major neck surgery has a considerable risk for postoperative laryngo-pharyngeal oedema and airway obstruction due to surgical manipulation or haematoma [ 1 ]. When patients undergoing these operations develop laryngeal oedema or airway obstruction and require reintubation after they have been extubated, reintubation may be very difficult or impossible through laryngoscopy because of the characteristics of these operations such as mandibular fixation with an archbar or as a result of anatomical changes. Extubation of a patient with risk factors for difficult tracheal reintubation is approached with concern, even in the experienced hands of the anaesthesiologist and critical care physician. Although all of the criteria used to predict successful extubation are generally satisfactory before extubation, none predict the adequacy of the airway once the endotracheal tube (ETT) has been removed [ 2 ]. Hence, acute respiratory distress can develop after extubation and mandate emergency tracheal reintubation. Mask ventilation and tracheal intubation may be difficult or impossible. Considerable time and an experienced physician are needed to secure a difficult airway with the use of alternative methods such as fibre-optic bronchoscope, retrograde] intubation or cricothyroidotomy. Re-establishing the airway in these patients can be extremely challenging, and often results in considerable morbidity and mortality [ 3 ]. In the study by Loudermilk and colleagues [ 2 ], the advantages of the use of a pediatric airway exchange catheter (PAEC) inserted before tracheal extubation of adult patients with a known or expected difficult airway were well shown. However, the routine use of PAEC as a rescue for reintubation after maxillofacial surgery has not been reported. The aim of this study was to determine the usefulness of routinely inserting the PAEC before tracheal extubation of adult patients undergoing major maxillofacial or neck surgery (Fig. 1 ). Methods Patients Thirty-six patients admitted to our intensive care unit (ICU) after maxillofacial or major neck surgery between January 2001 and May 2002 were routinely extubated with the use of a no. 11 PAEC (Cook Critical Care, Bloomington, IN), with the approval of the Institutional Review Board. Patients included in the study consisted of 13 post-operative patients with maxillofacial trauma, 14 patients who had undergone neck surgery (5 with hugely enlarged thyroid gland or tumor and 9 with larynx or tongue cancer), and 9 patients who had undergone maxillofacial cancer surgery. Written consent for publication of the photos of the patients was obtained. Technique A no. 11 PAEC is 83 cm in length and has a 4 mm external diameter and a 2.3 mm internal diameter with a hollow lumen. It is semi-rigid and made of radio-opaque polyurethane; there are six sideports in the distal 3 cm of the catheter. The patients were extubated when they became conscious and had normal body temperature and normal blood gases with an inspired oxygen concentration (FiO 2 ) of 0.4, a positive end expiratory pressure of less than 5 cmH 2 O and pressure support of less than 8 cmH 2 O. In addition, the haemodynamic status of the patients had to be stable before the decision to extubate was made. The PAEC was carefully inserted through the existing ETT before extubation, avoiding carinal irritation by placing it at the same depth as the ETT tip (20–22 cm orally or 27–30 cm nasally). The PAEC was not inserted against a resistance. After the ETT had been removed and the PAEC had been secured, humidified oxygen with a low flow of 1–2 l/min was insufflated via the lumen of the PAEC. Signs of respiratory failure and tolerance were also assessed. The PAEC was removed when it became clinically apparent that the need for tracheal reintubation was unlikely. We considered the ability of patients to manage secretions including cough and swallow functions in making the decision about extubation of the PAEC. A stable O 2 saturation and the extent of surgery were also important factors in this decision. The timing of removal of the PAEC was therefore different depending on various characteristics of patients and surgery. When patients failed to respond to tracheal extubation, the PAEC was used to facilitate the reintubation. Results Twenty-eight patients (77.8%) were men, and 8 (22.2%) were women. Ages ranged from 19 to 76 years, with a mean age of 52.6 ± 10.8 (all results are means ± standard deviation) years. An oral ETT was in place in 18 patients (50%) and a nasal ETT in 18 (50%). All patients had a cuff leak test before tracheal extubation. The median duration of endotracheal intubation after the operations was 1.2 days (range 2 hours to 10 days). After tracheal extubation with the PAEC, 4 of 36 patients (11.1%) required reintubation (Table 1 ). The reintubation of these four patients, who are discussed in detail as case reports below, was achieved over the PAEC and was easily accomplished on the first attempt without the need of an alternative method. We used the assistance of laryngoscope during the reintubation of two patients in whom the PAEC had been inserted orotracheally. In the other 32 patients who did not require reintubation, the PAEC was kept in the trachea for between 4 and 24 hours (mean 10.4 ± 4.2 (all results are means ± standard deviation) hours) and none of them required reintubation after the PAEC had been removed. Thirty-one patients had nasogastric tubes at the same time. The PAEC was well tolerated in 34 of 36 patients (94.4%). Two patients tried to remove the PAEC; they were therefore sedated for a few hours. We did not give any sedative drugs to the patients who could tolerate the PAEC. No adverse events were observed while the PAEC was being kept in the trachea. Case 1 The reason for reintubation of this male patient, who had undergone radical neck surgery for cancer and had been intubated easily by direct laryngoscopy before the operation, was excessive surgical bleeding and haematoma, which developed 2 hours after extubation. The patient was immediately taken to the operating room. He could not be ventilated effectively by bag-valve-mask during the induction of anaesthesia (fentanyl 2 μg/kg, propofol 2 mg/kg, vecronium 0.1 mg/kg) and his oxygen saturation decreased to 85%. He was reintubated orally over the PAEC with the assistance of a laryngoscope within a few seconds by using an 8 mm ETT. During observation with a laryngoscope, reintubation of this patient by direct laryngoscopy was thought to be nearly impossible because the glottis could not be seen as a result of the anatomic abnormality caused by haematoma. He was extubated again using the PAEC 24 hours after his second operation; the PAEC was removed again 6 hours after insertion. Case 2 The second patient (a male), who had also undergone neck surgery (unilateral dissection), was intubated with difficulty using a Fasttrach (intubating laryngeal mask airway) because of anatomical abnormalities, which developed as a result of previous operations and radiotherapy. He was extubated 4 hours after the operation in accordance with the criteria mentioned above. However, he required emergency reintubation 18 hours after extubation because he suffered acute respiratory distress following aspiration and bronchospasm. We found out from the history obtained from his relatives that the patient had already had a swallowing disorder before the operation and suffered from aspiration. Thus, we prolonged the presence of the PAEC. Reintubation of this hypoxic patient was urgently achieved over the PAEC with the assistance of a laryngoscope using a 7.5 mm ETT under sedation and neuromuscular relaxation. During laryngoscopic observation we could not see the glottis. In this patient, a surgical tracheotomy was performed later because of recurrent aspiration and the need for tracheal suction. Case 3 This patient (a female) was admitted to the ICU after she operation for maxillofacial trauma. She had been intubated nasally by direct laryngoscopy using a Magill forceps; she could not open her mouth after the operation because of inter-maxillary fixation (Fig. 2 ). Six hours after extubation her arterial CO 2 pressure increased, and she became confused as a results of hypoxaemia. She was reintubated nasally with a 7 mm ETT over the PAEC, with intravenous midazolam 0.05 mg/kg and fentanyl 1 μg/kg, without cutting the archbar. She was extubated with the use of the PAEC 2 days after her reintubation, and the PAEC was left in place for 8 hours. She did not need intubation again after the PAEC had been removed. Case 4 The fourth patient, a male, underwent maxillofacial reconstructive surgery for cancer after he had been intubated nasally over a flexible bronchoscope because of anatomical abnormalities in the oral route. He became hypoxic 4 hours after his extubation and required immediate reintubation. A serious pharyngeo-laryngeal oedema was thought to be the reason for hypoxia. His reintubation was easily achieved over the PAEC, with intravenous midazolam 0.05 mg/kg. He was extubated with the use of the PAEC 3 days after reintubation, and the PAEC was left in place for 6 hours. He did not require intubation again after removal of the PAEC. Discussion During the perioperative period, serious respiratory events due to inadequate airway management can develop, which can cause severe brain damage or death. Rosenstock and colleagues [ 4 ] reported that 60 of 284 complaints filed at the National Board of Patients' Complaints in Denmark over a period of 4 years were associated with perioperative respiratory complications, 50% of which resulted in death. Adverse outcomes associated with respiratory events constituted the single largest class of injury in the American Society of Anesthesiology Closed Claims Study (522 of 1541 cases; 34%). Death or brain damage occurred in 85% of these cases. Three mechanisms of injury accounted for three-quarters of the adverse respiratory events: inadequate ventilation (38%), oesophageal intubation (18%) and difficult tracheal intubation (17%) [ 5 ]. In previous studies, reintubation rates of 5–19% have been reported in surgical ICU patients [ 6 - 8 ]. In our study, 11% of the patients required reintubation because of surgical bleeding, pharyngo-laryngeal oedema, aspiration, and inability to manage secretions. The reintubation risk of our study patients was higher than general ICU patients because they had high risks in terms of airway obstruction due to surgical manipulation. Patients who are expected to have a difficult airway may remain intubated longer than necessary, simply for fear of the inability to reintubate. Before the use of the PAEC in our clinic, we usually restricted extubation of patients who had undergone maxillofacial surgery and were at risk of difficult reintubation to the daytime, when experienced physicians were available, rather than during the night, to provide safer conditions. Prolonged tracheal intubation not only increases the risk of complications but is also expensive because it requires respiratory therapy and more extensive monitoring [ 9 ]. The PAEC is a long, flexible and hollow tube designed to facilitate the exchange of an in situ ETT. The primary use of the PAEC (adult size, 16–18 F) has been as a tube exchanger in the critical care setting. It has been also used before the extubation of patients with a known difficult airway [ 10 ]. In the study of Loudermilk and colleagues [ 2 ], the use of the PAEC in 40 patients with risk factors for difficult reintubation, including a history of previous difficult intubation, airway edema secondary to surgical manipulation or volume resuscitation, morbid obesity, and an immobilized or unstable cervical spine, was well described. They reported that 3 of 40 patients (8%) had been easily reintubated with the use of PAEC, which is a reintubation rate similar to our results. Although our findings are similar to those in the study of Loudermilk, our study population consisted of a specific surgery group and we used PAEC as a routine procedure in this group without considering whether the patients had previously been intubated with difficulty. Various methods have been used to facilitate the reintubation of these patients such as a fibre-optic bronchoscope [ 11 ], rigid ETT guides [ 12 ] and retrograde intubation. When all of these methods fail, an urgent cricothyroidotomy or tracheotomy may be the only solution. The PAEC offers several advantages over these alternatives: first, it provides a method for the continuous administration of oxygen; second, it can be used as a stylet for tracheal reintubation; and third, it provides a method of ventilating the patient (jet ventilation) [ 13 ]. In patients whose reintubation was considered a risk and who were known to present difficult tracheal reintubation, elective tracheotomy has even been performed in many institutions [ 2 ]. Besides, there have been many cases reported who have undergone tracheotomy because of airway obstruction or other respiratory pathologies after neck surgery [ 14 , 15 ]. Intraoperative tracheotomy is a safe route to secure the airway in the postoperative period in patients undergoing maxillofacial or major neck surgery. However, tracheotomy is a considerably invasive method and can lead to serious complications including bleeding, pneumothorax, infection and tracheal stenosis. Furthermore, only about 10% of the patients undergoing maxillofacial or neck surgery require reintubation after their operations, and most of these patients can be extubated later. This means that performing the tracheotomy routinely is not necessary in most of these cases. However, sometimes tracheotomy can be unavoidable in a selected group, especially when the patients are expected to need prolonged mechanical ventilation or are at great risk of reintubation because of severe airway obstruction. Thus, both methods can be considered depending on patient characteristics. In addition to the operative factors, the patients should be meticulously evaluated before the operation in terms of respiratory capacity, neurological status and co-morbid factors. However, there are no strict criteria for a decision on tracheotomy or a trial extubation. For example, our case 2 would have benefited from an intraoperatively performed tracheotomy. Fortunately, we were able to reintubate this patient easily over the PAEC, and then decide to perform the tracheotomy. Although the PAEC is rigid enough to facilitate tracheal reintubation, not all patients' tracheas may be easily reintubated. Forceful insertion of the ETT should be avoided to minimize trauma to vital airway structures and to avoid kinking the PAEC. Direct laryngoscopy may also relieve the obstruction and identify its cause. We also used the assistance of the laryngoscope during the reintubation of two patients over the PAEC both to facilitate the intubation and to evaluate the anatomical structure of the upper airway with regard to the possibility of laryngoscopy. Gentle rotation of the ETT while trying to insert it may release the tip [ 16 ]. The PAEC should never be inserted against a resistance. Although the tip of the PAEC is rounded and blunt, perforations of the tracheo-bronchial tree during the insertion of these catheters have been reported [ 17 , 18 ]. In a study of patients requiring tracheal reintubation, 87% (34 of 39) required reintubation within the first 4 hours after extubation [ 19 ]. In our series, one patient required reintubation 2 h after extubation, two reintubations occurred within 6 h and the other 18 hours after extubation. This finding shows that the need for reintubation later than 4 hours after extubation is not rare. As it is impossible to know at what time patients may develop respiratory distress, the timing of removal of the PAEC can be decided only on an individual basis. We have no data to determine the optimal period for which the PAEC should be left in place. Potential complications of the prolonged use of PAEC are airway trauma and aspiration caused by incomplete glottal closure. One of our patients who underwent neck surgery and required reintubation after 18 hours of extubation aspirated before the reintubation, but this patient had already had swallowing dysfunction due to radiotherapy before the operation. We therefore considered that the aspiration was not associated with PAEC only, although it could have contributed to the development of aspiration. Besides, the presence of the PAEC in the trachea can cause the retention of tracheal secretion by inhibiting effective coughing, especially in patients with chronic pulmonary disease, smokers, or patients who stayed immobile for a long time before surgery. In these conditions, the PAEC should be left in place for as short a duration as possible. Conclusion The routine use of a PAEC in patients who have undergone maxillofacial or major neck surgery facilitates reintubation when necessary, and can be a life-saving method. It allows a safer trial of tracheal extubation and therefore can shorten the duration of intubation. We suggest that after these surgical procedures a PAEC be used routinely before tracheal extubation because it is difficult to predict which patients will require reintubation. Key messages • The PAEC is a long, flexable and hollow tube designed to facilitate the exchange of an in-situ endotracheal tube. • The routine use of the PAEC in patients who underwent maxillofacial or major neck surgery facilitates the reintubation when necessary, and can be a life-saving method. Competing interests The authors declare that they have no competing interests. Abbreviations ETT = endotracheal tube; ICU = intensive care unit; PAEC = pediatric airway exchange catheter

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1065058

The rules of the game: interprofessional collaboration on the intensive care unit team

Background The intensive care unit (ICU) is a nexus for interspecialty and interdisciplinary tensions because of its pivotal role in the care of the hospital's most critically ill patients and in the management of critical care resources. In an environment charged with temporal, financial and professional tensions, learning how to get results collaboratively is a critical aspect of professional competence. This study explored how team members in the ICU interact to achieve daily clinical goals, delineate professional boundaries and negotiate complex systems issues. Methods Seven 1-hour focus groups were conducted with ICU team members in two hospitals. Participants consisted of four nursing groups ( n = 27), two resident groups ( n = 6) and one intensivist group ( n = 4). Interviews were audio-recorded, anonymized and transcribed. With the use of a standard qualitative approach, transcripts were analyzed iteratively for recurrent themes by four researchers. Results Team members articulated their perceptions of the mechanisms by which team collaboration was achieved or undermined in a complex and high-pressure context. Two mechanisms were recurrently described: the perception of 'ownership' and the process of 'trade'. Analysis of these mechanisms reveals how power is commodified, possessed and exchanged as team members negotiate their daily needs and goals with one another. Conclusion Our data provide a non-idealized depiction of how health care professionals function on a team so as to meet both individual and collective goals. We contend that the concept of 'team' must move beyond the rhetoric of 'cooperation' and towards a more authentic depiction of the skills and strategies required to function in the competitive setting of the interprofessional health care team.

Introduction Interprofessional tensions can threaten the delivery of quality health care in a hospital setting. Such tensions have been documented in several clinical domains including internal medicine [ 1 - 3 ], pediatric wards [ 4 , 5 ], the operating room [ 6 - 8 ] and the intensive care unit (ICU) [ 9 ]. The ICU in particular is a nexus for interspecialty tensions because of its pivotal role in the care of the hospital's most critically ill patients and in the management of critical care resources. Within the hospital community, the ICU exists at the high-stakes intersection of emergency, surgery, internal medicine and palliative care, an intersection where the patient care resources are expensive, in scarce supply and a source of intense competition. Repeated calls have been made for improved collaboration, communication, congruence and equity within health care teams as ways of improving quality of care and protecting patient safety. Current notions of team-building advocate increasing flexibility in team structure, abolishing hierarchies and cultivating shared decision making [ 10 - 16 ]. Although these are important concepts, they can reflect a naive sense of the team as a unified entity rather than as a collection of individuals with distinct professional identities based on different models of care, skills, economic circumstances and political agendas. To foster optimal team function, we first need to understand better the forces governing the interactions between professions (for example, nurses and physicians) and between specialties (for example, the ICU team and external consultants) as they work together in an environment charged with professional, temporal and financial tensions. Previous work by our research group has described team dynamics in the ICU [ 9 ]. We found that the level of collaboration or conflict within the ICU team, and between the ICU and other specialties, fluctuated on the basis of six key catalysts: authority, education, patient needs, knowledge, resources and time. These findings provided insight into the divisive forces present even in high-functioning teams, and alerted us to the strategies that team members enact as they seek to balance individual needs with team goals. We also found that 'team', in the ICU, is not a unified body but rather is a complex and fluid entity composed of core and expanded groups. Membership in these groups is continually negotiated on the basis of relative professional roles, immediate needs and tacit 'rules of play'. In essence, to become empowered actors in the ICU, team members must progress beyond learning procedural steps to understanding the rules of the game: who has power on the team, how is that power commodified, how is it accessed, and in what circumstances is it applied? Understanding these rules can be the difference between knowing how to make something happen in principle (for example, ordering an X-ray) and being able to make it happen in practice (for example, getting an X-ray now ). Understanding the rules of the game is also essential if team members are to move beyond thinking as individuals to begin thinking as part of a team. The purpose of this study was to describe these tacit 'rules of the game'. We sought to determine how power is commodified and exchanged by ICU team members in their daily interactions as they work to achieve clinical goals, delineate professional boundaries, and problem solve around complex system issues. Methods In a follow-up to 4 months of ethnographic non-participant observations (phase 1, detailed methods and results previously reported [ 9 ]), seven 1-hour focus groups were conducted with ICU team members in two urban teaching hospitals in Toronto, Canada. Two hospitals were included because the participating intensivists and residents divide their time between the sites and because differences in the settings (for example, case types and case loads, nurse staffing patterns and hospital cultures) might affect team communication and collaboration. A semi-structured question script was derived to pursue recurrent patterns identified in the observational data. Participants consisted of a sample of four nursing focus groups ( n = 27), two resident groups ( n = 6 or 10 available individuals) and one intensivist group ( n = 4 of 8 available individuals). Residents and intensivists constituted a convenience sample of individuals who were able to accommodate the time for the focus group discussion. Within the nursing group, purposeful sampling was used to ensure some range in years of ICU experience and age in this population [ 17 ]. The sample was selected through consultation with the nurse managers of the units. The number of focus groups conducted was determined through theoretical sampling, in which data collection occurred alongside preliminary analysis, and collection ceased when no new themes were arising from the focus group discussions [ 18 ]. The study received institutional ethics approval, and informed consent was obtained from all participants. Focus group interviews were audio-recorded, anonymized and transcribed with standard linguistic conventions to yield about 140 pages of transcription for analysis. In the grounded theory tradition [ 19 ], transcripts were read iteratively by four researchers and were analyzed for emergent themes as well as for the themes identified in the analysis of the observational data. Both open coding (identification of primary themes) and axial coding (analysis of relationships among themes) were conducted. The combined expertise of the four analysts was essential to the coding process: one researcher was an intensivist experienced in qualitative research, one was an expert in team discourse, and the remaining two had conducted the observations in the first phase of the study. Emergent themes were revised and refined through the constant comparison of instances from the data set both by individual researchers and in a series of weekly 2-hour meetings during which the analysts compared interpretive memos and discussed relationships between categories. Discrepancies were given particular attention to ensure the validity of the analysis: they were considered by consulting specific instances in the transcripts, discussing their relationship to established themes, and reaching consensus as a group [ 20 ]. Results The phase 1 observation data provided insight into three areas: the shifting notion of team, the fluctuating levels of collaboration and tension on the team, and the catalysts underlying such fluctuations (previously reported) [ 9 ]. Thematic analysis of the focus group data extended our understanding of these three areas, in particular revealing team members' perceptions of the mechanisms by which collaboration is achieved or undermined. Two dominant mechanisms were recurrently described and were categorized in our analysis as 'the perception of ownership' and 'the process of trade'. The findings reported here describe these mechanisms as revealed by the focus group data and supported by the observational data; implications for team collaboration and conflict are emphasized. Perception of ownership This category included references to team members' perceived ownership of valued constructs or commodities, including specialized knowledge, technical skills, equipment, clinical territory and even the patient himself or herself. These constructs and commodities formed the basis of negotiation or exchange during interprofessional interactions. The title of 'ownership' rather than the more traditional concept of 'role' was selected to reflect the participants' emphasis on possession. Ownership was perceived as both collective (for example, ownership by the ICU team) and individual (for example, ownership by a nurse or by nursing as a profession). Shared perception of collective ownership was portrayed by participants as the foundation of the group's identity. It promoted collaboration between members of the ICU team and was often established by contrast with those outside the core team such as surgeons, internists, or nurses from the wards. For example, nurses explained the team's collective ownership of the patient in contrast to interlopers from outside the unit: ' We don't negotiate in the ICU because we are ultimately responsible for the patient, so there is no negotiating when you are in charge of that patient ' (Nurse FG1). Individual ownership was also a dominant issue and included instances where team members recognized their own or others' possession of valued commodities. For instance, respiratory therapists acted in a proprietary manner regarding the ventilator, and this ownership was recognized and respected by other team members. One resident acknowledged that: ' The RTs' role is probably essential, because, uh, as a medicine resident, we don't know much about the ventilators ... we don't have the time to learn the specifics that they know, so they contribute in areas that we– –we can't... ' (Resident FG1). In cases like this, the recognition of others' possession of knowledge and skills is part of the smooth collaborative functioning of the team. However, individual ownership can also create interdisciplinary tension when team members feel that their ownership of particular knowledge and skills is not recognized: Nurse: ' And we're the ones who do keep track because we're there 24 hours a day. It'll be like: "Well order a blood culture", well we did one just yesterday. Or "Order a thyroid test." They just did them 2 days ago. You know? ' (Nurse FG4). In both observations and focus group data, the designation of ownership was a complex mechanism and frequently a site of tension. In some cases, the allocation of ownership was defended by a particular group and in others, chafed at: Intensivist: ' At the end of the day the staff [intensivist] is the bottom line. I mean for better or for worse. I am not necessarily saying that it's the right thing but ... the amount of control you relinquish is really wholly dependent on how strong you feel these other members of the team are ' (Intensivist FG1). Nurse (describing a situation at morning rounds): 'The staff intensivist asked the nurse, are there any issues, any concerns for the patient going to the floor?" The nurse started up, and she was talking about blood pressure issues. The staff intensivist interrupts to say, "Oh well, that's a medical issue. No, I mean specifically a nursing issue. So shot her down immediately ' (Nurse FG2). The staff intensivist in the first example asserts his ultimate responsibility for patient care. In the latter example, however, the knowledge designated as nursing territory by the intensivist was perceived by the nurse as inappropriately constrained, signaling a conflict between the two professional domains. Although the recognition of others' ownership of commodities frequently facilitated smooth team function, it also served as a provocation for usurpation and theft. For instance, nurses reported situations in which residents sought nursing knowledge but later portrayed that knowledge as their own: ' They rely on our notes and our talking to them in the morning to give them the physical assessment of the patient but then they totally disregard you when it comes to rounds as part of the team as though they've done this assessment themselves and nothing you say is worthwhile ' (Nurse FG4). Participants' discussions of ownership illustrated key problems on an interprofessional team, problems that revolve around respecting the interface between individual and collective knowledge and the balance between individual and collective responsibility. Process of trade This second category captured instances in which team members traded valued commodities as they negotiated their collaborative work. Such trade commonly involved concrete, physical commodities, including equipment and resources, and abstract, social commodities, including respect, goodwill and knowledge. The trade of scarce physical resources was a catalyst for tension on the team. In many cases, this tension was amplified by its recurrence and by the infuriating smallness of some of the issues under debate: Nurse: ' I'll give you an example: I need a pump because my patient's blood pressure is dropping and some nurse is hoarding all of them and saying she needs it too. And I say, "I don't think you need it", so I just yank it out and get it because I know this is just a regular drip ' (Nurse FG1). Trade in such mundane resources was a commonplace ritual as team members negotiated to locate the items required for everyday patient care. In other cases, tension was amplified by the critical importance of the resources. Trade in beds, for example, was fraught with tension, particularly for trainees: Resident: ' There is always a shortage of nurses and they're always closing beds and we [trainees] sort of have to bear the brunt ... and get caught in a bed war ' (Resident FG1). Nurse: ' [There was] a new resident on call and the ER calls him, he accepts the patient. And then after he accepts the patient he comes to me to say, "Well, we have a patient", and I say, "No, you don't do that. You ask me first, do we have any beds?" Things like that. They're learning the rules ' (Nurse FG2). As the latter example illustrates, the trade in physical resources is governed by implicit, social rules, such as who can authorize a trade. Trainees frequently had difficulty in recognizing and negotiating these implicit rules. Alongside the trade of concrete resources was trade in more abstract commodities. For the nursing group, the most dominant currency for trade was 'respect', which they described themselves expecting in return for information, knowledge, resources and goodwill. The failure of other team members to present the currency of respect was often met with revenge strategies in the form of an embargo of trade. For instance, a nurse might refrain from offering her knowledge if appropriate respect was not proffered first: Nurse: ' [Consultants to the ICU should] introduce themselves, to say what service they're from, and to ask some questions about the patient as you're the primary caregiver. And ... then they would learn so much more and it would save a lot of time, instead of digging through all this information ... they're flipping, flipping, trying to find bloodwork, but they're not asking me, so I'm not going to help, you know? You find it yourself ' (Nurse FG2). Such trade of knowledge for goodwill occurred not only among team members but also between the ICU team and consulting teams. This critical sort of trade was recognized and discussed by all team members in the study. Failure to engage in such trade could mean that 'a good team approach was lost' (Nurse FG2). It could also be seriously detrimental to an individual team member's success. For instance, residents expressed that ' Your name can be ruined or made on one ... encounter, so ... you have to be very careful, because if you create one enemy you can end up having a tough time with a lot of people, and if they love you, then they love you mostly for whatever the time that you're here ... so it's a bit of a social game; you have to be careful ' (Resident FG1). The process of trade was a constant and at times difficult social game with potentially long-term consequences. The constancy of trade caused it to be a source of accumulated tension and perceived historical injustices, with a single trade event causing a ripple effect that might impact other patients, other team members, other hospital services, or other events later in time. For instance, based on experience, one nurse asserted that ' When you want to transfer a patient in a hurry there will be an obstruction there ... you know there will be excuses. You know sometimes we feel like they're [ward nurses] prolonging it ... so I say, "Well, I'll call housekeeping for you." Of course they don't like that... ' (Nurse FG1). The environmental tensions endemic to the ICU served to make the successful negotiation of trade more difficult but also more essential. As one staff intensivist put it: '... we deal with a lot of conflict and you have to learn how to control yourself and how to become adept at conflict resolution. And not through intimidation and humiliation of the colleagues you have but honestly listening to them and trying to understand where they are coming from and trying to be respectful of them although ... that is tough sometimes when you are not feeling particularly patient or magnanimous towards these folks that you are talking with and, you know, you are tired, you're sleep deprived ... and you may be getting hassled from all sorts of people because of resource issues ' (Intensivist FG1). Discussion Our data depict team collaboration in a decidedly non-romanticized manner. The notion of team collaboration as rooted in the ownership and trade of commodities presents a stark contrast – and a strong challenge – to the established literature on creating medical teams, which emphasizes mutual support and shared goals, and minimizes competition and contest. What our participants describe as underlying 'rules' in the daily negotiation of individual and team activity, the literature has tended to portray as 'barriers' to teamwork [ 16 ]. Recent ethnographic studies of health professional teams suggest that the traditional conception of a stable, unified team does not account for the daily workings of teams in complex environments [ 6 - 9 ]. Further, this current research should caution us that adherence to the traditional ideal of 'team' may, in fact, constrain us from recognizing and promoting the functional mechanisms of group effort in the health care domain. As our results demonstrate, the forces of ownership and trade have a central role in the daily negotiations that constitute teamwork in the ICU setting. When these forces are ignored – that is, perceived ownership is not attended to, or one commodity is not offered in trade for another – tensions accumulate and collaboration becomes sluggish. When these forces are accommodated – for example, competition for ownership of resources is anticipated, or requests are accompanied by offers of trade – the team members navigate their competing interests more smoothly to act effectively together. From a sociological perspective, this is common sense. There are sound theoretical reasons for these rituals of ownership and trade, the most basic of which is that the 'team' is not a unified entity but rather a compilation of individuals with distinct professional identities: intensivist, nurse, respiratory therapist, resident, and so on. These professional identities are based in distinct models of care, different skill sets, diverse economic circumstances and competitive political agendas. A useful way of theorizing the construct of professional identity, particularly when diverse professions come in contact with one another, can be found in the theory of social structuration [ 21 ]. In this theory, professions or organizations are conceptualized as social systems, in which each professional's role is determined by its position in relation to others and by its access to certain commodities. These commodities include access to material resources ('economic capital'), access to levels of information ('cultural capital') and access to social connections and acknowledged forms of expertise ('social capital'). Structuration theory is especially useful because it recognizes that individuals both within a profession (such as nursing) and between professions (such as nursing and critical care medicine) are in the constant process of attempting to distinguish themselves and their profession and thus acquire more 'capital' so as to promote their ability to act ('agency') [ 22 ]. This notion of a profession and of an interprofessional team as a contested space is important, as it moves beyond a simplified notion of 'community' as a group with shared values [ 23 , 24 ] and allows us to theorize about important tensions in the formation of professional identity and the interaction between multiple professions. Acknowledging these tensions enables us to understand the way in which teams sustain the delicate balance between achieving a shared goal and competing for agency and status in the interprofessional setting. The forces of ownership and trade are products of the contested relations on an interprofessional team. The point is not to stamp out these forces or to overcome them, but rather to articulate their role in team collaboration, so that they can be more strategically harnessed by team members and, as a consequence, smooth team functioning can be promoted. Handled adeptly, these forces allow members of a team to get necessary clinical work done, even in the chaos of competing ambitions and interests that is the ICU team. As one nurse put it: 'It may be construed that you are demanding, but then if you don't demand sometimes you don't get it; it's just a matter of strategy' (Nurse FG1). Limitations This study is constrained by the design decisions underpinning it. Findings may reflect the attitudes of a subset of ICU team members, for instance those more interested in exploring these topics. Generalizability is not the goal of grounded theory research, which seeks instead to produce rich descriptions and theoretical explanations of situated processes. However, the explanatory utility of these findings may be explored and enhanced in future research in different centers or other interprofessional health care team contexts. Conclusions It is time that our understanding of team collaboration moved beyond the rhetoric of cooperation, and towards a more authentic depiction of the skills required to function in the competitive setting of the interprofessional health care team. Our intention is not to suggest a new rhetoric (of economics), but rather to shift our attention from idealized or abstracted depictions of teamwork, towards a grounded understanding of how collaboration is accomplished in daily practice. Knowing about perceptions of ownership, valued commodities and the rules of trade allows team members to shape outcomes and persuade people, to anticipate reactions and deflect obstructions, and to achieve individual goals while maintaining team cohesion. Efforts to improve teamwork must reflect such authentic, everyday 'rules of the game' if they are to affect how work gets done on health care teams in complex settings such as the ICU. These findings suggest educational implications relevant both to trainees and practising intensivists. In most training programs, professionalism and collaboration are part of an implicit, ad hoc curriculum largely consisting of role modeling and trial and error. As medical schools respond to recent calls to ensure competence in domains such as communication and collaboration [ 25 ], an understanding of authentic collaborative practice is essential to inform evidence-based curricula. For practising intensivists who may experience tension and difficulty in some team situations, understanding the rules of the game may assist them to analyze and improve their collaborative practice and, it is hoped, to improve the quality of care they provide to critically ill patients. Contributors All authors contributed to the design, conduct, analysis and interpretation of the research reported. LL and LH were Co-Principal Investigators and led the conceptual design of the study. SE and CE assisted with data collection and analysis, and with manuscript preparation. Key messages • In the daily negotiations that constitute inter-professional ICU teamwork, the ownership and trade of valued commodities play a central role. • When ownership and trade are appreciated and handled well, team members are able to anticipate reactions, deflect obstructions, and achieve individual goals while maintaining team cohesion. • Articulation of such authentic "rules of the game" is essential to the development of evidence-based curricula in collaborative practice. Competing interests None declared. Abbreviations ICU = intensive care unit.

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1065059

An international sepsis survey: a study of doctors' knowledge and perception about sepsis

Background To be able to diagnose and treat sepsis better it is important not only to improve the knowledge about definitions and pathophysiology, but also to gain more insight into specialists' perception of, and attitude towards, the current diagnosis and treatment of sepsis. Methods The study was conducted as a prospective, international survey by structured telephone interview. The subjects were intensive care physicians and other specialist physicians caring for intensive care unit (ICU) patients. Results The 1058 physicians who were interviewed (including 529 intensivists) agreed that sepsis is a leading cause of death on the ICU and that the incidence of sepsis is increasing, but that the symptoms of sepsis can easily be misattributed to other conditions. Physicians were concerned that this could lead to under-reporting of sepsis. Two-thirds (67%) were concerned that a common definition is lacking and 83% said it is likely that sepsis is frequently missed. Not more than 17% agreed on any one definition. Conclusion There is a general awareness about the inadequacy of the current definitions of sepsis. Physicians caring for patients with sepsis recognise the difficulty of defining and diagnosing sepsis and are aware that they miss the diagnosis frequently.

Introduction Sepsis is a major cause of death worldwide, with a large impact on mortality in the intensive care unit (ICU). It has been estimated that every day about 1400 patients die in ICUs as a result of sepsis [ 1 ]. Recent progress in sepsis research has been able to improve the knowledge about the basic pathophysiological processes of sepsis. However, in daily ICU practice it remains difficult to identify and treat sepsis, and its related conditions, adequately. Concerns remain about the lack of consistent definitions and understanding about sepsis among the global medical community [ 2 , 3 ]. The American College of Chest Physicians and the Society of Critical Care Medicine (ACCP/SCCM) proposed a definition of sepsis and related syndromes in 1991 [ 4 ]. Although these definitions were based on expert opinion, the recommendations have not found unequivocal acceptance. However, these definitions have since been used for research purposes investigating new therapeutic modalities, in essentially all intervention trials. To be able to diagnose and treat sepsis better it is important not only to improve knowledge about definitions and pathophysiology, but also to gain more insight into specialists' perception of, and attitude towards, the current diagnosis and treatment of sepsis. This knowledge is important for the development of strategies to improve consensus in defining sepsis criteria among the intensive care society. Moreover, the introduction of intensivists supporting critical care units has been shown to be associated with improved survival of septic patients [ 5 , 6 ]. Agreement among intensivists, as separate clinical specialists, in terms of their diagnosis of sepsis therefore also needs to be clarified. Our hypothesis was that although there is good awareness among physicians involved in treating septic patients, a fragmented view of the definitions of sepsis is present. To investigate these hypotheses an international survey was conducted among intensivists and other specialists involved in the diagnosis and treatment of sepsis. Materials and methods In an international survey 1058 physicians were interviewed for this study; they were interviewed after a random selection of 1100 physicians in Europe and the USA. Of these, 756 physicians were interviewed in France ( n = 150), Germany ( n = 155), Italy ( n = 150), Spain ( n = 151) and the UK ( n = 150). A further 302 physicians were interviewed in the USA. In each country equal numbers of intensive care and other specialists were interviewed. The specialist physicians included anaesthesiologists, cardiologists, endocrinologists, internists, nephrologists, pulmonologists, surgeons and emergency room physicians. The intensivists had to spend 50% or more of their time treating adults in the ICU, had to treat on average five or more ICU patients per month, had to treat two or more adult sepsis patients per month on average, and had to have worked for 2 years or more in the ICU. Otherwise they were classified as other physicians. The other specialists were also involved in the treatment of patients with sepsis, although on a less regular basis (fewer patients). They had to spend 10% or less of their time treating adult patients in the ICU and had to have been in practice for at least 2 years. It was intended that physicians spending between 10% and 50% of their time in the ICU should be excluded, but no physicians fulfilled this exclusion criterion. The study was conducted from November to December 2000. A recent study has shown a reduced mortality in patients with septic shock [ 7 ]. However, it was performed before the results of the present study were available. The survey was performed by telephone interview using trained staff of Yankelovich Partners. We list the questions asked in additional file 1 . All questions were grouped into three categories based on a model describing behaviour framework [ 8 ]. To implement sepsis definition guidelines effectively, first the physician's awareness of the problem should be raised, then agreement on the problem should be reached and finally the ability to implement the definition guidelines should be present. Statistics The data for this study are presented as means ± SEM or as percentages. Data were analysed with Student's t -test or χ 2 testing. P < 0.05 was considered statistically significant. The margin of error for the total group of physicians in this study was 3.0%, on the basis of the combined error values of all questions combined. Results Respondent profile Most physicians (83%) were male with an average age of 44.2 ± 0.3 years. The majority (57%) of these physicians were working in a non-teaching hospital. There was no difference between the intensivists interviewed and the other physicians with respect to gender, age distribution, percentage working in teaching hospitals, and percentage of practice based in hospital (Table 1 ). The intensivists worked on an average 77.2 ± 0.95% of their time in the ICU. The number of adult patients treated in the ICU per month by the intensivists was 60 ± 3; of these 16.5 ± 0.9 were septic patients. The intensivists had worked for 11.6 ± 0.3 years after residency on the ICU. Of the other physicians, interviewed 120 (23%) were anaesthesiologists, 26 (5%) cardiologists, 26 (5%) endocrinologists, 83 (16%) internists, 18 (3%) nephrologists, 48 (9%) pulmonologists, 32 (6%) surgeons, 119 (23%) emergency room physicians, and 57 (11%) oncologists. These physicians worked 4.0 ± 0.3% of the time on the ICU and had been 13.5 ± 0.4 years in practice since residency. Awareness of the problem of sepsis Three-quarters (767 of 1058) of all interviewed physicians agreed (strongly or somewhat) that sepsis is a leading cause of mortality compared with other conditions in intensive care. Of the intensivists, 78% considered sepsis as the leading cause in comparison with 67% of other physicians ( P < 0.0001). Nine in ten (934 of 1058) physicians agreed (strongly or somewhat) that sepsis is a significant financial burden on the health care system in their country. Among all physicians, 88% (937 of 1058) considered sepsis among the most challenging conditions that a doctor can treat. Two in five physicians (420 of 1058) had the impression that the incidence rate of sepsis has increased 'steadily' to 'dramatically' over the past 5 years, whereas 48% said that it remains stable. Two-thirds (285 of 420) thought that this increase is either 'extremely serious' or 'very serious'. Of the physicians surveyed, 77% reported the following major factors involved in this increase: an increased resistance of bacteria to antibiotics, an increased number of immuno-compromised patients, and a higher survival chance of post-surgical patients and patients with serious pathology. A majority (656 of 1058, 62%) of physicians believed that their definition of sepsis is commonly accepted within their speciality. More than four in five (905 of 1058, 86%) physicians agreed (strongly or somewhat) that the symptoms of sepsis can easily be misattributed to other conditions. There was concern (ranging from 'somewhat' to 'extremely concerned') about the lack of a common definition for sepsis in 67% (708 of 1058) of the physicians. Of the physicians who were concerned about the lack of a common definition, 83% (199 of 708) stated that it is at least somewhat likely that the diagnosis of sepsis is missed. This figure was 53% (29 of 350) for the physicians who were not concerned about the lack of a common definition for sepsis. Although physicians are divided over whether the lack of a common definition for sepsis hinders proper diagnosis, they are not divided over whether a common definition would be a significant step towards better treatment. Agreement on definitions of sepsis In general, physicians' definitions of sepsis were fragmented. When defining sepsis, only 22% (114 of 529) of the intensivists and 5% (26 of 529) of the other physicians gave the definition of the ACCP/SCCM consensus statement ( P < 0.0001). Fewer than one-fifth (17%) of the physicians agreed on any one definition for sepsis, and six different definitions were mentioned by at least 1 in 10 physicians. This was not different between intensivists and other physicians. Moreover, physicians were divided as to whether sepsis is a systemic response (46%, 490 of 1058) as opposed to a syndrome (36%, 380 of 1058). One in ten physicians (103 of 1058), of both the intensivists and the other physicians, said that sepsis is a disease. Among physicians, 71% (751 of 1058) said that fever is a sign or symptom that must be present to diagnose sepsis rather than any other factor. Aside from fever, no one symptom was listed by a majority of physicians as a sign or symptom that must be present to diagnose sepsis. Tachycardia was only cited by 29%, leukocytosis or leukopenia by 20%, hypothermia by 14%, and tachypnoea by 9% of physicians. Ability to diagnose sepsis and communicate about sepsis Four in five physicians (911 of 1058) agreed (strongly or somewhat) that patients need better monitoring to diagnose sepsis at the earliest possible stage. In addition, 84% (890 of 1058) agreed (strongly or somewhat) that patients are often treated too late to reverse the onset of sepsis. According to the physicians, 46% of sepsis deaths are recorded as death by other diseases rather than death by sepsis. Bacterial culture results ranked as the most effective method for diagnosing sepsis by physicians; 80% found bacterial cultures either 'extremely' or 'very effective'. The second most effective method for diagnosing sepsis was haemodynamic monitoring. A significantly greater percentage of intensivists (74%, 393 of 1058) than the other physicians (66%, 350 of 1058) ranked haemodynamic monitoring as either extremely or very effective ( P = 0.002) for diagnosing sepsis. Two-thirds (65%, 684 of 1058) of physicians agreed that a physical examination of symptoms is an effective method. When speaking to the patients' relatives, 81% (858 of 1058) of physicians agreed that communicating a diagnosis of sepsis to the families of patients with sepsis is difficult. Therefore, more than four in five (85%, 899 of 1058) physicians said that they describe sepsis to patients' relatives as a complication arising from an underlying condition, as opposed to 10% who said they describe the diagnosis as sepsis. Discussion In the present age of intensive care, sepsis remains responsible for a considerable number of deaths in critically ill patients. This disease has a major impact on both health care and society resources. Despite an increased understanding of sepsis, so far no information has been presented about physicians' perception and knowledge of sepsis. This international survey was therefore conducted among physicians involved in treating septic patients. One of the main findings of this study is that there is a general awareness of the importance and impact of sepsis among the physicians interviewed. A vast majority of physicians consider sepsis a leading cause of mortality. Moreover, the physicians agree that sepsis is a commonly encountered condition with an increasing incidence. Two recent reviews summarised the published studies on the incidence and mortality rates reported for sepsis. In a review by Brun-Buisson [ 9 ], 25% of patients on the ICU develop sepsis, with incidence rates varying from 45 in 1000 hospital admissions to 494 in 1000 ICU admissions. In a review by Matot, sepsis occurred with a mean frequency of 22.4% [ 1 ]. In both reviews a clear division between definitions of sepsis and severe sepsis or septic shock was used. In the review by Brun-Buisson an additional 10–15% of patients developed septic shock [ 9 ]. In practice, however, a majority of physicians agree that it is at least somewhat likely that the diagnosis of sepsis is being missed frequently. One of the remarkable findings of this study is the lack of agreement on the definition of sepsis. A new set of definitions was proposed by the consensus conference of the ACCP/SCCM in 1992 [ 4 ] to improve the bedside recognition of sepsis, to permit early intervention and to differentiate infectious from non-infectious conditions. However, only a small percentage of physicians report the ACCP/SCCM criteria for the definition of sepsis. Not more than one-fifth agree on any one definition. This is consistent with the fact that a majority of physicians were concerned that there is no common definition of sepsis and a large proportion of physicians (for non-intensive care physicians even 41%) believe that other physicians within their speciality define sepsis differently from themselves. This perceived lack of a common definition might also explain why a significant number of physicians believe that sepsis is missed as a diagnosis. Indeed, the recommendations from the International Sepsis Forum recognise that in the past different definitions of sepsis were used interchangeably, which led to confusion [ 10 ]. When looking at the precise criteria that must be present according to the physicians interviewed, a wide variety of signs and symptoms were given. The one factor most frequently quoted was fever; the second most frequent answer was hypotension. This is of interest, given the fact that intensivists, in this survey, considered themselves extremely knowledgeable about the definition of sepsis and in the distinction between sepsis, severe sepsis and septic shock. Both the use of only one criterion and the use of hypotension are not at all consistent with the consensus definitions established in 1992 [ 4 ]. This misunderstanding with regard to the consensus criteria is consistent with the perception, among most physicians surveyed, of a lack of clear definitions for sepsis. The lack of agreement on the definitions of sepsis criteria has an influence on the ability of physicians to diagnose and communicate about sepsis. The physicians in this survey were not content about the diagnostic tools they have for the diagnosis of sepsis. Most physicians agreed that better monitoring tools are needed to diagnose sepsis at the earliest possible time. Although a large percentage of physicians surveyed considered bacterial cultures and haemodynamic monitoring very effective for diagnosing sepsis, they also reported a high degree of interest in the investigation of other, more sensitive tools. Another aspect of this survey was the differences found between intensivists and other specialists with less involvement in ICU care, indicating a difference in patient numbers with sepsis. Recent studies investigated the effects of specialised ICU staffing on outcome [[ 5 , 6 , 11 ], 12]. The results of these studies suggested that the presence of intensive care physician staffing is associated with a decreased length of ICU stay and with decreased costs, complications and mortality. However, it remained relatively unclear whether the institution of specialised ICU staffing had its effects on agreement, awareness and ability to diagnose sepsis. This survey showed that in general the intensivist seems to be more aware of issues involved for critically ill patients with sepsis. More intensivists consider sepsis a leading cause of mortality and a significant financial burden on the health care system. Moreover, they more frequently have the impression that the incidence is increasing. However, although awareness seems to be higher in specialised ICU staff, agreement on the definitions of sepsis is just as scattered as with non-ICU specialists. As a consequence the ability of intensivists and other specialists to diagnose sepsis is more or less comparable. Moreover, the ability of physicians to communicate the diagnosis of sepsis to the patients' relatives is equally problematic. Two conclusions can be drawn from this survey, despite the limitations of a telephone survey. First, many doctors cannot define sepsis in accordance with the previously published consensus criteria. Second, sepsis is perceived as a leading cause of death in ICUs. The incidence of sepsis is high, and in addition physicians believe that the diagnosis of sepsis is often missed. This survey lends support to the idea that definitions of sepsis should be reviewed and that education is required, for both physicians and the public, for a better standardisation of clinicians' definition and diagnosis of sepsis. Key messages • The current awareness of physicians concerning the impact which sepsis has on resources is widespread. • Physicians are concerned that lack of agreement on the definitions of sepsis may lead to underestimating of the incidence of sepsis. • The lack of agreement on the definitions of sepsis criteria has its influence on the ability of the physicians to diagnose and communicate about sepsis. Competing interests The author(s) declare that they have no competing interests. Abbreviations ACCP = American College of Chest Physicians; ICU = intensive care unit; SCCM = Society of Critical Care Medicine. Supplementary Material Additional File 1 A PDF file containing a list of questions from the international sepsis survey. Click here for file

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1065063

Ventilator associated pneumonia: comparison between quantitative and qualitative cultures of tracheal aspirates

Introduction Deferred or inappropriate antibiotic treatment in ventilator-associated pneumonia (VAP) is associated with increased mortality, and clinical and radiological criteria are frequently employed to establish an early diagnosis. Culture results are used to confirm the clinical diagnosis and to adjust or sometimes withdraw antibiotic treatment. Tracheal aspirates have been shown to be useful for these purposes. Nonetheless, little is known about the usefulness of quantitative findings in tracheal secretions for diagnosing VAP. Methods To determine the value of quantification of bacterial colonies in tracheal aspirates for diagnosing VAP, we conducted a prospective follow-up study of 106 intensive care unit patients who were under ventilatory support. In total, the findings from 219 sequential weekly evaluations for VAP were examined. Clinical and radiological parameters were recorded and evaluated by three independent experts; a diagnosis of VAP required the agreement of at least two of the three experts. At the same time, cultures of tracheal aspirates were analyzed qualitatively and quantitatively (10 5 colony-forming units [cfu]/ml and 10 6 cfu/ml) Results Quantitative cultures of tracheal aspirates (10 5 cfu/ml and 10 6 cfu/ml) exhibited increased specificity (48% and 78%, respectively) over qualitative cultures (23%), but decreased sensitivity (26% and 65%, respectively) as compared with the qualitative findings (81%). Quantification did not improve the ability to predict a diagnosis of VAP. Conclusion Quantitative cultures of tracheal aspirates in selected critically ill patients have decreased sensitivity when compared with qualitative results, and they should not replace the latter to confirm a clinical diagnosis of VAP or to adjust antimicrobial therapy.

Introduction The incidence of nosocomial pneumonia in mechanically ventilated patients ranges from 9% to 68%, and mortality rates range from 33% to 71% [ 1 , 2 ]. In the EPIC (European Prevalence of Infection in the Intensive Care) study [ 3 ], ventilator-associated pneumonia (VAP) was the most frequent infection acquired in the intensive care unit (ICU), accounting for 45% of all infections in European ICUs. The diagnosis of VAP is a challenge for the clinician because the presentation is variable, and other causes of fever and chest infiltrates may occur in these patients. Clinical/radiological evaluations provide the only criteria that permit timely diagnosis. Early institution of adequate antibiotic therapy is associated with decreased mortality, at least in the more severely ill patients. Culture results are currently used to guide adjustment or withdrawal of antibiotic therapy rather than to decide whether to treat. The practice of changing therapy with culture results has resulted in reduced consumption of antibiotics. Conversely, studies have shown that over-treatment with antibiotics may select organisms such as Pseudomonas aeruginosa and Acinetobacter calcoaceticus [ 4 , 5 ]. The value of endotracheal aspirates for diagnosing VAP is controversial, but there is a growing body of evidence showing an important role for these cultures. Recent studies have consistently shown that outcome in VAP may not be influenced by whether cultures are obtained by bronchoscopy or from tracheal aspirates collected at the bedside. Furthermore, a cost-effectiveness analysis [ 6 ] strongly supported the employment of tracheal aspirates in the management of VAP. Although the use of tracheal aspirates in VAP management is increasing, there are few data regarding the usefulness of quantitative as opposed to qualitative cultures. Some studies [ 7 , 8 ] suggested that quantitative cultures should be used in order to avoid false-positive results, but little is known about the sensitivity and specificity of quantitative culture findings in severely ill patients who have previously received broad-spectrum antibiotics. We conducted a prospective follow up of severely ill patients in a general ICU with a high rate of antibiotic use in order to evaluate the value of quantification of bacterial colonies in tracheal aspirates for diagnosing VAP. Methods Study protocol This study was conducted between March 2000 and January 2001 in a 28 adult bed medical/surgical critical care unit at the Hospital Israelita Albert Einstein – a major referral tertiary care centre. The ethics committee of our institution granted approval for this investigation. During the study period, every Monday morning all patients under mechanical ventilation for at least 48 hours were examined to determine whether they had VAP by three well trained intensivists and a respiratory therapist. We chose to evaluate all ventilated patients irrespective of the presence of VAP because on Mondays we routinely perform surveillance cultures of tracheal aspirates (in a search for multidrug-resistant pathogens and to determine contact precautions for such situations). We also aimed to include both patients with and without VAP based on clinical and radiological criteria. The diagnosis of VAP was confirmed if there was agreement between two of the three physicians using clinical/radiological criteria. On the same day, the respiratory therapist also provided a description of the appearance (purulence) of the tracheal secretions. Endotracheal secretions were collected using a standard procedure and endotracheal aspirates samples were sent for qualitative and quantitative culture. The research team was blind to culture results, but the physicians were aware of the patients' antibiotic consumption when they were evaluated. Clinical characteristics were recorded at every evaluation (not just at enrolment in the study). Diagnosis of ventilator-associated pneumonia For the purposes of the present study, VAP was diagnosed when a patient on mechanical ventilation for at least 48 hours developed a new or progressive pulmonary infiltrate on the chest radiograph in association with at least two of the following findings: râles or dullness to percussion on chest examination; new onset of purulent sputum or change in sputum character; decrease of at least 10% in arterial oxygen tension/fractional inspired oxygen ratio; leucocytes in excess of 12,000/mm 3 or under 4000/mm 3 ; positive blood cultures or pleural effusion cultures; and axilar temperature greater than 37.8°C or under 36.0°C in the absence of antipyretic treatment (excluding another site of infection). Tracheobronchial aspirate samples and microbiological processing Tracheobronchial secretions were collected by the respiratory therapist, following specimen collection guidelines, after tracheal instillation of 5 ml saline. The specimens were sent to the laboratory and cultivated within 1 hour of collection. A dilution of the tracheal aspirate was prepared and inoculated with a calibrated loop on chocolate agar and MacConkey agar. After overnight incubation in appropriate conditions, the plates were interpreted according to quantification of growth [ 9 , 10 ]. Qualitative cultures were considered positive when the growth of any micro-organism occurred and quantitative cultures were considered positive when the growth of 10 5 colony-forming units (cfu)/ml or more was observed. Sensitivity, specificity, positive predictive value and negative predictive values for qualitative and quantitative (10 5 cfu/ml and 10 6 cfu/ml) cultures from tracheal aspirates were calculated according to standard formulae. All samples were collected on the day of clinical and radiological evaluation. Results A total of 106 patients were prospectively evaluated during the study period. The mean age (± standard error) was 66.6 ± 18.3 years. A total of 88 patients (83.0%) were male and 18 (17.0%) were female. The mean Acute Physiology and Chronic Health Evaluation II score was 20.1 ± 6.5. Medical patients constituted the majority (60.38%) compared with surgical patients (39.62%; Table 1 ). Among medical patients, 30 (28.2%) were neurological and 21 (19.8%) were cancer patients. In these 106 patients, a total of 314 clinical evaluations were conducted and endothracheal aspirates collected, corresponding to 42.3 ± 36.5 days (mean ± standard error) of mechanical ventilation. In 95 of these evaluations the radiological or laboratory investigations for VAP were incomplete at the time of clinical evaluation, and so these evaluations were excluded. Therefore, a total of 219 evaluations in 106 patients were included in the analysis. Thirty-eight (17.4%) evaluations were classified as 'with VAP' in 33 patients and 181 (82.6%) were classified as 'without VAP' in 73 patients (Table 2 ). The overall concordance between the first two observers for a diagnosis of VAP in the total population was high (94%). Within the VAP group, the overall concordance between the first two observers was 86.9%. Qualitative and quantitative analyses For qualitative analysis, among all 219 evaluations, 168 (76.7%) yielded cultures that were positive for at least one agent. In the VAP group, 31 of the 38 evaluations yielded positive cultures (81.6%). Thus, the sensitivity of qualitative cultures of tracheal aspirates was 81% and the specificity was 23%. The likelihood ratio for a positive test was 1.05 and the likelihood ratio for a negative one was 0.83. The positive predictive value was 18% and the negative predictive value was 86%. For quantitative analysis, among the 219 evaluations, 117 had = 10 5 cfu/ml in tracheal secretions (53.4%) and 49 had = 10 6 cfu/ml (22.4%). In the VAP group, 25 of the 38 evaluations had = 10 5 cfu/ml (65.8%) and 10 of them had = 10 6 cfu/ml (26.3%). Thus, for 10 5 cfu/ml the sensitivity was 65% and the specificity was 48%. The likelihood ratio of a positive test was 1.25 and the likelihood ratio of a negative test was 0.73. The positive predictive value was 21% and the negative predictive value was 87%. For 10 6 cfu/ml the sensitivity was 26% and the specificity was 78%. The likelihood ratio of a positive test was 1.18 and the likelihood ratio of a negative test was 0.95. The positive predictive value was 20% and the negative predictive value was 83% (Table 3 ). In the VAP group leucocytosis was present in 26 evaluations (68.4%) and fever in 24 (63.1%), and purulent endotracheal secretions were observed by the therapist in 22 (57.8%) evaluations. In four evaluations only (10.5%) was blood culture positive for the same agent as was isolated in endotracheal secretions (Table 4 ). Overall, in 96.8% of evaluations patients were receiving at least one antibiotic. Prescription of antibiotics for three or more days before data collection was high (86.7%). The most frequently administered antibiotics were glycopeptides (49.7%), antifungals (42.4%), third-generation cephalosporins (39.2%), or carbapenem (34.2%; Table 5 ). Considering all VAP episodes, the most frequently isolated agents were Staphylococcus aureus (15.7%), P. aeruginosa (15.7%) and Acinetobacter baumanii (7.3%). Fungi accounted for 13.3% of all agents isolated. In 18.4% of evaluations in the VAP group, no agent was recovered from the endotracheal aspirates (Table 6 ). Clinical observations Considering the population as a whole, in 59 evaluations (26.9%) patients had a tracheostomy. Stress ulcer prophylaxis was present at 210 of the 219 evaluations (96%), with H 2 -receptor blockers in 58.4%, proton pump inhibitors in 36.5% and sucralfate in 0.9%. Sepsis was diagnosed in 46 (21%) evaluations. Among the 38 evaluations classified as positive for VAP, tracheostomy was present in ten (26.3%). Previous lung disease was observed in six (15.7%) events. Ulcer prophylaxis was present in 100% of evaluations, with H 2 -receptor blockers in 22 (57.8%) and proton pump inhibitors in 16 (42.2%). Sepsis was diagnosed in 14 (36.8%) evaluations. Other clinical characteristics are listed in Table 2 . A total of 31 (29.2%) patients died during their hospitalization: 11 (33.3%) of the 33 patients in the VAP group and 20 (27.3%) of the 73 patients without VAP (not significant). Discussion VAP is the most frequent type of infection in ICU patients in Europe and Latin America (almost half of all nosocomial infections) [ 3 ] and ranks second in US ICUs [ 11 ]. The attributable mortality is higher in medical than in surgical patients, and rates vary according to the case mix and aetiological agent [ 12 ]. Inadequate or delayed antimicrobial treatment in VAP is an established independent predictor of death [ 13 ]. According to published data, changing an initial empirical treatment based on subsequent culture results may have either a beneficial effect (in terms of mortality, less antibiotic use, less days on antibiotics) [ 14 ] or no effect in more severely ill patients [ 15 ]. For this reason, efforts must be directed at choosing adequate empirical treatment as early as possible, which may be accomplished with a high degree of suspicion and adequate guidelines based on local antibacterial susceptibilities. In addition, adhering to ideal pharmacological principles (choosing continuous as opposed to intermittent administration, adjustment for renal and hepatic failures), reducing dosages when appropriate, and shortening the duration of treatment are presently standard of care for VAP. In order to avoid any delay in instituting antibiotic treatment, reliable diagnostic methods should be employed. Despite their variable sensitivity and specificity [ 16 ], clinical/radiological findings may currently be considered the best option, although rapid tests, such as the percentage of infected leucocytes on bronchial specimens, are promising in that they can provide rapid confirmation [ 17 ]. Culture results for bronchial or tracheal samples may be available late in the course of an episode of VAP and should not be used to decide whether to treat, especially in patients who are severely ill. On the other hand, culture results should be used to adjust (narrow or extend antibiotic spectrum) or withdraw empirical treatment – a practice that has been shown to be beneficial, with no increase in mortality, and that directs medical staff to seek other unsuspected foci of infection [ 18 ]. Although bronchoscopic samples increase the degree of confidence that a diagnosis of VAP is correct [ 14 ], endotracheal aspirates, despite their lack of consistency as a diagnostic tool [ 19 ], are widely employed in the management of VAP. Recent small trials have consistently shown that there is no advantage of using bronchoscopic methods over relying on tracheal aspirate cultures when mortality is an end-point [ 6 , 20 , 21 ]. Reduced costs and similar outcomes were reported using either quantitative or qualitative tracheal aspirates for guiding or deciding to interrupt antibiotic treatment for VAP [ 6 ]. This may be due to the high correlation between tracheal aspirates (both quantitative and qualitative) and bronchoscopic cultures when presence of VAP is highly probable [ 21 , 22 ]. However, the above-mentioned studies did not determine the value of quantification of micro-organisms in tracheal aspirate samples as compared with qualitative assessment. Quantification of micro-organisms in biological samples for the purpose of diagnosing infectious conditions is widely used, particularly for nosocomial infections. Regarding respiratory infections, bronchoscopic samples have established cutoff values (10 4 cfu/ml for bronchoalveolar lavage [BAL] fluid and 10 3 cfu/ml for protected brush specimen [PBS]) for improving diagnostic performance. On the other hand, use of these cutoff values has yielded conflicting results, and previous antibiotic treatment has great impact on these values. Souweine and coworkers [ 23 ] showed that the standard cutoff values of BAL and PSB would have to be lowered to 10 3 cfu/ml and 10 2 cfu/ml to retain diagnostic accuracy where antibiotics were previously administered, mainly when they are given in the preceding 24 hours. Only a small number of studies have evaluated the role of quantitative endotracheal cultures in the diagnosis of VAP. Albert and coworkers [ 24 ], studying 20 ventilated patients and using clinical/radiological parameters, found the threshold of 10 5 cfu/ml to have a sensitivity of 81%, specificity of 65%, positive predictive value of 55% and negative predictive value of 55%. In that study different cutoff values were not tested to evaluate the real usefulness of quantification. Jourdain and coworkers [ 25 ] studied a group of 57 patients with presumed VAP, 19 (33%) of whom were confirmed by PSB sample with more than 10 3 cfu/ml. Using quantification in this population, those investigators showed that the sensitivity of the test reduced considerably from 86% to 43% whereas specificity increased from 52% to 95% when a cutoff of 10 3 cfu/ml was compared with one of 10 7 cfu/ml. No data regarding previous use of antibiotics were available to explain the decreased sensitivity. We conducted a prospective follow up of severely ill patients with a high rate of antimicrobial use prior to diagnosis of VAP. Not surprisingly, the most frequent agents recovered were multidrug-resistant agents, such as methicillin-resistant S. aureus , P. aeruginosa and Acinetobacter spp. We found different levels of sensitivity (81%, 65%, 26%) and specificity (23%, 48%, 78%) for qualitative and quantitative (cutoffs 10 5 cfu/ml and 10 6 cfu/ml) findings, respectively, as was expected. However, the positive (18%, 21%, 20%) and negative (86%, 87%, 83%) predictive values obtained were very similar. Our data reveal sensitivity values for tracheal aspirates similar to those observed in the above-mentioned studies, although specificity values were lower. According to our data, use of the cutoff value 10 5 cfu/ml reduced the sensitivity of the test to levels too low to be useful in clinical practice, bearing in mind the proposed role of tracheal aspirates to guide antibiotic withdrawal or modification. Moreover, quantification did not improve predictive values for the purposes of diagnosing VAP at the time when a suspected case was evaluated. Patient characteristics may have an impact on the accuracy of diagnostic tests. Although there is broad correlation between the number of bacterial colonies in biological samples and the occurrence of infection as opposed to colonization, the exact bacterial count cannot be predicted in highly ill patients, for whom a lower inoculum may be sufficient for disease development. This has been observed for catheter-related infections in severely ill patients in a surgical ICU [ 26 ], in which true catheter-related bacteraemia was reported with fewer than 15 cfu on catheter tips. In our patient population there was a significant proportion of patients with renal failure, diabetes, cancer and sepsis – conditions that are known to be associated with immunosuppression. These decreased sensitivity values may also be explained by antimicrobial use. More than 95% of the patients studied were receiving antibiotics when the sample was collected for analysis, and the majority of them were broad-spectrum antibiotics (almost 50% had received glycopeptides and 35% carbapenems). About 80% had received them for longer than 72 hours. Decreased accuracy of quantification with samples obtained by bronchoscopy was reported by Soweine and coworkers [ 23 ]. BAL and PSB had significantly less sensitivity when the procedure was performed within 24 hours of antibiotic use than when antibiotics had not been given for longer than 72 hours. The impact of antibiotic use may be greater for tracheal aspirates, irrespective of the timing of administration; this may be due to the higher concentration of the antibiotic in upper tract secretions, although this point requires further investigation. Our study has a number of limitations. While we attempted to achieve a high degree of certainty in clinical/radiological parameters, with the participation of three experienced ICU physicians (with a high degree of correlation between them), no 'gold standard' technique was employed, such as bronchoscopic samples (although it remains controversial whether bronchoscopy samples can be regarded as the gold standard for VAP). Because of the low specificity of clinical judgement, we must consider the fact that we are studying a population in which VAP rate is over-estimated. This is supported by the rate of 18.4% of VAP diagnoses with a negative tracheal aspirate finding and a 13.3% rate of fungal isolates, which only rarely can be considered true causative agents. Thus, it is possible that we have false-positive rate of at least 31.7%, although technical problems with specimen collection cannot be ruled out. The virtual absence of a gold standard for VAP makes study designs that address the issue of diagnostic tests difficult. In accordance with our study design, we evaluated all patients with mechanical ventilation every week, irrespective of clinical suspicion of VAP. This strategy may have beneficial effects because we included in the same population patients who were likely and those who were unlikely to have definite VAP, but increasing the possibility of false-positive cases. Other study designs use populations selected because clinical/radiological judgement suggest the presence of VAP. In these studies, the control cases (no VAP) are defined as having negative bronchoscopic cultures, based on predetermined cutoff values. In these situations, problems with the lesser sensitivity of bronchoscopic samples in patients on antibiotics, and even the intrinsically low sensitivity of this diagnostic strategy when compared with histological criteria [ 27 ], increase the likelihood of including false-negative control individuals. In other words, with our study design we might have overestimated VAP, as compared with underestimating it with conventional study designs. For this reason we think that there is no ideal design for such studies, and studies that rely solely upon clinical/radiological parameters should not systematically be discarded. Furthermore, the use of bronchoscopy in our hospital is unreliable, as it may be in a large number of general ICUs. Tracheal aspirates have a definite role to play in the management of VAP, but only when correlated with clinical findings [ 28 ]. The use of quantitative results may be associated with under-diagnosis of VAP, leading to inappropriate changes to antibiotic regimens and, in some cases, antibiotic delay or withdrawal. Conclusion The severely ill and those who have previously received courses of broad-spectrum antibiotics – a population whose number is expected to increase in modern ICUs – may be targeted for use of qualitative findings rather than quantitative cultures of tracheal secretions for VAP management. Quantitative results may add costs and workload (in our laboratory it is five times more time consuming) and may then be of limited value in this group of patients, although enhanced specificity may be beneficial in terms of avoiding unnecessary treatment. In selected groups of severely ill patients, quantitative cultures of tracheal aspirates should not replace qualitative cultures for confirmation of diagnosis or management of antibiotic therapy. Key messages • Quantitative cultures of tracheal aspirates have increased specificity compared with qualitative analysis for diagnosis of VAP. • The sensitivity values for quantitative cultures of tracheal aspirates are significantly lower than those for qualitative cultures for VAP diagnosis in severely ill patients receiving prior antibiotics. • Quantitative cultures of tracheal aspirates should not replace qualitative cultures for the purpose of confirming a clinical diagnosis of VAP or adjusting antimicrobial therapy. Competing interests The authors declare that they have no competing interests. Abbreviations BAL = bronchoalveolar lavage; cfu = colony-forming unit; ICU = intensive care unit; PSB = protected specimen brush; VAP = ventilator-associated pneumonia.

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1065064

Prospective evaluation of an internet-linked handheld computer critical care knowledge access system

Introduction Critical care physicians may benefit from immediate access to medical reference material. We evaluated the feasibility and potential benefits of a handheld computer based knowledge access system linking a central academic intensive care unit (ICU) to multiple community-based ICUs. Methods Four community hospital ICUs with 17 physicians participated in this prospective interventional study. Following training in the use of an internet-linked, updateable handheld computer knowledge access system, the physicians used the handheld devices in their clinical environment for a 12-month intervention period. Feasibility of the system was evaluated by tracking use of the handheld computer and by conducting surveys and focus group discussions. Before and after the intervention period, participants underwent simulated patient care scenarios designed to evaluate the information sources they accessed, as well as the speed and quality of their decision making. Participants generated admission orders during each scenario, which were scored by blinded evaluators. Results Ten physicians (59%) used the system regularly, predominantly for nonmedical applications (median 32.8/month, interquartile range [IQR] 28.3–126.8), with medical software accessed less often (median 9/month, IQR 3.7–13.7). Eight out of 13 physicians (62%) who completed the final scenarios chose to use the handheld computer for information access. The median time to access information on the handheld handheld computer was 19 s (IQR 15–40 s). This group exhibited a significant improvement in admission order score as compared with those who used other resources ( P = 0.018). Benefits and barriers to use of this technology were identified. Conclusion An updateable handheld computer system is feasible as a means of point-of-care access to medical reference material and may improve clinical decision making. However, during the study, acceptance of the system was variable. Improved training and new technology may overcome some of the barriers we identified.

Introduction The rate of expansion of medical knowledge is increasing rapidly, and it is frequently difficult for clinicians to keep abreast of important new literature. For example, several recently published randomized controlled trials in critical care have demonstrated mortality benefits [ 1 - 5 ], but uptake of new knowledge into clinical practice is often delayed [ 6 - 8 ]. Improving access to this knowledge base at the point of care may lead to better clinical decision making, which could improve patient outcome, reduce costs and optimize bed utilization [ 9 ]. In critical care, rapid access to medical reference information may be particularly important in facilitating timely management decisions and avoiding errors [ 10 ]. Computing technology can allow point-of-care access to up-to-date medical reference material [ 11 ]. A study evaluating a mobile computerized cart to make evidence available to clinicians in an internal medicine setting [ 12 ] demonstrated that evidence-based medicine was more likely to be incorporated into patient care when the computerized system was used. Because of their portability, handheld devices may be more practical tools for disseminating knowledge to the point of care. Despite the popularity of handheld devices in medicine, few studies have evaluated the usefulness of this technology [ 13 ]. Before widespread dissemination of this type of technology can be encouraged, its impact must be thoroughly evaluated [ 14 ]. In the present study we evaluated whether it would be feasible and effective to provide updateable reference information from a central academic centre to handheld computers used by critical care specialists in community hospitals. Methods Study design, participants and setting A total of 17 intensivists at four community hospital intensive care units (ICUs) in the Greater Toronto Area participated in the present prospective interventional study. Intervention After training, each physician was equipped with a handheld computing device (Palm M505; Palm Inc., Milpitas, CA, USA) loaded with medical reference material pertinent to the critical care physician. This information included a customized critical care information handbook ('Critical Care'), which was previously developed for use by residents and physicians at our centre ( Additional file 1 ). Commercially available medical reference software was also incorporated, namely PEPID ED (PEPID LLC, Skokie, IL, USA) and MedCalc . The handheld devices were able to receive literature updates on a regular basis, using customized software (IqSync; Infiniq Software, Mississauga, Ontario, Canada), which accessed an internet-based server using either a connection via desktop computer or infrared data transfer to a telephone modem (Fig. 1 ). New information was sent to the handheld devices and appeared in a file called 'What's New'. These updates, provided every 2–3 weeks, comprised brief reviews of relevant new literature including a short summary, a commentary and the article abstract. All handheld devices were equipped with backup software that allowed the content to be rapidly restored in the event of a hardware failure (BackupBuddy VFS; Blue Nomad Software, Redwood City, CA, USA). The devices were also equipped with software capable of generating a log of the applications used (AppUsage; Benc Software Production, Slavonski Brod, Croatia). Between September and November 2002 the handheld devices were distributed to participating physicians, at which time they each received a 1-hour training session on the use of the handheld device and the internet link (Fig. 2 ). After training, the participants were able to utilize the devices in clinical practice for 12 months. We provided 24-hour support by telephone and e-mail, with a website for independent review. Outcome measures Feasibility Feasibility of the system was assessed by tracking physicians' use of the handheld device and tracking their access of the individual handheld applications during the study period. Physicians who updated their handheld computers at least once a month for 6 months were identified as 'regular users'. A qualitative assessment of the system was achieved through surveys and focus group methodology. Participants completed surveys at baseline to identify their prior familiarity with handheld devices, and at the end of the study period to evaluate subjectively the handheld reference system and the individual handheld applications. Survey data were scored on a 7-point scale, in which 'poor' scored 1 and 'excellent' scored 7. An independent company (The NRC+Picker Group, Markham, Canada) conducted the focus group evaluations at the end of the intervention period, to determine the perceived utility of the information system. Each hospital physician group participated in one focus group meeting. Information access Information sources that physicians accessed to make clinical decisions were evaluated during simulated patient care scenarios, completed in the physicians' own ICU utilizing a computerized patient simulator (SimMan; Laerdal Medical Corporation, Wappingers Falls, NY, USA). Each physician completed one scenario before the handheld device was introduced (baseline scenario) and one at the end of the intervention period (final scenario), when the handheld device could be used (Fig. 2 ). A small pool of five scenarios with equivalent complexity was developed, such that physicians would likely need to access information sources in order to make management decisions. The scenarios involved unusual but important conditions, namely thyroid storm, myasthenia gravis, methanol toxicity, malaria and methemoglobinaemia. They were allocated to study participants in such a way as to avoid participants from the same site receiving the same scenario at the same time point, and to avoid repetition of scenarios among individual participants. Each scenario concluded with the physician writing admission orders for the simulated patient. During the scenarios we tracked all medical reference sources utilized by the physicians, who were encouraged to use a 'think aloud' process [ 15 ]. An audiovisual recording was made of the scenarios for later analysis, and when the handheld was used real-time screen capture was incorporated into the recording ( Additional file 2 ). This allowed us to document which handheld applications were accessed, the time taken to access information and the time taken to complete the scenario. We developed an objective scoring system for the admission orders generated at each scenario. The admission orders were assigned a score (range 0–100) by a critical care physician (SM) and critical care pharmacist (LB), who were blinded as to whether the physician used the handheld device. The scenario-specific scoring system allocated points for all necessary diagnostic and therapeutic interventions, weighted according to relative importance. Negative points were given for potentially harmful orders. Data analysis Data are presented as median and interquartile range (IQR), and permutation tests were used for comparisons because numbers were small and not normally distributed. The differences between the final and baseline admission order scores and the time to completion of scenarios were calculated for each participant. A two-sample permutation test was used to compare these differences between the group of physicians who chose to use the handheld in the final scenario and those who did not use the device. Admission order scores obtained for each of the five scenarios were compared. Outcomes were considered statistically significant at α < 0.05. The SAS System for Window version 8.2 (SAS Institute, Inc., Cary, NC, USA) was used for all analyses. Focus groups were recorded, transcribed verbatim and subsequently analyzed. Themes were identified and unique perspectives on key issues noted [ 16 ]. Results Feasibility The handheld information system functioned well during the study period. Tracking of the deployment of handhelds identified 10 regular users (59%), four physicians (23%) who used the system variably and three physicians (18%) who never used their handheld device. The regular users accessed the personal information management applications more commonly (median 32.8 times/month, IQR 28.3–126.8) than the medical software (median 9/month, IQR 3.7–13.7; P = 0.028), although significant variation was noted (Table 1 ). Baseline survey data identified that, of the 17 critical care physicians participating, 12 (71%) had previous experience with handheld devices (nine had used the Palm operating system, and three had used Windows CE) for a median duration of 1 year (range 1 month to 3.8 years). Seven participants (41%) reported using handhelds for accessing medical information before the study. Of the 16 final survey respondents, seven (44%) felt that the handheld system had had a positive impact on their clinical practice. The handheld medical applications (Critical Care, What's New, Medcalc and PEPID) received similar ratings, with overall evaluation scores ranging from 4.1 to 5.3 on the 7-point scale. Four focus group meetings, involving a total of 13 participants (76%), identified the benefits and barriers to use of handhelds for information access, and made suggestions for improvement (Table 2 ). The overall impression of participants was that there is a role for handhelds for mobile information access, but that in situations away from the bedside other electronic media such as desktop computers were preferable. Information access Not all study physicians were able to participate in the simulated clinical scenarios on the pre-assigned day. Fourteen physicians (82.3%) participated in the baseline scenarios and 13 (76.5%) in the final scenarios. Information sources utilized during the baseline scenarios included the internet (50% of participants; e.g. Medline searches and electronic textbooks), textbooks (43%), telephoning colleagues, the ICU pharmacist or Poison Control Centre (71%), and other sources such as pocket guides (21%). In the final scenarios, the handheld device was used as the primary source of information by eight participants (62%; Table 3 ). Of 14 information searches on the handheld device, 11 searches (79%) were successful and the median time to access information was 19 s (IQR 15–40 s). The information sources of those participants not using the handheld device were similar to those in the baseline surveys (Table 3 ). Analysis of the time to completion of the clinical scenarios demonstrated no significant difference between those physicians who used the handheld and those who did not (12.92 min, IQR 10.73–16.62 min versus 15.5 min, IQR 12.85–22.72 min, respectively). Physicians who did not use their handheld device in the final clinical scenarios had similar scores to their baseline scenario scores (median 60.0, IQR 40.0–60.0 versus 58.0, IQR 44.5–70.5, respectively). In contrast, an improvement in the final scenario score as compared with the baseline score was noted for those participants who chose to use the handheld device (median 66.0, IQR 52.5–74.5 versus 44.8, IQR 30.5–54.5, respectively; P = 0.018; Fig. 3 ). When scores recorded for each of the five clinical scenarios were compared, no significant difference was noted, reducing the likelihood that scenario assignment influenced outcomes. Discussion This study demonstrates the feasibility of using an electronic knowledge translation system to provide high quality, regularly updated medical reference information from a central academic centre to multiple peripheral users. User acceptance of this technology was not uniform, with just over half of the participants using their handheld devices to access information on a regular basis. Nevertheless, the availability of point-of-care access to information may have improved the quality of clinical decision-making. Although mobile computing devices have potential beneficial roles to play in clinical medicine, few publications describe formal evaluation of this technology [ 13 ]. Because the present study was an early hypothesis-generating evaluation of this technology, multiple quantitative and qualitative outcomes were measured. We generated novel data on the use of handheld devices in a clinical situation, but the study has several limitations. The number of physicians involved was relatively small, with a significant proportion not utilizing the technology. The allocation of clinical scenarios was not randomized, because they were allocated predominantly to avoid using the same scenario at the same site and time point. However, the analysis performed compared participants who used the handheld with those who did not; because it was not known which participant would use the handheld at the time of allocation of scenarios, potential bias was minimized. Furthermore, the scenarios appeared to be equivalent in difficulty because no difference was noted when scores for the individual scenarios were compared. A confounding factor in the study was the outbreak of SARS (severe acute respiratory syndrome) from March to May 2003, which had a significant impact on the study ICUs [ 17 ]. Participants were advised to avoid using their handhelds during patient contact because of the potential to transmit infection, and this affected continuity of the study. Had we not encountered this event, utilization might have been higher. The lack of universal acceptance of this technology is not surprising and may be due to a number of factors, including inadequate training and the lack of familiarity with the technology [ 18 ]. Training is essential when introducing handheld computing technology [ 19 , 20 ] and, although all users underwent a training programme, the surveys and focus groups indicated a need for improvement. Familiarity with handhelds is increasing, with 33% of all Canadian physicians and 53% of under 35-year-olds using these devices in 2003, but these levels of utilization remain relatively low when compared with use of the internet, at 88% [ 21 ]. Increasing familiarity with the technology will probably increase acceptance of such a system. Other potential barriers to use of the handheld system may be addressed by the rapidly developing technology, including improved screen resolution, ease of data entry and wireless connectivity. Acceptance may be increased through the development of an all-in-one package on the handheld, allowing additional functionality such as decision support, billing, electronic prescribing and communication. The study demonstrated the potential role of an updateable handheld information system for knowledge translation in critical care. Rapid access to current clinical guidelines may be a valuable component of a comprehensive solution to reducing error and improving efficiency. Information access may be most beneficial in areas without full-time critical care physicians, particularly given the current imbalance between demand and supply with critical care physicians, which is expected to worsen [ 9 , 10 ]. Recent recommendations highlight the importance of leveraging information technology to standardize practice and promote efficiency in critical care [ 10 ]. Handheld information access alone is unlikely to change clinical practice, but it should be considered a component of an electronic knowledge translation system. In many situations other media, such as desktop or tablet computers, may be preferable for information access. Although the study was carried out in a critical care environment, such a system is probably applicable to other specialties in which clinicians are mobile and may not have ready access to a desktop computer (for example, anaesthesia, emergency medicine, home care). This study provides insight into the potential impact of this technology in improving health care outcomes [ 14 ]. Nevertheless, further study that builds on our findings is essential to determine how these new technologies can best be incorporated into the patient care setting. Conclusion A handheld computer system is feasible as a means of providing point-of-care access to medical reference material in the ICU. During this study acceptance of this system was variable, and improved training and more advanced technology may be required to overcome some of the barriers we identified. In clinical simulations, use of such a system appeared to improve clinical decision-making. Key messages • This study demonstrated that an updateable handheld computer information resource is a feasible means for providing point-of-care access to medical reference information in the ICU. • Acceptance of this system was variable and may be improved by enhanced training and newer technological innovations. • In clinical simulations, this system appeared to improve clinical decision making. Competing interests The author(s) declare that they have no competing intrests. Author contributions Stephen Lapinsky, Randy Wax and Thomas Stewart were responsible for study design. Stephen Lapinsky, Randy Wax, Randy Showalter and Carlos Martinez implemented the handheld system and collected study data. Sangeeta Mehta and Lisa Burry were responsible for data collection and interpretation. Stephen Lapinsky and David Hallet analyzed the data. The manuscript was written by Stephen Lapinsky, Randy Showalter and Thomas Stewart, with all authors participating in revisions and giving approval to the final draft for submission for publication. Abbreviations ICU = intensive care unit; IQR = interquartile range. Supplementary Material Additional File 1 Quicktime movie (video clip) providing a brief overview of the content of the handheld 'Critical Care' handbook, which is used as one of the medical reference sources in the present study. Click here for file Additional File 2 Quicktime movie (video clip) demonstrating a clinical simulation scenario, using the patient simulator Sim-Man. The physician can be seen accessing the handheld device, and utilization of the various information resources can be tracked. Click here for file

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1065065

Effects of lornoxicam on the physiology of severe sepsis

Introduction The purpose of the present study was to evaluate the effects of intravenous lornoxicam on haemodynamic and biochemical parameters, serum cytokine levels and patient outcomes in severe sepsis. Methods A total of 40 patients with severe sepsis were included, and were randomly assigned (20 per group) to receive either lornoxicam (8 mg administered intravenously every 12 hours for six doses) or placebo. For both groups the following were recorded: haemodynamic parameters (heart rate, mean arterial pressure), nasopharyngeal body temperature, arterial blood gas changes (pH, partial oxygen tension, partial carbon dioxide tension), plasma cytokine levels (IL-1β, IL-2 receptor, IL-6, IL-8, tumour necrosis factor-α), biochemical parameters (lactate, leucocytes, trombocytes, creatinine, total bilirubin, serum glutamate oxalate transaminase), length of stay in the intensive care unit, duration of mechanical ventilation and mortality. All measurements were obtained at baseline (before the start of the study) and at 24, 48 and 72 hours from the start of lornoxicam/placebo administration. Results No significant differences were found between the intravenous lornoxicam and placebo groups in major cytokines, duration of ventilation and length of intensive care unit stay, and inspired fractional oxygen/arterial oxygen tension ratio ( P > 0.05). Conclusion In these patients with severe sepsis, we found intravenous lornoxicam to exert no effect on haemodynamic and biochemical parameters, cytokine levels, or patient outcomes. Because of the small number of patients included in the study and the short period of observation, these findings require confirmation by larger clinical trials of intravenous lornoxicam, administered in a dose titrated manner.

Introduction Sepsis is defined as the systemic response to infection [ 1 , 2 ]. The deleterious effects of bacterial invasion of body tissues results from the combined actions of enzymes and toxins produced by the micro-organisms themselves, and the actions of endogenous cells in response to the infectious process. Despite advances in supportive care, mortality rates in patients with severe sepsis continue to exceed 30%. During sepsis vasoactive arachidonic acid metabolites of the cyclo-oxygenase (COX) pathway are released. In particular, thromboxane A 2 and prostacyclin have been found to be elevated in sepsis [ 3 , 4 ]. Thromboxane A 2 has been associated with bronchoconstriction, vasocontriction and platelet aggregation [ 3 ]. Prostacyclin, the predominant eicosanoid generated by activated endothelial cells, is a powerful vasodilator and antagonist of thrombosis [ 3 ]. Prostaglandin (PG)E 2 is among the most potent and inducible of the prostanoids that are produced in states of inflammation. Specifically, there is evidence to support roles for PGE 2 as a mediator of sepsis-induced immunosuppression, an inhibitor of proinflammatory cytokine expression from monocytes, and an inducer of IL-10 production [ 5 - 7 ]. Conversely, PGE 2 has been shown to mediate detrimental effects in sepsis, including vasodilation and increased vascular permeability [ 8 ]. In addition, its role as a mediator in fever induction and augmentation of pain is well established [ 9 ]. Several studies [ 10 - 12 ] conducted in endotoxin-challenged animals have found beneficial effects of nonselective COX inhibitors. These beneficial effects were felt to be mediated, in part, by mitigation of pathophysiological events in sepsis induced by PGs. COX exists as two isoforms – COX-1 and COX-2. The former is constitutively expressed, whereas COX-2 is expressed at low levels in most normal resting cells. Marked upregulation of COX-2 occurs in synoviocytes, macrophages and endothelial cells during stress and in inflammatory conditions such as sepsis. COX-2 expression is induced by a number of cytokines, including tumour necrosis factor (TNF) and IL-1, mitogens and growth factors, lipopolysaccharide (LPS), and other inflammatory stimuli [ 13 ]. Recent studies [ 14 , 15 ] provided evidence suggesting that selective COX-2 inhibitors have significant advantages over their nonselective counterparts. The specific benefits of COX-2 inhibitors include decreased gastrointestinal toxicity and bleeding [ 14 , 16 ]. As with other nonsteroidal anti-inflammatory drugs (NSAIDs), lornoxicam inhibits PG synthesis via inhibition of COX, but it does not inhibit 5-lipoxygenase. The ratio of inhibitory potency of human COX-1 to COX-2 for lornoxicam is 0.6 [ 17 ]. Lornoxicam was reported to be 100-fold more potent than tenoxicam in inhibiting PGD 2 formation in rat polymorphonuclear leucocytes in vitro , and it was more active than indomethacin and piroxicam in preventing arachidonic acid induced lethality in mice in vivo [ 17 ]. Lornoxicam also inhibited the formation of nitric oxide in RAW264.7 mouse macrophages stimulated with endotoxin, indicating an effect on inducible nitric oxide synthase [ 18 ]. It also exhibited marked inhibitory properties on endotoxin-induced IL-6 formation in THP1 monocytes, with less activity on TNF and IL-1β. It appears that lornoxicam, in addition to markedly inhibiting COX and inducible nitric oxide synthase, has a moderate effect on the formation of proinflammatory cytokines [ 19 ]. The purpose of the present study was to evaluate the effects of intravenous lornoxicam on serum cytokine levels, haemodynamic and biochemical parameters, and outcomes in humans with severe sepsis. Methods Patient population and study design The regional committee on medical research ethics approved the study. Written informed consent was obtained, directly from the patients wherever possible or from the next of kin. Critically ill patients with bacteriologically documented infections were included in the study as soon as they met at least two of the following criteria for sepsis, as defined by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference Committee [ 2 ]: temperature >38°C or <36°C; heart rate >90 beats/min; respiratory rate >20 breaths/min or arterial carbon dioxide tension <32 mmHg; and leucocyte count >12 × 10 9 cells/l or <4 × 10 9 cells/l. In addition, at least one of following conditions was required: hypoxaemia (arterial oxygen tension/fractional inspired oxygen ratio <250); oliguria (urine output <0.5 ml/kg body weight for 2 hours); lactic acidosis (lactate concentration >2 mmol/l); thrombocytopaenia (platelet count <100 × 10 9 /l); and a recent change in mental status without sedation. Patients who were younger than 18 years, had known or suspected hypersensitivity to COX inhibitors, or had received a COX inhibitor within 12 hours (or aspirin within 24 hours) were enrolled in another experimental protocol (not part of the present study), or were excluded if consent could not be obtained. Also excluded were patients with known or suspected brain death; those with advanced acute or chronic renal or hepatic failure; those who had received potent immunosuppressive drugs; those with gastrointestinal bleeding; those who were pregnant; and those with a known irreversible underlying disease, such as end-stage neoplasm. The Acute Physiology and Chronic Health Evaluation (APACHE) II score [ 20 ] and Sepsis-related (or Sequential) Organ Failure Assessment (SOFA) score [ 21 ] (Table 1 ) were employed to determine the initial severity of illness. If required, patients underwent surgical procedures before the start of the study. No invasive surgery was performed during the 72-hour study period. All patients were ventilated in volume-controlled mode (Puritan Bennett 7200; Carlsbad, CA) and received continuous analgesic sedation with midazolam and fentanyl. Ventilator settings, level of positive end-expiratory pressure and fractional inspired oxygen were kept constant during intravenous administration of lornoxicam or placebo. Antibiotic treatment was adjusted according to the results of bacteriological culture, such as blood culture or culture of samples taken from different body sites. In all participants fluid replacement was administered to maintain central venous pressure between 4 and 8 mmHg. No inotropic agent was administered during the study. Those patients who met the criteria for severe sepsis presented above were enrolled in the study within 8 hours of intensive care unit (ICU) admission. Protocol Randomization was done using a computer-steered permuted block design. The study was planned prospective, randomized, double blind, and placebo controlled. In order to perform the study in a double-blind manner, drug solution was administered to all patients by a nurse who had no knowledge of the study protocol, and follow up was done by an anaesthetist who also had no knowledge of the study protocol. Twenty patients received lornoxicam 8 mg (Xefo; Abdi Ýbrahim, Istanbul, Turkey), administered intravenously every 12 hours for a total of six doses. In the placebo group, also including 20 patients, saline was administered using the same volume and dosing regimen. Measurements All patients had arterial catheters placed (Abbott Transpac ® IV; Abbott, Sligo, Ireland) and central venous catheters placed via subclavian (Certofix trio V 720 7F×8"; Braun, Melsungen, Germany). Arterial blood samples were simultaneously withdrawn for measurements of pH, partial oxygen tension, partial carbon dioxide tension and arterial oxygen saturation (Medica Easy BloodGas; Massachusetts, USA). Central venous pressure, mean arterial pressure, heart rate and naso-opharyngeal temperature were continuously monitored (Space Labs Inc., Redmond, WA, USA). All measurements were obtained at baseline (before the start of the study) and again at 24, 48 and 72 hour after the start of infusion. Lactate, platelets, leucocytes, bilirubin, alanine aminotransferase and creatinine were determined at the same times (Vitalab Flexor, Dieren, The Netherlands). TNF-α, IL-1β, IL-2 receptor, IL-6 and IL-8 levels were measured at the same times. Venous blood was collected into a 10 ml sterile plain tube (without anticoagulant) before administration of any medications and stored at -20°C. Before assay, all samples were thawed to room temperature and mixed by gentle swirling or inversion. All sera were assayed on the same day to avoid interassay variation. TNF-α, IL-1, IL-2 receptor, IL-6 and IL-8 levels were measured using a solid-phase, two-site chemiluminescent enzyme immunometric assay method (Immulite TNF-α, Immulite IL-1β, Immulite IL-2 receptor, IL-6 Immulite and IL-8 Immulite; EURO/DPC, Llanberis, UK). The antibodies used in this procedure have no known cross-reactivities with other cytokines. The intra-assay and interassay coefficients of variation, respectively, for this procedure were as follows: for IL-1β, 2.8–4.9% and 4.8–9.1%; for IL-2 receptor, 2.9–3.7% and 6.1–8.1%; for IL-6, 3.6–6.2% and 5.4–9.6%; for IL-8, 3.6–3.8% and 5.2–7.4%; and for TNF-α, 2.6–3.6% and 4.0–6.5%. The lowest detectable limits of IL-1β, IL-2 receptor, IL-6, IL-8 and TNF-α were 1.5 pg/ml, 5 U/ml, 5 pg/ml, 2 pg/ml and 1.7 pg/ml, respectively. The duration of mechanical ventilation was recorded. Survival was defined as being alive at hospital discharge. Statistical analysis Repeated measures analysis of variance was used to evaluate the differences between and within groups from baseline. In the case of statistical significance, groups were tested by independent sample t-test to determine which difference was significant. Data are expressed as mean ± standard deviation. P < 0.05 was considered statistically significant. Results Patient characteristics Clinical and demographic characteristics of the patients are listed in Table 2 . Of the 40 patients included, 20 received intravenous lornoxicam and 20 received placebo. Fifteen patients had septic shock on admission (seven [35%] in the lornoxicam group and eight [40%] in the placebo group) and died while in the ICU. Baseline APACHE II scores (17.10 ± 3.58 and 18 ± 3.72 in the lornoxicam and placebo groups, respectively) and SOFA scores (5.90 ± 1.72 and 6.20 ± 2.2) were similar in the two groups ( P > 0.05). SOFA scores at 24 hours (5.50 ± 1.52 and 6.1 ± 1.2 in the lornoxicam and placebo groups, respectively), 48 hours (5.60 ± 1.6 and 6.0 ± 1.3) and 72 hours (5.72 ± 1.4 and 6.1 ± 1.6) were also similar ( P > 0.05). Infection was documented in all patients. Haemodynamic parameters and oxygen transport variables There were no significant differences between groups with respect to pH, partial oxygen tension, partial carbon dioxide tension, arterial oxygen tension/inspired fractional oxygen ratio and arterial oxygen saturation ( P > 0.05). No significant changes in mean arterial pressure and heart rate were found in either group (Table 3 ). There were no significant differences between groups in biochemical parameters (Table 4 ; P > 0.05). Outcomes Outcomes are listed in Table 2 . In the ICU, the overall mortality rates were 35% (seven patients out of 20) in the lornoxicam group and 40% (eight patients out of 20) in the placebo group ( P > 0.05). All of those who died did so while they were being mechanically ventilated. In the lornoxicam and placebo groups the mean durations of ventilation were 6.1 ± 2.4 and 5.8 ± 3.1 days, respectively ( P > 0.05). The length of ICU stay in lornoxicam treated survivors was not significantly different from that of placebo treated survivors (10.2 ± 7.1 versus 9.2 ± 8.4 days; P > 0.05). Plasma cytokine levels TNF-α, IL-1β, IL-2 receptor, IL-6 and IL-8 levels remained unchanged during the study (Table 5 ). Side effects Intravenous lornoxicam was well tolerated by all patients, and no side effects were noted during or after administration of lornoxicam. Discussion Systemic inflammatory response leading to postoperative organ dysfunction and sepsis remains a formidable clinical challenge and carries a significant risk for mortality. Sepsis and septic shock remain major causes of death in ICUs. A number of studies have examined the role of nonselective COX inhibitors both in animal models of sepsis and in patients with sepsis syndrome. Several studies [ 10 - 12 ] demonstrated beneficial effects of nonselective COX inhibition, predominantly in endotoxin-treated animals. However, subsequent studies [ 22 , 23 ] examining the role played by NSAIDs, particularly ibuprofen, in human sepsis trials have been disappointing. The present study was therefore conducted to determine whether COX inhibition is upregulated early after the onset of severe sepsis, and if so whether COX inhibition prevents the occurrence of septic shock. The arachidonic acid pathway is highly activated in macrophages, monocytes and other inflammatory cells, resulting in the formation of eicosonoids. PGs are involved in all phases of the inflammatory process, including fever and pain reactions, as well as in a large number of physiological functions, including intestinal motility, platelet aggregation, vascular tone, renal function and gastric secretion, among others. Two COX isoforms have been identified: COX-1 and COX-2. The former is a constitutive enzyme that is expressed in many cells as a house-keeping enzyme and stimulates homeostatic production of PGs. COX-2 is an inducible form of the enzyme that is expressed at the onset of inflammation by many cell types that are involved in the inflammatory response. NSAIDs act mainly through COX inhibitors, thus preventing the formation of proinflammatory prostanoids. Lornoxicam, a new member of the oxicam class of NSAIDs, inhibits PG synthesis via inhibition of COX, but it does not inhibit 5-lipoxygenase. Lornoxicam is at least 10 times more potent as an anti-inflammatory agent than piroxicam, and 12 times more potent as an analgesic than tenoxicam [ 17 , 19 ]. The primary pharmacological action of NSAIDs is, of course, to decrease the formation of PGs and thromboxanes by inhibiting COX, a key enzyme in the biochemical pathway that leads to formation of these potent mediators [ 24 ]. Accordingly, products of the COX pathway, sometimes referred to as 'prostanoids', have been implicated in the pathogenesis of the deleterious systemic consequences of serious infection and/or endotoxaemia. In addition, the toxic effects of TNF (thought to be one of the primary cytokines responsible for LPS-induced lethality) can be ameliorated by treating mice or rats with NSAIDs such as indomethacin or ibuprofen [ 25 ]. NSAIDs have been shown to increase release cytokines (TNF, IL-6, or IL-8) by stimulated mononuclear cells in vitro [ 26 , 27 ]. Complications of sepsis have been related to an intense host response based on a delicate equilibrium between various proinflammatory and anti-inflammatory mediators [ 28 ]. Overwhelming production of proinflammatory cytokines, such as TNF-α, IL-1β, IL-2 receptor, IL-6 and IL-8, may induce biochemical and cellular alterations either directly or by orchestrating secondary inflammatory pathways. Reddyl and coworkers [ 5 ] evaluated the effect of pretreatment with NS-398, a highly selective COX-2 inhibitor, on survival and inflammatory mediator production in two models of sepsis in mice (LPS challenge and peritonitis induced by caecal ligation and puncture [CLP]). They found that selective inhibition of COX-2 resulted in improvement in early survival in murine endotoxaemia but not in a more physiologically relevant model of abdominal sepsis (CLP). The early improvement in survival in endotoxin-challenged animals was not attributable to changes in inflammatory cytokine expression or organ-specific neutrophil sequestration. Pretreatment with NS-398 failed to improve long-term survival in either of the models studied, although in the endotoxaemia model administration of the COX-2 inhibitor had a modest salutary effect on early mortality. In addition, although treatment with NS-398 blocked LPS-induced increases in the circulating levels of immunoreactive PGE 2 , injection of the COX-2 inhibitor did not modulate plasma concentrations of TNF or the CXC chemokine KC. Knoferl and coworkers [ 29 ] also evaluated the effect of pretreatment with NS-398, that trauma/haemorrhage results in activation of Kupffer cells to release inflammatory mediators and it leads to immunosuppression. In vitro production of IL-6 by Kupffer cells after CLP was significantly reduced by in vivo NS-398 treatment. However, NS-398 had no effect on TNF-α levels in vivo or in vitro . Strong and coworkers [ 12 ] showed that administration of NS-398 for 24 hours after trauma improved survival when mice were subjected to CLP and puncture 7 days later. It is noteworthy that NS-398 exhibited protective effects in two models of sepsis characterized by infection in the setting of trauma-induced immunosuppression, whereas the drug was largely ineffective when sepsis was induced in immunocompetent animals. Dallal and coworkers [ 30 ] demonstrated that T-cell suppression during neonatal sepsis is accompanied by a decrease in IL-2 production. Such suppression was ameliorated by COX-2 inhibitor, suggesting a role for PGE 2 in suppressed T-cell-mediated immune function in neonatal sepsis. Arons and colleagues [ 22 ] compared the clinical and physiological characteristics of febrile septic patients with those of hypothermic septic patients, and compared plasma levels of cytokines TNF-α and IL-6 and thromboxane B 2 and prostacyclin between hypothermic septic patients and febrile patients. They administered ibuprofen but found that this drug had no effect on cytokine levels. Reddyl and coworkers [ 5 ] indicated that pharmacological inhibition of COX-2 has only very modest effects on outcome in experimental sepsis or endotoxaemia. Because these findings are discrepant with respect to those obtained with isoform nonselective agents, it is regrettable that those investigators did not include a 'positive control' arm in their studies to evaluate the effects of treatment with an agent such as indomethacin or ibuprofen in their laboratory's models of sepsis. In our study we did not observe any significant changes in systemic cytokine levels during NSAID administration in humans with severe sepsis. Cytokine levels in plasma do not necessarily reflect local synthesis of cytokines by cells. Many cells have surface receptors for these cytokines with high binding properties, and target cells and soluble receptors trap cytokines. Thus, cytokines released at the local level may remain undetected in plasma. In the present study we found plasma cytokine levels to remain unchanged over a period of 72 hours. Wang and coworkers [ 31 ] conducted a study to determine whether inhibition of PGI 2 synthesis prevents the hyperdynamic response in early sepsis in animals. Those investigators found that inhibition of PGI 2 production did not prevent the hyperdynamic and hypercardiovascular responses during early sepsis; hence, mediators other than PGI 2 appear to play a major role in producing the hyperdynamic response under such conditions. Fox and colleagues [ 32 ] postulated that the attenuated pulmonary and systemic vascular contractility observed in sepsis was secondary to the release of vasodilator PGs. They used the COX inhibitor meclofenamate to inhibit PG synthesis in a model of hyperdynamic sepsis, and found that meclofenamate had no effect on either the pulmonary or systemic response to phenylephrine infusion in septic animals. However, Wanecek and coworkers [ 11 ] demonstrated that endotoxin-induced pulmonary hypertension in the pig can be prevented with a combination of the nonpeptide mixed endothelin receptor antagonist bosentan and the COX inhibitor diclofenac. They found that the combination of bosentan and diclofenac induced systemic and pulmonary vasodilatation. During endotoxin shock, this drug combination efficiently counteracted pulmonary hypertension and improved cardiac performance, and splenic and renal blood flows. These favourable circulatory effects might have resulted in a reduction in both sympathetic nervous system activation and metabolic acidosis. In the present study we found that lornoxicam had no effect on the cardiovascular and pulmonary systems in severe sepsis in humans, but our study was designed to assess the effects of lornoxicam treatment given before septic shock but after systemic inflammatory response syndrome. For this reason we identified no serious cardiovascular and pulmonary system problems in the patients studied. Arons and coworkers [ 22 ] compared clinical and physiological characteristics of febrile septic patients with those in hypothermic septic patients, and compared plasma levels of cytokines TNF-α and IL-6, and thomboxane B 2 and prostacyclin between hypothermic septic patients and febrile patients. Those investigators found that ibuprofen treatment had a positive impact on vital signs, organ failure and mortality in hypothermic septic patients, and concluded that ibuprofen could substantially decrease mortality in this selected group of septic patients. In our study we found that lornoxicam had no effect on vital signs and mortality in patients with severe sepsis. The overall ICU mortality rate was 37.5% (15 patients out of 40) in total, and these deaths were all attributable to septic shock. However, all of the patients died after completion of the study. Lornoxicam has been shown to produce less gastric toxicity than its nonselective counterparts. This may be especially important in critically ill patients, who are at significantly greater risk for developing gastric ulceration. In addition, the lack of inhibitory effect on platelet function, which occurs with the use of COX-2 selective compounds, may decrease the incidence of bleeding complications [ 17 , 19 ]. In the present study we did not identify any lornoxicam related adverse effects. In summary, we found that intravenous lornoxicam had no effect on haemodynamic and biochemical parameters, cytokine levels, or patient outcomes in severe sepsis. Selective inhibition of COX-2 in sepsis requires further study. However, the findings reported here, indicating that lornoxicam lacks benefit in patients with severe sepsis, are disappointing. Key messages • Administration of intravenous lornoxicam appeared to confer no benefit in patients with severe sepsis. Competing interests The author(s) declare that they have no competing interests. Abbreviations APACHE = Acute Physiology and Chronic Health Evaluation; CLP = caecal ligation and puncture; COX = cyclo-oxygenase; ICU = intensive care unit; IL = interleukin; LPS = lipopolysaccharide; NSAID = nonsteroidal anti-infllammatory drug; PG = prostaglandin; SOFA = Sepsis-related (Sequential) Organ Failure Assessment; TNF = tumour necrosis factor.

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1065066

Early postoperative hyperglycaemia is not a risk factor for infectious complications and prolonged in-hospital stay in patients undergoing oesophagectomy: a retrospective analysis of a prospective trial

Introduction Treating hyperglycaemia in hospitalized patients has proven to be beneficial, particularly in those with obstructive vascular disease. In a cohort of patients undergoing resection for oesophageal carcinoma (a group of patients with severe surgical stress but a low prevalence of vascular disease), we investigated whether early postoperative hyperglycaemia is associated with increased incidence of infectious complications and prolonged in-hospital stay. Methods Postoperative glucose values up to 48 hours after surgery were retrieved for 151 patients with American Society of Anesthesiologists class I or II who had been previously included in a randomized trial conducted in a tertiary referral hospital. Multivariate regression analysis was used to define the independent contribution of possible risk factors selected by univariate analysis. Results In univariate regression analysis, postoperative glucose levels were associated with increased length of in-hospital stay ( P < 0.001) but not with infectious complications ( P = 0.21). However, postoperative glucose concentration was not found to be an independent risk factor for prolonged in-hospital stay in multivariate analysis ( P = 0.20). Conclusion Our data indicate that postoperative hyperglycaemia is more likely to be a risk marker than a risk factor in patients undergoing highly invasive surgery for oesophageal cancer. We hypothesize that patients with a low prevalence of vascular disease may benefit less from intensive insulin therapy.

Introduction Until recently hyperglycaemia after surgery was considered to be a benign phenomenon. However, in a landmark study, van den Berghe and coworkers [ 1 ] showed that treating transient postoperative hyperglycaemia with intensive insulin therapy in a surgical intensive care unit (ICU) dramatically reduces mortality and morbidity. Strict glucose control (target range between 4.4 mmol/l and 6.1 mmol/l) was responsible for a reduction in both ICU and in-hospital mortality, which was primarily attributed to the prevention of septic complications [ 1 ]. The population studied by van den Berghe and coworkers was diverse but consisted primarily of patients who underwent cardiac surgery (63%). Others have found beneficial effects of intensive insulin therapy in patients with obstructive vascular disease such as acute myocardial infarction and acute stroke, and in those who have undergone cardiovascular bypass surgery [ 2 - 7 ]. Strict glucose control is relatively time consuming for ICU personnel because of frequent glucose monitoring, and it may be hazardous because of the risk for hypoglycaemia. It is therefore important to determine which patient groups in the ICU are likely to benefit most or least from aggressively correcting hyperglycaemia. We investigated whether postoperative hyperglycaemia is a risk factor for postoperative infections and prolonged in-hospital stay in a cohort of patients undergoing resection for adenocarcinoma of the oesophagus (i.e. patients with a low prevalence of risk factors for insulin resistance and cardiovascular disease but who are subject to great postoperative stress). Methods Patients A total of 220 consecutive patients with adenocarcinoma of the oesophagus from two university hospitals in Amsterdam and Rotterdam were included in a previously reported randomized clinical trial investigating differences in short-term and long-term morbidity and mortality between two surgical approaches for resection of oesophageal adenocarcinoma [ 8 ]. Classification into American Society of Anesthesiologists (ASA) class 1 or 2 was a requirement for eligibility in that study. Only patients included in Amsterdam were included in the present analysis ( n = 160), because glucose values were taken only in a small proportion of the Rotterdam patients. In nine cases oesophageal resection was cancelled peroperatively because of distant dissemination of tumour, leaving 151 patients for this analysis. Data collection Glucose values were automatically determined with each arterial blood sample test (Ciba Corning 865; Chiron Diagnostics, Medford, MA, USA), and were collected retrospectively from laboratory reports. Forced expiratory volume in 1 s (FEV 1 ) expressed as percentage of the predicted value corrected for age and sex, and patient height (to calculate body mass index [BMI]) were collected retrospectively from preoperative lung function reports. Insulin use in the first 48 hours after surgery was determined retrospectively from ICU charts. In the prospective cohort patients were visited at least twice a week by one of the investigators to score postoperative complications. Postoperative infections were defined as signs of infection and positive culture [ 9 ]. History of cardiovascular disease, hypertension, weight loss, ASA class, postoperative occurrence of left ventricular failure and length of hospital stay were determined prospectively [ 8 ]. Patients were allowed to eat as they wished until 24 hours before surgery. Patients with more than 10% weight loss in the year preceding surgery received preoperative enteral tube feeding. Postoperatively, all patients received continuous tube feeding through a needle jejunostomy, starting 12–24 hours postoperatively, with 25 ml/hour tube feeding containing immunomodulatory nutrients (Impact ® ; Novartis, Basel, Switserland). As a general rule, patients received 30 ml glucose 5% intravenously during the first 48 hours after surgery and patients were treated with insulin when glucose values exceeded 12 mmol/l. Statistical analysis For each patient the mean postoperative glucose concentration was calculated using all available glucose measurements obtained until 48 hours postoperatively. For further analysis, mean postoperative glucose concentrations were divided into quartiles because of nonparametric distribution. Univariate regression analysis was used to select parameters associated with infectious complications and length of hospital stay. Parameters with P < 0.1 in univariate regression analysis were examined in multivariate analysis to define the independent contribution of each possible risk factor [ 10 ]. Postoperative glucose concentrations were automatically selected for multivariate analysis because it was the main aim of the study to determine their relationship with outcome. Logistic regression analysis was used for infectious complications, and linear regression analysis was used for length of stay. Because of nonparametric distribution, length of stay data were logarithmically transformed before regression analysis. Parameters included in the analysis Age, amount of preoperative weight loss, BMI and FEV 1 were entered into regression analyses as continuous variables. Postoperative glucose levels, insulin use within 48 hours after surgery, type of surgical procedure, sex, ASA class, history of hypertension, coronary artery disease, cardiac valve disease or arrhythmia, clinical staging of the tumour and presence of diabetes mellitus were entered as categorical variables. Results Preoperative characteristics are summarized in Table 1 . At least one postoperative glucose value could be retraced in 150 out of 151 cases (99%; median 7 glucose values per patient; range 1–21). A glucose level greater than 6.1 mmol/l was found in 97% of patients. During the first 48 hours after surgery, insulin was administrated to four patients with known diabetes mellitus and to five patients without diabetes mellitus, but insulin administration could not be retraced in one patient with known diabetes mellitus. At least one infectious complication occurred in 55 patients (36%) and more than one infection occurred in 15 patients (9.9%). Pneumonia occurred in 44 patients, wound infection in 15, urinary tract infection in six and sepsis in seven. Patients were admitted to the ICU for a median duration of 3 days (range <24 hours to 71 days). The median length of stay was 16 days (range 9–154 days). The incidences of postoperative left ventricular failure ( n = 13; 8.6%) and in-hospital death ( n = 5; 3.3%) were too low to allow for regression analysis. Postoperative glucose levels and postoperative infections According to univariate regression analysis, no association was found between postoperative glucose levels and infectious complications ( P = 0.21; Fig. 1a ) or between insulin administration and infectious complications ( P = 0.37; odds ratio [OR] 0.5, 95% confidence interval [CI] 0.1–2.4). Parameters associated with postoperative infections in univariate regression analysis were history of cardiac valve disease or arrhythmia ( P = 0.026; OR 11.5, 95% CI 1.35–98.2), FEV 1 per 10% increase ( P = 0.021; OR 0.78, 95% CI 0.63–0.96; OR per 10% of expected FEV 1 ), age per 10 years ( P = 0.069; OR 1.39, 95% CI 0.98–1.97) and duration of surgery per hour ( P = 0.059; OR 1.23, 95% CI 0.99–1.52). In the subgroup of patients with an ICU stay in excess of 5 days, there was no association between postoperative hyperglycaemia and infection ( P = 0.9 for trend; P = 0.8 by ? 2 analysis). Also in multivariate analysis, postoperative hyperglycaemia was not found to be a predictor of postoperative infection ( P = 0.28; OR 1.21, 95% CI 0.86–1.72; Table 2 ). Also, patients with at least one glucose value in excess of 10 mmol/l were not at greater risk for infections (data not shown). Postoperative glucose levels and length of stay In univariate analysis, a positive association was found between postoperative hyperglycaemia and length of hospital stay (P < 0.001; ß = 0.053; standard error [SE] of ß = 0.014), but not with insulin administration ( P = 0.5; ß = -0.56; SE of ß = 0.7). Other parameters associated with length of in-hospital stay were duration of surgery ( P < 0.001; ß = 0.050; SE of ß = 0.010), transthoracic procedure ( P < 0.001; ß = 0.119, SE of ß = 0.032), BMI ( P = 0.036; ß = 0.013; SE of ß = 0.006) and history of cardiac valve disease or arrhythmia ( P = 0.103; ß = 0.130; SE of ß = 0.079). After correction for these variables in multivariate analysis, mean postoperative glucose concentration was found not to be an independent risk factor for prolonged hospital stay ( P = 0.20; Table 3 ). Adding duration of ICU stay greater than 5 days as an interaction term was not statistically significant ( P = 0.12). Discussion In a cohort of patients undergoing highly invasive surgery for oesophageal cancer, we found that postoperative hyperglycaemia was present in almost all patients but that it was not associated with increased incidence of postoperative infections and length of hospital stay. Van den Berghe and coworkers [ 1 ] found that lowering postoperative hyperglycaemia with intensive insulin therapy significantly decreased morbidity and mortality in postoperative patients. Post hoc analysis revealed that both administration of insulin and, possibly to a greater degree, lower glucose levels contributed to better outcome [ 11 ]. However, it is unclear how the effect of intensive insulin therapy in surgical intensive care patients can be explained and which patient groups benefit most from intensive insulin therapy. We propose the following explanation for the seemingly contradictory findings of our study. The population evaluated in the study by van den Berghe and coworkers [ 1 ] consisted mainly of patients undergoing cardiovascular surgery. Transient or 'stress induced' hyperglycaemia was previously reported to be associated with a poor prognosis, primarily in patients with obstructive vascular disease such as those with acute myocardial infarction and acute stroke, and in those who have undergone cardiovascular bypass surgery and peripheral vascular surgery [ 12 - 16 ]. Few patients in our cohort suffered from (cardio)vascular disease because ASA class 1 or 2 was a prerequisite for inclusion in the study, and only 11% had a history of coronary artery disease. It could thus be hypothesized that, in a population with little vascular disease, high postoperative glucose levels are not associated with poor outcome. In response to surgery, both plasma glucose levels and free fatty acid (FFA) levels rise [ 17 ]. Pathophysiological mechanisms that may explain the relationship between stress induced hypermetabolism and poor outcome in patients with cardiovascular disease include the following: toxic effects of elevated FFA levels on the ischaemic myocardium [ 18 ]; elevated FFA levels and hyperglycaemia causing QT prolongation [ 19 ]; hyperglycaemia attenuating ischaemic preconditioning [ 20 ]; and hyperglycaemia causing reduced collateral coronary perfusion [ 21 ]. Haemodynamic effects of glucose and insulin may also play an important role in the pathophysiology of stress induced hypermetabolism. Hyperglycaemia has vasoconstrictive effects [ 22 ], which may aggravate tissue ischaemia, particularly in patients with obstructive vascular disease. Insulin has been reported to have vasodilatory effects, and part of the beneficial effect of intensive insulin therapy may be explained by increasing tissue perfusion [ 23 ]. Our data do not exclude the possibility that intensive insulin therapy or glucose–insulin–potassium infusions may still be beneficial in this particular subgroup of patients. The benefits of intensive insulin therapy may not solely be attributed to lowering hyperglycaemia, but may be mediated by the effect of insulin on protein and lipid metabolism, independent of its effects on glucose metabolism. In patients with sepsis and cancer, lower levels of insulin are needed to restore lipid levels than glucose levels [ 24 ]. Similarly, depleted protein storage and severe surgical stress after oesophageal resection may impair the immune response postoperatively and thus increase the risk for postoperative infection [ 25 ], which may be ameliorated by insulin. However, the administration of insulin was not associated with lower infection risk in our cohort. A shortcoming of the present study is that the number of glucose measurements taken in each patient was not standardized, because of the study's retrospective design. For some patients more glucose measurements were available than for others, and this may have influenced our results. However, glucose measurements were taken randomly with each arterial blood gas analysis, and because mean postoperative glucose levels were used, the relative weight of incidental extreme values was diminished. A strength of our cohort is its homogeneity. It represents a unique group of patients with high postoperative stress and a low frequency of risk factors for obstructive vascular disease. Conclusion Despite the limitations associated with the retrospective analysis of a prospective study, our data indicate that early postoperative hyperglycaemia is more likely to be a risk marker than a risk factor in a patient group encountering severe surgical stress but with a low prevalence of cardiovascular disease. We therefore suggest that the value of intensive insulin therapy, which is time consuming and potentially hazardous, needs further investigation in this particular patient group. Key messages • Postoperative hyperglycaemia after oesophagectomy was not found to be associated with postoperative infection risk. • Postoperative hyperglycaemia after oesophagectomy was found to be associated with longer duration of postoperative stay. However, when corrected for possible confounders, postoperative hyperglycaemia was not found to be an independent risk factor for longer duration of stay. • Strict glycaemic control may not be beneficial for patients after oesophagectomy. Competing interests The author(s) declare that they have no competing interests. Author contributions TMV participated in the design of the study, data collection, data analysis and writing of the manuscript. JHDV participated in data analysis and writing of the manuscript. JBH participated in the design of the study, data collection, data analysis and writing of the manuscript. FH participated in the design of the study and writing of the manuscript. JJvL participated in the design of the study, data collection and writing of the manuscript. JBLH participated in the design of the study, writing of the manuscript and coordinated the study. Abbreviations ASA = American Society of Anesthesiologists; BMI = body mass index; CI = confidence interval; FEV 1 = forced expiratory volume in 1 s; FFA = free fatty acid; ICU = intensive care unit; OR = odds ratio; SE = standard error.

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1065067

G-CSF and IL-8 for early diagnosis of sepsis in neonates and critically ill children – safety and cost effectiveness of a new laboratory prediction model: study protocol of a randomized controlled trial [ISRCTN91123847]

Introduction Bacterial infection represents a serious risk in neonates and critically ill paediatric patients. Current clinical practice is characterized by frequent antibiotic treatment despite low incidence of true infection. However, some patients escape early diagnosis and progress to septic shock. Many new markers, including cytokines, have been suggested to improve decision making, but the clinical efficacy of these techniques remains uncertain. Therefore, we will test the clinical efficacy of a previously validated diagnostic strategy to reduce antibiotic usage and nosocomial infection related morbidity. Methods All patients admitted to the multidisciplinary neonatal and paediatric intensive care unit of a university children's hospital will be included. Patients will be allocated either to routine sepsis work up or to the intervention strategy with additional cytokine measurements. Physicians will be requested to estimate the pre-test probability of sepsis and pneumonia at initial suspicion. In the treatment arm, physicians will receive raw cytokine results, the likelihood ratio and the updated post-test probability. A high post-test probability will suggest that immediate initiation of antibiotic treatment is appropriate, whereas a low post-test probability will be supportive of watchful waiting or discontinuing prophylactic empirical therapy. Physicians may overrule the suggestions resulting from the post-test probability. Conclusion This trial will ascertain the clinical efficacy of introducing new diagnostic strategies consisting of pre-test probability estimate, novel laboratory markers, and computer-generated post-test probability in infectious disease work up in critically ill newborns and children.

Introduction Bacterial infection is an important cause of mortality and morbidity in newborns and critically ill paediatric patients [ 1 , 2 ]. The high risks associated with untreated infection and the lack of accurate clinical or laboratory prediction methods result in a low threshold for initiating empirical antibiotic therapy. In neonatal and paediatric intensive care, antibiotic therapy is used in as many as 80% of patients, with an average of about 50% [ 3 ]. Only a minority of treated patients suffer from true infection. The majority receive antibiotics for 48–72 hours because clinical signs suggest possible infection and laboratory parameters are unable to rule out infection. In otherwise healthy newborns, this practice causes prolonged separation from the mother and increased the costs of care [ 4 , 5 ]. The high prevalence of unnecessary antibiotic therapy augments the risk for selecting resistant bacterial strains. Despite liberal antibiotic prescription, in some patients sepsis is not diagnosed until they have progressed to serious conditions such as septic shock. Several groups have suggested that measurement of cytokines may be done to facilitate early diagnosis [ 6 - 8 ]. We previously reported diagnostic test accuracy studies in which we derived a prediction model based on the measurement of plasma levels of granulocyte colony-stimulating factor (G-CSF) and IL-8, and tracheal aspirate levels of G-CSF [ 9 , 10 ]. If plasma cytokine concentrations rise above pre-specified thresholds, then serious bacterial bloodstream infection is highly likely. Gram-negative sepsis is practically excluded if plasma levels remain low. Although plasma measurements assist in ruling out life-threatening sepsis, localized infections such as ventilator-associated pneumonia [ 11 ] cannot be diagnosed on the basis of blood derived cytokine concentrations. However, we previously showed tracheal aspirate levels of G-CSF to assist in diagnosing ventilator-associated pneumonia [ 10 ], which is the most frequent reason for prescribing antibiotics in our unit [ 3 ]. We recently conducted validation studies for plasma measurements of IL-8 and G-CSF and tracheal aspirate levels of G-CSF, employing a new laboratory method that allows simultaneous determination of parameters from 50 μl blood or tracheal aspirate. We refined the fluorescent bead-based immunoassay to reduce the assay turnaround time from 4.5 hours to 2 hours, rendering it suitable for routine clinical use. To assess the clinical efficacy of the new diagnostic measures, we suggest that a randomized controlled trial be conducted comparing two management strategies. The control strategy will consist of routine management, with the exception that physicians are requested to provide a probability estimate for the presence of bacterial infection whenever a diagnostic work up (blood cultures or tracheal aspirate culture) is ordered. The intervention strategy will consist of cytokine measurement from the sample and provision of a result based post-test probability within a few hours after sample collection. The null hypothesis states that the management arms will not differ with respect to antibiotic utilization rate, measured as the number of days on systemic antibiotic treatment per 1000 days of hospitalization. The secondary null hypothesis states that the arms will not differ with respect to costs associated with hospital acquired septic shock. Methods Design The study is a multicentre randomized controlled trial comparing a new diagnostic treatment strategy for diagnosing bacterial infection versus standard care in critically ill newborns and children. During a 16-week period in 2003 we conducted a pilot study, which tested the intervention and data collection procedures, and led to modifications to the study design. The pilot study is outlined in detail below. In brief, physicians provide pre-test probabilities whenever they order a diagnostic work up for sepsis or ventilator-associated pneumonia (microbiological cultures). This includes any prescription of antibiotics. In the intervention arm, physicians are provided with cytokine results and the updated post-test probability. In the control arm no information is given. Eligibility criteria for participants All patients admitted to the interdisciplinary neonatal or paediatric intensive care unit (ICU) of the Children's Hospital of Zurich are eligible. Patients who are referred to other wards within 24 hours after admission will be excluded from data analysis, because in these patients the decision to stop antibiotic treatment is no longer the responsibility of participating intensivists. Setting The participating university hospital is the tertiary referral centre for Eastern and Southern Switzerland, and serves a population of approximately 3 million. The Department of Neonatology and Pediatric Intensive Care at the University Children's Hospital of Zurich contributes patients from its two ICUs, named unit A and unit B. Both units have average occupancy of 8–10 beds. The two units admit between 900 and 1000 patients annually, with the number of hospitalization days amounting to 5500 each year. The patient population in unit A includes infants of extremely low birth weight referred from other hospitals, critically ill children and adolescent patients, trauma victims and high-risk surgical patients. Unit B predominantly cares for infants and children who have undergone cardiac surgery. Intervention and controls Control strategy For patients randomized to the control arm, if the physician orders microbial cultures then they are obliged to document their best estimate of the probability that the patient has sepsis or pneumonia on two logarithmic visual-analogue scales (range 0–100%). This documentation is mandatory and must be marked on the laboratory form (Fig. 1 ). In the control arm blood or tracheal aspirate specimens are not analyzed; thus, physicians do not receive any information beyond routinely available data. Under the control strategy antibiotic treatment is managed according to current recommendations (cessation of therapy after 48 hours provided that blood cultures remain negative). Intervention strategy If patients are randomized to the intervention arm, then physicians are also obliged to document their best estimate of the probability that the patient has sepsis or pneumonia, again on two logarithmic visual-analogue scales (range 0–100%). This documentation is again mandatory and must be marked on the laboratory form (Fig. 1 ). In the intervention arm, blood or tracheal aspirate specimens are analyzed and results are returned to the unit before 1 p.m. Physicians receive the raw cytokine values as well as the calculated likelihood ratio and the post-test probability (Fig. 2 ). This information is provided in addition to routinely available data. Provided that the available post-test probability indicates absence of infection, physicians are encouraged to stop antibiotic treatment. It is suggested that antimicrobial therapy be continued if the post-test probability indicates infection. It is important to note that the protocol provides only 'suggestions', and that the final decision regarding therapy is left to the discretion of the responsible clinician. This is similar to clinical routine, in which diagnostic results may suggest alterations to treatment decisions but they do not dictate treatment. Randomization The units of randomization are calendar days. Randomization is generated through pre-specified assignment of 15 working days/month as intervention days. Physicians remain blinded to the allocation roster. Thirty minutes after the deadline for delivery of samples to the laboratory (10 a.m.), physicians are informed about the randomization status (control or intervention) of the day. In this way, physicians are able to adjust their decision making while they await test results if they so wish. Data collection Routine sepsis work up includes collection of blood cultures, other microbial specimens where appropriate, and measurements of white blood cell count, including differential and plasma levels of C-reactive protein. Routine surveillance for ventilator-associated pneumonia comprises microbiological examination of the tracheal aspirate, including cultures. As described above, physicians must provide two probability estimates, one for the presence of sepsis and one for pneumonia, whenever they order a sepsis or pneumonia work up. This ensures that clinicians state their estimate before knowledge of the test result. These estimates (pre-test probabilities) are integrated with cytokine concentrations derived from likelihood ratios for sepsis or pneumonia using Bayes' theorem. The algorithms for calculating post-test probabilities are presented in Table 1 . A study nurse records clinical data for both groups on the day preceding collection of culture specimens and on the following 6 days (Fig. 3 ). We will collect data on mortality, but this will not be included as a study outcome because of low mortality rates and the intended study size. Further data are collected from the hospital's database. This database contains all physician's reports, patient baseline data, routine laboratory results, pharmacology data, costs per patient and day of specific medications (e.g. fresh frozen plasma), and staff allocation. Cytokine measurement Blood samples are collected until 10 a.m. in EDTA-containing vacutainers. Immediately thereafter they are centrifuged at 3000 rpm for 10 min and plasma removed for cytokine analysis. Tracheal aspirate samples are obtained through the endotracheal tube using a sterile suction system (Medinorm AG, Quierschied, Germany). Samples are centrifuged at 10,000 rpm for 5 min and cell free supernatant removed for analysis. Cytokine concentrations (tracheal aspirate and plasma) are simultaneously determined using fluorescent latex beads linked to monoclonal antibodies (R&D Systems, Abington, UK) marked after incubation and coupling with a second phycoerythrin monoclonal antibody (sandwich technique) (R&D Systems, Abington, UK). Final measurement and analysis is done on a Cytomics™ FC 500 Series analyzer (Beckman Coulter Inc., Fullerton, CA, USA). Pilot study During a 16-week period in 2003, we conducted a pilot study in both paediatric ICUs. During this period, cytokine concentrations were available daily for all hospitalized patients so that the new laboratory marker could be implemented as part of routine diagnostic decision making. Clinical data were collected from all hospitalized patients for each day of their ICU stay by one of the investigators (TH). Several teaching sessions both for physicians and nurses were held to enhance the implementation process. The pilot study revealed that the diagnostic test performance (combined likelihood ratio derived from plasma levels of IL-8 and G-CSF; receiver operating characteristic [ROC] 0.88) was similar to that of a published study (ROC 0.85) [ 9 ]. However, because clinicians were certain about the presence or absence of infection in half of the episodes, potentially clinically useful test results were found in fewer than a third of all episodes. Thus, we designed the randomized controlled trial as a test to rule-in or rule-out suspected infection only. Objectives and hypotheses Our objectives are to achieve a clinically relevant reduction in overall antibiotic use and to reduce treatment costs caused by delayed diagnosis of nosocomial infection. In this study we will test the hypothesis that routine surveillance by determination of cytokine levels in plasma and tracheal aspirates will allow safe discontinuation of antibiotic therapy within 24 hours if the proposed laboratory prediction model indicates absence of infection. We regard a reduction in antibiotic exposure by 15% to be a clinically relevant effect. The second hypothesis we will test is whether early diagnosis reduces the morbidity and costs associated with hospital acquired infection. Ascertaining relevant indicators of morbidity and costs in all patients with culture proven bloodstream infection will operationalize this. Measures of outcome The primary outcome measure is the rate of systemic antibiotic use per 1000 days of hospitalization (see details under Sample size calculation and statistical considerations). Secondary outcome measures are as follows (for all episodes of hospital acquired infection with positive blood cultures for the first 7 days following initiation of antibiotics after adjusting for important possible patient confounders): number of days free from mechanical ventilation (an indicator of respiratory failure); number of days free from inotropic support (an indicator of circulatory failure); costs for specific expensive medications (e.g. fresh frozen plasma); and nurse allocation (an indicator of treatment intensity). Sample size calculation and statistical considerations At present, in the ICU antibiotic therapy is employed in 40% of patients, which represents a decline from our original survey conducted in 1998 (up to 80% of all patients) [ 3 ]. The expected reduction in antibiotic usage is 10–25%, with a clinically relevant reduction considered to be any reduction in excess of 10%. The minimum number of days of hospitalization in each arm required to detect a 10% reduction with a type I error under 5% and a power of 80% is 2300. The expected follow up rate is in excess of 90%. Because the unit of randomization is days and not individuals, an unknown intracluster (intraday) correlation coefficient must be considered. The standard χ 2 statistic, which assumes independence of individuals, may not be applicable. We may be forced to acknowledge the nested nature of the data (clustered randomized controlled trial) by using test statistics based on the generalized linear mixed model [ 12 ]. To safeguard against insufficient power we believe that the sample size must be increased to 25%, leading to a required accrual of 3000 hospitalization days per arm. Given the size of the participating units, this translates to a study duration of 24 months. All analyses will be carried out on an intention-to-treat basis. This means that any antibiotic treatment course will be allocated according to the randomization status of the day on which the decision to withhold or to continue had to be made. This requires us to perform three subgroup analyses: antibiotic prescription prevalence according to the day's randomization status; antibiotic free days following the 4 days after any microbiological work up; and antibiotic free days during the week following any initiation of antibiotics. Stopping rules Twelve months after initiating the trial, we will conduct an interim analysis at a two-sided P < 0.01 level. If the results indicate no trend toward a change (increase or reduction) in antibiotic treatment (curtailment from 48 to 24 hours) in prophylactic empirical therapy, and if there is no trend at the P < 0.1 level toward improved secondary outcomes, then the trial will be discontinued. The interim analysis implies that the result of the final analysis should be considered significant if P < 0.04. Discussion A variety of publications report excellent diagnostic performance of new markers of infection [ 13 , 14 ]. However, a theoretically useful test may not necessarily provide clinically useful information. Most test accuracy studies derive their results from a subgroup of potentially eligible patients who satisfy unanimously accepted criteria for acceptance as cases or controls. Unfortunately, this practice suffers from the potential overestimation of the test accuracy [ 15 ] and, even more importantly, it disregards any clinical information that is available apart from that pertaining to the test under question. In this randomized controlled trial we wish to assess the clinical efficacy of an innovative diagnostic procedure for the diagnosis of bacterial infection in newborns and critically ill children. It will evaluate whether this strategy results in a clinically relevant reduction in overall antibiotic usage, and whether the strategy is cost-effective by reducing treatment costs caused by delayed diagnosis of nosocomial infection. One of the possible limitations of the study is the required extended study duration of 24 months. It is conceivable that experience gained from patients in the intervention arm or other factors attributable to the conduct of the study (for example increased awareness by physicians because of more conscious decision making) will also affect the control arm. This might lead to an altered prescription pattern in the control group, which would reduce our ability to find a significant difference between the study arms. If the new test proves efficacious in clinical practice and is cost-effective, then it may become established as a routine marker of infection in this specific setting. Author's contributions JF initiated the project and is the principal investigator. JF, TH, SH, DN and OB participated in the design of the study. JF and TH wrote the protocol. TH carried out the pilot study under supervision of JF. TH implemented the project into clinical routine. JF will carry out statistical analyses. All authors read and approved the final manuscript. Key messages • Test accuracy should be evaluated prospectively with integrated bedside clinical information. • The presented design of this ongoing RCT addresses these demands and shall test whether an innovative diagnostic procedure results in a relevant reduction in unnecessary antibiotic utilization and whether this new strategy proves to be cost effective. Competing interests The author(s) declare that they have no competing interests. Abbreviations G-CSF = granulocyte colony-stimulating factor; ICU = intensive care unit; IL = interleukin; ROC = receiver operating characteristic.

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1065068

Case report: Greater meningeal inflammation in lumbar than in ventricular region in human bacterial meningitis

Differences in the composition of ventricular and lumbar cerebrospinal fluid (CSF) based on single pairs of samples have previously been described. We describe a patient that developed post-surgical recurrent meningitis monitored by daily biochemical and bacteriological CSF analysis, simultaneously withdrawn from lumbar space and ventricles. A 20-year-old Caucasian man was admitted to the ICU after a resection of a chordoma that extended from the sphenoidal sinus to the anterior face of C2. CSF was continuously leaking into the pharyngeal cavity after surgery, and three episodes of recurrent meningitis, all due to Pseudomonas aeruginosa O12, occurred. Our case showed permanent ventricular-to-lumbar CSF gradients of leukocytes, protein and glucose that were increased during the acute phase of meningitis, with the greatest amplitude being observed when bacteria were present in both ventricular and lumbar CSF. This might suggest a greater extent of meningeal inflammation in the lumbar than in the ventricular region. Our case also showed that the increase in intravenous antibiotics (cefepim from 8 to 12 g/day and ciprofloxacine from 1.2 to 2.4 g/day) led to an increase in concentration in plasma but not in CSF.

Introduction Bacterial meningitis and ventriculitis remain the most frequent complication in neurosurgery. Diagnosis is based almost exclusively on biochemical and bacteriological analysis of cerebrospinal fluid (CSF) withdrawn either by puncture in the lumbar space or through an external drain located either in the lumbar or ventricular space. It is established that CSF infection is strongly suspected in the presence of a positive CSF culture and/or of a CSF : serum glucose ratio of less than 0.6 and/or of a CSF leukocyte count of more than 11/mm 3 in the lumbar space [ 1 ]. Differences in the composition of ventricular and lumbar CSF, based on single pairs of CSF samples, were previously described [ 2 - 4 ]. These studies showed a rostrocaudal gradient of leukocytes and protein and an inverse gradient of glucose in the first CSF withdrawn in patients with a confirmed diagnosis of meningitis. However, the time course of a ventricular-to-lumbar gradient of leukocytes, glucose and protein, during the occurrence and the relief of meningitis, remains unknown. Here we describe a patient who developed, after surgery for a chordoma of the clivus, three episodes of recurrent meningitis due to Pseudomonas aeruginosa O12. The last two episodes were monitored by daily biochemical and bacteriological analysis of CSF withdrawn in parallel from the lumbar space and ventricles by external lumbar drainage (ELD) and external ventricular drainage (EVD). Case report A 20-year-old Caucasian man with no medical history was admitted for elective surgery of a chordoma that extended from the sphenoidal sinus to the anterior face of C2. The first surgical step consisted of a subtotal removal of the tumour by a transfrontal approach. An EVD was inserted at day 1 (D1) because of the appearance of hydrocephalia. At D10, the second approach consisted in a transoral resection of the tumour with a reconstruction of the pharyngeal wall with skin taken from the arm. However, the wall was not totally occlusive, with a continuous CSF leak into the pharyngeal cavity. Seven days later (D17), the patient developed meningitis with fever and a white blood cell count of 13,800/mm 3 . CSF withdrawn through the ventricular drain showed CSF leukocytes at 830/mm 3 , a CSF protein concentration of 0.99 g/l and a CSF glucose concentration of 3 mmol/l (for a glycaemia of 6 mmol/l). A Ps. aeruginosa O12 resistant to almost all antibiotics except ceftazidime and polymyxin B, similar to that repeatedly found in the oral cavity, grew in CSF culture. It was therefore decided to replace the ventricular drain with another in the controlateral hemisphere for two purposes: first, to withdraw CSF to reduce CSF leakage by the fistula, and second, to perform a biochemical and bacteriological analysis. Antibiotherapy was started with intravenous (i.v.) ceftazidime (6 g/day for 2 days, followed by 8 g/day for 25 days) combined with amikacin and polymyxin B both in the ventricles. A clear improvement in the meningitis allowed us to perform the third and last approach (at D42): an occipito-cervical fixation procedure with EVD removed. Three days later (D45), the patient developed a new episode of hydrocephalia. It was therefore decided to introduce ELD rather than EVD. Twelve days later (D57), the patient developed a second episode of meningitis: fever, lumbar CSF leukocytes at 14,000/mm 3 , a CSF protein concentration of 1.88 g/l and a glucose concentration of 0.9 mmol/l (for a glycaemia of 6 mmol/l). A CSF culture found the same bacteria as in the first episode of meningitis. This second episode was considered to be related to the persistent pharyngeal fistula. The ELD was replaced with a new one and EVD was added because of the suspicion of an additional obstruction in the 4th ventricle related to post-surgical oedema. Meningitis was treated with an increasing dose of i.v. ciprofloxacin (from D61 to D95: 1.2 g continuously over 24 hours for 4 days, followed by 2.4 g over 24 hours for a further 31 days) and i.v. cefepim (from D61 to D95: 4 × 2 g/day for 4 days to 4 × 3 g/day for a further 31 days; see below for the inhibitory minimal concentration and the plasma and CSF concentrations of antibiotics) and amikacine and polymyxin B colistine both administered directly into the ventricles. The third episode of meningitis appeared at D66 with identification of the same Ps. aeruginosa O12 in CSF culture, increased CSF protein and decreased CSF glucose levels in both ELD and EVD. Antibiotics were kept constant and, despite negative cultures, ELD and EVD were replaced with new drains. Interestingly, since this last episode of meningitis, the pharyngeal fistula disappeared, which indicated the end of pharyngeal contamination of CSF. The patient improved rapidly and was discharged home at D108. No further episode of meningitis during the next 3 years, nor any toxic effect related to the high doses of antibiotics, was observed. It is noteworthy that repetitive cerebral computed tomography scans showed no empyema. Figure 1 shows the time course of the following parameters: leukocyte counts, glucose and protein concentrations, measured in parallel in CSF from EVD and ELD, for 17 days (D57 to D73) corresponding to the second and third episodes of meningitis. Figure 1 shows strikingly that the leukocytes and the protein concentration were always higher and the glucose concentration was always lower in ELD than in EVD. Interestingly, the highest ventriculo-lumbar CSF gradients in leukocytes, protein and glucose concentration were present at the very acute phase of meningitis, when Ps. aeruginosa O12 was present in the meningeal cavity. Our case also showed that the increase in the amount of antibiotics given did increase their concentration in plasma but not in CSF. Indeed, i.v. cefepim was increased from 8 to 12 g/day and i.v. ciprofloxacin from 1.2 to 2.4 g/day from D64 to D95. This induced a persistent increase in plasma cefepim concentration from 46 μ g/ml to more than 60 μg/ml and plasma ciprofloxacin concentration from 0.2 μg/ml to more than 1.0 μg/ml. However, only a transient increase in cefepim concentration (D63, 7 μg/ml; D73, 15 μg/ml; D81 and D95, less than 9 μg/ml) and no increase in ciprofloxacin concentration (0.4–0.5 μg/ml from D63 to 95) were seen in lumbar and ventricular CSF. It is noteworthy that the inhibitory minimal concentrations of cefepim and ciprofloxacin for Ps. aeruginosa O12 were 16 and 0.25 mg/ml, respectively. Discussion Our case report followed ventriculo-lumbar CSF gradients in leukocytes, protein and glucose concentration during two episodes of post-operative recurrent meningitis due to Ps. aeruginosa O12. It showed the presence of a rostrocaudal gradient of leukocytes and protein and an inverse gradient of glucose. This confirmed previous work that showed greater leukocytes and protein concentration in lumbar than in ventricular CSF in patients with a central neural system infection, mostly after neurosurgery [ 2 , 4 ]. However, patients from those studies each had only one pair (ventricular and lumbar) of measurements within a 24-hour interval [ 2 ] and glucose concentration in CSF was measured in only six patients [ 4 ]. We extend previous studies by showing that the greatest amplitude of ventricular-to-lumbar gradients for all measured parameters (leukocytes, protein and glucose concentration) were seen during the very acute phase of meningitis, when bacteria were present in the meningeal cavity. The mechanisms of such ventricular-to-lumbar gradients are unknown. Our data strongly suggest a compartmentalization of meningeal inflammation in the ventricular and lumbar area. Indeed, similar bacteria, here Ps. aeroginusa O12, in similar quantities, seemed to induce a greater alteration of meningeal permeability with greater leukocyte and protein concentrations and a lower glucose concentration in the lumbar than the ventricular CSF region. Although still debatable, the decrease in glucose concentration in CSF seems to be less related to a 'leukocyte-induced glucose consumption' but rather to a meningeal shift of glucose metabolism to anaerobic glycolysis, as indicated by the concomitant increase in CSF lactate concentration and/or a decrease in meningeal glucose transport [ 5 ]; the latter is probably directly related to the degree of meningeal inflammation. An alternative explanation of the existence of a rostrocaudal gradient of leukocytes is that leukocytes from ventricular CSF might fall by gravity to lumbar CSF. However, as explained above, a greater concentration of leukocytes cannot by itself explain a greater protein concentration and a lower glucose concentration in lumbar CSF. Accordingly, our study suggests that meningeal inflammation was greater in the lumbar than the ventricular region in our patient with CSF infection due to a pharyngeal fistula. Recurrent meningitis led us to increase the antibiotic dosage to achieve a better concentration in CSF [ 6 ]. Surprisingly, only a transient increase in CSF cefepim concentration and no change in CSF ciprofloxacine concentration were observed despite a more than 50% increase in plasma concentrations of both antibiotics. The transient increase in cefepim in CSF paralleled that of protein in CSF and could be related to the transient alteration in meningeal permeability. In summary, this case report shows that the maximal rostrocaudal gradient of leukocytes, protein and glucose was seen in the very acute phase of meningitis. This strongly suggests a greater alteration in the meningeal barrier and very probably a greater meningeal inflammation in the lumbar than the ventricular regions. Key messages • The paper describes a patient that developed, after surgery for a chordoma of the clivus, three episodes of recurrent meningitis due to Ps. aeruginosa O12. • Episodes were monitored by biochemical and bacteriological daily analysis of CSF withdrawn in parallel from lumbar space and ventricles by external lumbar and ventricular damamge. • We observed a permanent ventricular-to-lumbar CSF gradients of leukocytes, protein and glucose that increased during the acute phase of meningitis, with the greatest amplitude observed when bacteria was present in both ventricular and lumbar CSF. • This may suggest a greater extent of meningeal inflammation in lumbar than in ventricular region Abbreviations CSF = cerebrospinal fluid; D1, day of insertion of EVD; ELD = external lumbar drainage; EVD = external ventricular drainage; ICU = intensive care unit; i.v. = intravenous. Competing interests The author(s) declare that they have no competing interests. Author's contributions WN and AM coordinated the data analysis and drafted the manuscript. JM, LR and J-F T participated in bacteriological analysis. A-C L and BG participated in analysis of clinical data. DP helped to draft the manuscript. All authors read and approved the final manuscript

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1065070

Extravascular lung water assessed by transpulmonary single thermodilution and postmortem gravimetry in sheep

Introduction Acute lung injury is associated with accumulation of extravascular lung water (EVLW). The aim of the present study was to compare two methods for quantification of EVLW: transpulmonary single thermodilution (EVLW ST ) and postmortem gravimetric (EVLW G ). Methods Eighteen instrumented and awake sheep were randomly assigned to one of three groups. All groups received Ringer's lactate (5 ml/kg per hour intravenously). To induce lung injury of different severities, sheep received Escherichia coli lipopolysaccharide 15 ng/kg per min intravenously for 6 hours ( n = 7) or oleic acid 0.06 ml/kg intravenously over 30 min ( n = 7). A third group ( n = 4) was subjected to sham operation. Haemodynamic variables, including EVLW ST , were measured using a PiCCO plus monitor (Pulsion Medical Systems, Munich, Germany), and the last measurement of EVLW ST was compared with EVLW G . Results At the end of experiment, values for EVLW ST (mean ± standard error) were 8.9 ± 0.6, 11.8 ± 1.0 and 18.2 ± 0.9 ml/kg in the sham-operated, lipopolysaccharide and oleic acid groups, respectively ( P < 0.05). The corresponding values for EVLWI G were 6.2 ± 0.3, 7.1 ± 0.6 and 11.8 ± 0.7 ml/kg ( P < 0.05). Ranges of EVLWI ST and EVLWI G values were 7.5–21.0 and 4.9–14.5 ml/kg. Regression analysis between in vivo EVLW ST and postmortem EVLW G yielded the following relation: EVLW ST = 1.30 × EVLW G + 2.32 ( n = 18, r = 0.85, P < 0.0001). The mean bias ± 2 standard deviations between EVLW ST and EVLW G was 4.9 ± 5.1 ml/kg ( P < 0.001). Conclusion In sheep, EVLW determined using transpulmonary single thermodilution correlates closely with gravimetric measurements over a wide range of changes. However, transpulmonary single thermodilution overestimates EVLW as compared with postmortem gravimetry.

Introduction Acute lung injury (ALI) of septic and non-septic origin is a frequent cause of mortality in critically ill patients. During ALI, the inflammatory process in the lungs may increase the microvascular pressure and permeability, resulting in an accumulation of extravascular lung water (EVLW) and development of pulmonary oedema [ 1 ]. However, it is difficult to estimate the amount of oedema fluid at the bedside. Clinical examination, chest radiography and blood gases have proven to be of limited value in quantifying pulmonary oedema [ 1 - 3 ]. Several techniques to assess EVLW have therefore been developed. Among the various methods for measurement of EVLW, thermo-dye dilution has been used most frequently [ 4 - 8 ]. In animal models of lung oedema, this method has been evaluated by comparison with postmortem gravimetry, which is supposed to be the 'gold standard' of EVLW measurements [ 7 - 9 ]. In critically ill patients, fluid management guided by thermo-dye measured EVLW was associated with improved clinical outcome [ 10 ]. Hence, EVLW has been suggested to play a role as an independent predictor of the prognosis and course of illness [ 6 , 8 , 10 ]. However, the thermo-dye dilution method is relatively time consuming, cumbersome and expensive. For these reasons, the method has not gained general acceptance [ 4 , 5 , 7 ]. Use of a technique based on injection of a single thermo-indicator that can be detected using an indwelling arterial catheter was an appealing concept. Recent experimental and clinical studies have shown that EVLW assessed by single thermodilution (ST) exhibits good reproducibility and close agreement with the thermo-dye double indicator technique [ 11 , 12 ]. The ST method is simpler to apply, less invasive and more cost effective; all of these factors make it more suitable for use at the bedside. However, to date, this new method has been sparsely evaluated against gravimetry [ 13 , 14 ], and further validation is needed. Thus, the aim of the present study was to evaluate the accuracy of the ST technique by comparing it with that of postmortem gravimetry (EVLW G ) in conscious sheep, in which ALI was induced either by lipopolysaccharide (LPS) or by oleic acid (OA). Both of these models of ALI are reproducible and have been extensively described [ 7 , 9 , 11 , 15 , 16 ]. Methods Surgical preparation and measurements The study was approved by the Norwegian Experimental Animal Board and conducted in compliance with the European Convention on Animal Care. Eighteen yearling sheep weighing 27.5 ± 0.4 kg were instrumented, as a modification to previously described techniques [ 16 - 19 ], by inserting introducers into the left external jugular vein and common carotid artery. After 1–4 days of recovery, sheep were placed in an experimental pen. A thermodilution catheter (131HF7; Edwards Life Sciences, Irvine, CA, USA) was introduced into the pulmonary artery and a 4-Fr thermistor-tipped catheter (PV2014L16; Pulsion Medical Systems, Munich, Germany) into the carotid artery. The catheters were connected to pressure transducers (Transpac ® III [Abbott, North Chicago, IL, USA] and PV8115 [Pulsion Medical Systems], respectively). Mean pulmonary arterial pressure (PAP), pulmonary arterial occlusion pressure (PAOP) and right atrial pressure (RAP) were displayed on a 565A Patient Data Monitor (Kone, Espoo, Finland) and recorded on a Gould Polygraph (Gould Instruments, Cleveland, OH, USA). Heart rate, mean systemic arterial pressure, cardiac index (CI), systemic vascular resistance index, extravascular lung water index (EVLWI) assessed using the single thermodilution technique (EVLWI ST ), pulmonary vascular permeability index (PVPI), global end-diastolic volume (GEDV) index (GEDVI), intrathoracic blood volume (ITBV) index (ITBVI) and blood temperature were determined at 1-hour intervals using a PiCCO plus monitor (Pulsion Medical Systems). Every value reported here is the mean of three consecutive measurements, each consisting of a 10 ml bolus of ice-cold 5% dextrose injected into the right atrium randomly during the respiratory cycle. To estimate EVLW we used the following formula [ 12 ]: EVLW ST (ml) = ITTV - ITBV (where ITTV is the intrathoracic thermal volume). During clinical application of ST by means of the PiCCO monitor, ITBV is calculated as 1.25 × GEDV, the coefficient 1.25 being derived from critically ill patients [ 12 ]. However, in our previous investigations in sheep [ 17 - 19 ], in which ITBV was measured directly using the thermal-dye dilution technique, we found the coefficient to be 1.34 [ 14 ]. Thus, in the present study we used the corrected values of ITBVI, EVLWI ST and PVPI, based on the following equation: ITBVI = 1.34 × GEDVI. Blood samples were drawn from the systemic arterial (a) and pulmonary arterial (v) lines and analyzed every two hours for blood gases and haemoglobin (Rapid 860; Chiron Diagnostics Corporation, East Walpole, MA, USA). The pulmonary vascular resistance index (PVRI), venous admixture (Qs/Qt), oxygen delivery index (DO 2 I) and oxygen consumption index were calculated as described previously [ 16 , 19 , 20 ]. Experimental protocol After establishing a stable baseline at time 0 hours, awake and spontaneously breathing sheep were randomly assigned to three experimental groups: a sham operated group ( n = 4); a LPS group ( n = 7), receiving an intravenous infusion of Escherichia coli O26:B6 LPS (Sigma Chemical, St. Louis, MO, USA) at 15 ng/kg per min for 6 hours; and an OA group ( n = 7), in which sheep were subjected to an intravenous infusion of OA (Sigma Chemical) 0.06 ml/kg mixed with the animal's blood. The duration of the infusion of OA was 30 min. During the experiment, all animals received a continuous infusion (5 ml/kg per hour) of Ringer's lactate, aiming to maintain intravascular volume at baseline levels. After the last measurements, at 2 hours in the OA group and at 6 hours in the sham-operated and the LPS groups, the sheep were anaesthetized and killed with a lethal dose of potassium chloride. Then, postmortem EVLWI (EVLWI G ) was determined by gravimetry, as previously described [ 21 - 24 ]. Statistical analysis For each continuous variable, normality was checked using the Kholmogorov-Smirnov test. Data are expressed as mean ± standard error of the mean, and assessed by analysis of variance followed by Scheffe's test or test of contrasts, when appropriate. To evaluate the relationship between EVLWI ST and EVLWI G , we used linear regression and Bland-Altman analysis. P < 0.05 was considered statistically significant. Results All animals survived until the end of the experiments. At baseline no significant differences were found between groups, as shown in Figs 1 and 2 , and Tables 1 and 2 . In the sham-operated sheep, all variables remained unchanged throughout the study. Haemodynamic and extravascular lung water measurements Figure 1 and Table 1 show that LPS and OA induced marked increments in PAP and PVRI, peaking at 1 hour and subsequently decreasing gradually to values significantly above the respective baselines and the corresponding values in the sham-operated group. PAOP and RAP also rose in both the LPS and the OA groups ( P < 0.05; data not shown). In parallel, LPS increased EVLWI ST transiently by 20–35% ( P < 0.05; Fig. 1 ). After OA administration, EVLWI ST rose to a maximum of 84% above baseline ( P < 0.01). At the end of the experiment, EVLWI ST in the OA group had increased by 6.4 ml/kg and 9.3 ml/kg relative to the LPS and the sham-operated groups, corresponding to increments of 54% and 104%, respectively ( P < 0.05). PVPI increased by 40% after LPS administration and by 90% after OA ( P < 0.05; Fig. 1 ). GEDVI and ITBVI varied within 10–15% of baseline with no intergroup differences. As shown in Table 1 , LPS caused tachycardia and a rise in CI accompanied by a slight increase in mean arterial pressure whereas systemic vascular resistance index decreased ( P < 0.05). In contrast, in the OA group CI declined and systemic vascular resistance index increased relative to baseline ( P < 0.05). Oxygenation and gas exchange LPS caused significant increments in mixed venous oxygen saturation, DO 2 I and Qs/Qt (Fig. 2 ). OA decreased both arterial and venous oxygenation and reduced DO 2 I ( P < 0.05). Oxygen consumption index did not change significantly (not shown). LPS caused a transient reduction in arterial carbon dioxide tension and a rise in pH ( P < 0.05; Table 2 ). After OA, pH decreased ( P = 0.04). The haemoglobin concentration as well as the body temperature rose only in the LPS group ( P < 0.05). Linear regression and Bland-Altman analysis As shown in Fig. 3 , the regression analysis between EVLW ST and postmortem EVLW G yielded the following relation: EVLWI ST = 1.30 × EVLW G + 2.32 ( n = 18, r = 0.85, P < 0.0001). Notably, the mean EVLWI ST at the end of experiment was higher than EVLWI G : 13.6 ± 1.1 ml/kg versus 8.7 ± 0.7 ml/kg ( P = 0.0005). Ranges of EVLWI ST and EVLWI G values were 7.5–21.0 ml/kg and 4.9–14.5 ml/kg. According to the Bland-Altman analysis, the mean difference between EVLWI ST and EVLWI G was 4.91 ml/kg, with upper and lower limits of agreement (± 2 standard deviations) of +9.99 ml/kg and -0.17 ml/kg, respectively (Fig. 4 ). The difference between methods increased with increasing values of mean EVLWI ( n = 18, r = 0.64; P = 0.005); the regression line equation was as follows: EVLWI ST - EVLWI G = 0.89 × ([EVLWI ST + EVLWI G ]/2) + 6.82. Postmortem gravimetry As shown in Fig. 5 , EVLWI G in the OA group increased by 4.7 ml/kg and 5.6 ml/kg relative to the LPS and the sham-operated groups, amounting to increments by 65% and 90%, respectively ( P = 0.001). Discussion The present findings confirm that, in sheep, EVLW measured using the single transpulmonary thermodilution technique correlates closely with EVLW determined using postmortem gravimetry. However, EVLWI ST overestimates EVLWI G , with the degree of overestimation increasing with the severity of ALI. A number of experimental and clinical studies focused on the potential role of EVLW as a guide to diagnosis and treatment of critically ill patients [ 3 , 6 - 14 , 25 , 26 ]. During pulmonary oedema, accumulation of EVLW occurs before any changes take place in blood gases, chest radiogram and, ultimately, pressure variables. In addition, the latter variables are nonspecific diagnostic tools that are influenced by a variety of factors [ 2 , 4 , 5 , 8 ]. Thus, Boussat and coworkers [ 3 ] recently demonstrated that, in sepsis induced ALI, commonly used filling pressures such as PAOP and RAP are poor indicators of pulmonary oedema. Rather than those measures, they recommended direct measurement of EVLW. Consistent with this, we found that EVLW, in contrast to RAP, correlates with markers of lung injury in human septic shock [ 26 ]. Victims of ALI, regardless of pathogenesis, have a significantly higher EVLW than do other patients [ 6 , 26 ]. Hence, measurement of EVLW supports the diagnosis and may even improve clinical outcomes when used cautiously in combination with treatment protocols that are known to hasten the resolution of pulmonary oedema [ 10 , 25 ]. Instrumented awake sheep represents a stable experimental model for measuring cardiopulmonary variables, as demonstrated in the sham-operated group in the present study as well as by other investigators [ 15 , 27 ]. The model can be used to assess different interventions during ALI. Consistent with previous investigators [ 15 , 17 , 27 ], we observed that infusion of LPS and OA caused pulmonary hypertension, increased EVLW and impaired gas exchange. Despite increments in PAP, PAOP and PVRI, both ITBV and GEDV remained constant whereas PVPI (an index of microvascular permeability, calculated as the ratio of EVLW to pulmonary blood volume) increased significantly. Thus, the haemodynamic responses to LPS and OA are not purely hydrostatic but may also manifest as noncardiogenic permeability pulmonary oedema [ 13 , 15 - 18 , 27 , 28 ]. In the present study lung oedema was significantly more severe in the OA group than in the LPS group, which is consistent with the findings of other investigators [ 29 ]. In fact, OA causes acute haemorrhagic alveolitis, which may lead to acute endothelial and alveolar necrosis and a severe proteinaceous oedema [ 30 ]. In contrast, the LPS-induced ALI is initiated by accumulation of granulocytes and lymphocytes in the pulmonary microcirculation that results in more moderate damage to endothelial cells and lung oedema [ 31 ]. Lung injury in the LPS group was accompanied by a hyperdynamic circulatory state, which was manifested by systemic vasodilation and increments in CI and DO 2 I toward the end of the experiment. In contrast, in the OA group we observed cardiac depression and systemic vasoconstriction. This is consistent with previous investigations of LPS and OA [ 18 , 27 , 30 , 32 ]. Thus, ovine models exhibit a scatter of cardiopulmonary changes from normal in the sham-operated group to mild or moderate ALI in endotoxaemic sheep and moderate to severe ALI in animals subjected to OA. The significant correlation of EVLWI ST and EVLWI G observed in the present study is consistent with findings of Katzenelson and coworkers [ 13 ], who validated EVLWI ST versus postmortem gravimetry in dogs [ 13 ]. However, those investigators did not specifically assess the relationship between EVLWI ST and EVLWI G in sepsis-induced ALI. In addition, their study was performed in anaesthetized and mechanically ventilated animals; hence, further investigation of the correlation in a conscious state was required. Recently, ST has been evaluated against the thermo-dye dilution method in both experimental and clinical settings [ 11 , 12 ]. The studies revealed a close agreement between the techniques. Thus, we believe that injection of cold saline can provide valuable information about the EVLW content and the severity of pulmonary oedema. During ALI, both ST and postmortem gravimetry demonstrated similar relative increases in EVLWI as compared with sham-operated animals. However, we noticed that ST overestimates the absolute values of EVLWI compared with the gravimetric technique – a discrepancy that increased with progression of pulmonary oedema. This finding could be accounted for by heat exchange of the thermal indicator with extravascular intrathoracic structures, such as the walls of the large vessels and the myocardium, and by recirculation of the indicator [ 8 ]. In addition, the coefficients for calculation of EVLWI ST and ITBV may vary with weight and age, as well as between animal species [ 11 ]. Consequently, in the experimental setting EVLWI ST requires a specific correction. In the present study we replaced the coefficient 1.25 used in humans in the ITBVI equation (i.e. ITBVI = 1.25 × GEDVI) with the recalculated 'ovine' coefficient 1.34 [ 14 ], which is based on 426 measurements in 48 animals [ 17 - 19 ]. In contrast to ST, the thermo-dye dilution technique runs the risk of underestimating EVLW in comparison with gravimetry [ 4 ]. This underestimation increases during ALI caused by instillation of hydrochloric acid into the airways, and has been explained by redistribution of pulmonary blood flow away from the oedematous areas. The redistribution is thought to prevent indicator diffusion and consequently to prevent detection of oedema [ 7 ]. In addition, detection of EVLW by thermo-dye dilution can be impaired by changes in CI as well as by positive end-expiratory pressure during mechanical ventilation [ 8 , 28 ]. Compared with other techniques for assessment of EVLW, ST may underestimate EVLW during pulmonary oedema due to intratracheal instillation of saline, although it is an accurate method in normal lungs [ 33 ]. However, intratracheal instillation of saline can also be criticized because a proportion of the fluid is rapidly absorbed and obscured from detection [ 34 ]. Notably, the use of postmortem gravimetry as the reference method for evaluating pulmonary oedema also has limitations [ 21 , 33 ]. For example, the method only allows one measurement and is therefore of no use in following variations over time. The application of gravimetry is limited almost exclusively to experimental studies. The comparison of gravimetric measurement with results of other techniques for determination of EVLW can be influenced by the duration from death to removal of the lungs and by pathophysiological changes in the lungs after cardiac arrest. Thus, the gravimetric technique can underestimate the real value of EVLWI because of partial reabsorption of fluid before excision of the lungs. Conclusion The determination of EVLW by ST in sheep correlates closely with gravimetric measurements over a wide range of changes, and thus it may potentially be of benefit in quantifying lung oedema in critically ill patients. However, compared with postmortem gravimetry, single transpulmonary thermodilution overestimates the absolute values of EVLW. Thus, further studies are warranted to evaluate the accuracy of this method for managing ALI in humans. Key messages • In sheep, extravascular lung water assessed by transpulmonary single thermodilution correlates closely with gravimetric measurements over a wide range of changes. • Despite a moderate overestimation of the extravascular lung water content compared with post-mortem gravimetry, single thermodilution can be a useful tool for assessment of pulmonary oedema during ALI. Competing interests This study was supported by Helse Nord (Norway), project number 4001.721.132; departmental funds, the Department of Anesthesiology, University Hospital of North Norway; and Pulsion Medical Systems (Germany). Author contributions MYK participated in the design of study, performed statistical analysis, and drafted the manuscript. VVK participated in the design of study, performed statistical analysis, and prepared the figures. VVK and KW participated in the design of study. LJB participated in the design of study and provided coordination. All authors read and approved the final manuscript. Abbreviations ALI = acute lung injury; CI = cardiac index; DO 2 I = oxygen delivery index; EVLW = extravascular lung water; EVLWI = extravascular lung water index; GEDV = global end-diastolic volume; GEDVI = global end-diastolic volume index; ITBV = intrathoracic blood volume; ITBVI = intrathoracic blood volume index; LPS = lipopolysaccharide; OA = oleic acid; PAOP = pulmonary arterial occlusion pressure; PAP = pulmonary arterial pressure; PVPI = pulmonary vascular permeability index; PVRI = pulmonary vascular resistance index; Qs/Qt = venous admixture; RAP = right atrial pressure; ST = single thermodilution.

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1065072

Discomfort and factual recollection in intensive care unit patients

Introduction A stay in the intensive care unit (ICU), although potentially life-saving, may cause considerable discomfort to patients. However, retrospective assessment of discomfort is difficult because recollection of stressful events may be impaired by sedation and severe illness during the ICU stay. This study addresses the following questions. What is the incidence of discomfort reported by patients recently discharged from an ICU? What were the sources of discomfort reported? What was the degree of factual recollection during patients' stay in the ICU? Finally, was discomfort reported more often in patients with good factual recollection? Methods All ICU patients older than 18 years who had needed prolonged (>24 hour) admission with tracheal intubation and mechanical ventilation were consecutively included. Within three days after discharge from the ICU, a structured, in-person interview was conducted with each individual patient. All patients were asked to complete a questionnaire consisting of 14 questions specifically concerning the environment of the ICU they had stayed in. Furthermore, they were asked whether they remembered any discomfort during their stay; if they did then they were asked to specify which sources of discomfort they could recall. A reference group of surgical ward patients, matched by sex and age to the ICU group, was studied to validate the questionnaire. Results A total of 125 patients discharged from the ICU were included in this study. Data for 123 ICU patients and 48 surgical ward patients were analyzed. The prevalence of recollection of any type of discomfort in the ICU patients was 54% ( n = 66). These 66 patients were asked to identify the sources of discomfort, and presence of an endotracheal tube, hallucinations and medical activities were identified as such sources. The median (min–max) score for factual recollection in the ICU patients was 15 (0–28). The median (min–max) score for factual recollection in the reference group was 25 (19–28). Analysis revealed that discomfort was positively related to factual recollection (odds ratio 1.1; P < 0.001), especially discomfort caused by the presence of an endotracheal tube, medical activities and noise. Hallucinations were reported more often with increasing age. Pain as a source of discomfort was predominantly reported by younger patients. Conclusion Among postdischarge ICU patients, 54% recalled discomfort. However, memory was often impaired: the median factual recollection score of ICU patients was significantly lower than that of matched control patients. The presence of an endotracheal tube, hallucinations and medical activities were most frequently reported as sources of discomfort. Patients with a higher factual recollection score were at greater risk for remembering the stressful presence of an endotracheal tube, medical activities and noise. Younger patients were more likely to report pain as a source of discomfort.

Introduction Being admitted to an intensive care unit (ICU) can be considered a stressful life event, the reason for admission being a critical or even life-threatening condition. The ICU stay itself may also be stressful. Some patients report vivid recollections [ 1 - 3 ] whereas others have a poor or even no recollection at all of their stay on the ICU. No recollection at all of the ICU study ranges from 23% to 38% among postsurgical patients [ 4 ]. Various authors have reported that patients had unpleasant recollections after a stay on an ICU. Patients recalled discomfort arising from anxiety, pain, thirst, sleeplessness, disorientation, shortness of breath, inability to move, painful medical interventions and the presence of an endotracheal tube [ 5 ]. Turner and coworkers [ 6 ] specifically mentioned arterial blood gas sampling and endotracheal suctioning. However, recollection of discomfort during the ICU stay is inseparably connected to the quality of recollection itself: events considered stressful at the time may not be remembered; conversely, recollections of stressful events may not be based on actual experiences. Jones and coworkers [ 7 ] investigated patients' estimation of the duration of their ICU stay in order to evaluate the accuracy of their memories. The patients' recall of events was generally poor, and 41% of them felt that they had been confused at some time during their stay in the ICU. To our knowledge, there is no literature investigating whether the recollection of discomfort is related to the accuracy of recollection of facts as such, and for what sources of discomfort this holds true. The purpose of this study was to describe the incidence of discomfort reported by ICU patients, the sources of their discomfort, the factual recollection of ICU patients and ward patients, and determinants of the recollection of discomfort in ICU patients. Methods Consecutive ICU patients, who were older than 18 years and who had undergone intubation for longer than 24 hours, were included in the study. During mechanical ventilation patients received sedation by continuous infusion of midazolam (range 1–4 mg/hour) and fentanyl (range 50–150 µg/hour), with the degree of sedation given depending on their clinical requirements. The patients participated in a study comparing routine endotracheal suctioning with minimally invasive airway suctioning. The study was approved by the medical ethics committee of the University Hospital. The Acute Physiology Age and Chronic Health Evaluation (APACHE) II score was used to quantify the severity of illness [ 8 ] and was recorded on the day of admission to the ICU. All ICU patients participated in a structured in-person interview, using a standardized questionnaire, within three days after discharge from the ICU to the ward. The reference group consisted of postsurgical ward patients, matched for age and sex. Data from the reference group were obtained in a structured telephone interview conducted within three days after discharge from hospital. In the questionnaire, all patients were asked to give answers to 14 questions concerning the ICU environment (lighting, timing of ward rounds, number of fellow ICU patients), the nursing staff (uniform, male/female) and personal care (clothing, position of intravenous drip, washing and toilet activities). Patients from the ICU group were asked whether they remembered any discomfort during their stay on the ICU, and if they did then they were asked to specify the sources of discomfort that they remembered. The questions regarding recollection of facts were first asked as open questions. Two points were given for each correct answer to these open questions. Patients who were unable to answer the open questions were presented with four multiple choice answers. One point was given for each correct answer to the multiple choice questions. Summation of the points resulted in a total score for factual recollection, providing an indication of the level of factual recollection. The range for the total score was 0–28 points. Statistical analysis SPSS version 10 (SPSS Inc., Chicago, IL, USA) was used to perform all analyses. To assess the reliability of the questionnaire, a Cronbach's alpha was calculated. Differences between the ICU group and the reference group were analyzed using the ? 2 test for categorical variables and the t-test for normally distributed intervals or ratio scale variables. Differences between patients who recalled discomfort and those who recalled no discomfort were analyzed using the ? 2 test in case of categorical variables, the Mann–Whitney test for ordinal variables and the t-test for normally distributed intervals or ratio scale variables such as age. To analyze potential determinants of discomfort, logistic regression was performed. The presence or absence of discomfort was entered as the dependent variable, and independent variables were as follows: age, sex, APACHE II score (only in ICU patients), length of stay in the ICU or ward, factual recollection score and duration of tracheal intubation. Correlation coefficients between factual recollection score and age were calculated using a Spearman's test for categorical variables. From the logistic regression analysis, odds ratios (ORs) were calculated for all independent variables in the equation. The OR expresses the odds in the group with the condition relative to the other group without the condition. To an extent, the OR can be considered a measure of relative risk. An OR greater than 1 indicates a higher risk and an OR below 1 indicates a lower risk in the group with the condition relative to the group without the condition. Results A total of 125 patients discharged from the ICU were included in this study. Two patients were unable to respond to the questions. Patient characteristics are summarized in Table 1 . In the population studied the prevalence of any discomfort recalled after discharge from the ICU was 54% ( n = 66). The sources of discomfort identified by these 66 patients are summarized in Table 2 . Six patients were disorientated at the time of the interview, but were able to recall discomfort. The median (min–max) factual recollection score was 15 (0–28) in the ICU patients and 25 (19–28) in the reference group; the difference between the groups was highly significant ( P < 0.001). Analyses of reliability of the questionnaire for the ICU patients revealed a Cronbach's alpha of 0.86, indicating high reliability. Items of factual recollection by ICU patients and the reference group, in descending order of being identified correctly, are listed in Table 3 . ICU patient characteristics are summarized in Table 4 separately for the group that recalled any discomfort and the group that did not recall any discomfort. Significant differences were found between the two groups in factual recollection, age and duration of intubation. Logistic regression analysis of determinants of recollection of discomfort confirmed that factual recollection was indeed an independent factor in predicting recollection of discomfort. The calculated OR was 1.1 ( P < 0.001), with a correct percentage in regression analysis of 68%. This implies that the risk for recalling discomfort was 1.1 times higher for each factual recollection point. Age also was a determinant of recollection of discomfort. The calculated OR was 0.97 ( P = 0.006; correct percentage in regression analysis 66%). This implies that the risk for recalling discomfort was lower by a factor of 0.97 for each year of advancing age. The duration of intubation appeared not to be independently related to recollection of discomfort. Factual recollection appears to be inversely related to age. Analysis of the relationship between factual recollection score and age in the ICU group revealed that the correlation coefficient was -0.352 ( P < 0.001); in the reference group it was -0.327 ( P = 0.023; Fig. 1 ). Finally, recollection of pain appeared to be related to age (OR 0.936, P = 0.002; correct percentage in regression analysis 94%). This implies that younger patients reported more recollection of discomfort in the form of pain. Discussion The results of the present study show that a considerable proportion (54%) of patients discharged from the ICU had a recollection of discomfort during their stay in the ICU. The presence of an endotracheal tube, medical interventions, noise and experiences of hallucination were among the sources of discomfort most frequently reported. To our knowledge, this study is the first to evaluate the association between recollection of discomfort and intact factual recollection. In a study conducted by Rose and coworkers [ 9 ] in 50 patients, 60% remembered endotracheal suctioning and 52% remembered extubation as unpleasant experiences. In a study by Turner and coworkers [ 6 ], arterial blood gas sampling and tracheal suctioning were recalled by 48% and 44% of the patients. Although those two studies did not investigate the prevalence of discomfort per se , we conclude that their findings are similar to ours, in that discomfort was recalled by 54% of ICU patients. Within the context of ICU patients' recollections, a memory of an (stressful) event raises the question of whether this recollection is based on reality or fantasy/imagination. In the present study we found the degree of factual recollection to be an important determinant of discomfort, in the sense that more discomfort was reported by those with better factual recollection. Each item of factual recollection that was scored correctly increased slightly the risk for recollection of discomfort. Factual recollection and recollection of discomfort therefore appeared to be related. In an ICU many factors contribute to impairment in memory: critical illness itself, the use of benzodiazepines and opioids, and the common occurrence of delirious states. When a patient's health is improving or when sedative agents are reduced below effective levels, patients tend to remember more regarding factors, mostly unpleasant, in the ICU. Jones and coworkers [ 10 ] described many causes of amnesia during severe illness, including large dosages of sedative medication and withdrawal syndromes. Because levels of sedation strongly influence the function of memory, a weak point in our study is that no sedation score was recorded to enable us to evaluate the effects of sedatives on patient recollection. It should also be noted that we did not look for objective signs of postdischarge psychological distress or examine their relationship to memories of stressful events, either real or perceived. We merely wished to improve our understanding of discomfort by taking into account the confounding role of memory. The presence of an endotracheal tube, medical activities, and noise and bustle were the sources of discomfort remembered most frequently (Table 2 ). This finding is comparable with those of other studies. In a group of 68 ventilated medical patients, Turner and coworkers [ 6 ] found a prevalence of recollection of endotracheal suctioning of 44%, and in 26 mainly surgical patients those investigators found a prevalence of recollection of endotracheal suctioning of 47% [ 11 ]. In a mixed surgical/medical group of cardiac patients ( n = 50), Rose and colleagues [ 9 ] found a 60% prevalence of recollection of endotracheal suctioning during the ICU stay. The reason for discomfort relating to the endotracheal tube may be endotracheal suctioning. While intubated, patients are regularly suctioned via the endotracheal tube in order to maintain airway patency. The strong mechanical stimuli resulting from endotracheal suctioning may explain why the endotracheal tube is remembered as a prominent source of discomfort. In a previous study [ 12 ], we investigated recollection of endotracheal suctioning with two methods of suctioning: routine endotracheal suctioning and minimally invasive airway suctioning. In the case of routine endotracheal suctioning, a 49 cm suction catheter was passed into the lower airways. With minimally invasive airway suctioning the suction catheter did not enter the lower airways and suctioning was limited to the endotracheal tube. A significantly lower prevalence of recollection of airway suctioning was found in the minimally invasive airway suctioning group (20%) than in the routine endotracheal suctioning group (41%; P < 0.001). Our findings show that discomfort resulting from the endotracheal tube and its handling can be reduced by changing the procedure. Hallucinations were another source of discomfort. In the total ICU patient group ( n = 123), 24 (20%; 95% confidence interval 13–23%) of patients experienced hallucinations. This finding is comparable with that of an earlier and smaller study conducted by Holland and coworkers [ 2 ], who found that 10% of patients reported hallucinations. In a more recent study, Ely and colleagues [ 13 ] found that 81.7% of ICU patients developed delirium at some stage in their ICU stay. Delirium was an important variable, contributing as an independent predictor to higher 6-month mortality and longer hospital stay. Delirium was defined as 'a disturbance in consciousness characterized by an acute onset and fluctuating course of impaired cognitive functioning so that a patient's ability to receive, process, store and recall information is strikingly impaired'. Clearly, the presence of delirium by this definition does not imply the presence of hallucinations. The exact percentage of patients who recalled hallucinations was not stated in the report by Ely and coworkers. In studies conducted by Puntillo [ 14 ] and Holland and coworkers [ 2 ], pain was reported as a source of discomfort as well. In a post-cardiac surgery population ( n = 24), Puntillo [ 14 ] described awareness of pain during the ICU period as a significant problem. Holland and coworkers [ 2 ] reported that, in a group of postsurgery patients ( n = 21), 71% had a recollection of pain. In our study of mainly surgical ICU patients, only 12% indicated that pain was a source of discomfort. Differences in type of sedation and pain medication, number of patients, inclusion criteria and type of questionnaire used are possible explanations for the low recollection of pain in the present study as compared with previous ones. A standardized score to assess recollection in this type of patient was lacking at the time our study was performed. We developed a factual recollection questionnaire that may represent a reliable new tool for acquiring information regarding recollection of facts in post-ICU patients. Analysis of reliability revealed a high Cronbach's alpha, and the descriptive data of our score showed a significant difference between ICU patients and the reference group. These findings are hardly surprising in view of the considerable differences between groups in severity of illness and consumption of hypnotics and sedatives. Further studies are needed to determine the validity and reliability of this instrument. Jones and coworkers [ 15 ] have since proposed a similar tool (Intensive Care Unit Memory tool), which has been validated in a number of settings [ 4 , 16 ]. Both good factual recollection and younger age increased the risk for discomfort. Factual recollection and age were inversely associated with each other, but this association was weak. The association of increasing age with reduction in memory function is widely recognized [ 17 , 18 ]. Although factual recollection and recollection of discomfort appear to be related, increasing the level of sedation is not necessarily the best way to prevent discomfort. Not only will deep sedation lead to increased length of stay in the ICU and prolonged ventilator dependency [ 19 ] but it may also have an adverse effect on the rate of post-traumatic stress disorder experienced by patients after their discharge from the ICU [ 10 ]. It has been proposed by various authors that factual recollection helps to offset the emotional impact of delusional memories [ 10 , 19 ] and may actually help to avoid adverse psychological outcomes in this type of patient. The development of drugs that can eliminate the emotional impact of stressful events in the ICU, while preserving mental clarity and memory, might offer the best way to avoid long-term psychological distress. Meticulous treatment of delusional states will also contribute to this end. Conclusion In a series of patients discharged from the ICU, 54% recalled discomfort. The most frequent sources of discomfort cited were presence of an endotracheal tube, hallucinations and medical interventions. The median factual recollection score for ICU patients was significantly lower than the median factual recollection score for ward patients who had not been in an ICU environment. Younger patients were at greater risk for remembering pain as source of discomfort. Patients with better factual recollection had greater recollection of discomfort. Factual recollection and age were inversely related, but this relationship was weak. Discomfort thus appears to be a serious problem for patients in an ICU environment. Its prevalence is probably underestimated because retrospective assessment of the degree of discomfort when the patient has been discharged from the ICU is seriously handicapped by global or partial amnesia, caused by critical illness, delusional states and the use of drugs. However, the fact that discomfort is not always remembered does not imply that the patient has not suffered during his or her stay in the ICU. Reduction in discomfort should remain a focus of attention for both researchers and clinicians caring for critically ill patients. Key messages • Discomfort is a serious problem; 54% of ICU patients experienced discomfort. • Endotracheal tube, hallucinations and medical interventions were cited as sources of discomfort. • Patients with a higher factual recollection have greater recollection of discomfort. Abbreviations APACHE = Acute Physiology and Chronic Health Evaluation; ICU = intensive care unit; OR = odds ratio. Competing interests The author(s) declare that they have no competing interests. Author's contributions JvdL designed the study, performed data collection, data entry, statistical analysis and wrote the manuscript. CvdS, BL, BD and JZ participated in the design of the study. CvdS, BL, JG and JZ participated in the statistical analysis and writing the manuscript.

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1065073

Cardiovascular stability during arteriovenous extracorporeal therapy: a randomized controlled study in lambs with acute lung injury

Introduction Clinical application of arteriovenous (AV) extracorporeal membrane oxygenation (ECMO) requires assessment of cardiovascular ability to respond adequately to the presence of an AV shunt in the face of acute lung injury (ALI). This ability may be age dependent and vary with the experimental model. We studied cardiovascular stability in a lamb model of severe ALI, comparing conventional mechanical ventilation (CMV) with AV-ECMO therapy. Methods Seventeen lambs were anesthetized, tracheotomized, paralyzed, and ventilated to maintain normocapnia. Femoral and jugular veins, and femoral and carotid arteries were instrumented for the AV-ECMO circuit, systemic and pulmonary artery blood pressure monitoring, gas exchange, and cardiac output determination (thermodilution technique). A severe ALI (arterial oxygen tension/inspired fractional oxygen <200) was induced by lung lavage (repeated three times, each with 5 ml/kg saline) followed by tracheal instillation of 2.5 ml/kg of 0.1 N HCl. Lambs were consecutively assigned to CMV treatment ( n = 8) or CMV plus AV-ECMO therapy using up to 15% of the cardiac output for the AV shunt flow during a 6-hour study period ( n = 9). The outcome measures were the degree of inotropic and ventilator support needed to maintain hemodynamic stability and normocapnia, respectively. Results Five of the nine lambs subjected to AV-ECMO therapy (56%) died before completion of the 6-hour study period, as compared with two out of eight lambs (25%) in the CMV group ( P > 0.05; Fisher's exact test). Surviving and nonsurviving lambs in the AV-ECMO group, unlike the CMV group, required continuous volume expansion and inotropic support ( P < 0.001; Fisher's exact test). Lambs in the AV-ECMO group were able to maintain normocapnia with a maximum of 30% reduction in the minute ventilation, as compared with the CMV group ( P < 0.05). Conclusion AV-ECMO therapy in lambs subjected to severe ALI requires continuous hemodynamic support to maintain cardiovascular stability and normocapnia, as compared with lambs receiving CMV support.

Introduction Neonatal, pediatric, and adult extracorporeal membrane oxygenation (ECMO), using venoarterial or venovenous modes, have been practised for over 3 decades [ 1 - 5 ]. These modes of ECMO are known to activate the inflammatory cascade [ 6 , 7 ], but the long-term cardiopulmonary outcome (10–15 years follow-up period) and neurodevelopmental outcome (at age 5 years) are relatively comparable to those in control individuals [ 8 - 10 ]. Patients who now receive ECMO therapy may also be different from patients treated in the 1980s and early 1990s because the alternative therapies have improved [ 11 ]. A search for safer modes of bypass therapy, including arteriovenous (AV)-ECMO, is warranted because of the cardiovascular and cerebral autoregulatory complications that are common during ECMO operations [ 12 , 13 ]. This new mode of ECMO therapy may have some advantages over conventional venoarterial ECMO or venovenous ECMO techniques because the AV-ECMO technique appears simpler and may involve fewer operational complications [ 14 ]. The first investigators to conduct AV-ECMO trials, Kolobow and coworkers [ 15 ] studied eight normal and conscious lambs (age 1–8 days) for periods up to 96 hours. They described reductions in hemoglobin concentrations during AV-ECMO therapy, showing some mild postmortem pulmonary pathology in a few cases. In a later study, those investigators [ 16 ] also designed a carbon dioxide membrane lung, which was used to reduce ventilation in spontaneously breathing or sedated animals subjected to controlled mechanical ventilation. They suggested that a carbon dioxide membrane lung could ideally be operated in an AV mode without using a pump. The AV shunt of the AV-ECMO circuit requires adequate blood flow from the systemic circulation, which may require an increase in cardiac output (CO). Animal models of AV-ECMO without acute lung injury (ALI) show clinically acceptable cardiorespiratory stability [ 17 - 21 ], whereas models with ALI usually require inotropic and fluid support [ 13 , 22 - 26 ]. Conrad and coworkers [ 27 ], following a series of preclinical studies [ 14 , 23 - 25 ], evaluated the safety and efficacy of AV-ECMO therapy in a phase I clinical study. They treated eight patients (five males and three females, aged 21–67 years), who had acute respiratory failure and hypercapnia, with AV-ECMO over a 72-hour period. They found no significant changes in hemodynamic variables, whereas arterial carbon dioxide tension (PaCO 2 ) was significantly reduced from 90.8 ± 7.5 mmHg to 51.8 ± 3.1 mmHg after 2 hours of AV-EMCO therapy [ 23 ]. At the same time, minute ventilation was reduced from a baseline of 6.92 ± 1.64 l/min to 3.00 ± 0.53 l/min. AV-ECMO technique applied in the presence of ALI requires reasonable hemodynamic stability to permit an extracorporeal AV shunt sufficient for carbon dioxide clearance. Recently, we demonstrated that lambs with normal lungs are able to maintain effective CO and provide efficient ventilator support with a relatively moderate AV shunt of 15% [ 17 ]. The aim of the present study was to determine the cardiovascular support needed to maintain hemodynamic stability and the minute ventilation needed to maintain normocapnia in lambs subjected to severe ALI and treated with AV-ECMO (AV shunt flow of up to 15%) or conventional mechanical ventilation (CMV; AV shunt flow of 0%). Methods Surgical procedures The experimental protocol for this study was approved by the Institutional Animal Care and Use Committee of the Mount Sinai Hospital Research Institute (Miami Beach, FL, USA). Seventeen lambs (aged 2–6 weeks, weight 3.6–12.7 Kg) and their ewes were transported to the laboratory at least 3 days before the experiments began. On the day of an experiment, an intravenous line was established, and anesthesia was induced (initial dose 50 mg/kg ketamine intravenously) and maintained throughout the experiment (5 mg/kg per hour intravenous ketamine). A 2% xylocaine solution was used to provide local anesthesia at the incision sites. A while after induction of anesthesia (30–45 min), a tracheotomy was performed and the lambs were connected to a ventilator (Adult Star Infrasonics, Inc., San Diego, CA, USA) at a fractional inspired oxygen (FiO 2 ) of 1.0. Animals were then paralyzed with an intravenous bolus of 1.0 mg/kg vecuronium bromide, followed by 0.1 mg/kg per hour. To establish an ECMO circuit, one internal jugular vein and one carotid artery were cannulated using neonatal ECMO catheters (Medtronic Bio-Medicus, Inc., Eden Prairie, MN, USA). A femoral vein was then cannulated using a 5 Fr Swan–Ganz catheter (Baxter Health Care Co., Critical Care Division, Irvine, CA, USA) for periodic measurement of CO employing the thermodilution technique (Oximetrix-3, CO Computer; Abbott Critical Care System, North Chicago, IL, USA) and for continuous recording of the mean pulmonary artery pressure (PAP). A femoral artery was cannulated for continuous monitoring of the mean arterial pressure (MAP; Datascope 2001; Datascope Co., Paramus, NJ, USA) as well as periodic blood sampling for gas analyses. A bolus of 200 U/kg heparin was administered intravenously, followed by a maintenance infusion of 200 U/kg per hour. Normothermia (38 ± 0.5°C) was maintained throughout the experiments. Lactated Ringer's solution (5 ml/kg per hour) was provided for fluid replacement. Procedures before injury One hour after the completion of all invasive procedures, pre-ALI baselines were determined for all investigated variables. Arterial blood samples, corrected for body temperature, were measured using a blood gas analyzer (ABL-30; Radiometer, Copenhagen, Denmark). The same samples were used to measure arterial hemoglobin concentration and hemoglobin–oxygen saturation (Hb-O 2 ) using a hemoximeter (OSM-3; Radiometer). CO was determined by the thermodilution technique using the indwelling Swan–Ganz catheter and a CO computer (Oximetrix-3; Abbot Critical Care System). Minute ventilation was measured using a neonatal respiratory monitor (Bicore Neonatal Respiratory System, Model CP-100; Bicore, Irvine, CA, USA). The ventilator tidal volume was set at 7 ml/kg body weight and positive end-expiratory pressure was set at 4 cmH 2 O. The peak inspiratory pressure was maintained below 30 cmH 2 O. Because arterial hypercapnia may affect the cardiovascular system [ 28 ], maximizing the ability of the heart to drive the AV shunt, we elected to maintain the PaCO 2 between 30 and 45 mmHg, rather than allowing permissive hypercapnia to occur. Acute lung injury model To establish a model of severe ALI, in a preliminary study we used the above surgical procedures without AV lines in two lambs. This was accomplished with three consecutive saline lavages (5 ml/kg saline for each). The third lung lavage was followed by an intratracheal instillation of a single dose of 2.5 ml/kg 0.1 N HCl. This procedure resulted in substantial increases in the alveolar–arterial oxygen gradient and an average 60% increase in PAP with relatively stable CO over an 8-hour study period (Fig. 1 ). Saline lavage followed by tracheal instillation of HCl was used in all animals administered CMV and AV-ECMO therapy. This combination may result in surfactant deficiency (caused by the saline lavage), and cellular injury and edema (caused by pulmonary exposure to acid). Post-acute lung injury procedures In our ALI model significant arterial hypercapnia developed (data not presented), which was adjusted to relative normocapnia by changes in the respiratory frequency. Based on our preliminary results in the ALI model, we allowed a 90-min interval before determination of a postinjury baseline in order to stabilize gas exchange and hemodynamic parameters. During this recovery period, arterial blood gases were determined every 15 min. A postinjury baseline for all variables was then determined (time 0). At this stage, lambs were consecutively assigned either to continued CMV treatment or to AV-ECMO plus CMV therapy. Group I These lambs received continuous CMV support during a 6-hour study period with a closed AV shunt ( n = 8). All hemodynamic, and arterial and venous mixed blood gas exchange variables were recorded every 2 hours. The oxygen content of both arterial and mixed venous blood was determined for calculation of oxygen consumption as a product of oxygen delivery (the difference between arterial oxygen content and mixed venous blood oxygen content) and CO (Fick's equation). Oxygen extraction was calculated using the differences between the measured values of arterial Hb-O 2 and venous Hb-O 2 saturation. After completion of the study period the lambs were euthanized by lethal dose of pentobarbital (100 mg/kg intravenously). Group II In this treatment group a set of baseline values were obtained during CMV with a closed AV shunt ( n = 9). Subsequently, lambs were subjected to 6 hours of AV-ECMO plus CMV (AV-ECMO therapy) with a maximum AV shunt of 15% (calculated from CO measured during postinjury baseline). The AV-ECMO circuit was established using a hollow fiber oxygenator (Minimax; Medtronic, Inc. Minneapolis, MN, USA) primed with fresh maternal blood (150–200 ml). To test the efficiency of AV-ECMO as compared with that of CMV in terms of carbon dioxide clearance, we attempted to maintain relative normocapnia in both groups. This required changes in minute ventilation that were achieved by modifying the respiratory rate while maintaining peak inspiratory pressure below 30 cmH 2 O. To control the flow rate through the AV shunt, a clamp was placed on the arterial side of the AV-ECMO circuit and the flow was continuously measured (Medical Volume Flow Meter; Transonic Systems Inc., Ithaca, NY, USA). Carbon dioxide clearance during an AV-ECMO operation is dependent on the gas flow through the oxygenator. The efficacy of carbon dioxide removal and oxygenation of the Minimax hollow fiber oxygenator were previously studied in our laboratory using 15% AV shunt during stepwise decreases in minute ventilation and oxygenation with gas flow of 1 l/min [ 17 ]. This gas flow was approximately four times the maximum blood flow through the AV shunt and maintained normocapnia with a 50% reduction in minute ventilation [ 17 ]. In the present study, the oxygenator's gas flow was kept constant at 1 l/min of 100% oxygen and was controlled by an in-line gas regulator (Servo pressure limited system; Hudson RCI, Temecula, CA, USA). To ensure proper performance of the oxygenators during AV-ECMO therapy, the post-oxygenator partial oxygen tension and partial carbon dioxide tension were measured at 2 and 6 hours during the study period. Resuscitative measures The outcome measures in our study were the degree of cardiovascular support needed to maintain hemodynamic stability and the minute ventilation needed to maintain normocapnia during both CMV and AV-ECMO therapy. A number of resuscitative measures were used to maintain hemodynamic stability during both CMV and AV-ECMO trials. These included the following: boluses of 10 ml/kg per hour of lactated Ringer's solution, which were provided if MAP fell below 60 mmHg; infusion of dopamine (5 μg/kg per min) and epinephrine (adrenaline; 0.5–2 μg/kg per min) to maintain MAP above 60 mmHg, given if this MAP was not achieved with fluid resuscitation; and 1 mEq/kg sodium bicarbonate, which was given if the base excess was below –5 mmol/l despite institution of other resuscitative measures. The end-point for resuscitation was deemed to have occurred when all of the above measures failed and the MAP fell below 30 mmHg for a period of 15 min. This cutoff point was selected empirically because below this level of MAP the AV-ECMO animals could not maintain an AV shunt of over 5% of baseline CO. Statistical analyses All values are expressed as mean ± standard deviation. Differences in specific variables after establishment of postsurgery baseline (60 min after completion of surgery) and post-ALI baseline (90 min after injury), both within the same group at different times and between the CMV and AV-ECMO groups, were evaluated using two-tailed unpaired t-tests. Data from the surviving lambs in the same group over the 6-hour study period were evaluated using analysis of variance (ANOVA), followed by Dunnett multiple comparisons test. For this analysis, we used the postinjury baselines in each variable as controls. Differences in each parameter among the surviving lambs in CMV and AV-ECMO groups and a group of nonsurvivors in the AV-ECMO category were evaluated using ANOVA, followed by Bonferroni multiple comparison test for comparable time periods. The use of resuscitative measures (lactated Ringer's, dopamine, epinephrine and bicarbonate) in all lambs after time zero and in the surviving lambs in the CMV and AV-ECMO groups, as well as mortality (death before completion of the 6-hour study period), were compared using Fisher's exact test. All resuscitative measures before baseline (time zero) were excluded from data analyses. P < 0.05 was considered statistically significant. Results Pre- and post-acute lung injury baselines These data were collected in all animals (survivors and nonsurvivors) before assignment to the CMV or the AV-ECMO groups (Table 1 ). No significant differences were found between the preinjury values of lambs that were later randomized to CMV and AV-ECMO groups. After ALI, all lambs required significant increases in minute ventilation in order to achieve relative normocapnia (Table 1 ). Comparison of postinjury PaCO 2 and pH between the two treatment groups revealed statistically significant differences in favor of the AV-ECMO group (Table 1 ). ALI created a arterial oxygen tension (PaO 2 )/FiO 2 ratio of less than 200 (also representing PaO 2 ) in both groups. After ALI, the PAP was significantly increased by approximately 50% in both groups. There were no significant differences in the postinjury baselines of MAP, PAP, and CO between the groups. The average body weight, measured before surgical procedures, was not significantly different between lambs consecutively assigned to CMV and those that were assigned to AV-ECMO (6.3 ± 1.7 kg versus 8.5 ± 2.8 kg, respectively). However, the four surviving lambs in the AV-ECMO group had significantly greater body weight than the five nonsurviving lambs (11.0 ± 2.2 kg versus 6.5 ± 1.3 kg; P < 0.05, by two-tailed unpaired t-test). Conventional mechanical ventilatory support versus arteriovenous extracorporeal membrane oxygenation therapy The data presented in Tables 2 and 3 , and Figs 1 and 2 are from the surviving lambs only. Six out of eight lambs (75%) in the CMV group and four out of nine lambs (44%) in the AV-ECMO group survived the 6-hour study period after ALI. Three of the five nonsurviving lambs in the AV-ECMO group died within 45–90 min and two others died after 4 hours, despite a combination of resuscitative measures. On average, the surviving lambs in both groups had stable CO and MAP during the 6-hour study period (Tables 2 and 3 ). The four surviving lambs in the AV-ECMO group were able to maintain CO and MAP with varying degrees of hemodynamic support. This also allowed for a relatively stable AV shunt flow (14.8 ± 0.4% of the CO, measured at 0, 2, 4, and 6 hours) and a significant reduction of 25–30% in minute ventilation, as compared with the CMV group (Fig. 2 ). There were no significant differences between the PaCO 2 in CMV and AV-ECMO treated lambs during the study period, but the alveolar–arterial oxygen gradient was consistently higher in the AV-ECMO group (Fig. 3 ). The last measurements of MAP, PAP, and PaO 2 , which were obtained in four out of the five nonsurviving lambs in the AV-ECMO group, were 33.5 ± 9.3, 36.0 ± 6.3, and 53.7 ± 9.2 mmHg, respectively. These values were significantly lower than those recorded in the surviving lambs in either the AV-ECMO or the CMV group (Tables 2 and 3 ; ANOVA followed by Bonferroni multiple comparison test). Gas exchange of the oxygenators remained stable within the 6 hours of the study period. For example, the postoxygenator partial oxygen tension was 282 ± 8 mmHg and 282 ± 7 mmHg at 2 and 6 hours, respectively, and the postoxygenator partial carbon dioxide tension was 19.7 ± 5.1 mmHg and 21.0 ± 5.0 mmHg at 2 and 6 hours of AV-ECMO therapy. Hemodynamic stability Analysis of the use of resuscitative measures as indicators of hemodynamic stability between the CMV and AV-ECMO groups revealed that significantly more lambs in the AV-ECMO group (including survivors and nonsurvivors) were resuscitated than in the CMV group (Table 4 ; P < 0.001, Fisher's exact test). However, there was no significant difference in 'mortality' between AV-ECMO and CMV groups within the 6-hour period of study ( P > 0.05, Fisher's exact test). Discussion The cardiovascular effects of AV-ECMO have been studied in adult and neonatal animal models [ 14 - 26 ]. It has been suggested that the resistance of the membrane oxygenator, hemodynamic stability, and the number, size and length of the conducting cannula, as well as the viscosity of the blood, will all affect the exogenous flow rate [ 22 ]. In the present study we utilized a low resistance membrane oxygenator, minimized the length of the conducting cannulae, and attempted to maintain MAP above 60 mmHg by using various resuscitative measures (Table 4 ). These measures in the AV-ECMO group failed to sustain hemodynamic stability in five out of nine lambs (56%), whereas the survivors (44%) were able to maintain normocapnia with a maximum of 30% reduction in minute ventilation over a 6-hour period of study (Fig. 2 ). The latter implies that AV-ECMO therapy, providing an AV shunt flow of up to 15% of the CO, may be able to reduce ventilator-induced lung injury in hypercapnic respiratory failure. However, in acute respiratory failure or acute respiratory distress syndrome with high intrapulmonary right-to-left shunt, extracorporeal blood flow in the range of 5–15% of CO may not be sufficient to provide adequate arterial oxygenation. The reasons for the relatively poor performance of AV-ECMO therapy in our lamb model, as compared with the findings of studies conducted in adult animals [ 14 , 23 , 25 ], may be related to a number of factors. These possibilities are considered below. First, differences between our model and other experimental models of ALI could account for differences between our findings and those of other studies. The present model may create a noncardiogenic pulmonary edema, which could be associated with loss of intravascular volume. Such conditions may require prolonged fluid and positive inotropic treatments to support a sufficient AV shunt flow. In comparison, Zwischenberger and coworkers [ 6 , 25 ] used an adult sheep model, in which acute respiratory distress syndrome was induced by smoke inhalation and 40% third degree burns. Sheep were then ventilated for 2 days before randomization to CMV and AV-ECMO (AV shunt of 11–14%) groups for a period of 7 days. There were no deaths in the AV-ECMO group ( n = 8), as compared with only three survivors in the CMV group ( n = 8). That model [ 6 , 25 ] demonstrates that perhaps a longer period of CMV support is needed to achieve relative cardiovascular stability before subjecting animals with severe ALI to the additional stress of an AV shunt. How may a short recovery period after ALI affect hemodynamic stability during an AV-ECMO operation? ALI leads to the release of a variety of bioactive materials, including proinflammatory cytokines and reactive oxygen species [ 29 ]. The addition of an ECMO circuit to animals with ALI is known to stimulate the generation of inflammatory mediators, leading to further deterioration in cardiovascular function [ 6 , 7 , 16 ]. Zwischenberger and coworkers [ 6 ] studied the pathophysiology of ovine smoke inhalation lung injury after a relatively short recovery interval of 6 hours during both conventional ECMO therapy and CMV in female sheep. Those investigators demonstrated that animals treated with smoke and ECMO had significantly increased circulating thromboxane B 2 levels and oxygen free radical activity, and a significant increase in lung wet:dry weight ratios. They suggested that an ECMO operation could potentiate the pathophysiology of smoke inhalation injury and lead to initial deterioration in native lung function [ 6 ]. Therefore, despite the simplicity of AV-ECMO procedures, as compared with conventional ECMO [ 14 , 30 ], it could be still subject to free radical generation because of presence of the membrane oxygenator. Thus, the addition of an AV shunt after ALI may further compromise the cardiovascular system. A second factor that could account for the discrepancy between our findings and those of other investigators is that the AV shunt opening in our study led to a mortality rate in the smaller lambs, resulting in a difference between the body weights of the surviving lambs in two groups. This implies that smaller (and presumably younger) lambs with ALI could be more vulnerable to the presence of an AV shunt than relatively larger or older animals. Thus, studies concerning the safety and efficacy of neonatal AV-ECMO therapy should use animals with a narrow age range (1–7 days in lambs). The third factor is whether the ALI in the CMV and AV-ECMO therapy groups was equal in severity. Whether the severity of ALI was different between the groups may be indirectly evaluated by comparing the indices of pre- and post-injury gas exchange. Our data indicate that pulmonary performance before starting AV-ECMO therapy was comparable with that observed in the CMV group (Table 1 ). The degree of lung injury was not significantly worsened during the 6-hour study period, as judged by lack of significant changes in alveolar–arterial oxygen gradient in the surviving lambs subjected to CMV or AV-ECMO therapy (Fig. 3 ). Study limitations The outcome measures in this study were the degree of hemodynamic stability and the minute ventilation required to maintain relative normocapnia, while comparing CMV support with AV-ECMO therapy. Our study was not designed to evaluate mortality as an ultimate clinical outcome. A greater number of lambs would have been required to demonstrate significant differences in mortality between the CMV and AV-ECMO groups. However, the more than 50% mortality rate in the AV-ECMO group may raise questions about the clinical and/or statistical significance of our findings. Technically, we failed to use a narrow range of age and body weight in our lambs. However, the average body weights in lambs consecutively randomized to CMV support and AV-ECMO therapy were not significantly different (Table 1 ). Conclusion Our study indicates that cardiovascular support is required to maintain hemodynamic stability during application of AV-ECMO therapy in lambs with severe ALI. In this model, AV-ECMO therapy with continuous cardiovascular support and an AV shunt flow of 15% of CO can provide a maximum 30% reduction in minute ventilation. We suggest that AV-ECMO with cardiovascular support [ 30 ] could be suitable for use in ALI of mild severity, in which permissive hypercapnia is not an acceptable treatment [ 28 , 31 ]. Key messages • Continuous hemodynamic support is required during AV-ECMO in lambs subjected to severe ALI. • By using a shunt flow of up to 15% of CO, AV extracorporeal therapy in lambs with severe ALI can reduce minute ventilation by 25–30%. • Neonatal patients with severe ALI and hemodynamic instability may not be suitable candidates for AV-EMCO therapy. Abbreviations ALI = acute lung injury; ANOVA = analysis of variance; AV = arteriovenous; ECMO = extracorporeal membrane oxygenation; CMV = conventional mechanical ventilation; CO = cardiac output; FiO 2 = fractional inspired oxygen; Hb-O 2 = hemoglobin–oxygen saturation; MAP = mean arterial pressure; PaCO 2 = arterial carbon dioxide tension; PaO 2 = arterial oxygen tension; PAP = pulmonary artery pressure. Competing interests The author(s) declare that they have no competing interests. Author's contributions BRT, JBS and DT completed the proposal writing and experimental design. DT and BRT participated in research coordination, data analysis and presentation. JG, HF, YM, and JLO conducted all experimental aspects of the study. BRT, DT, JBS, and JW prepared the manuscript.

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1065074

The effect of interruption to propofol sedation on auditory event-related potentials and electroencephalogram in intensive care patients

Introduction In this observational pilot study we evaluated the electroencephalogram (EEG) and auditory event-related potentials (ERPs) before and after discontinuation of propofol sedation in neurologically intact intensive care patients. Methods Nineteen intensive care unit patients received a propofol infusion in accordance with a sedation protocol. The EEG signal and the ERPs were measured at the frontal region (Fz) and central region (Cz), both during propofol sedation and after cessation of infusion when the sedative effects had subsided. The EEG signal was subjected to power spectral estimation, and the total root mean squared power and spectral edge frequency 95% were computed. For ERPs, we used an oddball paradigm to obtain the N100 and the mismatch negativity components. Results Despite considerable individual variability, the root mean squared power at Cz and Fz ( P = 0.004 and P = 0.005, respectively) and the amplitude of the N100 component in response to the standard stimulus at Fz ( P = 0.022) increased significantly after interruption to sedation. The amplitude of the N100 component (at Cz and Fz) was the only parameter that differed between sedation levels during propofol sedation (deep versus moderate versus light sedation: P = 0.016 and P = 0.008 for Cz and Fz, respectively). None of the computed parameters correlated with duration of propofol infusion. Conclusion Our findings suggest that use of ERPs, especially the N100 potential, may help to differentiate between levels of sedation. Thus, they may represent a useful complement to clinical sedation scales in the monitoring of sedation status over time in a heterogeneous group of neurologically intact intensive care patients.

Introduction The majority of mechanically ventilated patients in the intensive care unit (ICU) require sedation to reduce their anxiety and to increase their tolerance of the tracheal tube and mechanical ventilation. The choice of sedative drugs and the way in which they are administered may have an important impact on patient outcome and cost of care [ 1 ]. Excessively deep sedation will prolong ventilator dependence and length of stay in the ICU, which can be avoided by careful monitoring and interruption to sedative infusions [ 2 ]. Differentiation between adequate comfort and excessive sedation requires the use of clinically relevant sedation scales; however, these are not suitable for application during deep sedation or muscle relaxation. Other methods to assess the level of sedation in the clinical setting are therefore needed. Growing knowledge of the depressive effects of sedative drugs on the central nervous system has led to increasing interest in a possible correlation between neurophysiological indices and the level of sedation. The most commonly used neurophysiological indices in the assessment of sedation are electroencephalogram (EEG) and auditory evoked potentials (AEPs), which measure different aspects of brain functioning. The evoked potentials show whether the central nervous system responds systematically to an auditory stimulus, and they may thus be considered a direct measure of the responsiveness of the brain. In contrast, the EEG signal, if not associated with a sensory stimulus, will only reflect the ongoing background electrical activity of the brain. In other words, if the patient is not stimulated and the level of sedation is measured using indices derived from the EEG signal, then it can be speculated that those indices may only be used as predictors of whether the patient will actually react to a given stimulus, but they provide no measure of responsiveness. AEPs may therefore provide a more accurate tool with which to assess the level of sedation. Within the AEPs, the middle-latency AEPs (10–50 ms after the stimulus) are mainly evoked by the physical features of the auditory stimulus. Their presence establishes the integrity of the afferent auditory pathway and confirms that basic auditory signal processing is taking place in the primary auditory cortex (Fig. 1a ). The long-latency AEPs, or event-related potentials (ERPs; >50 ms after the stimulus), result from deeper processing of the auditory stimulus and are generated by areas of cortex at and beyond the primary projection area. ERPs may therefore be better indicators of the effect of sedative drugs on the mental state than are middle-latency AEPs. The most prominent ERP component is N100, which appears about 100 ms after the onset of stimulus and reflects the simultaneous activation of several different brain regions, indicating detection of a change in acoustic surroundings (Fig. 1b ) [ 3 ]. Another ERP component, namely mismatch negativity (MMN), is elicited by infrequently presented stimuli that differ in some physical dimension from the standard stimuli and reflects the brain's automatic auditory change detection mechanism, which depends on the integrity of auditory sensory memory (Fig. 1c ) [ 4 ]. Appearance of MMN indicates that several brain regions are activated simultaneously. The fact that MMN reflects widespread brain activation may explain why sedative drug induced changes in the MMN have been shown to be a better marker of mental state than are the respective changes in the middle-latency AEPs [ 5 ]. ERPs have exhibited graded changes with increasing doses of sedative drugs in volunteers and surgical patients [ 6 , 7 ], but to date only few data are available concerning the use of ERPs for monitoring sedation level in the ICU. Despite the known superiority of ERP parameters over EEG parameters for monitoring sedation level, in this preliminary pilot study we hypothesized that both ERPs and EEG may be used to assess the level of sedation in a heterogeneous group of neurologically intact intensive care patients. Methods The study protocol was approved by the local ethical committee and written informed consent was obtained from each patient or from the next of kin. We measured EEG and ERPs in a heterogeneous group ( n = 19; 13 males and six females; age 65 ± 11 years) of mechanically ventilated patients presenting with a range of surgical and medical conditions requiring intensive care but with no known organic brain dysfunction (Table 1 ). Patients who were known to have impaired hearing were excluded from the study. Sedation was administered following the modified Brook protocol [ 1 ]. Repeated midazolam boluses were initially used to induce and maintain sedation. If the obtained sedation level was still considered inadequate, then propofol infusion was begun and midazolam administration discontinued. The optimal depth of sedation for each patient was determined on clinical grounds, independent of the study, and was assessed using the sedation–agitation scale (SAS; Table 2 ) [ 8 ]. At the time of the first EEG and ERP recordings, patients were receiving propofol sedation (infusion rate 1.91 ± 0.88 mg/kg per hour) and the duration of the infusion had exceeded 8 hours in all patients (31 ± 29 hours). Discontinuation of sedation was then considered necessary so that the patients could be weaned from the ventilator or so that their neurological status could be evaluated. Propofol infusion was interrupted, and the measurements were repeated once the sedation had subsided and the patients were able to follow commands (i.e. to open their eyes and squeeze their hand). Apart from propofol, no sedative drugs other than opioids were allowed during the 8 hours preceding the measurements or during the study period (Table 1 ). Electroencephalogram and event-related potential recording The EEG signal was recorded using Ag/AgCl electrodes placed on the scalp according to the international 10–20 system. Two electrode locations (frontal [Fz] and central [Cz]) were used. Both electrodes were referred to the right mastoid, and the electrode–skin impedances were kept below 5 kO. The EEG signal was amplified and digitized continuously at 279 Hz using EMMA (ERP measuring machine; developed and custom-made in the Department of Clinical Neurophysiology, Kuopio University Hospital, Kuopio, Finland). Background EEG was recorded for 5 min during sleep and/or while the patients lay motionless with their eyes closed. Auditory stimulation was then set to 'on' so that ERPs could be recorded. The stimulation was applied according to an oddball paradigm, which consisted of 85% standard (800 Hz) and 15% deviant (560 Hz) stimuli, with an interstimulus interval of 1 s. The duration of each stimulus was 84 ms, including 7 ms rise and fall times. Altogether 600 stimuli were delivered through earphones to the right ear for each measurement, corresponding to a recording time of about 10 min. The stimulus intensity was set at 75 dB. Electroencephalogram analysis The background EEG, measured before auditory stimulation, was band pass filtered using a finite impulse response-type filter employing cutoff frequencies of 0.5 and 32 Hz (Matlab, version 6.12; The Mathworks Inc., Natick, MA, USA). Then, the filtered EEG signal (5 min long) was cut into 5 s epochs with 50% overlap. Serious artifacts were excluded by checking the maximum amplitude for each epoch; if the amplitude was greater than 100 µV then the epoch was excluded. The appropriateness of artifact rejection was manually confirmed. For each EEG epoch, first the root mean squared (RMS) total power was calculated. Then, the epoch was subjected to power spectral density estimation, using Welsh's averaged periodogram method [ 9 ], and the spectral edge frequency 95% (SEF95) was computed from the power spectral density using a frequency range of 0.5–32 Hz. The mean of the RMS and SEF95 values of the accepted epochs were then individually computed. Event-related potential analysis The EEG signal recorded during the auditory stimulation was first filtered using a finite impulse response-type filter using cutoff frequencies of 1 and 20 Hz, and then transformed to epochs from -100 ms to +900 ms relative to the onset of each stimulus. After removing artifactual epochs (rejection level ± 100 µV), the individual responses to standard and deviant stimuli were averaged. The N100 component was defined as a maximum negative deflection appearing 80–150 ms from the stimulus onset. The amplitude and the latency of the prominent N100 components in response to standard stimuli were manually scored with respect to the pre-stimulus baseline. The MMN was obtained by subtracting first the waveform elicited by the standard stimuli from the one resulting from the deviant stimuli. The MMN was then computed from the difference curve (deviant standard) as the mean amplitude between 100 and 250 ms [ 10 ]. Statistical analysis We carried out exploratory analyses to determine which EEG and ERP parameters changed significantly in response to interruption to sedation. For this purpose, Wilcoxon signed rank test (nonparametric paired sample test) was applied to the N100 amplitude and latency values (in response to standard stimuli), MMN, RMS power and SEF95 values measured before and after interruption to sedation. Moreover, Kruskal–Wallis test (nonparametric counterpart of one-way analysis of variance) was used to test whether the ERP and the EEG parameters differed among the sedation levels present during propofol infusion. The effect of the total duration of propofol infusion on the studied parameters was assessed using Spearman's correlation coefficient. The recording channels Fz and Cz were studied separately. Data are expressed as mean ± standard deviation, unless otherwise indicated. All statistical analyses were done using the SPSS software (SPSS for Windows, version 11.0; SPAA Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant. Results During propofol infusion the sedation level for each patient was determined on clinical grounds. It varied from deep sedation (SAS score 2) to light sedation (SAS score 4). All patients were responsive and cooperative (SAS score 4) within 30 min after discontinuation of propofol. Weaning and extubation were successful in 10 patients, whereas sedation was electively restarted in the remaining nine patients. Of the ERP recordings, 2% and 5% were discarded as artifact during and after sedation, respectively. Accordingly, 8% and 20% of the background EEG recordings were discarded. Effect of interruption to propofol infusion The EEG parameters (RMS power and SEF95) and ERP parameters (N100 and MMN) measured before and after interruption to sedation did not differ between those patients who proceeded to weaning and extubation and those in whom sedation was restarted. The RMS power increased after interruption to sedation (Fz and Cz, P < 0.05; Fig. 2a,2b ), whereas the SEF95 values exhibited only a tendency toward a decrease (not significant; Fig. 2c,2d ). The amplitude of the N100 component (in response to standard stimuli) increased at both frontal (Fz, P < 0.05) and central recording sites (Fig. 3a,3b ). The latency of the N100 component (in response to standard stimuli) and the MMN did not change in response to interruption to propofol infusion. The MMN mean amplitude, which should be a negative value while awake, exhibited both positive and negative values after sedation had subsided (Fig. 3c,3d ). Effect of sedation level During propofol infusion, seven patients were deeply sedated (SAS score 2), seven patients were moderately sedated (SAS score 3) and five patients were lightly sedated (SAS score 4). The level of sedation did not influence EEG parameters. The amplitude of the N100 component (in response to standard stimuli) differed between sedation levels (Fz and Cz, P < 0.05), in contrast to N100 latency and MMN (Fig. 3 ). Both negative and positive MMN mean amplitudes were obtained independently of sedation level (Fig. 3c,3d ). Patient characteristics and duration or rate of propofol infusion did not differ among sedation level groups. Effect of propofol infusion duration None of the ERP and EEG parameters correlated with the total duration of propofol infusion. Discussion ERPs have exhibited graded changes with increasing doses of sedative drugs in volunteers and surgical patients [ 6 , 7 ], but to date no parallel studies have been conducted in severely ill patients. We assessed ERPs together with EEG parameters in a heterogeneous group of intensive care patients under sedation with propofol. The range of doses of sedative and analgesic drugs varied widely, but despite this our preliminary data suggest an association between clinical level of sedation and neurophysiological parameters. Our main findings were that the amplitude of the standard N100 component differed among the sedation levels during propofol sedation, and that the amplitude of the standard N100 in the frontal area as well as the RMS power increased in response to interruption to propofol infusion. We selected RMS power and SEF95 to describe the changes in the EEG spectrum related to the interruption to propofol infusion. The RMS power represents the total power of the signal and the SEF95 is the frequency below which 95% of the power in the EEG spectrum resides. Sedative doses of propofol have been shown to produce an increase in total, delta and beta activity in the EEG signal, especially in the Cz and Fz regions [ 11 - 13 ]. In our study the total power of the EEG signal was inversely related to sedation, increasing after interruption to propofol infusion. However, the SEF95 decreased in many patients under the same circumstances. This suggests that awakening was not paralleled by a prominent increase in the high frequency range, probably due to the decrease in beta activity related to interruption to propofol infusion. Administration of opioids might also have markedly modified the EEG pattern as compared with that observed during isolated propofol infusion. Identifiable ERPs may indicate an increased risk for auditory perception during general anaesthesia [ 14 , 15 ] and a positive outcome in coma patients [ 16 , 17 ]. During propofol sedation, the N100 component has been reported to decrease in amplitude and to delay in latency as compared with recording before the beginning of propofol infusion [ 5 ]. As sedation subsides, the opposite (amplitude increase and latency shortening) has been observed in surgical patients recovering from postoperative propofol sedation [ 7 ]. In the present study the N100 amplitude recovered similarly as the level of sedation subsided, although the amplitude values were markedly smaller than those of the surgical patients both during sedation and after sedation had subsided. Moreover, the MMN exhibited a large inter-individual variability and many patients had a positive MMN mean amplitude (Fig. 3c,3d ), suggesting that MMN was not present or could not be reliably measured. In our earlier study conducted in surgical patients [ 7 ], the MMN was present at comparable sedation levels. The small N100 amplitude and the absence of the MMN could have resulted from the use of medication other than propofol and opioids during the study period. We cannot exclude the presence of some level of sedative potentiation or side effects resulting from this medication, which might have affected the results. In all patients benzodiazepines were discontinued for a minimum of 8 hours before measurements were taken. However, some degree of residual sedative effect due to potentially impaired metabolism might have influenced our findings. Clifford and Buchman [ 18 ] reported that the combination of benzodiazepine and fentanyl affected information processing in response to novel and standard stimuli in a different manner than the combination of propofol and fentanyl in intensive care patients. Nevertheless, both of these drug combinations globally reduced the amplitudes of the responses to all stimuli as the sedative drug dose increased, in a manner similar to that in our study. We also speculate that, because of the short time allowed after propofol discontinuation, patients were still under influence of this drug during the later measurements. Thus, ERP parameters might not have had enough time to recover, even if the patients were awake and able to follow simple commands (SAS score 4). We did not study the effect of opioids on the ERPs in more detail because subanaesthetic doses of fentanyl [ 19 ] and remifentanil [ 20 ] have been shown not to attenuate the N100 component. In the intensive care setting, EEG parameters and ERPs are influenced not only by the administration of sedative drugs but also by the underlying illness, which may cause considerable changes in functioning of the sensory pathways [ 21 ]. Diagnosis and reason for intensive care varied considerably in our population. We excluded patients with known organic brain dysfunction from the study, but it is possible that some of the patients suffered from mild subclinical neurological deficits. However, because all patients woke up and were able to follow commands, we believe that possible brain dysfunction did not have a significant effect on our results. Moreover, no differences could be found in neurophysiological parameters between extubated patients and those whose sedation was continued electively. The statistical methods we applied deserve comment. We conducted exploratory analyses to determine which EEG and ERP parameters changed significantly because of interruption to sedation. Performing multiple comparisons, as we did, is known to increase the risk for type I error (i.e. obtaining significant differences by chance). However, because of both the exploratory nature of our analysis and the controversy concerning the Bonferroni method, we opted not to use this adjustment [ 22 , 23 ]. Furthermore, the heterogeneity of our patient group limits the power of statistical analysis. To overcome this limitation, we presented individual data points and used statistical analysis only to show trends in our findings. Conclusion In a group of intensive care patients, with heterogeneous diagnosis and reasons for intensive care, assessment of the level of sedation using spectral EEG alone may not be sufficiently accurate. Concomitant use of ERPs, especially the N100 component, which requires widespread activity and functional integrity of the brain, may provide better distinction between sedation levels. Neurophysiological methods may thus be useful complements to clinical sedation scales in the monitoring of sedation status over time in intensive care patients under controlled sedative drug administration. Key messages • The EGG alone may not be sufficiently accurate in the assessment of sedation levels in intensive care unit patients. • Concomitant use of ERPs, especially the N100 potential, may help to differentiate between sedation levels. • Neurophysiological methods may offer a complement to clinical sedation scales in neurologically intact intensive care patients. Abbreviations AEP = auditory evoked potential; Cz = central region; EEG = electroencephalogram; ERP = event-related potential; Fz = frontal region; ICU = intensive care unit; MMN = mismatch negativity; RMS = root mean squared; SAS = sedation–agitation scale; SEF95 = spectral edge frequency 95%. Competing interests The author(s) declare that they have no competing interests. Author's contributions HYP, SN, IK, JP and ER participated in the interpretation of the results and writing of the manuscript. HYP and SN performed data collection, data entry and statistical analysis.

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1065077

Perioperative factors determine outcome after surgery for severe acute pancreatitis

Introduction There is evidence that postponing surgery in critically ill patients with severe acute pancreatitis (SAP) leads to improved survival, but previous reports included patients with both sterile and infected pancreatic necrosis who were operated on for various indications and with different degrees of organ dysfunction at the moment of surgery, which might be an important bias. The objective of this study is to analyze the impact of timing of surgery and perioperative factors (severity of organ dysfunction and microbiological status of the necrosis) on mortality in intensive care unit (ICU) patients undergoing surgery for SAP. Methods We retrospectively (January 1994 to March 2003) analyzed patients admitted to the ICU with SAP. Of 124 patients, 56 were treated surgically; these are the subject of this analysis. We recorded demographic characteristics and predictors of mortality at admission, timing of and indications for surgery, and outcome. We also studied the microbiological status of the necrosis and organ dysfunction at the moment of surgery. Results Patients' characteristics were comparable in patients undergoing early and late surgery, and there was a trend toward a higher mortality in patients who underwent early surgery (55% versus 29%, P = 0.06). In univariate analysis, patients who died were older, had higher organ dysfunction scores at the day of surgery, and had sterile necrosis more often; there was a trend toward earlier surgery in these patients. Logistic regression analysis showed that only age, organ dysfunction at the moment of surgery, and the presence of sterile necrosis were independent predictors of mortality. Conclusions In this cohort of critically ill patients operated on for SAP, there was a trend toward higher mortality in patients operated on early in the course of the disease, but in multivariate analysis, only greater age, severity of organ dysfunction at the moment of surgery, and the presence of sterile necrosis, but not the timing of the surgical intervention, were independently associated with an increased risk for mortality.

Introduction Morbidity and mortality after surgery for severe acute pancreatitis (SAP) remain considerable, despite the introduction of new strategies to reduce infectious complications [ 1 , 2 ], such as antibiotic prophylaxis, early enteral nutrition [ 3 ], and the recognition of complications such as abdominal compartment syndrome in severely ill patients [ 4 ]. There is limited evidence in the literature that postponing surgery beyond the initial phase of the disease leads to improved survival. Mier and colleagues [ 5 ] randomized 36 patients to early versus late surgery, and stopped the study after an interim analysis showed that patients operated on early had a higher mortality. This finding has been confirmed by others in retrospective studies. Hungness and colleagues [ 6 ] found a trend toward an increased mortality in 14 of 26 patients who were operated on within the first two weeks of diagnosis. Hartwig and colleagues [ 7 ] found in a review of 62 surgically treated patients that those operated on within three days had a higher mortality rate (53% versus 22%, P = 0.02). In contrast, Fernández-del Castillo and colleagues [ 8 ] found a similar mortality rate in their patients when either operated on early or later than 6 weeks after admission. There are conflicting data on the impact of timing of surgery on mortality, and the different definitions used for early surgery, ranging from three days to six weeks, makes comparing the data in the literature difficult. All studies that reported increased mortality in patients undergoing early surgery included patients operated on for a range of indications (such as absence of clinical improvement after 3–5 days, persistent pancreatitis, infected necrosis, pancreatic abscess and sepsis syndrome) at different stages of the disease. It is not clear to what extent the severity of illness at the moment of surgery or the microbiological status of the necrosis were confounding factors and were a bias in finding increased mortality rates for early surgery. In this paper we report our study on the impact of the timing of surgical intervention and perioperative factors (severity of organ dysfunction and microbiological status of the necrosis) on mortality in patients undergoing surgery for SAP. Materials and methods Data collection We retrospectively (January 1994 to March 2003) analyzed all patients admitted with SAP to the intensive care unit (ICU) of the Ghent University Hospital, a tertiary referral centre with a total of 1060 beds. SAP was defined in accordance with the criteria described by the International Symposium on Acute Pancreatitis [ 9 ]. Patients were identified from the hospital registry with the use of the International Classification of Diseases (ICD-9-CM) code for acute pancreatitis' (577.0). Preoperative data collected included age, sex, etiology, use of antibiotics, C-reactive protein level, Ranson score and Acute Physiology And Chronic Health Evaluation (APACHE) II score [ 10 ] on admission. Time to the first surgical intervention, the severity of organ dysfunction at the day of the first surgical intervention (as assessed by the sepsis-related organ failure assessment (SOFA) score [ 11 ]), length of stay in the ICU and in the hospital, and mortality were retrieved from the patient's file. The occurrence of organ dysfunction during the ICU stay was recorded, and organ dysfunction was defined as follows (based on a score of 2 or more in the SOFA scoring system): (1) cardiovascular dysfunction was defined as hypotension requiring vasoactive medication; (2) renal dsyfunction, serum creatinine above 2.0 mg/dl; (3) respiratory dyscfunction, the need for mechanical ventilation or a PaO 2 /FIO 2 ratio of less than 300. Microbiological data collected included peroperative cultures from the initial surgical intervention, and fine-needle aspirates (FNAs), when available. Infected pancreatic necrosis was defined as the presence of microorganisms in cultures obtained at the first operation or in cultures of a FNA of the pancreatic necrosis without previous surgery; consequently, sterile pancreatic necrosis was defined as negative cultures from intraoperative cultures, independently of infections occurring later in the course of the disease. Mortality was defined as in-hospital mortality. The study was approved by the local ethical committee. Study design Patients treated surgically early in the course of the disease were compared with patients who underwent delayed surgical intervention. Early surgery was defined as surgery within 12 days of diagnosis, as described in the prospective trial by Mier and colleagues [ 5 ]. Furthermore, we compared patients with sterile pancreatic necrosis with patients with infected necrosis, and survivors with non-survivors, using univariate and multivariate analysis techniques. Patient management All patients were admitted to the ICU before or after surgical treatment and were treated by the same surgical team. The use of antibiotic prophylaxis was left to the discretion of the attending ICU physician. Enteral nutrition was started as early as possible. Computed tomography (CT) scanning and FNA of the pancreatic necrosis was performed on an individual patient base, namely when the clinical condition of the patient was suggestive of infection of the pancreatic necrosis. Indications for surgery were a documented infection of pancreatic necrosis (as evidenced by positive cultures from FNA), a deterioration of the clinical condition of the patient, unresolving pancreatitis or suspected pancreatic infection without proof on FNA or CT scan. Surgical intervention consisted of necrosectomy through a midline laparotomy as described by Beger and colleagues [ 12 ]. The pancreas was debrided using blunt dissection, and two to four large-calibre drains were inserted in the retroperitoneum. Continuous postoperative lavage of the retroperitoneum was started initially at a rate of 500–1000 ml/h, and progressively decreased, on the basis of the general condition of the patient, inflammatory parameters (C-reactive protein), and the macroscopic aspect of the drain effluent. Statistical analysis Statistical analysis was performed with SPSS for Windows 11.0.1 ® (SPSS, Chicago, IL, USA). Continuous variables were compared by using Student's t -test or the Mann–Whitney U -test where appropriate. Categorical data were compared with the ? 2 or Fisher Exact test. A double-sided P value of less than 0.05 was considered statistically significant. Parameters found to be different in survivors and non-survivors in univariate analysis with a P value of 0.25 or less were entered in a logistic regression model with mortality as the dependent variable, to identify factors available at the moment of surgery that were independently associated with mortality. Results Patients Of 124 patients with SAP, 56 (35 male, 21 female) were treated surgically. The mean age of the patients was 56 years (SD 13.5). The cause of the pancreatitis was biliary tract stones in 19 patients (33.9%), alcohol in 21 (37.5%), trauma in 6 (10.7%), hyperlipemia in 1 (1.8%) and idiopathic in 9 (16.1%). Thirty-nine patients (69.6%) were referred from other hospitals; for three patients the first surgical intervention was performed in the referring hospital 1 day before referral ( n = 2) or on the day of referral ( n = 1). Early versus late surgical intervention Twenty-two patients (39.2%) were operated on within the first 12 days of diagnosis of pancreatitis (median 5 days, interquartile range 3–9), and 34 (60.8%) later than 12 days after admission (median 20 days, interquartile range 17–31). Age and gender distribution were comparable in both groups (Table 1 ). Disease severity, assessed by Ranson and APACHE II scores on admission in these patients, was not different; neither was the SOFA score at the day of surgery. Indications for surgery in patients operated on early were different from those operated on later in the course of the disease. In patients operated on early, deterioration of multiple organ dysfunction syndrome (MODS) was the indication for surgical intervention in 41% of the patients, compared with 9% in the late surgery group. Overall, the length of stay in the hospital was significantly longer for the patients who underwent surgery late in the course of the disease, even after censoring the patients who died in both groups. Duration of ICU stay was not different. There was a trend toward a higher mortality in the early surgery group (55% versus 29%, P = 0.06). Microbiological status of necrosis and mortality In 26 (46.4%) patients, intraoperative cultures confirmed the diagnosis of infected pancreatic necrosis. Microorganisms isolated from the necrosis are listed in Table 2 . Gram-negative and Gram-positive microorganisms were present in comparable numbers (38.9%); seven patients had fungal infections at the first operation. In 10 patients more than one organism was isolated. Thirty of 56 patients (54%) had sterile pancreatic necrosis at the moment of the first surgical intervention. Patient characteristics, severity of disease, and the timing of surgery were not different in patients with sterile or infected pancreatic necrosis (Table 3 ). There was a trend toward a higher occurrence rate of organ failure in patients with sterile pancreatic necrosis, and mortality was significantly higher in patients with sterile necrosis (57%) than in patients with infected necrosis (19%) ( P = 0.004). Especially in patients undergoing early surgery, mortality was significantly higher in patients with sterile pancreatic necrosis (85% versus 11%, P = 0.001) In the patients who underwent delayed surgery, there was no difference in mortality between patients with sterile pancreatic necrosis and those with infected pancreatic necrosis (35% in patients with sterile pancreatic necrosis and 23% in patients with infected pancreatic necrosis, P = 0.71). Factors influencing outcome after surgical intervention Overall mortality in our patients was 39.2% (22 of 56 patients). Table 4 summarizes differences between survivors and non-survivors. In univariate analysis, patients who died were older, had higher APACHE II scores on admission, higher SOFA scores on the day of surgery, more often sterile necrosis, and more often organ dysfunction during their ICU stay, and were operated more often because of MODS. There was also a trend toward earlier surgical intervention in patients who died. The following variables were entered in a logistic regression analysis: age, SOFA score on the day of surgery, the presence of sterile pancreatic necrosis at surgery, and interval from diagnosis to surgical intervention as a continuous variable. SOFA score at the day of surgery was preferred to APACHE II score on admission and deteriorating MODS as an indication for surgery, because it better describes the severity of illness at the moment of surgery, and the difference in univariate analysis was more significant. In multivariate analysis, only age, SOFA score at the moment of surgery, and the presence of sterile necrosis were associated with mortality (Table 5 ). Discussion It has been suggested that postponing surgery beyond the initial phase of the disease leads to improved survival [ 5 - 7 ]. In this analysis of 56 patients undergoing surgery because of SAP, we found that disease severity at the moment of surgery, age, and the presence of sterile necrosis, but not early surgery, determined mortality. The trend toward an increased mortality in patients operated on within 12 days of diagnosis, found in univariate analysis, was apparently confounded by perioperative factors. Disease severity at admission has long been recognized as an important factor determining outcome in patients with SAP, irrespective of surgical intervention. So far, Ranson score at admission and C-reactive protein levels at 48 hours [ 13 , 14 ] have proven to be the best predictors of disease severity; more recently, the APACHE II score [ 15 ] and determination of the individual Ranson parameters [ 16 ] at 48 hours showed improved predictive value compared with admission scores. In patients undergoing surgery for SAP, perioperative organ dysfunction affects outcome. Connor and colleagues [ 17 ] reported that a high postoperative APACHE II score was the only factor associated with mortality in a group of moderately ill patients (initial APACHE II score 9) undergoing pancreatic necrosectomy. Hungness and colleagues [ 6 ] reported higher organ failure scores and more advanced age in patients who died after surgery for SAP. The present study further confirms these findings. The reason for this increased mortality is not clear. Surgical intervention by itself in the early phase of the disease is a possible explanation for the high mortality rate in patients undergoing early surgery, and has been suggested by several authors [ 6 , 7 ], but the evidence for this is indirect. It seems plausible that in the early stage of the disease, when there is peripancreatic and retroperitoneal inflammation, surgery is often difficult, with increased blood loss. Patients with severe organ dysfunction might also be more prone to other complications that could arise from the surgical intervention, such as gastrointestinal ischemia or blood loss. Another possibility is that in these patients other complications – that have only recently been recognized – were involved, and were left untreated for too long. Intra-abdominal hypertension and abdominal compartment syndrome are increasingly described in patients with SAP [ 4 , 18 ], and can lead to multiple organ dysfunction. Other problems such as relative adrenal insufficiency [ 19 ], which is increasingly recognized in patients with septic shock [ 20 ] or high-risk surgical patients [ 21 ], might be involved. The fact that sterile necrosis is a risk factor for mortality in patients undergoing surgery is an important finding. At first sight this might be in sharp contradiction of the fact that infected pancreatic necrosis has been associated with increased mortality in several studies. It should be kept in mind that this undoubtedly is true for patients with SAP as a whole, and that this analysis included only patients who were operated on. The findings of the present study are in line with the current concept that patients with sterile pancreatic necrosis do not need surgery, although this is still advocated by some experts in the field. Several authors have reported mortality rates below or about 10% when managing these patients non-operatively [ 22 - 24 ]. Le Mée and colleagues reported that, in most of their patients, organ dysfunction was reversible if necrosis remained sterile [ 25 ]. These and our results suggest that in patients with suspected infection of the pancreatic necrosis, the presence of microorganisms should be actively sought with ultrasound-guided or CT scan-guided FNA before surgical intervention is considered [ 26 ]. Our study could not reproduce the negative impact of early surgery on outcome after adjustment for other factors that were associated with increased mortality in univariate analysis. The often-used strategy to postpone surgery in patients with SAP is based on limited data. Mier and colleagues [ 5 ] randomized 36 patients with SAP to early (within 48–72 hours) versus late surgery (later than 12 days). Mortality in the group that underwent early debridement was 56%, 3.4-fold that in the control group, a result that halted the trial. This finding has also been reported by other investigators, but the definition of early surgery should be carefully considered, because the use of different time frames makes it very difficult to compare the evidence available in the literature. Fernandez-del Castillo and colleagues [ 8 ] analyzed 64 patients operated on with a technique of closed packing, and found that mortality in patients operated on within the first six weeks after onset of the disease was not different from mortality in patients operated on later than six weeks. This study included patients with pancreatic abscesses, a disease that has a different clinical course and prognosis from that of patients who require surgery for infected pancreatic necrosis. Patient selection and the definition of early surgery make it very difficult to compare this study with ours. Hartwig and colleagues [ 7 ] found a significantly higher mortality in patients operated on within 72 hours (53% versus 22%, P = 0.02) in 136 patients treated between 1980 and 1997, about half of them surgically. During the study period, indications for surgery gradually shifted from a lack of clinical improvement after 2–3 days to a suspicion of infected necrosis, resulting in patients being operated on later, and lower mortality rates. Over all, operating less, and if necessary, as late as possible, markedly improved outcome. From the data available in the literature, the advice to postpone surgery by default beyond the first 2–3 weeks seems to be based on unblinded, unadjusted, or retrospective analyses. A similar process has been observed with the use of prophylactic antibiotics. The use of these became widespread on the basis of limited evidence, but the benefit could not be demonstrated in a controlled randomized trial [ 27 ]. Although we agree that there are several pathophysiological considerations in deferring surgery, such as those described above, we did not find any evidence that the timing of surgery by itself influenced outcome. Conclusion Our data suggest that not the timing of the surgical intervention, but rather perioperative factors, determine mortality in critically ill patients undergoing necrosectomy for SAP. We found that mortality was associated with greater age, increasing severity of organ dysfunction, as expressed by the SOFA score at the moment of surgery, and the presence of sterile necrosis. In future studies on the effect of timing of surgery, the severity of organ dysfunction and microbiological status at surgery should be evaluated as possible confounding variables. Key messages • In a series of 56 patients who were treated surigically for severe acute pancreatitis, no effect of the timing of surgery was found if perioperative factors such as severity of illness and microbiological status of the necrosis were considered. Abbreviations APACHE = Acute Physiology And Chronic Health Evaluation; CT = computed tomography; FNA = fine-needle aspirate; ICU = intensive care unit; MODS = multiple organ dysfunction syndrome; SAP = severe acute pancreatitis; SOFA = sepsis-related organ failure assessment. Competing interests The author(s) declare that they have no competing interests. Authors' contributions JDW, UH and FC were responsible for the conception and design of the study. JDW and SB acquired a substantial portion of the data. JDW, EH and DV performed the analysis and interpretation of data. JDW and DV drafted the manuscript. FC, PP, BDH, JDC and EH undertook critical revision of the manuscript for important intellectual content. EH and SB were responsible for statistical expertise. FC performed supervision and took overall responsibility for all aspects of the project or study. All authors read and approved the final manuscript.

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1065097

Bispectral index versus COMFORT score to determine the level of sedation in paediatric intensive care unit patients: a prospective study

Introduction Most clinicians give sedatives and analgesics according to their professional experience and the patient's estimated need for sedation. However, this approach is prone to error. Inadequate monitoring of sedation and analgesia may contribute to adverse outcomes and complications. With this in mind, data obtained continuously using nonstimulating methods such as bispectral index (BIS) may have benefits in comparison with clinical monitoring of sedation. The aim of this prospective observational trial was to evaluate the use of electroencephalographic (EEG) BIS for monitoring sedation in paediatric intensive care unit (PICU) patients. Methods Forty paediatric patients (<18 years) were sedated for mechanical ventilation in a cardiac surgical and general PICU. In each paediatric patient BIS and COMFORT score were obtained. The study protocol did not influence ongoing PICU therapy. BIS and corresponding COMFORT score were collected three times for each patient. Measurements with the best starting EEG impedances were analyzed further. Deep sedation was defined as a COMFORT score between 8 and 16, and light sedation as a score between 17 and 26. Biometric and physiological data, and Pediatric Risk of Mortality III scores were also recorded. Results There was a good correlation (Spearman's rho 0.651; P = 0.001) between BIS and COMFORT score in the presence of deep sedation and low starting impedance. Receiver operating characteristic (ROC) analysis revealed best discrimination between deep and light sedation at a BIS level of 83. Conclusion In the presence of deep sedation, BIS correlated satisfactorily with COMFORT score results if low EEG impedances were guaranteed.

Introduction Most paediatric intensive care unit (ICU) patients need sedative and analgesic drugs during mechanical ventilation [ 1 ]. Sedatives and analgesics are given to improve comfort, to reduce pain, to facilitate aggressive ICU therapy (i.e. mechanical ventilation or insertion of intravascular lines) and to avoid accidental removal of medical devices. Most clinicians give sedatives and analgesics according to their professional experience and the patient's estimated need for sedation. Inadequate monitoring of sedation and analgesia may contribute to adverse outcomes and complications [ 2 ]. Only a few clinical scores have been validated for estimating the level of sedation in paediatric ICU patients. The best evaluated score is the COMFORT score [ 3 ]. The COMFORT score consists of eight categories and can evaluate a child's behaviour and physiological responses to discomfort, fear and pain in approximately 2 min. The COMFORT score is age independent because age-adapted physiological parameters are used. Apart from the examination of muscular tone, calculation of the COMFORT score does not require any stimulation of the patient. The COMFORT score can be divided into three groups. A score of 8–16 points corresponds to deep sedation, 17–26 indicates light sedation and 27–40 indicates inadequate sedation [ 4 ]. Common concerns about clinical sedation scales are that the employed parameters are susceptible to subjective interpretation and that information about the level of sedation can only be obtained intermittently. Deeper levels of sedation are difficult to assess using clinical sedation scores. Sufficient information cannot be obtained regarding over-sedation, which is associated with adverse outcomes, prolonged ICU stay and increased costs [ 5 - 7 ]. Additionally, patients treated with muscle relaxants cannot be evaluated using clinical rating scores. In paediatric ICU patients, neuromuscular blocking agents are used in approximately 6–16% of ventilatory support days [ 8 ]. A point of criticism regarding the COMFORT score is that physiological parameters such as haemodynamic indices and heart rate, which contribute to the score (Table 1 ), can be influenced by ICU therapy. Therefore, objective tools with which to measure the level of sedation are urgently needed so that over-sedation can be avoided and the level of sedation adjusted if muscle relaxants are given. With this in mind, data obtained continuously using nonstimulating methods such as bispectral index (BIS) may have benefits in comparison with clinical monitoring of sedation. BIS is a processed electroencephalographic (EEG) parameter that provides a measure of sedative levels on a relative scale [ 9 - 11 ]. For various agents (e.g. propofol and midazolam) it has been shown that the BIS may correlate with dose-dependent levels of anaesthesia [ 10 , 12 - 14 ] and ICU sedation [ 15 - 18 ]. Level of sedation and changes in memory function correlated well with BIS in volunteers [ 19 ]. The BIS monitor was initially designed to measure the level of consciousness in adults during anaesthesia. In paediatric patients age-specific changes in EEG activity could complicate interpretation of BIS measurements [ 20 ]. Nevertheless, the first reports of use of BIS in paediatric anaesthesia were promising [ 21 - 23 ]. Recently, McDermott and coworkers [ 24 ] investigated the use of BIS during sedation in children undergoing elective diagnostic or therapeutic procedures. Those investigators found good correlation between BIS and the University of Michigan Sedation Scale. In a paediatric intensive care unit (PICU) setting, four groups found a good to moderate correlation between BIS and clinical scores [ 25 - 28 ]. Unfortunately, those studies used clinical scores that have not been evaluated in paediatric patients [ 25 , 27 ], the investigators were not sufficiently blinded to the BIS results [ 25 , 26 , 28 ], or uncontrolled repeated measurements in one patient were included in the study [ 25 , 27 , 28 ]. The objective of the present study was to evaluate use of the BIS to monitor levels of sedation in paediatric ICU patients in a blinded and prospective manner, and to compare it with use of the COMFORT score. To avoid bias by repeating analysis of data from the same patient, each patient was included only once in the statistical analysis. In addition, we wished to focus on the age of the children and on quality of EEG data. Methods Following agreement from the local ethics committee and once informed consent from the legal guardians (parents) had been obtained, we studied 50 paediatric patients admitted to our PICU. All included patients met primary inclusion (age <18 years, requirement for mechanical ventilation) and exclusion criteria (brain trauma, any kind of end-stage disease, use of muscle relaxants or persistent postoperative relaxation according to train-of-four monitoring, intractable agitation). After enrolment patients were excluded if correct attachment of electrodes was impossible, impedances did not comply with the quality requirements of the manufacturer (<10 kΩ, signal quality index >0.8, less than threefold deviation between electrode impedances), or recording could not be repeated at least twice. The sedation and/or analgesic regimens were not controlled in the study and were administered in accordance with standard operating procedures in the PICU. After PICU admission, the study was started when all examinations on admission were completed and all sources of irritation were eliminated. Electrodes were placed at F7, F8 and Fp (reference), and one electrode was placed beside Fp (ground) in accordance with the international ten-twenty system [ 29 ]. Electrode sites were abraded using conventional alcohol swabs. Paediatric silver–silver–chloride self-adhesive ECG electrodes were applied (Blue Sensor Neonatal™ Medicotest, Friedberg, Germany). An Aspect A-1000™ monitor (software version 3.12; Aspect Medical Systems, Natick, MA, USA) was used to compute the BIS. As recommended by the manufacturer, electrode impedances were kept below 10 kΩ, deviations among electrode impedances less than threefold, and signal quality index above 0.8. Group assignment Patients were assigned post hoc into groups according to their level of sedation as measured using the COMFORT scale (deep sedation, light sedation, inadequate sedation) and into age groups (≤ 6 months and > 6 months of age). This age limit was used because in children younger than 6 months old [ 30 ] synchronization of the EEG is limited. Synchronization is among the columns of the BIS algorithm [ 11 ]. Study protocol The study protocol is summarized in Fig. 1 . After PICU admission, the first recording was started immediately after all necessary manipulations (examinations, laboratory specimens) were completed. If the data were of good quality (see above), then BIS values were sampled for 1 min and the median was calculated. A second investigator, who was blinded to the BIS results, assessed the patient clinically using the COMFORT scale [ 3 ]. Every patient was assessed three times, with a minimal interval of 1 hour between measurements. The set with the best impedance values was chosen for calculation of further statistics. To interpret the significance of the data quality, we also calculated the correlation between BIS and COMFORT score using the data couples with the poorest starting impedances. For each patient the following were also recorded: age, sex, medical diagnosis, Pediatric Risk of Mortality III score [ 31 ], medications administered, blood gases and temperature. Statistical analysis Correlations between the BIS and the COMFORT score were calculated for each group using the Spearman's rank order correlation coefficient. Because BIS and COMFORT score were classified on an ordinal scale, regression analysis and confidence intervals could not be calculated. Therefore, we calculated the coefficient of determination (r 2 ). The ability of BIS to discriminate between sedation levels as classified using the COMFORT scale was tested using 'receiver operating characteristic' (ROC) statistics. The cut-off point was determined at the point of greatest sensitivity and specificity for discrimination. A logistic regression model using the BIS was developed to predict sedation levels of the patients (deep versus light sedation) in accordance with the COMFORT classification. Data are expressed as mean ± standard deviation in the case of a normal distribution and interval scaling level, and as median (range) if the data were not distributed normally or in case of ordinal scaling level. The χ 2 and Fisher's exact tests were used to analyze categorical data and Mann–Whitney U-test for data on at least ordinal level. Data analysis was performed using SPSS statistical software (version 8.0; SPSS Inc., Chicago, IL, USA) and Microsoft Excel with Analyze It™ (version 1.48; Analyze-it Software Ltd, Leeds, UK) modification. Results A total of 53 paediatric intensive care patients met our primary inclusion criteria. Three patients were excluded because of inability to achieve lead impedances with less than threefold deviance with the first set. In 10 patients recording could not be repeated at least twice because extubation was performed during the period of study. Finally, we enrolled 40 PICU patients into the study. According to COMFORT scoring no child was inadequately sedated, resulting in two COMFORT groups (i.e. deep sedation and light sedation). Patient characteristics are presented in Table 2 . On comparing the sedation groups, we found that the parameters diagnosis ( P = 0.162), Pediatric Risk of Mortality III score ( P = 0.891), sex ( P = 0.770) and age ( P = 0.716) did not differ significantly between groups. We also found no significant differences in arterial carbon dioxide tension ( P = 0.750), body temperature ( P = 0.879) and type of medication (benzodiazepines, P > 0.99; opioids, P = 0.650; propofol, P = 0.286; ketamine, P > 0.99). For those data couples with the best impedance levels (3.3 ± 1.8 kΩ, range 0.3–7.8 kΩ), BIS and COMFORT score correlated significantly for all patients ( n = 40, P = 0.001; Spearman's rho: r = 0.651, r 2 = 0.42; Fig. 2 ) and for patients without ketamine (n = 38, P = 0.001; Spearman's rho: r = 0.668; r 2 = 0.45). The correlation between BIS and COMFORT for data couples with the worst impedances (5.1 ± 2.2 kΩ, range 1.9–9.9 kΩ) was poor ( P = 0.05; Spearman's rho: r = 0.387, r 2 = 0.15). All further results reported are for the data couples with the best impedance levels. Our results showed a significant correlation ( n = 29, P = 0.003; Spearman's rho: r = 0.525, r 2 = 0.28) for deeply sedated patients (COMFORT score 8–16) and for patients who had not received ketamine ( n = 27, P = 0.002; Spearman's rho: r = 0.565, r 2 = 0.32) whereas no correlation was found in the group with light sedation (COMFORT score 17–26; n = 11, P = 0.956; Spearman's rho: r = 0.019, r 2 < 0.01). ROC analysis identified the BIS index level that distinguished best between deep and light sedation (groups classified by COMFORT scores). The calculated cut-off point between the groups was at a BIS of 83, which had a sensitivity of 75.9% and a specificity of 81.8% (area under the curve 0.834, 95% confidence interval 0.699–0.968; Fig. 3 ). The logistic regression model using BIS as an explanatory variable was able to predict sedation according to COMFORT score correctly for 80% of the children. This total percentage is derived from correct predictions in 90% of deeply sedated patients (COMFORT score <17, n = 29) and in 55% of lightly sedated patients (COMFORT score 17–26, n = 11). In patients younger than 6 months ( n = 21, range 0.7–5.7 months) BIS and COMFORT score correlated significantly better ( P < 0.001 in the younger group versus P = 0.041 in the older group; n = 19, range 6.3 months–16 years) and with a higher correlation coefficient (Spearman's rho: in younger patients r = 0.781, r 2 = 0.61 versus in older children r = 0.473, r 2 = 0.22; Fig. 4 ). The correlation was slightly better in older children when the two patients who had received ketamine were excluded from the analysis (Spearman's rho: n = 17, P = 0.030; r = 0.527, r 2 = 0.28). Discussion Most sedation scoring systems use responses to stimuli [ 32 ] and/or patient appearance and physiological variables [ 3 ] to estimate the level of sedation. These scores must be interpreted subjectively or, in case of physiological parameters, can be influenced by ICU therapy. In contrast to clinical scoring systems, the BIS system generates information continuously and objectively. The BIS monitor was developed to assess intraoperative depth of anaesthesia and to avoid awareness in adults. Data from studies that were not blinded sufficiently [ 25 , 26 , 28 ] or that employed scores that have not been evaluated in the PICU setting [ 25 , 27 ] suggested a moderate correlation between BIS and clinical scoring systems. The aim of this prospective, blinded study was to determine whether BIS is a useful tool for assessing the level of sedation in critically ill paediatric patients. For statistical calculation only one data set (BIS versus COMFORT score) per patient was evaluated. Therefore, bias introduced by including multiple observations from one patient was avoided. In the study we compared BIS with the COMFORT score, which was previously validated in the PICU setting [ 3 , 4 ]. The study indicated that there was a moderate correlation between BIS and corresponding COMFORT scores (r 2 = 0.42). Subanalysis revealed a distinctly better coefficient of determination during deep sedation (r 2 = 0.28) than with light sedation (r 2 < 0.01) for the assessment tools. This was confirmed by the binary logistic regression findings, which revealed a good ability of BIS to predict deep levels of sedation (90%). According to COMFORT score, BIS could predict this level among lightly sedated children in only 55% of the cases. The overall percentage of correct prediction was 80%. Using ROC analysis we found a BIS value of 83 to distinguish best between light and deep sedation as assessed using the COMFORT score. Probably because of the exclusion of agitated children, we did not observe under-sedation in the study. We were therefore unable to differentiate further between lightly sedated and under-sedated children. This could be interpreted as an investigational bias. Furthermore, poor EEG data quality could cause a large number of movement artifacts, resulting in low signal quality, and we cannot exclude the possibility that movement artifacts contributed to the lower coefficient of determination for lighter sedated children than for children under deeper sedation. This was reported in adult settings [ 33 , 34 ]. Our data support the findings of previous studies [ 25 - 28 ]. Berkenbosch and colleagues [ 25 ] compared BIS with three simultaneously measured clinical sedation scores (Ramsay Sedation Score [RSS], Tracheal Suctioning Score, and Pediatric Intensive Care Unit Sedation Score) in paediatric patients (age 5.7 ± 6.1 years, range 1 month–20 years). None of the studied scores was clinically validated for use in paediatric ICU patients. The BIS monitor correlated moderately with clinically assessed sedation levels (r 2 = 0.12, 0.08 and 0.21, respectively). BIS was found to differentiate reliably between adequate and inadequate sedation (cut-off BIS 70), but it was relatively insensitive in differentiating between adequate and over-sedation (cut-off BIS 50). Critical aspects in the study conducted by Berkenbosch and coworkers are that different individuals performed the sedation assessments, which might have resulted in considerable interobserver variability. The nurses assessing the level of sedation were not formally blinded to the BIS results. Multiple sets of data derived from single patients were included in same analysis, which might have influenced the results. Twenty-four patients were included in the study, but measurements were repeated 18 ± 14 times per patient. Crain and colleagues [ 26 ] also used the COMFORT score to estimate the level of sedation. Those investigators studied 31 patients (age 53 ± 11 months, median 25 months; no range presented) and selected each patient's lowest and highest BIS measurement for further investigation. The direct coefficient of determination between BIS and COMFORT score was only moderate (r 2 = 0.26), which reflected their finding that some patients exhibited good correlation whereas others did not. After grouping BIS results into four levels of sedation, a high coefficient of determination (r 2 = 0.89) with the COMFORT score resulted. Grouping of BIS values was conducted according to results formerly derived from adult data, but it is not proven whether this procedure is appropriate in children. Aneja and coworkers [ 27 ] studied 24 patients without neuromuscular blockade, comparing BIS with RSS. They found a high and significant coefficient of determination (r 2 = 0.77; age 6.3 ± 2.9 years, range 1–16 years). The calculated cut-off point for distinguishing between over-sedation (RSS 6) and comfortable sedation (RSS 2–5) by ROC analysis was a BIS level of 42. Under-sedation (RSS 1) was found at a BIS level in excess of 76. Another component of that study dealt with patients receiving neuromuscular blockade (age 8.4 ± 3.7 years, range 0.5–19 years). According to BIS, the authors observed a significant number of patients suffering from inadequate sedation, which would not have been detectable by clinical investigation. Limitations of the study were the inclusion of multiple observations per patient and that the RSS has not been validated for use in paediatric patients, as mentioned above. Courtman and coworkers [ 28 ] recently compared BIS and COMFORT score in critically ill children (mean age 3.9 ± 4.5 years). Those investigators found a moderate coefficient of determination (r 2 = 0.26) between BIS and COMFORT score in 25 neurologically normal children and a weak coefficient of determination (r 2 = 0.06) in 15 children who were classified as being neurologically abnormal. In that study BIS could discriminate between light and deep levels of sedation. In conformance with the findings of Berkenbosch and coworkers [ 25 ], Courtman and colleagues found that BIS was unable to discriminate between deep and very deep levels of sedation. The significance of this study is also limited by the inclusion of multiple observations per patient. In our study the quality of EEG impedance appeared to have a major impact on correlation between BIS and COMFORT score. Although within the limits recommended by the manufacturer, we found only a low coefficient of determination (r 2 = 0.15) between COMFORT score and corresponding BIS in case of higher impedances (5.1 ± 2.2 kΩ). This emphasizes the importance of good data quality. On the basis of our experience, we would advise use of impedance values of less than 5 kΩ. Unexpectedly, correlation between the methods was better in children younger than 6 months. Patient basic data do not explain this finding. The impact of age on BIS is still debated, with divergent findings reported in the anaesthesia literature [ 21 , 35 ]. Davidson and coworkers [ 35 ] compared BIS with the corresponding consciousness level during emergence from anaesthesia in a prospective, blinded manner in children (≥1 year old) and infants (<1 year old) undergoing elective circumcision. BIS increased significantly as sevoflurane concentrations decreased in children, but a similar relationship was not demonstrated in infants. Adult EEG sensors were used in that study for all patients, which could partly account for the difference in findings between that study and ours. There are no additional data in the paediatric ICU literature. Because different scoring systems are used for clinical estimation of the level of sedation, it is difficult to compare our findings with those of other investigators. The end-points of sedation are neither defined consistently nor comparable between the studies mentioned above. In our study COMFORT scores corresponded to a wide range of BIS values. In other words, the BIS index does not always reflect the expected clinical/subjective level of sedation. This observation is in agreement with the experience of other groups who compared neurophysiological parameters (i.e. BIS, somatosensory evoked magnetic fields, evoked potentials) with clinical sedation scores in adult patients [ 36 - 38 ] and with the other paediatric ICU studies [ 25 - 28 ]. This could be related to the fact that neurophysiological parameters and clinical sedation scores measure different attributes. BIS automatically matches certain EEG patterns to clinical states that are found in adult volunteers under sedation or anaesthesia. In contrast, clinical scores are used to summarize the investigator's impression of whether the patient is comfortable in the ICU setting. An extreme example would be the patient who is awake with a high BIS score but who appears to be completely comfortable. The limitations of clinical scores in estimating the level of sedation have been discussed broadly [ 32 ]. In addition to these limitations, in the case of the COMFORT score there is a specific limitation caused by the inclusion of physiological data (heart rate and blood pressure). When physiological parameters are used in a heterogeneous population such as paediatric ICU patients, it is difficult to define reference data. If changes in physiological parameters caused by sedatives are to be interpreted, then a standardized sedation regimen and co-medication are needed. In our opinion this is not the case in the ICU setting. The majority of children included in the present study were postoperative cardiac surgical patients. Most of them had reduced cardiac function and required catecholamines. In this group of patients in particular, haemodynamic variability could not be attributed solely to sedative drugs. The COMFORT score cannot distinguish between very deep stages of sedation, whereas with neurophysiological methods such as the BIS this might be possible. EEG measurements often fail to provide correct values with very light or absent sedation because motor activity and skeletal muscle tone increase. However, this situation will never fall within the domain of electrophysiological monitoring because clinical estimation will be sufficient in unsedated or only lightly sedated patients in the ICU setting. Data from the investigations cited above and our data suggest that the BIS monitor might be useful in the case of moderate to deep levels of sedation. Our group found that BIS levels below 83 had good correlation with clinical estimation. Berkenbosch and coworkers [ 25 ] suggested that a moderate level of sedation could be achieved at BIS values between 50 and 70, and deep sedation at levels below 50. This corresponds well with the data presented by Aneja and coworkers [ 27 ], who found that a BIS above 76 indicated inadequate, light sedation and that a BIS below 46 marked very deep sedation or over-sedation. Whether BIS can detect over-sedation remains controversial. Berkenbosch and coworkers [ 25 ] and Courtman and colleagues [ 28 ] found that BIS could not discriminate between deep and very deep levels of sedation. In our opinion, this finding could indicate that clinical scores are not useful for identifying very deep levels of sedation. Few data exist for paralyzed PICU patients. In this situation clinical scores are not applicable because they need muscular activity to be present to rate the level of sedation. Aneja and coworkers [ 27 ] stated that the RSS and bedside nurse assessments are inadequate for monitoring the depth of sedation in paralyzed children, and concluded that BIS is a useful adjunct for assessing sedation in paralyzed patients. In our opinion use of electrophysiological monitoring tools such as the BIS is imperative in paralyzed patients to prevent levels of sedation that are too light or too deep. Furthermore, the terms 'under-sedation' and 'over-sedation' should be used carefully because the level of sedation required depends on the individual patient's needs. Bearing this in mind, clinician should adapt the level of sedation to the demand of the patient. When deeper levels of sedation are needed, BIS can help by avoiding undesired levels of sedation. In situations when muscular paralysis is necessary, BIS could make a valuable contribution because established clinical tools fail to measure the level of sedation in this setting. Conclusion Our data indicate that an impedance level below 5 kΩ is needed for valid interpretation of BIS values in PICUs. At such impedance levels the BIS index correlates well with the COMFORT sedation score, in particular in children with deeper levels of sedation. Discrimination between light and moderate sedation with high sensitivity and specificity was possible at a BIS level of 83. The BIS monitor provides continuous measurements of the level of sedation without having an impact on patient comfort. It may be a useful adjunct in clinical routine and may be especially helpful in certain situations when clinical estimation fails (e.g. if muscular paralysis is necessary). Key messages • In daily clinical practice of paediatric ICU therapy, the BIS monitor provides continuous measurements of sedation level without having an impact on the patient's comfort. • In certain situations when clinical estimations fail (i.e. if muscular paralysis is necessary), electrophysiological monitoring tools such as BIS should be considered imperative to prevent inadequately light or inadequately deep sedation. • In the case of deep sedation, BIS correlated satisfactory with the COMFORT score results if low EEG impedances were guaranteed. Abbreviations BIS = bispectral index; EEG = electroencephalography; ICU = intensive care unit; PICU = paediatric intensive care unit; ROC = receiver operating characteristic; RSS = Ramsay Sedation Score. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors contributed to the design, conduct, analysis and interpretation of the research reported. CS and WK were principal investigators and led the conceptual design of the study. MS and GN assisted with data collection and analysis, and with manuscript preparation.

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Cellular infiltrates and injury evaluation in a rat model of warm pulmonary ischemia–reperfusion

Introduction Beside lung transplantation, cardiopulmonary bypass, isolated lung perfusion and sleeve resection result in serious pulmonary ischemia–reperfusion injury, clinically known as acute respiratory distress syndrome. Very little is known about cells infiltrating the lung during ischemia–reperfusion. Therefore, a model of warm ischemia–reperfusion injury was applied to differentiate cellular infiltrates and to quantify tissue damage. Methods Fifty rats were randomized into eight groups. Five groups underwent warm ischemia for 60 min followed by 30 min and 1–4 hours of warm reperfusion. An additional group was flushed with the use of isolated lung perfusion after 4 hours of reperfusion. One of two sham groups was also flushed. Neutrophils and oedema were investigated by using samples processed with hematoxylin/eosin stain at a magnification of ×500. Immunohistochemistry with antibody ED-1 (magnification ×250) and antibody 1F4 (magnification ×400) was applied to visualize macrophages and T cells. TdT-mediated dUTP nick end labelling was used for detecting apoptosis. Statistical significance was accepted at P < 0.05. Results Neutrophils were increased after 30 min until 4 hours of reperfusion as well as after flushing. A doubling in number of macrophages and a fourfold increase in T cells were observed after 30 min until 1 and 2 hours of reperfusion, respectively. Apoptosis with significant oedema in the absence of necrosis was seen after 30 min to 4 hours of reperfusion. Conclusions After warm ischemia–reperfusion a significant increase in infiltration of neutrophils, T cells and macrophages was observed. This study showed apoptosis with serious oedema in the absence of necrosis after all periods of reperfusion.

Introduction Ischemia–reperfusion injury in lung tissue is a common problem in medical practice, with sometimes severe consequences such as acute respiratory distress syndrome (ARDS) and a high mortality rate for the patient. Some causes of warm ischemia–reperfusion injury are cardiopulmonary bypass during cardiac surgery and pulmonary sleeve resection. In contrast, lung transplantation is the main example of partial cold ischemia–reperfusion injury. Neutrophils are known to be one of the cell types responsible for tissue damage in many ways. First, they are able to deliver toxic radicals that damage pulmonary endothelium directly or indirectly by activating caspase-3, which results in apoptosis [ 1 , 2 ]. Second, they can damage pulmonary endothelium and parenchyma by delivering elastase and other proteases [ 3 ]. Third, the cell membrane of activated neutrophils becomes rigid and adhesion between neutrophils and endothelial adhesion molecules occurs, resulting in sequestration and a 'no-reflow phenomenon' [ 4 , 5 ]. The role of neutrophils in pulmonary ischemia–reperfusion injury has also been investigated in experiments in which neutrophil depletion was induced and by the inhibition of tissue infiltration. The role of the neutrophil is currently still controversial [ 6 - 9 ]. The role of macrophages has been investigated in several transplantation models [ 3 , 10 , 11 ]. Eppinger and colleagues have specified chemical mediators of reperfusion injury by using antibodies against cytokines. Although some mediators seemed to be required during the early phase of ischemia–reperfusion injury, only tumor necrosis factor-α (TNF-α) is involved in the evolution of late ischemia–reperfusion injury. These cytokines are released from activated macrophages probably as a result of acute lung reperfusion [ 10 ]. These results suggest a role for macrophages in the early reperfusion phase and a role for activated and recruited neutrophils in the late reperfusion phase [ 3 ]. Currently, the role of lymphocytes in ischemia–reperfusion injury remains unclear. Qayumi and colleagues concluded that upregulation of MHC II on peripheral lymphocytes is related to the degree of damage caused by ischemia–reperfusion [ 12 ]. Apoptosis, necrosis and alveolar oedema, representing alveolar permeability, are morphological changes of ischemia–reperfusion-induced lung injury. Fischer and colleagues were the first to describe apoptosis of specifically type II alveolar pneumocytes resulting from pulmonary ischemia–reperfusion in a human lung transplantation study [ 13 ]. In summary, little is known about the role of neutrophils, T cells and macrophages in ischemia–reperfusion injury. In preparation for studies investigating the specific role of infiltrating cells, the aim of this study was to specify the type of infiltrating cells and their sequence after 1 hour of warm ischemia followed by 30 min to 4 hours of reperfusion in a model of acute lung injury, which was defined by quantifying apoptosis and alveolar oedema. Materials and methods Animals Male inbred Wistar rats (mean weight 225 g), obtained from Iffa Credo (Brussels, Belgium), were used for all experiments. Animals were treated in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23, revised 1985). The rats were transported in sterile conditions, housed in suspended mesh-wired cages and fed ad libitum with a standard pellet diet (standard rat chow; Hope Farms, Woerden, The Netherlands). The Ethical Committee of the University of Antwerp approved the experimental protocols. Study design Fifty rats were randomized into eight groups. Five groups underwent 1 hour of warm lung ischemia followed by 30 min, 1, 2, 3 and 4 hours of reperfusion, respectively ( n = 7 in each group). One sham group underwent the identical surgical procedure without ischemia–reperfusion ( n = 4). To find out whether adhesion of the inflammatory cells had occurred, the lungs in one extra group were flushed with 6% buffered hetastarch after 1 hour of ischemia and 4 hours of isolated lung perfusion ( n = 7) [ 14 ]. This group was compared with a sham group, which was also flushed ( n = 4) (Fig. 1 ). Induction of ischemia–reperfusion Anesthesia was induced by 4% isoflurane in a mixture of oxygen (O 2 ) and nitrous oxide (N 2 O) in a ratio of 1:3 for 4 min. Intubation was performed with a 16-gauge Insyte-W catheter using translaryngeal illumination in accordance with the technique described by Hendriks [ 14 ]. After the rats had been connected to the ventilator, the N 2 O : O 2 ratio was set to 1:1 and the concentration of isoflurane was titrated to 0.5–1.5% according to muscle relaxation, heart rate and pupil size. To prevent thrombosis in lung vasculature during ischemia, 100 IU/kg heparin was infused into the left femoral vein 5 min before the left lung hilum was clamped. After left posterolateral thoracotomy through the fourth intercostal space, a rib retractor was placed to luxate the left lung anteriorly. Ischemia was induced by clamping the left lung hilum with two occluding curved microvascular clamps (Kleinert-Kurz WK65145) without further dissection. One clamp was placed in a cranial–caudal direction and the other clamp was placed laterally in the opposite direction. In a separate experiment, four rats received intravenous and bronchial injection of methylene blue solution to test the vascular and bronchial occlusion obtained by the microvascular clamps. Complete vascular and bronchial occlusion was achieved. To simulate physiological circumstances, the thoracotomy incision was closed in layers after the introduction of a 16-gauge catheter connected to a 50 ml syringe into the left chest cavity. When animals recovered, the chest tube and endotracheal tubes were removed. Ten minutes before reperfusion, anesthesia was induced and rats underwent a left thoracotomy with the use of the same incision as described above. Reperfusion occurred on removal of the clamps. The left thoracotomy was closed as described above. Ten minutes before the end of reperfusion time anesthesia was induced and the rat underwent a left thoracotomy for the third time followed by left pneumonectomy. Ten seconds before the rat was killed, maximal inflation of the left lung was achieved by occlusion of the expiratory ventilation cannula for 3 s to prevent inter-animal variation of inflation of the left lung. After reperfusion all rats underwent intramuscular injection of tramadol for pain control. To prevent cooling, rats were placed on a warm-water pad during the operation and under a heating light during both ischemia and reperfusion. Rectal temperature was measured before clamping of the left lung hilum and before killing and was held constantly between 36.8 and 37.4°C. Rats in the sham group underwent an identical surgical procedure except for clamping the left lung hilum. Rats in this group were killed 1 h after anterior luxation of the left lung. Flush procedure To study cellular adhesion to the endothelium, lungs of one more group were flushed after 4 hours of isolated lung perfusion with buffered starch. This procedure has been extensively described previously [ 15 , 16 ]. In brief, after ischemia–reperfusion, the pulmonary artery and vein were clamped with curved microvascular clamps. A 16-gauge angiocatheter was placed through the chest wall. A PE-10 perfusion catheter (Clay Adams, Parsippany, NJ, USA) was introduced into the chest through the angiocatheter and secured by a 4/0 silk suture after insertion into the pulmonary artery. Perfusate (6% buffered starch) was delivered through this catheter for 4 min at 0.5 ml/min. In addition, a pulmonary venotomy was performed to discard the venous effluent. Killing and tissue storage At killing, the left lung was taken out of the rat and cut caudal–cranially into four pieces. The lateral sample was fixed in metacarn for 4 hours at room temperature (23°C) and stored in 70% ethanol at 4°C. Directly after killing, the weight of the medial sample was measured and the sample was put into an oven at 65°C for 5 days to assess the wet : dry ratio as a parameter for lung oedema. The middle samples were fixed in chloroform calcium for 90 min at room temperature and then stored in buffer (10 ml of distilled water, 1 g of CaCl 2 , 0.121 M cacodylate) at 4°C until further processing. Killing was performed by a cut down of the superior caval vein. Sample processing Tissue samples for light-microscopic investigations were dehydrated with propan-2-ol, cleared with toluene and embedded in paraffin wax. Sections 4 μm thick were stained with hematoxylin/eosin stain (H&E) for neutrophil count. Immunohistochemistry was applied for macrophage and T cell visualization. After deparaffination, endogenous peroxidase was blocked by incubation in 0.9% H 2 O 2 for 15 min. The sections were incubated overnight with CD-3-specific antibody 1F4 (Pharmingen, Becton Dickinson, Erembodegem, Belgium) or with antibody ED-1 (Serotec, Diagnostic Products Cooperation, Humbeek, Belgium) directed against lysosomal membrane glycoprotein on macrophages. Incubation for 30 min with secondary biotinylated horse anti-mouse antibody (Vector, Burlingame, CA, USA) was followed by incubation for 1 hour with peroxidase-labeled avidin–biotin complex (Vector). The slides were developed in 3,3-diaminobenzidine with 0.03% H 2 O 2 or 3-amino-9-ethylcarbazole (AEC) with 0.006% H 2 O 2 for 30 min. Finally, counterstaining was performed in methyl green and Haemaluin Carazzi to reveal T cells and macrophages, respectively. Light-microscopy investigation All slides were evaluated in random order. The first field was chosen at random and the next fields in accordance with a standard pattern. Neutrophils were counted in 20 fields per slide (0.95 mm 2 per slide, magnification ×500). Macrophages were counted in 30 fields per slide (5.65 mm 2 per slide, magnification ×250). T cells were counted in 20 fields per slide (1.54 mm 2 per slide, magnification ×400). Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated (TdT) dUTP nick end labelling (TUNEL) staining. Deparaffinization was performed as described above. After decalcification with 3% citrate dissolved for 1 hour at 37°C, sections were incubated with TdT (Roche, Brussels, Belgium) in combination with fluorescein isothiocyanate-labelled dUTP nucleotides (AP Biotech, Roosendaal, The Netherlands) for 1 hour at room temperature. Furthermore, incubation with anti-fluorescein isothiocyanate (Dako, Glostrup, Denmark) peroxidase was performed followed by subsequent washes and the specimens were stained in AEC and counterstained with Haemaluin Carazzi. Only cells with TUNEL-positive nuclear and no cytoplasmic staining were considered to be apoptotic. Cells containing positive cytoplasmic staining were not counted. TUNEL-positive fragments closely ordered in a group were defined as apoptotic bodies. Apoptotic bodies and cells were both counted in 20 fields per slide (0.23 mm 2 per slide, magnification ×800). The occurrence of necrosis was investigated in H&E by a pathologist (EvM) who did not have any knowledge of details of the study. Oedema was twice assessed blindly at H&E and was graded, ranging from mild, moderate to severe. Mild oedema was defined as no to slight exudation within the alveolar space (Fig. 2a ). Severe oedema was defined as easily recognizable full exudation in the alveolar space (Fig. 2b ); moderate oedema was defined as being between mild and severe. Statistics All statistics were performed with SPSS 9.0 for Windows. Cellular infiltrates and apoptosis were evaluated statistically with the Kolmogorov–Smirnov test to confirm normal distribution. Analysis of variance and Student's t -test were applied to compare data obtained from the different reperfusion periods with the sham groups. Graded oedema frequencies were analyzed with the χ 2 test by comparison of the reperfusion groups with the sham groups. Statistical significance was accepted at P < 0.05. Results Cellular infiltrations Neutrophils (H&E, magnification ×500) A significant increase in neutrophils was observed after 30 min to 4 hours reperfusion compared with the sham group ( P < 0.01) (Fig. 3 ). After 4 hours of reperfusion followed by flushing, significantly more neutrophils were counted than in the flushed sham group ( P = 0.003), whereas no significant difference was observed compared with 4 hours of reperfusion without flushing ( P = 0.10). Macrophages (ED-1, magnification ×250) Significantly more macrophages were counted after 30 min of reperfusion ( P = 0.0002), 1 hour ( P = 0.004) and 2 hours ( P = 0.007) of reperfusion compared with the sham group (Fig. 4 ). A significant decrease was observed after 1 hour of reperfusion compared with 30 min of reperfusion ( P = 0.01). After 3 hours ( P = 0.06) and 4 hours ( P = 0.61) of reperfusion no significant increase in macrophages was observed compared with the sham group. T cells (1F4, magnification ×400) A fourfold increase of T cells was observed after 30 min of reperfusion ( P = 0.0002) compared with the sham group (Fig. 5 ). This increase was also significant after 1 hour of reperfusion ( P = 0.004). From 2 hours to 4 hours no significant increase was observed. Injury evaluation Apoptosis (TUNEL, magnification ×800) and necrosis (H&E) Significantly more apoptotic cells were seen after 1 hour ( P = 0.03), 2 hours ( P = 0.01), 3 hours ( P = 0.04) and 4 hours ( P = 0.00004) of reperfusion (Fig. 6 ). The number of apoptotic bodies was significantly higher after 4 hours of reperfusion ( P = 0.0006). Necrosis was not observed in any group. Oedema (H&E) Histological examination showed significantly more alveolar oedema after 30 min, 2, 3 and 4 hours of reperfusion ( P < 0.0001) compared with the sham group (Fig. 7a ). However, after 1 hour of reperfusion, oedema was not significantly increased compared with the sham group. The wet : dry ratio was significantly increased in all groups (30 min, P < 0.05; 2 hours, P < 0.01; 3 hours, P < 0.001; 4 hours, P < 0.01) except for 1 hour of reperfusion (Fig. 7b ). Discussion In this study a significant increase in neutrophils was observed after 1 hour of warm ischemia followed by 30 min to 4 hours of reperfusion. A first peak was shown after 30 min of reperfusion and a second peak after 3 hours of reperfusion. Furthermore, after 4 hours of reperfusion, significantly more neutrophils were observed after pulmonary artery flushing than in the flushed sham group. This resulted in flushing of cells that did not adhere to the endothelium. These results suggest activation and adhesion of neutrophils to the endothelium. Our observations are partly in contrast with results of Eppinger and colleagues, who showed a bimodal pattern of lung injury after 90 min of warm ischemia, with a first peak after 30 min of reperfusion and a second peak after 4 hours of reperfusion [ 17 ]. In their report, myeloperoxidase activity, representing neutrophil sequestration, diminished during the reperfusion time course. Neutrophil depletion did not have a protective effect on microvascular permeability after 30 min of reperfusion but the authors did show a protective effect after 4 hours, suggesting an early neutrophil-independent phase and a late neutrophil-dependent phase [ 17 ]. The observation of late neutrophil-dependent lung injury is indirectly related to our observation that significantly more neutrophils were counted after flushing of non-adhesive cells, suggesting activation of these cells. The role of macrophages has been investigated only in transplantation models [ 3 , 10 , 11 ]. Our data show significantly more macrophages after 30 min to 2 hours of reperfusion, which is in accordance with data from Eppinger. Using the permeability index Eppinger showed an attenuation of reperfusion injury using antibodies against monocyte chemoattractant protein-1, TNF-α and interferon-γ, suggesting that reduced early reperfusion injury is probably due to suppression of macrophage function [ 10 ]. A recent report by Maxey and colleagues confirmed the central role of macrophages in early reperfusion injury. They demonstrated significantly less lung injury in TNF-α-deficient mice after 1 hour of ischemia and 1 hour of reperfusion, suggesting that TNF-α is a key initiating factor in acute lung injury [ 18 ]. Fiser has made a distinction between the role of donor macrophages on the one hand and the role of recipient macrophages on the other. Activation of donor macrophages could be the initial consequence of ischemia and early reperfusion. In reaction to activation, donor macrophages deliver cytokines, chemotactic agents and proteolytic enzymes responsible for early reperfusion injury [ 3 , 11 ]. Subsequently, early lung injury activates the inflammatory mechanisms of the recipient [ 10 ]. Beside augmentation of neutrophils and macrophages, our study also showed a fourfold ( P = 0.0002) increase in T cells after 30 min to 1 hour of reperfusion, followed by a rapid attenuation. Because of the short duration of reperfusion it is unlikely that local proliferation of lymphocytes occurred, suggesting that chemotaxis is responsible for these observations. However, it is not clear that activation of these cells happened because of the rapid attenuation after 2 hours of reperfusion. This finding implies that the early augmentation of lymphocytes is just a non-specific inflammatory reaction on early reperfusion injury. The role of T cells was investigated recently in a model of mouse lung perfusion with fresh blood [ 19 ]. The interaction between allogenic blood lymphocytes and vascular endothelial cells is correlated with high expression of mRNA of both adhesion molecules and TNF-α in the perfused lung, suggesting that antigen-dependent activation of lymphocytes had occurred [ 19 ]. To our knowledge the present study is the first to show apoptosis in the absence of necrosis in lung tissue after warm ischemia–reperfusion. An explanation for the absence of necrosis after 4 hours of reperfusion might derive from the length of reperfusion. Experiments with longer reperfusion periods will be necessary to confirm this hypothesis. The number of apoptotic bodies is significantly increased after 1–4 hours of reperfusion, whereas the number of apoptotic cells is significantly increased after 4 hours of reperfusion. The tendency of apoptosis to increase is in accordance with observations of Fischer and colleagues in a human transplantation study with 1–5 hours of cold ischemia that showed significant increases in the number of apoptotic cells after reperfusion, in a time-dependent manner [ 13 ]. In particular, alveolar type II pneumocytes seemed to be apoptotic [ 13 ]. Stammberger and colleagues reported a peak of apoptotic cells after 18 hours of cold ischemia and 2 hours of reperfusion followed by a quick decrease in apoptotic cells as a function of reperfusion time [ 20 ]. The rapid attenuation of apoptotic cells is probably due to the occurrence of apoptosis after 6–12 hours of preservation and especially necrosis after 18–24 hours of preservation as described by Fischer and colleagues [ 21 ]. Furthermore, an inverse correlation of the occurrence of necrosis with oxygenation was shown, implying the necessity of preventing necrosis [ 21 ]. This study showed an identical pattern of alveolar oedema in a function of time by using a histological examination (H&E) and assessment by wet : dry ratio. An important increase of alveolar oedema was observed after 30 min, 2, 3 and 4 hours of reperfusion. We do not have an explanation for the absence of significant oedema after 1 hour of reperfusion. However, a bimodal pattern of lung injury reported by Eppinger and colleagues [ 17 ] is confirmed by our results. Using the vascular permeability of 125 I-labeled bovine serum albumin, Eppinger and colleagues showed an increased presence of serum albumin in bronchoalveolar lavage after 90 min of warm ischemia followed by a first peak after 30 min of reperfusion and a second peak after 4 hours of reperfusion, indicative of damage to the normal vascular/airway barrier [ 17 ]. Pulmonary ischemia results histologically in alveolar oedema due to changing permeability at the blood/air barrier after only 30 min of reperfusion. Apoptotic cells appear after 4 hours of reperfusion in a warm model of ischemia–reperfusion and after 6–9 hours of reperfusion in a transplantation model, whereas necrosis is observed after 18–24 hours of reperfusion related to an inverse correlation with oxygenation [ 21 ]. It may be noticed that these observations are related to a clinical feature known as ARDS. Clinical ARDS is characterized by acute hypoxemic respiratory failure due to non-cardiogenic pulmonary oedema caused by increased permeability of the alveolar capillary barrier, resulting in mortality ranging from 35% to 44% [ 22 ]. On the basis of the results of this study, research has to be focused on how cellular infiltrates are involved in the occurrence of ARDS and in what manner intervention might diminish the damaging effect of pulmonary ischemia–reperfusion. Conclusion This study has shown a significant increase in neutrophils after 30 min to 4 hours of reperfusion as well as after reperfusion followed by flushing. Macrophages doubled in number in lung tissue after ischemia–reperfusion. A fourfold increase in T cells in lung tissue after 1 hour of warm ischemia and 30 min of reperfusion was observed. Furthermore, apoptosis in the total absence of necrosis was shown together with important alveolar oedema. Key messages • Significant early increase of T-cells macrophages and neutrophils after 1 hour of ischemia and 4 hours of reperfusion • Significant late increase of neutrophils after 1 hour of ischemia and 4 hours of reperfusion. • Significant apoptosis and lung oedema in the absence of necrosis after 1 hour of ischemia and 4 hours of reperfusion. Abbreviations AEC = 3-amino-9-ethylcarbazole; ARDS = acute respiratory distress syndrome; H&E = hematoxylin/eosin stain; TNF = tumor necrosis factor; TUNEL = TdT-mediated dUTP nick end labelling. Competing interests The author(s) declare that they have no competing interests. Authors' contributions BVP and JH performed all surgical procedures under the supervision of PVS. BVP and VP performed histological analyses of the lung specimens under the supervision of EvM and MDB. VP also performed statistical analyses. BVP drafted the manuscript and was advised by JK. All authors read and approved the final manuscript.

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1065104

Effect of magnesium sulfate administration on blood–brain barrier in a rat model of intraperitoneal sepsis: a randomized controlled experimental study

Introduction Permeability changes in the blood–brain barrier (BBB) and their possible contribution to brain edema formation have a crucial role in the pathophysiology of septic encephalopathy. Magnesium sulfate has been shown to have a protective effect on BBB integrity in multiple experimental models. In this study we determine whether magnesium sulfate administration could have any protective effects on BBB derangement in a rat model of sepsis. Methods This randomized controlled experimental study was performed on adult male Sprague–Dawley rats. Intraperitoneal sepsis was induced by using the infected fibrin–thrombin clot model. To examine the effect of magnesium in septic and sham-operated rats, a dose of 750 μmol/kg magnesium sulfate was given intramuscularly immediately after surgery. Control groups for both infected and sham-operated rats were injected with equal volume of saline. Those rats surviving for 24 hours were anesthetized and decapitated for the investigation of brain tissue specific gravity and BBB integrity by the spectrophotometric assay of Evans blue dye extravasations. Another set of experiments was performed for hemodynamic measurements and plasma magnesium level analysis. Rats were allocated into four parallel groups undergoing identical procedures. Results Sepsis significantly increased BBB permeability to Evans blue. The dye content of each hemisphere was significantly lower in the magnesium-treated septic rats (left hemisphere, 0.00218 ± 0.0005; right hemisphere, 0.00199 ± 0.0007 [all results are means ± standard deviation]) than in control septic animals (left hemisphere, 0.00466 ± 0.0002; right hemisphere, 0.00641 ± 0.0003). In septic animals treated with magnesium sulfate, specific gravity was higher (left hemisphere, 1.0438 ± 0.0007; right hemisphere, 1.0439 ± 0.0004) than in the untreated septic animals (left hemisphere, 1.0429 ± 0.0009; right hemisphere, 1.0424 ± 0.0012), indicating less edema formation with the administration of magnesium. A significant decrease in plasma magnesium levels was observed 24 hours after the induction of sepsis. The dose of magnesium that we used maintained the baseline plasma magnesium levels in magnesium-treated septic rats. Conclusions Magnesium administration attenuated the increased BBB permeability defect and caused a reduction in brain edema formation in our rat model of intraperitoneal sepsis.

Introduction Patients with severe sepsis often manifest symptoms of encephalopathy. Acute alterations in mental status, which occur fairly frequently in septic patients, have been shown to be associated with poor prognosis [ 1 ]. However, not much is known about the exact mechanism of brain injury in sepsis. Studies have suggested that septic encephalopathy might involve a disturbance of plasma and brain neutral amino acid transport across the blood–brain barrier (BBB), similar to those seen in porto-systemic encephalopathy. This process has been related to the breakdown of the BBB because patients with septic encephalopathy have high protein levels in the cerebrospinal fluid [ 2 ]. Recently, derangements in the BBB causing perivascular edema have been demonstrated in sepsis-induced pigs [ 3 ]. Protective effects of magnesium sulfate (MgSO 4 ) against BBB breakdown after severe insulin-induced hypoglycemia have been reported in animals [ 4 ]. Similar effects of magnesium on BBB were also evident in a diffuse traumatic brain injury model in rats [ 5 - 7 ]. In summary, MgSO 4 was shown to have a protective effect on BBB integrity in multiple experimental models. We hypothesized that MgSO 4 will also protect against BBB derangements observed in sepsis and tested the hypothesis in a rat model of sepsis induced by an intraperitoneally inserted infected fibrin–thrombin clot. Methods One hundred and twenty-six male Sprague–Dawley rats weighing 320–440 g were used in this study. Rats were purchased from the Institute for Experimental Research and Application (Istanbul Medical Faculty), and were cared for before and during all stages of the experimental protocol in compliance with the applicable institutional guidelines and regulations of the Institute for Experimental Medicine Research and Application. Rats were prepared for surgery under anesthesia with intramuscular 100 μg/g ketamine (Parke-Davis, Morris Plains, NJ, USA) and 20 μg/g xylazine hydrochloride Rompun 2% (Bayer, Munich, Germany) and allowed to breathe spontaneously. The loss of corneal reflex and no movement in response to a painful stimulus confirmed maintenance of adequate anesthesia for the experimental procedure. The rats were subsequently randomized into one of four groups: sham control (C), sham control MgSO 4 -treated (C-Mg), septic (S) and septic with MgSO 4 (S-Mg). Intraperitoneal sepsis was induced with the infected fibrin–thrombin clot model described by Mathiak and colleagues [ 8 ]. Fibrin–thrombin clots were formed by adding 2 ml of 1% sterile fibrinogen solution, 1 ml of a bacterial suspension (1.8 × 10 9 colony-forming units/ml [infected] or vehicle [sterile 0.9% NaCl]) and 160 μl (100 units/ml) of sterile human thrombin to a 5 ml syringe. The resulting clot was then incubated at room temperature for 30 min before implantation into the abdominal cavity. The Escherichia coli strain was isolated from an intra-abdominal collection from a patient with secondary peritonitis. The bacteria were inoculated into a brain heart infusion broth (DIFCO Laboratories, Detroit, MI, USA) and incubated overnight at 35°C. The count of E. coli was adjusted to 1.8 × 10 9 colony-forming units/ml with McFarland standard 6. After making a 0.5 cm midline abdominal incision, the peritoneum was opened and the prepared clot was injected into the peritoneal cavity directly from the syringe. Sham-operated rats had a sterile clot injected into their peritoneal cavity. To examine the effect of magnesium in septic and sham-operated rats, a dose of 750 μmol/kg MgSO 4 was given intramuscularly immediately after surgery. Control groups for both infected and sham-operated rats were injected with an equal volume of saline. After surgery, the animals were given 50 μl/g per hour of saline subcutaneously and were allowed to wake up while breathing spontaneously. They were returned to their cages and were allowed free access to water. Those rats surviving for 24 hours after the surgery were anesthetized and decapitated for the investigation of brain tissue specific gravity (SG) and BBB integrity. We used the method described by Mikawa and colleagues [ 9 ] to determine BBB integrity by Evans blue (EB) dye. EB dye (4 ml/kg, 2%) was administered intravenously and allowed to circulate for 60 min. The animals were then perfused with saline through the left ventricle at a pressure of 110 mmHg until colorless fluid was obtained from the right atrium. Afterwards, the brains were removed and dissected. Each hemisphere was weighed and the samples were then homogenized in 3.5 ml phosphate-buffered saline and vortex-mixed for 2 min after the addition of 2.5 ml of 60% trichloroacetic acid to precipitate protein. The samples were then cooled for 30 min and centrifuged for 30 min at 1000 r.p.m. The absorbance of the supernatants for EB dye was measured at 610 nm with a spectrophotometer. EB dye content is expressed as μg/mg of brain tissue against a standard curve. The method defined by Marmarou and colleagues was used for the determination of SG [ 10 ]. We obtained 1 mm 3 samples taken from the right and left hemispheres of each animal. Samples were placed into linear density gradient columns of kerosene and bromobenzene. A calibration curve was determined for each column by using anhydrous K 2 SO 4 solutions of known SG (1.045, 1.040, 1.035 and 1.025). Brain tissue SG values were subsequently determined with this calibration curve. Another set of experiments were performed for hemodynamic measurements and plasma magnesium level analysis. These rats were allocated into four parallel experimental groups with identical procedures. Right femoral artery catheterization was performed under general anesthesia for blood pressure monitoring and blood sampling. Blood samples (0.5 ml) were taken for the determination of plasma magnesium levels at baseline (T0) and 24 hours (T24) after the induction of sepsis, and an equal volume of saline was given. Mean arterial pressure was recorded at baseline and 2, 3, 4, 8, 12 and 24 hours after the surgical procedure. Four of 12 rats in group S and 3 of 11 rats in group S-mg died within 24 hours of the induction of sepsis. Data for these rats were excluded from the study. We continued to enter rats with a balanced randomization sequence until we had eight surviving rats for each group. Statistical analysis The results are expressed as means ± standard deviation. EB dye content, brain tissue SG, serum magnesium levels, mean arterial pressures and heart rates were compared among four groups with a Kruskal–Wallis analysis of variance followed by Dunn's multiple comparisons test. A Mann–Whitney U -test and a Friedman nonparametric repeated-measures test were used for within-group comparisons. Paired serum magnesium levels were compared within each group by using a Wilcoxon signed rank test. Mortality rate was compared between septic groups receiving and not receiving magnesium with a χ 2 test. A probability ( P ) of less than 0.05 was considered significant. Results Thirteen of 29 rats in group S and 10 of 26 rats in group S-Mg died within 24 hours after the induction of sepsis, whereas all of the rats in groups C and C-Mg survived. The mortality rate was not statistically different between septic rats receiving and not receiving magnesium (in the experimental groups, χ 2 = 0.229, P = 0.632; in the monitoring groups, χ 2 = 0.100, P = 0.752). Both groups of septic rats appeared ill as demonstrated by exudates around nose and eyes, tachypnea and decreased spontaneous movement. Sham-operated rats seemed grossly normal and were active within their cages. Changes in mean arterial pressure are summarized in Figure 1 . A significant decrease was observed 2 hours after the induction of sepsis in groups S and S-Mg. No further changes in blood pressures were observed with the administration of magnesium in the control and sepsis groups. Plasma magnesium levels were comparable between groups at baseline (Table 1 ). A significant decrease in plasma magnesium levels was observed 24 hours after the induction of sepsis. An intramuscular dose of 750 μmol/kg MgSO 4 maintained the baseline plasma magnesium levels in magnesium-treated septic rats. Quantitative estimation of the EB dye revealed that sepsis significantly increased BBB permeability as measured by EB extravasations into brain tissue. In the S-Mg group, BBB permeability was significantly decreased in comparison with the S group (Table 2 ). The SG of both hemispheres taken from sepsis-induced rats were significantly less than the sham-operated rats, indicating the formation of brain edema after the induction of sepsis (Table 3 ). Brain tissue SG measurements in the magnesium-treated septic rats were significantly higher than in the untreated sepsis group. Within-group comparisons indicated no difference between the right and left hemispheres. Discussion The results of the present study demonstrate that treatment with magnesium immediately after experimental sepsis attenuated BBB permeability and the extent of brain edema formation. Alterations of BBB permeability with subsequent brain edema formation are common features of septic encephalopathy. Several hypotheses for the pathogenesis of septic encephalopathy have been discussed in the literature: metabolic derangement, direct bacterial invasion of the central nervous system, the effect of endotoxin on the brain, or altered cerebral macrocirculation and microcirculation [ 11 - 16 ]. Recent evidence implicates the changes in the BBB permeability that favor brain edema formation in the pathophysiology of septic encephalopathy [ 3 , 17 ]. In our model the BBB permeability defect induced by sepsis, as demonstrated by the EB dye extravasation technique, is consistent with previous reports demonstrating a loss of BBB integrity as a result of a septic challenge; however, the change in the SG representing brain tissue edema formation was relatively minor. Although the small change in SG that we obtained in the sepsis group reached statistical significance, indicating some amount of edema formation with the induction of sepsis, it is not possible to relate the edema formation to the disturbed integrity of the BBB. Our results are consistent with previous reports on the integrity of the BBB and the role of a permeability defect in the formation of cerebral edema using other models of cerebral damage [ 18 - 20 ]. In our previous experimental study we evaluated the effects of magnesium on brain edema formation and BBB breakdown after closed-head trauma in rats [ 5 ]. Our results of BBB breakdown by the measurement of EB dye extravasation were comparable with those that we obtained in our sepsis model; however, the changes in SG were higher in the traumatic brain injury model than in our sepsis model. This might be explained by the different mechanisms causing BBB breakdown and edema formation in trauma and sepsis. The discrepancy between brain edema and BBB permeability defect in sepsis might also indicate a low grade of permeability defect due to the complex cascade of sepsis, which is not enough to create edema as such in trauma. Another possible explanation might be that the quantitative determination of BBB permeability defect by EB dye extravasation is more sensitive than the SG method for determining brain edema. Other methods have been used to determine BBB damage in septic encephalopathy. In rodents with sepsis, colloidal iron dioxide [ 21 ], 14 C-labelled amino acids [ 22 ] and 125 I-labelled albumin [ 23 ] have been shown to pass from the circulation into the brain parenchyma in a similar manner to that seen in portosystemic encephalopathy. However, there is no evidence in the literature to suggest that this damage is related to edema formation in sepsis. Most recently, morphologic changes have been showed in the frontal cortex of a pig model of sepsis [ 3 ]. Fecal peritonitis resulted in severe perimicrovessel edema that was associated with swelling and rupture of astrocyte endfeet. Although this was suggested as evidence for the breakdown of the BBB, the ultrastructure of intercellular tight junctions seemed morphologically intact in pigs with sepsis. The authors have suggested that some other mechanism might be involved in the formation of edema. It is not known whether edema formation is related to BBB breakdown or other factors in sepsis. The exact mechanism and the relation between BBB breakdown and edema formation in sepsis-induced brain injury need to be further evaluated by more sensitive methods. A major finding of the present study is that magnesium administration attenuates the increase in BBB permeability and edema formation. The exact mechanism of magnesium's beneficial effect on the integrity of the BBB is unclear. However, magnesium can affect many aspects of the mediator cascade that can cause a permeability defect in the BBB. Alternatively, magnesium can act directly on the BBB. Magnesium's cytoprotective effect to reduce the profound breakdown of the BBB was first demonstrated in a rat model of severe insulin-induced hypoglycemia [ 4 ]. In this study it was speculated that magnesium might exert this effect through suppression of the endothelial cells. It was suggested that even before magnesium reaches the brain site it interacts with the endothelial cells forming the BBB and inhibits their activation [ 4 , 24 , 25 ]. To our knowledge, the present data are the first to show the positive effects of magnesium on sepsis-induced BBB permeability changes. Although this might have clinical significance, a contrary suggestion could be that increasing the integrity of the BBB might also have negative effects in terms of antibiotic emergence when the clinical situation is complicated with encephalitis or meningitis. In our model of intra-abdominal sepsis, the cultures of brain specimens taken after the experiment were all sterile (data not shown). One of the major pitfalls in the interpretation of the data was the difficulty of establishing a dose response for magnesium. In our present study the dose and the timing of magnesium administration were chosen with reference to our previous experiments on traumatic brain injury [ 5 ]. This dose of magnesium was determined as an optimum dose showing the best neurologic outcome in a traumatic brain injury model [ 26 ]. Plasma magnesium levels decreased significantly with the induction of sepsis and returned to nearly control levels with the dose of magnesium that we administered. However, it is known that the plasma magnesium level does not represent tissue magnesium content, and the lack of correlation between plasma magnesium and total body magnesium content in healthy subjects has already been reported [ 27 ]. More recently, it was demonstrated that free magnesium levels in brain tissue is a sensitive method that reflects magnesium homeostasis in a traumatic brain injury model [ 28 ]. Although we do not know to what extent the plasma magnesium levels represent brain tissue levels in the present study, our data show that significant beneficial effects are achievable with the dose administered. However, future studies will be needed to establish a dose response by measuring free magnesium levels in brain tissue for the effects of magnesium therapy in sepsis-induced brain injury. Conclusion This investigation shows that sepsis increases BBB permeability and leads to the formation of brain edema in septic rats. Magnesium administration attenuated the increased BBB permeability and caused a reduction in brain edema formation in our rat model of intraperitoneal sepsis. The precise mechanisms and the pharmacodynamics of magnesium administration in sepsis-induced brain injury need further investigation. Key messages • Sepsis causes BBB permeability defect. • Magnesium attenuates the increased BBB permeability associated with sepsis. Abbreviations BBB = blood–brain barrier; EB = Evans blue; SG = specific gravity. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors were responsible for study design and implementation of the experiment. Study data were collected by TE, DA and FE. Results were analyzed by FE and TE. The manuscript was written by FE and TE; all authors participated in revisions and gave approval to the final draft for submission for publication.

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1065106

Differentiating midazolam over-sedation from neurological damage in the intensive care unit

Introduction Midazolam is used routinely to sedate patients in the intensive care unit (ICU). We suspected that midazolam over-sedation was occurring in the ICU of the Guy's and St. Thomas' Trust and that it could be difficult to differentiate this from underlying neurological damage. A sensitive assay for detecting midazolam and 1-hydroxymidazolam glucuronide (1-OHMG) in serum was developed and applied in the clinical setting. Methods In the present study we evaluated a series of cases managed in a mixed medical, surgical and trauma ICU. Serum was collected from 26 patients who received midazolam, were 'slow to wake' and in whom there was suspicion of neurological damage. Patient outcome was followed in terms of mortality, neurological recovery and neurological damage on discharge. Results Out of 26 patients, 13 had detectable serum levels of midazolam and/or 1-OHMG after a median of 67 hours (range 36–146 hours) from midazolam cessation. Of these 13 patients in whom midazolam/1-OHMG was detectable, 10 made a full neurological recovery. Of the remaining 13 patients with no detectable midazolam/1-OHMG, three made a full neurological recovery; 10 patients were subsequently found to have suffered neurological damage ( P < 0.002), eight of whom died and two were discharged from the ICU with profound neurological damage. Conclusion These findings confirm that prolonged sedation after midazolam therapy should be considered in the differential diagnosis of neurological damage in the ICU. This can be reliably detected by the assay method described. The effects of midazolam/1-OHMG persist days after administration of midazolam has ceased. After prolonged sedation has been excluded in this patient group, it is highly likely that neurological damage has occurred.

Introduction Midazolam is an intravenous sedative that is commonly used during ventilation in critical illness. It is often regarded as the sedative of choice in the intensive care unit (ICU). According to the findings of our recent electronic survey (93% respondents) [ 1 ], midazolam is still routinely used in the UK as a sedative in ICUs. When used as a single dose, midazolam's pharmacological characteristics appear favourable, with a rapid onset of action and a short plasma elimination half-life. Midazolam is 94–98% bound to plasma albumin and has a volume of distribution of 1.7 l/kg in healthy individuals [ 2 ]. It is extensively metabolized first via cytochromes p450, 3A4 and 2B6 to 1-hydroxymidazolam, before undergoing glucuronidation to form 1-hydroxymidazolam glucuronide (1-OHMG), which has sedative properties and is excreted in the urine [ 3 , 4 ]. A wide interpatient variability in the pharmacokinetic properties of midazolam in critically ill patients with multiple organ failure has been reported [ 5 ], which can lead to prolonged sedation after midazolam therapy is stopped. However, there are other important causes of patients being 'slow to wake'; of these, it is most important to identify severe neurological damage. Patients with multiple organ failure are at high risk for neurological damage because they frequently have episodes of hypotension and dysrhythmia, and may have significant coagulopathy during the course of their critical illness. We suspected that some patients in our ICU, particularly those with renal impairment, were becoming over-sedated with midazolam and the active metabolite 1-OHMG, and that this was complicating the neurological assessment of 'slow to wake' patients. We previously developed a rapid assay for measuring midazolam and its glucuronide metabolite simultaneously [ 1 ]. This short report describes the usefulness of this assay for identifying midazolam over-sedation and its potential use as a predictor of eventual neurological recovery. Methods The assay was available for clinical application in the ICU. To differentiate between midazolam over-sedation and neurological damage, consultant intensivists requested detection of midazolam and 1-OHMG in serum. This request was normally made during the morning ICU ward round. The patients studied were those who had received intravenous midazolam therapy by continuous infusion either before (e.g. in operating theatres) or during the course of their ICU admission, and who were 'slow to wake' and in whom there was clinical suspicion of neurological damage. Arterial blood (2 ml) was collected from each patient via an in situ arterial catheter. The time of sample collection and the midazolam administration history, including cessation time, were recorded. A specific assay utilizing high-performance liquid chromatography coupled to mass spectrometric detection was used for simultaneous detection and quantification of midazolam and 1-OHMG [ 1 ]. Mass spectrometry allowed identification of midazolam and 1-OHMG individually based on their isotopic patterns. The studies were performed on the basis of clinical need, and in all cases they were requested by the consultant intensivist, normally during the morning ward round. The quantified serum level of midazolam and 1-OHMG could be reported to the medical team after a minimum of 2 hours so that they could consider the findings in their decisions regarding further clinical intervention. In practice, morning requests were available for interpretation by the evening round. Unit characteristics The ICU at Guy's and St. Thomas' National Health Service Trust is a 30-bed, level 3 unit that serves a mixture of medical, surgical, trauma, oncology and haematology patients. It has an average of 100 admissions per calendar month. For the year from March 2003 to April 2004, the mean Acute Physiology and Chronic Health Evaluation II score (day 1) was 18.5 ± 7.3, with a hospital mortality of 32.5% and a median length of stay of 5 days (variance 189.5, maximum 246). Patient characteristics All patients appeared to be deeply sedated at the time that the sample was taken, with a Glasgow Coma Scale score of less than 5. They were considered 'slow to wake' from either a pharmacological and neurological cause if, in the absence of a focal neurological deficit, consciousness did not return within 36 hours of stopping sedation. Patients were deemed to have regained consciousness if they both opened their eyes and moved their limbs in response to commands. Studies were conducted in 26 patients who had received midazolam sedation therapy by continuous intravenous infusion and in whom neurological damage was considered clinically possible (e.g. a hypoxic event was noted during cardiac surgery). The mean age of these patients was 63 ± 16 years, and the median time from cessation of midazolam therapy to serum collection was 67 hours (range 36–146 hours). The median daily midazolam dose was 4 mg/hour (range 2–20 mg/hour). The reasons for ICU admission are described in Table 1 . We followed the clinical outcomes of these patients in terms of mortality, neurological recovery and neurological damage on discharge. If no midazolam or 1-OHMG was detected, then a series of standard clinical and diagnostic tests was undertaken to determine whether neurological damage was likely. These included the response to painful stimuli and computed tomography of the head. In patients in whom midazolam or 1-OHMG was detected, tests were deferred until either the patients awoke or levels became undetectable. Results Midazolam and/or 1-OHMG were detected in the serum of 13 of the 26 patients (referred to as the midazolam-positive group). Of these 13 patients, 10 made a full neurological recovery; nine of these patients were discharged from the ICU and one later died as a result of critical illness but with intact neurological function. The remaining three patients died without regaining consciousness as a result of neurological damage. In contrast, neurological damage was observed in 10 of the remaining 13 patients who had no detectable serum concentrations of midazolam and/or 1-OHMG (midazolam-negative group). Midazolam-positive patients were significantly less likely to have experienced neurological damage (χ 2 test [degrees of freedom = 1]: P < 0.002). Twelve of the midazolam-positive patients had serum midazolam concentrations between 16 and 650 ng/ml, with a median value of 30 ng/ml, whereas the remaining patient's level exceeded the upper limit of the assay (3000 ng/ml). 1-OHMG was detected at a mean of 6800 ± 3432 ng/ml (range 3121–11,525 ng/ml) in the serum of six of the 13 midazolam-positive patients. All six of these patients exhibited a degree of renal impairment (defined as serum creatinine >130 μmol/l; Table 1 ), four of whom required renal replacement therapy in the form of continuous venovenous haemofiltration (employing an ultrafiltration rate of between 1500 and 3000 ml/hour). 1-OHMG was not detected in any of the midazolam-negative patients. Of the 13 midazolam-negative patients, eight died without regaining consciousness as a result of neurological damage, and two were discharged from the ICU with significant neurological impairment and required prolonged neurological rehabilitation. None of these 10 patients had responded appropriately to painful stimuli when in the ICU. In addition, in seven of these patients structural neurological damage was detected by computed tomography scan. Only three out of 13 patients in this group of midazolam-negative patients left the ICU with no neurological deficit. Other sedative and opiate agents Out of 26 patients, 15 were administered fentanyl by continuous intravenous infusion at a dosage between 0 and 300 μg/hour. In the 15 patients the fentanyl infusion was ceased at a minimum of 56 hours and a maximum of 120 hours before sample collection. In 25 of the 26 patients we could find no documented evidence of administration of sedative and opiate agents for a minimum of 36 hours before serum sample collection. The remaining patient, in the midazolam-negative group, was receiving 30 mg/day of the sedating antihistamine chlorphenamine; this was one of the three patients who were discharged from the ICU with neurological function intact. Discussion In this study, midazolam with or without 1-OHMG was detected in half of the 'slow to wake' patients, in whom testing was requested after a mean time from therapy cessation of 3 days. In one patient, in whom there was no record of midazolam administration in the ICU, a level of 200 ng/ml was recorded. It later transpired that a large dose of midazolam had been administered in the operating theatre more than 96 hours earlier. Detection of 1-OHMG in renal impairment confirmed that 1-OHMG accumulates in the presence of renal failure. Furthermore, its presence in high serum concentrations (3121–11,525 ng/ml) in the face of midazolam levels below the therapeutic range, normally quoted in the critically ill of 100–1000 ng/ml [ 5 ], while the patient remained deeply sedated concurs with earlier reports [ 3 , 4 ] that 1-OHMG has a sedative effect and contributes to prolonged sedation in renal impairment. Other investigators have reported the presence of 1-OHMG in the absence of midazolam [ 3 , 4 ], but we did not observe this and suspect that it was because the assay we used is able to detect very low concentrations of midazolam. Our findings suggest that serum levels of midazolam and/or 1-OHMG in 'slow to wake' patients may be used to aid differentiation between prolonged sedation and neurological damage. Patients found to be midazolam positive using this rapid assay were significantly less likely to have suffered neurological damage. Correct discrimination between neurological damage and prolonged sedation was made for 20 out of 26 patients, indicating a high degree of accuracy. Clearly, the possibility that midazolam-positive patients also have neurological damage remains and must be excluded if these patients do not awaken when serum concentrations of benzodiadepines have fallen to undetectable levels. Additionally, in the midazolam-negative group three patients were discharged with neurological function intact. This of course does not exclude a neurological cause of the coma that had fully resolved on discharge. One patient was receiving the sedating antihistamine chlorphenamine (30 mg/day intravenously) and did not regain full consciousness until it was stopped. In the remaining two patients no other clinical cause of the coma was apparent. The only other agent used routinely in these patients that could have significantly contributed to their reduced level of consciousness was the intravenous opiate fentanyl. Although fentanyl is known to accumulate in critical illness [ 6 ], we could find no evidence of accumulation for longer than 36 hours [ 7 ], and, because our group of patients had not received the drug for more than 2 days before sampling, it was not thought to contribute to the patients being 'slow to wake'. Arguably, the most important finding is that over three-quarters of the 'slow to wake' patients with no detectable serum midazolam/1-OHMG either died or were discharged from the ICU with profound neurological damage, whereas more than three-quarters of those with detectable midazolam/1-OHMG went on to make a full recovery. This observation suggests that prolonged sedation occurs after midazolam therapy and that it can be difficult to differentiate this from neurological damage in the acutely ill patient. The exclusion of midazolam or its metabolite 1-OHMG should be confirmed either by assay detection, as we describe, or by using the short-acting benzodiazepine antagonist flumazenil before a formal diagnosis of neurological damage is made. There are reports [ 3 , 8 ] in the literature of successful reversal of benzodiazepine sedation in critical illness using flumazenil, but we rarely use it in our unit because we find it to be nonspecific, short acting and able to induce seizures [ 9 ]. We recommend that use of alternatives to midazolam be considered in this patient group whenever possible, and that if its use is considered essential then steps should be taken to exclude the continuing presence of the drug or its metabolite before an opinion regarding neurological damage is formed. These findings have led to a change in prescribing practice in our ICU. We no longer use midazolam for sedation, and our sedation policy is now based on administering propofol or lorazepam. This view is also supported by the Society of Critical Care Medicine's most recently published guidelines [ 10 ], which recommend use of lorazepam for sedating most patients via intermittent or continuous infusion and use of propofol for short-term sedation, and that midazolam be reserved for rapid control of agitated patients and for short-term sedation. As a consequence, we were unable to conduct a more formal study of midazolam's role in over-sedation or extend the study to a larger group of patients. Conclusion The results of this investigation confirm that prolonged sedation from midazolam or 1-OHMG should always be considered in the differential diagnosis of neurological damage in critically ill patients who have received midazolam. This can be accurately detected using the assay method described. The sedative effects of midazolam/1-OHMG can persist for days after stopping administration of midazolam. If prolonged sedation can be excluded in these patients, then it is highly likely that neurological damage has occurred. Key messages • In some patients midazolam is metabolized to its glucuronide, which has sedative properties. • Prolonged sedation resulting from this metabolite should be considered when making a differential diagnosis of neurological damage in 'slow to wake' patients. • Measurement of midzolam and its metabolite in slow to wake patients will aid the differential diagnosis in these patients. Abbreviations ICU = intensive care unit; 1-OHMG = 1-hydroxymidazolam glucuronide. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All authors participated in the study design, interpretation of results and manuscript preparation. CMK also performed data collection and analyses.

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1065107

Determinants of the cuff-leak test: a physiological study

Introduction The cuff-leak test has been proposed as a simple method to predict the occurrence of post-extubation stridor. The test is performed by cuff deflation and measuring the expired tidal volume a few breaths later ( V T ). The leak is calculated as the difference between V T with and without a deflated cuff. However, because the cuff remains deflated throughout the respiratory cycle a volume of gas may also leak during inspiration and therefore this method (conventional) measures the total leak consisting of an inspiratory and expiratory component. The aims of this physiological study were, first, to examine the effects of various variables on total leak and, second, to compare the total leak with that obtained when the inspiratory component was eliminated, leaving only the expiratory leak. Methods In 15 critically ill patients mechanically ventilated on volume control mode, the cuff-leak volume was measured randomly either by the conventional method (Leak conv ) or by deflating the cuff at the end of inspiration and measuring the V T of the following expiration (Leak pause ). To investigate the effects of respiratory system mechanics and inspiratory flow, cuff-leak volume was studied by using a lung model, varying the cross-sectional area around the endotracheal tube and model mechanics. Results In patients Leak conv was significantly higher than Leak pause , averaging 188 ± 159 ml (mean ± SD) and 61 ± 75 ml, respectively. In the model study Leak conv increased significantly with decreasing inspiratory flow and model compliance. Leak pause and Leak conv increased slightly with increasing model resistance, the difference being significant only for Leak pause . The difference between Leak conv and Leak pause increased significantly with decreasing inspiratory flow ( V ' I ) and model compliance and increasing cross-sectional area around the tube. Conclusion We conclude that the cross-sectional area around the endotracheal tube is not the only determinant of the cuff-leak test. System compliance and inspiratory flow significantly affect the test, mainly through an effect on the inspiratory component of the total leak. The expiratory component is slightly influenced by respiratory system resistance.

Introduction In mechanically ventilated patients the frequency of post-extubation stridor is estimated to range between 4% and 22% [ 1 - 3 ]. Post-extubation stridor is usually due to laryngeal edema or decreased cross-sectional area of trachea, although vocal-cord dysfunction and overdose of sedative drugs may be also the cause. Nevertheless, this complication may result in emergency re-intubation in rather difficult circumstances with increased morbidity and mortality. The cuff-leak test has been proposed as a simple method of predicting the occurrence of this complication [ 4 - 7 ]. This test consists of deflating the balloon cuff of the endotracheal tube to assess the air leak around the tube during expiration by measuring the expiratory tidal volume with and without a deflated cuff [ 4 - 6 ]. A relatively large difference between these two values indicates that the cross-sectional area of the tracheal and/or upper airways is large enough to render the occurrence of post-extubation stridor, and therefore the possibility of re-intubation due to airway obstruction, unlikely [ 4 - 7 ]. Obviously the cuff-leak test is not useful if vocal cord dysfunction or overdose of sedative drugs is the cause of post-extubation stridor. Typically the cuff-leak test is performed during volume control ventilation (using a tidal volume of 10 ml/kg) by deflating the cuff, whereas the expired tidal volume is measured a few breaths later [ 4 - 7 ]. The leak is calculated as the difference between the expiratory tidal volume with and without a deflated cuff [ 4 - 7 ]. However, because most ventilators in the intensive care unit do not compensate for leaks, it is possible that during inspiration with a deflated cuff a portion of the total amount of the predetermined volume given by the ventilator may leak around the endotracheal tube. In this case, the difference between expiratory tidal volume with and without a deflated cuff represents a total leak consisting of an inspiratory and an expiratory component. This total leak may depend on various factors such as the cross-sectional area around the endotracheal tube, inspiratory flow and respiratory system mechanics, which may affect either the inspiratory component or the expiratory component or both, therefore contributing to the poor performance of the cuff-leak test in identifying patients with post-extubation stridor, reported by some studies [ 8 ]. The aims of this physiological study were, first, to examine the effects of various variables, such as cross-sectional area around the endotracheal tube, inspiratory flow and respiratory system mechanics on total leak, and second, to compare the total leak with that obtained when the inspiratory component was eliminated, leaving only the expiratory leak. The inspiratory leak was eliminated by deflating the cuff at end-inspiration, a manoeuvre that guarantees that the ventilator delivers all the predetermined gas volume into the lung. Methods Clinical study Fifteen mechanically ventilated patients (aged 65 ± 19 years [mean ± SD]; seven males, eight females) were prospectively studied. All were orotracheally intubated (low-pressure cuff endotracheal tube, diameter 8.0 ± 0.5 mm, tube length 28 ± 1 mm), hemodynamically stable without vasoactive drugs, lightly sedated with propofol and with a PaO 2 /F i O 2 of more than 250 mmHg. The study was approved by the Hospital Ethics Committee, and informed consent was obtained from the patients or their families. Flow ( V ') at the airway opening was measured with a heated pneumotachograph (model 3700; Hans-Rudolf, Kansas City, KS, USA) and a differential pressure transducer (Micro-Switch 140PC; Honeywell Ltd, Montreal, Ontario, Canada), both placed between the endotracheal tube and the Y-piece of the ventilator. Flow was electronically integrated to provide volume. Airway pressure ( P aw ; Micro-Switch 140PC; Honeywell Ltd) was measured from a side port between the pneumotachograph and the endotracheal tube. Each signal was sampled at 150 Hz (Windaq Instruments Inc., Akron, OH, USA) and stored on a computer disk for later analysis. Initially the patients were placed on volume control mode (Puritan-Bennett 840, Lenexa, KS, USA) with no flow compensation, heavily sedated (propofol–fentanyl) to achieve a Ramsay scale of 6 and paralyzed with cis -atracurium. Inactivity of respiratory muscles was confirmed with the use of standard criteria [ 9 ]. Tidal volume ( V T ) was set to 10 ml/kg given with a constant inspiratory flow rate of 1 litre/s. No end-inspiratory pause was applied. External positive end-expiratory pressure (PEEP) was set to zero while ventilator frequency was adjusted such as to achieve zero intrinsic PEEP, confirmed by end-expiratory occlusion [ 10 ]. When the patients were stable on volume control, the (baseline) expiratory V T was measured by averaging five consecutive breaths ( V T,baseline ). The absence of a leak was verified by an end-inspiratory occlusion of 10 s and observing a constant P aw after 3 s of occlusion. Thereafter, the cuff-leak test was performed randomly, either using the conventional method or by deflating the cuff at the end of a 3 s end-inspiratory pause. The conventional method consisted of balloon cuff deflation and measuring the expiratory tidal volume four breaths later ( V T,defl ). Five such trials were performed to obtain an average value of V T,defl . The difference between V T,baseline and V T,defl was defined as the cuff-leak volume obtained by the conventional method (Leak conv ). When the cuff was deflated at the end of the end-inspiratory pause only the following expiratory tidal volume was measured ( V T,pause ). Again five such trials were performed. The difference between V T,baseline and V T,pause was defined as the cuff-leak volume obtained by deflating the cuff during end-inspiratory pause (Leak pause ). The mechanics of the respiratory system were measured by using the occlusion technique [ 10 - 12 ]. In each patient at least five breaths with a satisfactory plateau were analyzed and the mean values were reported. Respiratory system static inflation end-inspiratory compliance ( C rs ), minimum ( R int ) and maximum ( R rs ) resistance of the respiratory system and the difference between R rs and R int (Δ R ) were computed according to standard formulas and procedures [ 11 , 12 ]. In all patients ΔLeak was calculated as the difference between Leak conv and Leak pause . Assuming that the difference between peak inspiratory P aw (Δ P aw,peak ) between methods was entirely due to different end-inspiratory lung volume, the predicted ΔLeak was calculated by the product of Δ P aw,peak and C rs . Lung model study To examine the effects of various variables on cuff-leak volume measurement, a two-chamber test lung (Michigan Instruments Inc., Grand Rapids, MI, USA) was used [ 13 ]. Each chamber was connected to a common tube representing the trachea by a tube with varying resistance. The compliance of each chamber was also variable. The two chambers were connected to a ventilator (Puritan-Bennett 840) via a cuffed endotracheal tube 8 mm in diameter inserted into the common tube. Small plastic bands were inserted between the endotracheal tube and the common tube to create controlled leaks when the balloon cuff was deflated. Two levels of leak were created, simulating two different cross-sectional areas around the endotracheal tube (large and small). The cross-sectional area around the endotracheal tube was quantified by cuff deflation during the end-inspiratory pause time and observation of the rate of pressure drop when an inspired tidal volume of l litre was used and total model compliance was 50 ml/cmH 2 O. The rate of pressure decrease was about 10 and 5 cmH 2 O/s with large and small cross-sectional areas, respectively. The absence of leak with the cuff inflated was confirmed by end-inspiratory occlusion and demonstration of a constant plateau P aw . V T was set at 0.6 litre (given with constant flow rate) and external PEEP to zero throughout. Ventilator frequency was adjusted so that no dynamic hyperinflation was observed. The absence of dynamic hyperinflation was verified by end-expiratory occlusion and no intrinsic PEEP demonstration [ 10 ]. Two protocols were performed. In the first (protocol A), the effects of inspiratory flow ( V ' I ) on cuff-leak volume measurement as well as the interaction between V ' I , cross-sectional area around the endotracheal tube and model mechanics were studied. At small and large cross-sectional area around the endotracheal tube and three combinations of model mechanics, representing normal (model airway resistance, R = 8 cmH 2 O/litre per second; model airway compliance, C = 50 ml/cmH 2 O), restrictive ( R = 8 cmH 2 O/litre per second, C = 20 ml/cmH 2 O) and obstructive pattern ( R = 16 cmH 2 O/litre per second, C = 100 ml/cmH 2 O), V ' I was varied between 0.6 and 1 litre/s and cuff-leak volume was measured either by the conventional method or by deflating the cuff at the end of a 3 s end-inspiratory pause as described above. The effects of model mechanics on cuff-leak volume were further studied in a separate protocol (protocol B). At a constant cross-sectional area around the endotracheal tube (large) and an inspiratory flow of 0.6, each method of cuff-leak volume measurement was studied at three levels of R and C , resulting in nine combinations of system mechanics ( R = 8, 16 and 32 cmH 2 O/litre per second and C = 20, 50 and 100 ml/cmH 2 O). Similarly to protocol A, at each combination of model mechanics the cuff-leak volume was measured either by the conventional method or by deflating the cuff at the end of a 3 s end-inspiratory pause. Data were analyzed with a paired t -test and a multi-factorial analysis of variance for repeated measurements, where appropriate. When the F value was significant, Tukey's test was used to identify significant differences. Linear regression analysis was performed with the least-squares method. P < 0.05 was considered statistically significant. Data are expressed as means ± SD. In the lung model study, means ± SD for the variables were determined from a total of 10 measurements. Results Clinical study Baseline ventilator settings and respiratory mechanics are shown in Table 1 . When the cuff remained deflated throughout the respiratory cycle, P aw,peak of the analyzed breaths (24.0 ± 6.6 cmH 2 O) was significantly lower than that of the breath in which the cuff was deflated at the end of the inspiratory pause (26.7 ± 7.1 cmH 2 O); the mean Δ P aw,peak averaged 2.6 ± 2.6 cmH 2 O (range 0.5–8.2 cmH 2 O). As expected, P aw,peak of the breaths in which the cuff was deflated at the end of the inspiratory pause was similar to the corresponding value of the baseline. In all patients Leak conv was higher than Leak pause , averaging 188 ± 159 ml (32 ± 25% of V T,baseline ) and 61 ± 75 ml (10 ± 12% of V T,baseline ), respectively ( P < 0.05; Fig. 1 ). There was a significant linear relationship between Leak conv and Leak pause ( y = - 12.3 + 0.39 x , r = 0.84, P < 0.05; Fig. 1 ). The observed ΔLeak averaged 127 ± 105 ml. There was a significant linear relationship between Δ P aw,peak and the observed ΔLeak ( y = 64.8 + 26.2 x , r = 0.66, P < 0.05) and between the predicted and observed ΔLeak ( y = 13.14 + 0.73 x , r = 0.69, P < 0.05). There was no relationship between observed ΔLeak and respiratory system mechanics ( R int , R rs , Δ R and C rs ), the time constant of the respiratory system and V T,baseline . Model study Protocol A For a given condition, Leak conv was significantly higher than Leak pause (Table 2 ). For a given cross-sectional area, and independently of model mechanics, Leak pause was not affected by V ' I , whereas Leak conv increased significantly with decreasing V ' I (Table 2 ). Independently of the cross-sectional area around the endotracheal tube with simulated restrictive respiratory system disease and at a V ' I of 0.6 litre/s, Leak conv was significantly higher than the corresponding values with simulated normal mechanics and obstructive respiratory system disease. ΔLeak increased significantly with decreasing V ' I and increasing the size of the cross-sectional area around the endotracheal tube (Fig. 2 ). The effect of V ' I on ΔLeak was significantly higher with simulated restrictive respiratory system disease and large cross-sectional area around the endotracheal tube (Fig. 2 ). Protocol B Similarly to protocol A, and independently of model mechanics, Leak conv was significantly higher than Leak pause (Table 3 ). For a given R , Leak conv increased significantly with decreasing C , whereas Leak pause remained constant. For a given C , Leak pause and Leak conv tended to increase slightly with the highest resistance, the difference being significant only for Leak pause . ΔLeak was not affected by model resistance, whereas it increased significantly with decreasing compliance (Fig. 3 ). Discussion The main findings of this study were as follows. First, because in mechanically ventilated patients the expiratory leak volume is about 30% of the sum of inspiratory and expiratory leaks (total leak), the inspiratory leak significantly affected the results of the cuff-leak test. Second, the cross-sectional area around the endotracheal tube is not the only determinant of cuff-leak test. Third, respiratory system compliance and inspiratory flow affect the test significantly, mainly through an effect on the inspiratory component. Fourth, the expiratory component is slightly influenced by respiratory system resistance. To avoid the confounding factors of respiratory muscle activity and dynamic hyperinflation on the calculation of cuff-leak volume, the patients were paralyzed and ventilated with settings that permitted the respiratory system to reach passive functional residual capacity at the end of expiration. Similarly, in the lung model the ventilator settings were such that dynamic hyperinflation was not observed. Therefore, for a given experimental condition the inspired tidal volume entirely determined the total expired volume. Finally, contrary to other studies [ 5 ], cuff-leak volume was measured by comparing the expired tidal volume with and without a deflated cuff. In this case the difference between inspired and expired tidal volume due to gas exchange and the different temperature and humidity of inspired and expired gas were not an issue. By deflating the cuff at the end of the inspiratory pause we guaranteed that the ventilator delivered all of the predetermined gas volume into the lung, as indicated by the similar peak P aw between the breaths used to calculate the cuff-leak volume. Because inactivity of respiratory muscles and absence of dynamic hyperinflation were ensured, any difference in expired volume with and without a deflated cuff should be entirely due to gas leak around the endotracheal tube during expiration (pause cuff leak). In contrast, when the cuff-leak volume was measured with the conventional method, a fraction of gas volume delivered by the ventilator might leak around the endotracheal tube during inspiration. In that case the measured cuff-leak volume is the total leak consisting of an inspiratory and expiratory component. The design of this study did not permit us to measure with accuracy the inspiratory leak. This is because pause cuff leak is not similar to expiratory leak obtained with the conventional method because end-inspiratory lung volume and thus elastic recoil pressure at the beginning of expiration differ substantially between the two methods of cuff leak determination. The pause cuff leak should be higher than the expiratory component of the total leak, because end inspiratory lung volume and elastic recoil pressure were considerably higher when pause cuff leak was obtained. Both in clinical and model study the cuff-leak volume determined with the conventional method (Leak conv ) was always higher than that obtained by cuff deflation at end-inspiratory pause, which eliminated the inspiratory component of total leak (Leak pause ). It follows that the inspiratory component is an important determinant of the cuff-leak test. It is of interest to note that in patients Leak conv was about threefold Leak pause whatever the amount of the total leak. In Protocol A of the lung model study, for a given cross-sectional area, the system mechanics and inspiratory flow considerably affected Leak conv ; Leak conv increased significantly with decreasing compliance and inspiratory flow. In contrast, neither system compliance nor inspiratory flow influenced Leak pause , which remained relatively constant. As a result ΔLeak increased significantly with decreasing compliance and inspiratory flow. The constancy of Leak pause suggested that the expiratory component of the total leak was also unaffected by changes in system compliance and inspiratory flow. It follows that respiratory system compliance and inspiratory flow have an important impact on cuff-leak test, mainly through an effect on the inspiratory component. The increased inspiratory leak with decreasing system compliance is predictable because the stiffness of the respiratory system causes a greater fraction of inspiratory flow to deviate to atmosphere though the free space between the endotracheal tube and the trachea. Similarly, the increased inspiratory leak with low inspiratory flow was also expected. The free space between the endotracheal tube and trachea represents a low-resistance pathway and, because for a given tidal volume low inspiratory flow is associated with longer inspiratory time, the inspiratory leak should increase, a situation resembling that of bronchopleural fistula in which high inspiratory flows are recommended so as to reduce the amount of air leaking through the fistula [ 14 ]. Thus the cuff-leak volume calculated by the conventional method does not solely reflect the cross-sectional area of the trachea and/or the upper airways but is influenced by other factors such as respiratory system mechanics and inspiratory flow. In protocol B of the lung model study, a slight increase in cuff-leak volume at the highest resistance value was observed with both methods. As a result, ΔLeak was not influenced by model resistance, indicating that system resistance affected mainly the expiratory component of the total leak. Although the factors underlying the above increase are not clear, the flow velocity profile during expiration could account for these findings. Nevertheless the difference was relatively small (less than 25 ml or less than 4% of V T ), making the clinical significance of this finding questionable. Furthermore the increase in expiratory leak was observed at very high values of resistance that preclude the weaning process, making the performance of the cuff-leak test clinically irrelevant. We should note that in patients the cuff leak was determined at the relatively high constant inspiratory flow of 1 litre/s. Although the effect of flow was not studied in our patients, the model study indicates that overestimation should be higher at low flow. Nevertheless, high inspiratory flow is recommended in patients with obstructive lung disease ventilated on volume control so as to reduce dynamic hyperinflation [ 15 ]. In contrast with the model study, in the clinical study there was no relationship between observed ΔLeak and respiratory system mechanics ( R int , R rs , Δ R and C rs ), the time constant of the respiratory system and V T,baseline . Differences in cross-sectional area of the trachea and upper airways between patients might obscure any relationship between these variables and ΔLeak. Studies suggest that leak volume, as obtained by the conventional method, may predict the occurrence of post-extubation stridor and might thus identify the subset of patients at risk of re-intubation due to upper airway obstruction [ 4 , 5 , 7 ]. However, the cut-off point of leak volume differed substantially between studies. In addition, the positive predictive value was quite low, indicating that the results of the cuff-leak test should not be used to postpone the extubation but might be particularly useful to exclude significant laryngeal edema [ 4 , 5 , 7 , 16 ]. In contrast, other authors concluded that the cuff-leak test is inaccurate [ 8 ]. Indeed, a cuff-leak volume (measured conventionally) of more than 300 ml has been observed in three patients who developed post-extubation stridor after cardiac surgery [ 8 ]. Although these different results between studies might be due to the populations studied, our study indicates that the respiratory system mechanics and inspiratory flow, factors influencing the inspiratory leak that were not taken into account, might to some extent contribute to the poor performance of the cuff-leak test. A measured conventional cuff-leak volume of less than 15.5% [ 4 ], 12% [ 7 ] or 10% of predetermined V T [ 6 ] has been used to identify patients at risk for post-extubation stridor. In our study with the conventional method, 5 of 15 patients had a cuff-leak volume less than 15.5% of predetermined V T , whereas with the pause method 11 patients demonstrated true cuff-leak volume less than this threshold (10 patients had a cuff-leak volume less than 12%). The purpose and design of our study were such that they did not permit us to examine whether by eliminating the inspiratory leak it would be possible to improve the predictive value of the cuff-leak test. The number of patients was small and the cuff-leak volume was not determined on the day of extubation, but the patients were examined under highly controlled conditions. The aim of the study was not to propose a new method of cuff leak determination but to examine factors affecting the total cuff-leak volume obtained by the conventional method. Our results clearly showed that the cuff-leak test (particularly its inspiratory component) is influenced by factors other than the cross-sectional area of the trachea and/or the upper airways and thus the above-mentioned cut-off points of cuff-leak volume should be re-evaluated. Conclusion Our study has shown that the cross-sectional area around the endotracheal tube is not the only determinant of the cuff-leak test. Respiratory system mechanics and inspiratory flow are other important determinants of the cuff-leak test, mainly through an effect on the inspiratory component of the total leak, complicating its interpretation. Key messages • Cross-sectional area around the endotracheal tube is not the only determinant of the cuff leak test. • Respiratory system mechanics and inspiratory flow are the other important determinants of the cuff leak test, complicating its interpretation. Abbreviations C = model airway compliance; C rs = end-inspiratory static compliance of the respiratory system (ml/cmH 2 O); ΔLeak = difference between Leak conv and Leak pause ; Δ P aw,peak = difference between peak inspiratory P aw between methods; Δ R = difference between R rs and R int ; Leak conv = cuff-leak volume obtained by the conventional method; Leak pause = cuff-leak volume obtained when the cuff was deflated at the end of the end-inspiratory pause; P aw = airway pressure; PEEP = positive end-expiratory pressure; R = model airway resistance; R int = minimum resistance of the respiratory system; R rs = maximum resistance of the respiratory system; V ' = flow at the airway opening; V ' I = inspiratory flow; V T = expired tidal volume; V T,baseline = expiratory V T measured by averaging five consecutive breaths; V T,defl = expiratory V T measured when cuff was deflated; V T,pause = expiratory tidal volume measured at the end of the end-inspiratory pause. Competing interests The author(s) declare that they have no competing interests. Authors' contributions GP designed the study and performed the statistics. CA collected the data from patients and from the model. EM and EK participated in data collection. DG designed the study, evaluated the data and drafted the manuscript. All authors read and approved the final manuscript.

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1065109

Pro-atrial natriuretic peptide is a prognostic marker in sepsis, similar to the APACHE II score: an observational study

Introduction Additional biomarkers in sepsis are needed to tackle the challenges of determining prognosis and optimizing selection of high-risk patients for application of therapy. In the present study, conducted in a cohort of medical intensive care unit patients, our aim was to compare the prognostic value of mid-regional pro-atrial natriuretic peptide (ANP) levels with those of other biomarkers and physiological scores. Methods Blood samples obtained in a prospective observational study conducted in 101 consecutive critically ill patients admitted to the intensive care unit were analyzed. The prognostic value of pro-ANP levels was compared with that of the Acute Physiology and Chronic Health Evaluation (APACHE) II score and with those of various biomarkers (i.e. C-reactive protein, IL-6 and procalcitonin). Mid-regional pro-ANP was detected in EDTA plasma from all patients using a new sandwich immunoassay. Results On admission, 53 patients had sepsis, severe sepsis, or septic shock, and 68 had systemic inflammatory response syndrome. The median pro-ANP value in the survivors was 194 pmol/l (range 20–2000 pmol/l), which was significantly lower than in the nonsurvivors (median 853.0 pmol/l, range 100–2000 pmol/l; P < 0.001). On the day of admission, pro-ANP levels, but not levels of other biomarkers, were significantly higher in surviving than in nonsurviving sepsis patients ( P = 0.001). In a receiver operating characteristic curve analysis for the survival of patients with sepsis, the area under the curve (AUC) for pro-ANP was 0.88, which was significantly greater than the AUCs for procalcitonin and C-reactive protein, and similar to the AUC for the APACHE II score. Conclusion Pro-ANP appears to be a valuable tool for individual risk assessment in sepsis patients and for stratification of high-risk patients in future intervention trials. Further studies are needed to validate our results.

Introduction Affecting about 700,000 people annually, sepsis accounts for 210,000 deaths each year in the USA, and both of these figures are likely to increase [ 1 , 2 ]. Sepsis is not an homogenous disease; rather, it is a complex clinical syndrome with distinct immunological features [ 3 , 4 ]. The ambiguity of clinical findings and unclear risk stratification in sepsis have been major problems in sepsis intervention trials [ 5 ]. The effectiveness of anti-inflammatory treatment correlates with risk for death and severity of disease [ 6 ]. Thus, the prognosis of a septic patient may contribute significantly to the success of any intervention [ 5 ]. Within this context, there is need for biomarkers to tackle the challenges of sepsis monitoring and treatment [ 7 ]. Members of the natriuretic peptide family are established markers of congestive heart failure [ 8 - 10 ]. Defending against hypertension and salt and water retention, they antagonize the renin–angiotensin–aldosterone system, including effects on renal tubule sodium reabsorption, vascular tone and cell growth. Atrial natriuretic peptide (ANP) is predominantly produced in the atrium of the heart and comprises 98% of natriuretic peptides in the circulation [ 11 ]. Recently, both ANP and pro-ANP have attracted interest as new markers in the field of sepsis [ 12 - 16 ]. Mature ANP is derived from carboxyl-terminal amino acids 99–126 of the prohormone (pro-ANP), which is 126 amino acids in length [ 11 ]. The amino-terminal portion of pro-ANP (termed NT-pro-ANP, or pro-ANP 1–98 ) is secreted at the same molar ratio as ANP. Because it has a much longer half-life than has mature ANP, it has been suggested that pro-ANP 1–98 is a more reliable analyte [ 17 ]. However, results from various competitive immunoassays and high-performance liquid chromatography analyses indicate that pro-ANP 1–98 may be subject to further fragmentation [ 18 , 19 ]. Consequently, sandwich immunoassays for pro-ANP 1–98 might underestimate actual levels of pro-ANP, and immunoassays for measurement of mid-regional pro-ANP may have an advantage [ 20 ]. In the present study we aimed to evaluate the prognostic value of mid-regional pro-ANP levels in a well defined cohort of medical intensive care unit (ICU) patients as compared with those of other biomarkers (i.e. IL-6, C-reactive protein [CRP] and procalcitonin [PCT]) and a physiological score (Acute Physiology and Chronic Health Evaluation [APACHE] II). Methods Patients In the present study we evaluated plasma samples from a cohort of 101 consecutive critically ill patients admitted to the medical ICU of the University Hospital of Basel, Switzerland. The primary end-point of this study was the prognostic value of endocrine dysfunction in critically ill patients ('PEDCRIP' study). The characteristics of the study population, study design, diagnostic criteria and levels of various markers of inflammation and infection were reported in detail elsewhere [ 21 - 24 ]. Briefly, over a 9-month period 101 consecutive patients, including neutropenic and immunosuppressed patients, admitted to the medical ICU were included. Patients were followed until hospital discharge or death. Data were collected on admission (i.e. during the first 24 hours), on day 2, and on the day of discharge from the ICU or on the day of death. At these time points (a total of 276 plasma samples), patients were either very sick or in a stable condition and ready for discharge to a medical ward, respectively. In patients who died within 24 hours after admission, only data from admission were collected ( n = 5). Vital signs, clinical status and severity of disease parameters (APACHE II score) were assessed daily. The APACHE II score was calculated by means of maximal daily deviations of 12 physiological variables from normal plus correction for age and various chronic illnesses. A pulmonary artery catheter was not routinely inserted. When feasible, consent was obtained from conscious patients before enrolment; otherwise, consent was obtained from the next of kin. The study protocol had been granted approval by the hospital institute's ethical review board. Patients were classified at the time of blood collection into those with systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis and septic shock, which were defined according to international criteria [ 25 , 26 ]. Infection was diagnosed according to standardized criteria or, in case of uncertainty, by an infectious disease specialist. This was done retrospectively on the basis of review of complete patient charts, results of microbiological cultures, chest radiographs and, when available, autopsy reports. An isolated micro-organism was considered to be pathogenic if it was identified within a 24-hour period before or after the onset of the systemic response. Colonization with bacteria (e.g. in a patient with a bladder catheter but without leucocyturia) or positive blood cultures at autopsy were disregarded. Microbiological tests were requested and antibiotic therapy prescribed by physicians on duty according to the usual practice, without interference from the research team. Although optimal fluid resuscitation was done in the initial treatment phase in all patients, 31% of septic patients needed additional treatment with intravenous noradrenaline (norepinephrine). The mean dose of noradrenaline on admission was 8.7 ± 12.1 μg/min, on day 2 it was 10.1 ± 10.9 μg/min and on the day of discharge/death it was 47.2 ± 35.2 μg/min ( P < 0.001). Nonsurvivors from severe sepsis and septic shock needed higher doses of noradrenaline than did survivors (5.7 ± 7.8 μg/min versus 30.5 ± 28.1 μg/min; P < 0.001). Overall, 23 of the 101 patients died (22.8%). The majority of patients who died suffered from multiple organ failure (56.5%), defined as failure of two or more vital organs. Assays Results of the routine blood analyses (i.e. complete blood count, serum chemistry including CRP, blood gas analyses) were known and recorded. Blood was obtained from an indwelling arterial or venous catheter. Plasma was separated from the blood samples at the time of blood draw and frozen at -70°C until assayed. Measurement was done in a blinded manner as a batch analysis. Mid-regional pro-ANP (epitopes covering amino acids 53–90) was detected in EDTA plasma from all patients with a new sandwich immunoassay (BRAHMS Seristra ® LIA; BRAHMS AG, Hennigsdorf/Berlin, Germany), as described in detail elsewhere [ 20 ]. As a modification to the published assay, the calibration was changed from a synthetic peptide to pro-ANP in human serum. This modification to the initial description increased the precision and dynamic (i.e. signal to noise ratio) of the assay, and allowed measurement of pro-ANP in serum and plasma (with EDTA, heparin, or citrate). Briefly, patient samples (1:40 dilution of 5 μl plasma in incubation buffer) or standards were added in duplicate to antibody-coated tubes (directed at pro-ANP peptide 73–90) and incubated for 30 min at room temperature. After five washings with 1 ml washing buffer, 200 μl tracer was added, containing acridinium ester-labelled anti-pro-ANP antibody (directed at peptide 53–72), followed by 30 min incubation at room temperature. Tubes were washed three times with 1 ml washing buffer, and detection was performed in a luminometer (1 s detection time per sample). Relative light units of the chemiluminescence assay were expressed in pmol/L pro-ANP, as calculated from a calibration curve (4–1800 pmol/l) that was included in every analytical run. The lower detection limit of the assay is 4.3 pmol/l and the functional sensitivity of the assay (interassay coefficient of variation <20 %) is 11 pmol/L pro-ANP. The 97.5th percentile in 325 healthy individuals was 163.9 pmol/l (median 45 pmol/l), with no difference between sexes [ 20 ]. PCT was measured using the LUMITest ® PCT (BRAHMS AG), following the manufacturer's instructions. CRP was determined using en enzyme immunoassay (EMIT; Merck Diagnostica, Zurich, Switzerland). A serum level greater than 5 mg/l was considered abnormally elevated. Serum IL-6 concentrations were measured using a commercially available quantitative sandwich enzyme immunoassay (Pelikine Compact™; CLB, Amsterdam, The Netherlands), with a limit of detection at 0.6 ng/l. Statistical analysis Data in the text are expressed as mean ± standard deviation. Frequency comparison was done by χ 2 test. Two-group comparisons were performed using the Mann–Whitney U-test. For multigroup comparisons, Kruskal–Wallis one-way analysis of variance was used with Dunn's post-test evaluation. Levels that were nondetectable were assigned a value equal to the lower limit of detection for the assay. All testing was two-tailed, and P < 0.05 was considered statistically significant. Correlation analyses were performed by using Spearman rank correlation. Results Descriptive characteristics of the patients The mean age of the 101 patients (55 men and 46 women) included in the study was 57 ± 15 years (range 23–86 years) and the mean APACHE II score on admission was 22 ± 8. The median length of stay in the medical ICU was 4 days (range 0.2–60 days) and the mortality rate was 23%. More detailed baseline characteristics of the study population are described elsewhere [ 21 ]; however, to allow better understanding of the study results, the principal diagnoses of patients are summarized in Table 1 and the sites of infection in Table 2 . Sepsis was diagnosed in 58% of patients (on admission in 53 patients; five additional patients developed sepsis during their stay in the ICU). The principal site of infection was the lung (Table 2 ). In 38 (66%) of the 58 patients with infections, the responsible micro-organism was identified and 14 patients (24%) had bacteraemia. There was no difference in mortality between patients with and those without infection. Of the 53 patients admitted with sepsis, severe sepsis, or septic shock, 13 (25%) died; 10 (21%) of the 48 patients without infection on admission died. Pro-atrial natriuretic peptide and severity of the disease Figure 1a shows the distribution of pro-ANP values according to severity of infection (i.e. SIRS, sepsis, severe sepsis and septic shock) and serum PCT concentrations. Depending on the clinical severity of the infection, pro-ANP values exhibited a gradual increase from the group with SIRS to the group with septic shock ( P < 0.001). Similarly, circulating pro-ANP levels showed a similar gradual increase when categorized based on PCT levels (Fig. 1b ). Post-test analysis revealed a significant difference ( P < 0.001) between patients without SIRS, SIRS, sepsis and severe sepsis as compared with patients with septic shock. There was no significant difference between patients with severe sepsis and those with septic shock. Accordingly, patients with PCT levels greater than 10 ng/ml and greater than 1 ng/ml had significantly higher pro-ANP levels than did patients with PCT levels of 0.5–1 ng/ml and under 0.5 ng/ml ( P < 0.001). Pro-ANP levels correlated with serum IL-6 levels (r = 0.22; P < 0.001), and with serum and urine osmolarity (r = 0.55 and r = -0.43, respectively; P < 0.001), but not with serum sodium (r = 0.03; not significant) and only weakly with urine sodium concentrations (r = -0.17; P < 0.01). Pro-atrial natriuretic peptide and outcomes in patients with sepsis, severe sepsis and septic shock Figure 2 shows all pro-ANP values in survivors and nonsurvivors with sepsis, severe sepsis or septic shock, measured during their stay in the ICU. Thereby, patients were grouped by clinical diagnosis of sepsis according to international guidelines (panels a and c) or by circulating PCT level in excess of 1 ng/ml (panels b and d). The median pro-ANP value in the nonsurvivors was significantly greater than in the survivors, independent of grouping used. This difference in pro-ANP values was clear on the first day of admission to the ICU ( P < 0.001). In contrast, the difference between the survivors and nonsurvivors on the first day of admission was not significant for PCT ( P = 0.38 and P = 0.05, respectively), CRP, or IL-6 (data not shown for CRP and IL-6). Similarly, in patients without infections pro-ANP values were not higher in nonsurvivors than in survivors (all time points: 197.2 ± 361.5 pmol/l versus 226.0 ± 183.4 pmol/l, P = 0.7; on admission: 221.5 ± 209.7 pmol/l versus 161.3 ± 132.1 pmol/l, P = 0.3). To define an optimal decision threshold for pro-ANP values in septic patients, we performed receiver operating characteristic (ROC) plot analysis, including only data from patients with sepsis, severe sepsis, or septic shock obtained within the first 48 hours after admission to the ICU. Sensitivity was calculated among those patients who did not survive sepsis, and specificity was assessed among those patients who were discharged from the ICU. For comparison, the same ROC plot analysis was performed with CRP, PCT, IL-6 and APACHE II score. Table 3 shows the area under the ROC curve (AUC) for all parameters, including the 95% confidence interval. The AUC for pro-ANP was 0.88, which was significantly higher than the AUCs for PCT and CRP, and similar to the AUC for the APACHE II score (0.86). ROC curves are shown in Fig. 3 . Again, patients were grouped by clinical diagnosis of sepsis according to international guidelines (panel a) or by circulating PCT levels in excess of 1 ng/ml (panel b), yielding comparable results. The optimal threshold for pro-ANP was 530 pmol/l. At this cut-off, the sensitivity for correct prediction of death in the ICU was 86.7% and the specificity was 88.6%. Considering a prevalence of 33% for death in the ICU as a result of sepsis, the positive predictive value (PPV) of pro-ANP was 72.2% with a negative predictive value (NPV) of 95.1%. None of CRP, PCT, or IL-6 had similarly high values for sensitivity, specificity, PPV and NPV. The APACHE II score was also predictive for prognosis but yielded lower values as compared with pro-ANP. At an APACHE II threshold of 30, the sensitivity was 73.3% and the specificity was 95.6% (PPV = 84.6 %, NPV = 91.5 %). At a cut-off of 25, which was recommended by the US Food and Drug Administration for the use of Xigris ® , sensitivity was 80.0%, specificity 75.6%, PPV 48.0% and NPV 91.4%. Because PPV and NPV are dependent on the prevalence of the disease, Table 4 shows the relative likelihood with the prevalence independent likelihood ratio for different cut-offs. Discussion ANP and pro-ANP are markers for congestive heart failure [ 8 - 10 ], but their pathophysiological and prognostic significance in severe sepsis and septic shock is not yet understood. In the present study we found a significant increase in mid-regional pro-ANP in the plasma of sepsis patients as compared with patients without sepsis and healthy individuals. This increase was most marked in those patients with sepsis who did not survive their disease. Importantly, on the first day of admission to the ICU, pro-ANP, but not other markers of infection and inflammation such as CRP and PCT, were significantly increased in nonsurvivors as compared with survivors, suggesting that pro-ANP levels represent a new and valuable prognostic tool in patients with sepsis. At a threshold of 530 pmol/l, pro-ANP had a sensitivity of 86.7% for death in the ICU with sepsis, with a specificity of 88.6%; these figures were not reached by any of the other tested biomarkers. As is generally recommended, we diagnosed sepsis, severe sepsis and septic shock using well defined and widely accepted clinical guidelines [ 25 , 26 ]. However, true gold standards for the diagnosis of infections do not exist, and clinical classification of critically ill patients is not 100% certain despite the use of these guidelines, not only in sepsis trials but also in routine bedside use [ 27 , 28 ]. An ideal sepsis marker should permit early diagnosis, should provide information about the course of disease, and should help to differentiate bacterial from noninfectious and viral causes of systemic inflammation. It was shown that PCT has some of these features and is helpful in diagnosing septic conditions [ 29 - 31 ]. Therefore, we also classified pro-ANP levels according to circulating PCT levels, which are not subject to the uncertainty associated with clinical sepsis definitions. Importantly, the prognostic value of pro-ANP was similar independent of the classification system used, which suggests that our findings are reproducible. Thus, pro-ANP is of prognostic value in critically ill septic patients, in contrast to PCT, which is predominantly a diagnostic parameter. The first observations that ANP may play a role during endotoxic shock came from animal studies in which ANP was elevated within 2–6 hours after lipopolysaccharide injection [ 32 , 33 ]. Subsequent studies in critically ill humans showed an association of ANP with various cardiac physiological parameters [ 34 , 35 ]. The use of different assays might be responsible for part of the inconsistency in reported findings over recent years. Whereas Berendes and coworkers [ 14 ] found no association of ANP values with severity of the disease or mortality in critically ill patients, Hartemink and coworkers [ 13 ] found a strong association of ANP levels with myocardial depression in septic shock and with lethal outcome in 14 patients. A similar association of cardiac depression in septic shock was described for NT-pro-ANP in 17 patients [ 12 ]. Unfortunately, a limitation of our study is that cardiac indices were not routinely assessed by pulmonary artery catheter. Therefore, the precise mechanisms of pro-ANP release in patients with sepsis remain unknown. Nevertheless, a cardiac origin of natriuretic peptides makes an association with septic cardiac dysfunction likely. In addition, apart from volume overload, osmolarity rather than sodium concentration is associated with pro-ANP release, as suggested by regression analyses in our patients. Based on our findings and recent reports in the literature [ 36 ], in critically ill patients increased levels of natriuretic peptide are not specific for decompensated heart failure. In this context, the increase in ANP levels in septic shock may be potentiated by IL-6 elevation [ 15 ]. A recent study in meningococcal sepsis provided conclusive evidence that IL-6 is directly involved in myocardial depression [ 37 ]. Accordingly, in the present study IL-6 levels were correlated with pro-ANP levels, albeit relatively weakly. IL-6 had lower value in terms of outcome prediction than did mid-regional pro-ANP, which may be due to differences in the half-life of the molecules. The half-lives of both IL-6 and mature ANP are short, and measurement of those markers in septic patients does not allow a direct conclusion to be drawn regarding the level of production. We recently developed a sandwich immunoassay for the detection of a mid-regional fraction of pro-ANP in plasma [ 20 ]. This fragment has a much longer half-life in plasma, and because it is produced in equimolar concentrations to the mature hormone, it mirrors true production of ANP. Furthermore, it is possible that mid-regional pro-ANP exerts a physiological effect on its own, as is described for other fragments of NT-pro-ANP [ 38 ] and fragments of other prohormones, such as pro-adrenomedullin amino-terminal 20 peptide [ 39 ]. Measurement for ANP or fragments of NT-pro-ANP is potentially influenced by other factors, such as sex, age and kidney function, as is discussed elsewhere for brain-type natriuretic peptides [ 40 , 41 ]. Indeed, we observed a significant correlation of circulating pro-ANP levels with serum osmolarity and creatinine. Measurements in nonseptic patients with kidney failure revealed mostly normal pro-ANP values, and it is therefore possible that the observed elevation in pro-ANP and creatinine in this study is a result of kidney failure related to sepsis. Sepsis is a complex syndrome, and the immunological and biochemical situation may vary considerably between individual patients [ 3 , 4 ]. In the past almost all intervention trials failed to show any benefit from therapy for sepsis, and sepsis intervention has been termed the 'graveyard for pharmaceutical companies' [ 7 , 42 ]. Reasons for this may be found in immunological heterogeneity and insufficient patient stratification in those trials [ 5 ]. The need for markers that permit better stratification of patients with different stages of sepsis is underlined by the ongoing discussion concerning recombinant human activated protein C (drotrecogin alpha; Xigris ® ) [ 42 - 45 ]. The US Food and Drug Administration approved recombinant human activated protein C only for those patients with an APACHE II score in excess of 24, and thus only for those patients with the greatest risk for dying [ 28 , 46 , 47 ]. The APACHE II score – a complex algorithm – was not originally developed for individual outcome prediction in sepsis patients [ 48 ]. Despite its limitations, outcome predictors such as the extensively evaluated APACHE II score are helpful in identifying those septic patients who are at high risk for death and who are more likely to benefit from intervention [ 6 ]. In the present study the prognostic value of pro-ANP levels was comparable to that of APACHE II score. Importantly, mid-regional pro-ANP it is easier to determine than a physiological score and mirrors distinct pathophysiological changes that occur in sepsis. Conclusion In septic patients, we found that APACHE II score and mid-regional pro-ANP level on admission to a medical ICU had similar ability to predict outcome. The results of our study are novel and of interest because they may help to improve stratification of septic patients. Our findings are descriptive in nature and warrant validation in future prospective studies, including measurement of cardiac indices or evaluating patients who have undergone surgery. If our findings are confirmed, then mid-regional pro-ANP might become a new and useful additional prognostic marker for individual risk assessment in sepsis, and may represent a helpful tool for patient stratification in future intervention trials. Key messages • In septic patients mid-regional pro-ANP levels on admission to a medical ICU had a similar ability to predict outcome as did the APACHE II score. • Pro-ANP levels appear to be a useful tool for individual risk assessment in septic patients and for stratification of high risk patients in future intervention trials. • Because our findings are descriptive in nature, further prospective studies are warranted to validate our results. Abbreviations ANP = atrial natriuretic peptide; APACHE = Acute Physiology and Chronic Health Evaluation; AUC = area under the curve; CRP = C-reactive protein; ICU = intensive care unit; IL = interleukin; NPV = negative predictive value; NT = amino terminal; PCT = procalcitonin; PPV = positive predictive value; ROC = receiver operating characteristic; SIRS = systemic inflammatory resonse syndrome. Competing interests NG, JS and AB are employees of BRAHMS AG, the manufacturer of the pro-ANP assay (BRAHMS Seristra ® LIA; BRAHMS AG, Hennigsdorf/Berlin, Germany). BM has served as a consultant and received payments from BRAHMS AG to attend meetings related to the trial and for travel expenses, speaking engagements and research. Authors' contributions BM conceived the study, collected the data, drafted the protocol and supervised the writing of the manuscript. NGM, JS and AB were involved in assay development. NGM and MCC conducted statistical analyses and wrote the report. All authors read and approved the final manuscript.

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1065112

Timing of tracheostomy as a determinant of weaning success in critically ill patients: a retrospective study

Introduction Tracheostomy is frequently performed in critically ill patients for prolonged intubation. However, the optimal timing of tracheostomy, and its impact on weaning from mechanical ventilation and outcomes in critically ill patients who require mechanical ventilation remain controversial. Methods The medical records of patients who underwent tracheostomy in the medical intensive care unit (ICU) of a tertiary medical centre from July 1998 to June 2001 were reviewed. Clinical characteristics, length of stay in the ICU, rates of post-tracheostomy pneumonia, weaning from mechanical ventilation and mortality rates were analyzed. Results A total of 163 patients (93 men and 70 women) were included; their mean age was 70 years. Patients were classified into two groups: successful weaning ( n = 78) and failure to wean ( n = 85). Shorter intubation periods ( P = 0.02), length of ICU stay ( P = 0.001) and post-tracheostomy ICU stay ( P = 0.005) were noted in patients in the successful weaning group. Patients who underwent tracheostomy more than 3 weeks after intubation had higher ICU mortality rates and rates of weaning failure. The length of intubation correlated with the length of ICU stay in the successful weaning group (r = 0.70; P < 0.001). Multivariate analysis revealed that tracheostomy after 3 weeks of intubation, poor oxygenation before tracheostomy (arterial oxygen tension/fractional inspired oxygen ratio <250) and occurrence of nosocomial pneumonia after tracheostomy were independent predictors of weaning failure. Conclusion The study suggests that tracheostomy after 21 days of intubation is associated with a higher rate of failure to wean from mechanical ventilation, longer ICU stay and higher ICU mortality.

Introduction Tracheostomy is among the most frequently performed procedures in critically ill patients, being done in about 24% of patients in medical intensive care units (ICUs) [ 1 ]. The most common indication for tracheostomy in the ICU is need for prolonged mechanical ventilation [ 2 , 3 ]. Tracheostomy has several advantages over endotracheal intubation, including lower airway resistance, smaller dead space, less movement of the tube within the trachea, greater patient comfort and more efficient suction [ 4 , 5 ]. Although recent studies have suggested that tracheostomy can be a safe procedure in the ICU [ 6 , 7 ], tracheostomy has also been found to lead to serious complications, including tracheal stenosis, increased bacterial colonization and haemorrhage [ 8 , 9 ]. Many critically ill patients' families have been hesitant in authorizing tracheostomy because of cosmetic issues and speech problems. Because there are no definitive guidelines available, the timing of tracheostomy depends on clinical conditions, physician judgement and communication with families. The judgement of the attending physician can be influenced by the patients' likelihood of extubation, life expectancy and other clinical conditions, including haemodynamic status, oxygenation, consciousness level and ability to protect the airway. There is little consensus on the timing of tracheostomy. In the 1989 American College of Chest Physicians (ACCP) Consensus Conference on Artificial Airways in Patients Receiving Mechanical Ventilation [ 10 ], it was concluded that the appropriate duration of translaryngeal intubation could not be defined. It was suggested that if the anticipated need for mechanical ventilation is longer than 21 days then tracheostomy is preferable. For mechanical ventilation that is anticipated to last between 10 and 21 days, the decision was left to the physician, and daily assessment was recommended. Recent ACCP guidelines [ 11 ] suggest that tracheostomy should be considered after an initial period of stabilization on the ventilator, when it becomes apparent that the patient will require prolonged ventilator assistance. Maziak and coworkers [ 12 ] reviewed five reports on the timing of tracheostomy and concluded that there was insufficient evidence to conclude that the timing of tracheostomy alters the duration of mechanical ventilation. However, there is still a lack of data on the relationship between the timing of tracheostomy and weaning from mechanical ventilation for patients in the medical ICU. Therefore, we investigated the timing of tracheostomy and other factors that might influence weaning from mechanical ventilation and outcomes of patients admitted to the medical ICU. Methods Patients Over a period of 36 months (from July 1998 to June 2001), all adult patients admitted to the medical ICU of National Taiwan University Hospital – a 1500-bed tertiary medical centre that accommodates tracheostomy within the ICU – were considered for inclusion in the study. Patients were excluded if the tracheostomy was performed in an emergency setting because of difficulties with the airway or other causes. Tracheostomy was performed using standard surgical techniques at bedside in the ICU, and no patients underwent percutaneous tracheostomy. The timing of tracheostomy depended on the attending physician's decision. Indications to initiate an attempt to wean a patient from mechanical ventilation included stable haemodynamic status, improved oxygenation (arterial oxygen tension [PaO 2 ]/fractional inspired oxygen [FiO 2 ] ratio >150), controlled infection and lack of need for further intervention. The weaning process was begun with synchronized intermittent mandatory ventilation with pressure support. Then, patients underwent continuous positive airway pressure with pressure support, or intermittent T-piece for a spontaneous breathing trial when clinical conditions improved. Successful weaning was defined as weaning from mechanical ventilation for more than 72 hours. Patients were transferred to long-term care settings once tracheostomy and the weaning process were completed if there was no other active clinical disease. Data collection The indications for intubation were defined as any major problem(s) that necessitated intubation. The underlying disease of the patients, including diabetes mellitus, hypertension, congestive heart failure, chronic renal insufficiency, chronic obstructive pulmonary disease and malignant disease with lung metastasis, were ascertained through chart reviews. Medical records were analyzed for age, sex, underlying disease and cause of intubation, Acute Physiology and Chronic Health Evaluation (APACHE) II score [ 13 ], duration of mechanical ventilation, complications of tracheostomy, pneumonia after tracheostomy, length of ICU stay, and mortality in the ICU and hospital. APACHE II scores were calculated using clinical data, which were available from the first 24 hours of intensive care. Clinical data within 72 hours before tracheostomy, including PaO 2 /FiO 2 ratio, peripheral white blood cell (WBC) counts, haemoglobin, creatinine and albumin, were also recorded and analyzed. Old age was defined as age above 65 years. Anaemia was defined as haemoglobin below 10 g/dl, and leucocytosis was defined as a WBC count above 11,000/ μl before tracheostomy. Renal insufficiency was defined as creatinine above 1.5 mg/dl, and poor oxygenation as PaO 2 /FiO 2 ratio below 250. Complications of tracheostomy, including bleeding, air leakage, pneumothorax, subcutaneous emphysema, cardiopulmonary arrest, dislodgement of the tube, obstruction, tracheal stenosis, granuloma, tracheo-oesophageal fistula and tracheomalacia, were recorded. Complications that occurred within 7 days after tracheostomy were defined as early complications; those occurring later were considered late complications. Severity of bleeding after tracheostomy was classified as follows: minor if there was only minimal blood clot over the wound or if new onset bloody sputum was noted on the next day of the tracheostomy; moderate if bleeding needed external compression and component therapy or surgical management; and massive if the bleeding resulted in obvious haemodynamic change. The clinical definition of post-tracheostomy pneumonia used was as follows [ 14 ]: new and persistent radiographic opacity found after the tracheostomy had been removed and within 48 hours into the weaning period; positive sputum culture; and three of body temperature above 38°C, WBC count above 15,000/μl, increased airway secretions, or worsening gas exchange. Statistical analysis Values are expressed as mean ± standard deviation (continuous variables) or as a percentage of the group from which they were derived (categorical variables). Only variables with complete data were analyzed in the study. Differences in the groups, including sex, underlying diseases and associated medical conditions, indications for intubation, occurrence of post-tracheostomy pneumonia, successful weaning and mortality, were analyzed using χ 2 test. Other variables, including age, sex, APACHE II score, the length of ICU stay, PaO 2 /FiO 2 ratio, peripheral WBC count, haemoglobin, albumin and weaning period, were analyzed by an independent t-test. The correlations between the intubation period and the length of ICU stay were analyzed using a Pearson bivariate correlation test. The correlations between successful weaning and potentially influential factors, including old age, sex, presence of comorbidities, indications for intubation, leucocytosis, anaemia, thrombocytopenia, renal insufficiency, poor oxygenation, post-tracheostomy pneumonia and timing of tracheostomy, were analyzed using the Kaplan–Meier method with a log rank test. Censoring was performed for those patients who died during mechanical ventilation. A Cox regression model was applied for multivariate analysis with variables that were significantly associated with successful weaning in the univariate analysis. P < 0.05 was considered statistically significant. Results Clinical characteristics From July 1998 through June 2001, a total of 167 patients who underwent tracheostomy in the medical ICU were included in the study. Four patients were excluded because of emergent tracheostomy due to difficult airway ( n = 3) or laryngeal oedema ( n = 1). Thus, 163 patients were included (93 male and 70 female; mean age 70 years, range 19–104 years; Table 1 ). The indications for intubation in the 163 patients were classified into four categories: pulmonary ( n = 107), infectious ( n = 18), neurological ( n = 28) and circulatory ( n = 10) disease. The most common cause of intubation was pneumonia with respiratory failure ( n = 81 [73%]). The mean APACHE II score within the first 24 hours after ICU admission was 20.0 ± 7.2. The mean duration of intubation was 18.5 ± 10.9 days (range 1–62 days). Complications The most common early complication of tracheostomy was bleeding (moderate bleeding in 11 [6.7%] and minor bleeding in 46 [28.2%]), followed by subcutaneous emphysema (3 [1.8%]; in two this occurred together with bleeding and in one it occurred together with air leakage) and obstruction (3 [1.8%]). The most common late complication was bleeding (4 [2.5%]), followed by air leakage (3 [1.8%]) and tracheal stenosis (2 [1.2%]). The incidence of complications did not differ significantly between the successful weaning and failure-to-wean groups (early complications: 38.5% versus 37.6%, P = 1.0; late complications: 6.4% versus 9.4%, P = 0.6). No patient died during the procedure operation or because of complications of tracheostomy. Timing of tracheostomy and outcomes The patients were divided in two groups according to weaning outcome. Seventy-eight patients were successfully weaned from mechanical ventilation, and 85 patients failed to wean. The clinical characteristics, including sex, age, APACHE II score and previous comorbid conditions, were similar between the groups (Table 1 ). The most frequent reason for intubation was pulmonary disease (107 [65.6%]), followed by neurological disease (28 [17.2%]). The indications for intubation in the two groups were also similar, except that more neurological disease was noted in the successful weaning group (Table 2 ). Hypoalbuminaemia, anaemia, leucocytosis and impaired gas exchange were noted before tracheostomy. Pre-tracheostomy albumin, creatinine and haemoglobin levels were similar between groups, but the failure-to-wean group was noted to have higher WBC counts ( P = 0.05), lower platelet counts ( P = 0.005) and poor PaO 2 /FiO 2 ratio ( P = 0.003; Table 3 ). After tracheostomy, 109 patients (66.9%) developed nosocomial pneumonia. The average number of post-tracheostomy ventilator days was 27.3. Higher rates of post-tracheostomy pneumonia ( P = 0.05) and longer post-tracheostomy mechanical ventilation periods ( P = 0.001) were noted in the failure-to-wean group (Table 4 ). Shorter intubation periods ( P = 0.02), length of ICU stay ( P = 0.001) and post-tracheostomy ICU stay ( P = 0.005) were noted in the successful weaning group (Table 4 ). The overall ICU mortality was around 19%. ICU mortality is summarized in Fig. 1 . Regarding the relationship of timing of tracheostomy to successful weaning, an intubation period in excess of 21 days was associated with decreased rate of successful weaning (31.5% versus 56%, P = 0.004) and increased ICU mortality (27.8% versus 14.7%, P = 0.057). The intubation period exhibited a correlation with length of ICU stay in the successful weaning group (r = 0.70, P < 0.001; Fig. 2 ). We used day 21 as a cut-off point to define early and late trachostomy, in accordance with the clinical observations summarized in Fig. 1 . Early tracheostomy was defined as tracheostomy performed within 21 days after intubation ( n = 110); late tracheostomy was defined as tracheostomy performed later than this ( n = 53). The early tracheostomy patient group had a higher rate of successful weaning (56.4% versus 30.2%, P = 0.002) and lower ICU mortality (14.5% versus 28.3%, P = 0.05), but there were no differences between early and late tracheostomy groups in terms of hospital mortality (44.5% versus 54.7%, P = 0.25) or occurrence of nosocomial pneumonia during the weaning period (43.6% versus 60.4%, P = 0.06). The patients who underwent early tracheostomy also had shorter post-tracheostomy ICU stays (10.8 versus 14.2 days, P = 0.04) and weaning periods (19.0 versus 44.3 days, P < 0.001). In univariate analysis using the Kaplan–Meier method with log-rank test, reasons for intubation (pulmonary disease [ P = 0.03] and lack of neurological disease [ P < 0.01]), thrombocytopenia ( P = 0.03), poor oxygenation before tracheostomy ( P < 0.001), post-tracheostomy pneumonia during the weaning period ( P < 0.001) and late tracheostomy ( P < 0.001) were correlated with lower rates of successful weaning. A Cox regression model applied to the multivariate analysis showed that late tracheostomy, poor oxygenation and post-tracheostomy pneumonia during the weaning period were independent predictors of unsuccessful weaning (Fig. 3 ). Discussion The present study demonstrated that patients who underwent tracheostomy and failed to wean from mechanical ventilation had longer intubation periods before tracheostomy. Timing of tracheostomy was correlated with length of ICU stay in the successful weaning group. The type of ICU may also have an impact on the timing of tracheostomy. In surgical ICUs most patients do not have chronic lung disease or severe lung injury. These patients usually undergo tracheostomy early if they underwent a major surgical procedure and failed to extubate within several days after the operation. Previous studies [ 15 - 18 ] conducted in surgical ICUs have shown that tracheostomy performed within 1 week after intubation may be beneficial in lowering rates of pneumonia, and in shortening the duration of mechanical ventilation and length of ICU stay. However, other studies reported a higher incidence of ventilator-associated pneumonia [ 19 , 20 ] and longer length of ICU stay [ 21 ] in association with tracheostomy. In a neurological ICU, tracheostomy is usually performed if there is a depressed level of consciousness and poor ability to protect the airway. A recent study [ 22 ] demonstrated that early tracheostomy in patients in a medical ICU shortened the length of hospital stay and lowered hospital costs. The present study demonstrated that late tracheostomy may predispose to failure to wean and ICU mortality, especially when the intubation period is longer than 3 weeks. We also found that the duration of intubation before tracheostomy was correlated with length of ICU stay in patients who weaned successfully. There were no obvious differences in terms of age, sex, APACHE II score, or underlying disease between the successful weaning and failure-to-wean groups, except for more neurological disease in the successful weaning group. However, in the 3 days before tracheostomy, higher WBC count, lower platelet count and lower PaO 2 /FiO 2 ratio were noted in the failure-to-wean group. These observations suggest that leucocytosis, low platelet count and severity of respiratory failure before tracheostomy might have had a greater impact on outcome than initial presentation at ICU admission. A longer intubation period was noted in those patients who failed to wean, indicating that, like the pre-tracheostomy conditions mentioned above, late tracheostomy may predispose to poor weaning outcome. A prolonged intubation period may impair the local barrier and bronchial hygiene, increasing the risk for bacterial colonization. Also, it may result in a higher rate of post-tracheostomy pneumonia – an association that was found in the failure-to-wean group. Ely and coworkers [ 23 ] demonstrated that prolonged intubation with mechanical ventilation was associated with increased hospital mortality and was independent of severity of illness. In the present study we found that prolonged intubation was associated with prolonged ICU stay. Delaying tracheostomy might not have been beneficial in these patients. Reasons for intubation, poor pre-tracheostomy conditions, prolonged intubation and post-tracheostomy pneumonia were found to influence ventilator weaning in univariate analysis. However, in multivariate analysis we found that only late tracheostomy, pre-tracheostomy poor oxygenation and post-tracheostomy pneumonia during the weaning period were independent predictors of unsuccessful weaning. This finding suggests that timing of tracheostomy has an impact on ventilator weaning, as well as other clinical events. The 1989 ACCP Consensus Conference on Artificial Airways in Patients Receiving Mechanical Ventilation [ 10 ] suggested that tracheostomy is preferable if the anticipated need for mechanical ventilation is for more than 21 days. Recent ACCP guidelines [ 11 ] encourage early tracheostomy after patient stabilization if the patient needs prolonged mechanical ventilation. Our data support the suggestion of the earlier ACCP guidelines [ 10 ] that, when tracheostomy is performed more than 3 weeks after intubation, rates of ICU mortality and failure to wean increase. The incidence of complications in adults who have undergone tracheostomy varies from 6% to 51% [ 4 , 24 , 25 ]. In the present study, the early complication rate was 38% and the late complication rate was 8% during hospitalization. The major early complication was minor to moderate bleeding from surgical wounds, which did not cause obvious clinical deterioration. We found tracheostomy to be a relatively safe procedure for airway management in patients who needed prolonged mechanical ventilation. There are some limitations to the study. This retrospective study lacks baseline pulmonary function data before tracheostomy, which might have influenced the duration of weaning. Poor patient condition on admission to the medical ICU might have influenced the decision to perform a tracheostomy late. Conclusion In this study we found that performance of tracheostomy more than 21 days after intubation was associated with prolonged weaning periods and low rates of successful weaning. It might also result in prolonged ICU stay. If one waits longer than 21 days, then it may be better to forego tracheostomy altogether. Key messages • We found that performance of tracheostomy more than 21 days after intubation was associated with prolonged weaning periods and low rates of weaning. • Late tracheostomy might also result in prolonged ICU stay; if one waits longer than 21 days, then it may be better to forego tracheostomy altogether. Abbreviations ACCP = American College of Chest Physicians; APACHE = Acute Physiology and Chronic Health Evaluation; FiO 2 = fractional inspired oxygen; ICU = intensive care unit; PaO 2 = arterial oxygen tension; WBC = white blood cell. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CLH participated in the study design and drafted the manuscript. KYC conceived the study, participated in its design and helped to draft the manuscript. JSJ, CJY and PCY participated in study design.

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1065114

Aerosolized colistin for the treatment of nosocomial pneumonia due to multidrug-resistant Gram-negative bacteria in patients without cystic fibrosis

Introduction The clinical and economic consequences of the emergence of multidrug-resistant Gram-negative bacteria in the intensive care unit (ICU) setting, combined with the high mortality rate among patients with nosocomial pneumonia, have stimulated a search for alternative therapeutic options to treat such infections. The use of adjunctive therapy with aerosolized colistin represents one of these. There is extensive experience with use of aerosolized colistin by patients with cystic fibrosis, but there is a lack of data regarding the use of aerosolized colistin in patients without cystic fibrosis. Methods We conducted the present study to assess the safety and effectiveness of aerosolized colistin as an adjunct to intravenous antimicrobial therapy for treatment of Gram-negative nosocomial pneumonia. We retrospectively reviewed the medical records of patients hospitalized in a 450-bed tertiary care hospital during the period from October 2000 to January 2004, and who received aerosolized colistin as adjunctive therapy for multidrug-resistant pneumonia. Results Eight patients received aerosolized colistin. All patients had been admitted to the ICU, with mean Acute Physiological and Chronic Health Evaluation II scores on the day of ICU admission and on day 1 of aerosolized colistin administration of 14.6 and 17.1, respectively. Six of the eight patients had ventilator-associated pneumonia. The responsible pathogens were Acinetobacter baumannii (in seven out of eight cases) and Pseudomonas aeruginosa (in one out of eight cases) strains. Half of the isolated pathogens were sensitive only to colistin. The daily dose of aerosolized colistin ranged from 1.5 to 6 million IU (divided into three or four doses), and the mean duration of administration was 10.5 days. Seven out of eight patients received concomitant intravenous treatment with colistin or other antimicrobial agents. The pneumonia was observed to respond to treatment in seven out of eight patients (four were cured and three improved [they were transferred to another facility]). One patient deteriorated and died from septic shock and multiple organ failure. Aerosolized colistin was well tolerated by all patients; no bronchoconstriction or chest tightness was reported. Conclusion Aerosolized colistin may be a beneficial adjunctive treatment in the management of nosocomial pneumonia (ventilator associated or not) due to multidrug-resistant Gram-negative bacteria.

Introduction Nosocomial pneumonia due to multidrug-resistant Gram-negative bacteria, such as certain Pseudomonas aeruginosa and Acinetobacter baumannii strains, is among the most serious complications that occur in the intensive care unit (ICU) setting. Mortality, morbidity and health care costs are substantially increased by this type of infection [ 1 - 3 ]. Increasing rates of resistance among Gram-negative bacteria to most classes of antimicrobial agents have frequently led to clinical failure of currently employed therapies. Lack of development and introduction into clinical practice of new antibiotics to combat multiresistant Gram-negative bacteria have stimulated renewed interest in the use of the older antibiotic colistin. Outcomes in patients with ventilator-associated pneumonia (VAP) due to multidrug-resistant Gram-negative bacteria are poor [ 1 ]. Intravenous colistin was recently used to treat such infections. Notably, a recent study [ 4 ] compared intravenous colistin (21 patients) with imipenem (14 patients) in the treatment of VAP due to multidrug-resistant A baumannii . Mortality rates were similar: 61.9% among patients treated with intravenous colistin and 64.2% among patients treated with imipenem. In patients with cystic fibrosis, aerosolized colistin has successfully been used to treat acute pulmonary exacerbations of infection or initial colonization with P aeruginosa strains [ 5 , 6 ]. However, there is a lack of data regarding the use of aerosolized colistin in patients without cystic fibrosis. A few reports have indicated that aerosolized colistin may be a beneficial additional therapeutic intervention in the management of nosocomial pneumonia (whether ventilator associated or not) [ 7 - 10 ]. In addition, a few old reports of the use of aerosolized polymyxin B yielded controversial results. Feeley and coworkers [ 11 ] reported that use of polymyxin B aerosol in seriously ill patients is associated with increased incidence of pneumonia due to polymyxin-resistant organisms. However, Klastersky and colleagues [ 12 ] found endotracheal administration of polymyxin B plus aminosidin to be a useful alternative regimen to endotracheal gentamicin for the prevention of lung infections. We present data from our recent experience with aerosolized colistin for the treatment of pneumonia due to multidrug-resistant Gram-negative bacteria in eight ICU patients. Methods Design of the study and patient population Patients who received colistin (Colomycin ® , Forest Laboratories, Kent, UK, or Colistin ® , Norma, Athens, Greece) for treatment of infections with multidrug-resistant Gram-negative bacteria from 1 October 2000 to 31 January 2004 at 'Henry Dunant' Hospital (a 450-bed tertiary care centre in Athens, Greece) were identified from the pharmacy electronic database. Medical records, specifically nursing records of medication administration, were retrospectively reviewed for all patients in order to identify those who received aerosolized colistin. One milligram of the colistin formulations used is approximately equal to 12,500 IU (Forest Laboratories, Kent) or 13,333 IU (Norma, Athens). Administration of aerosolized colistin for the treatment of nosocomial pneumonia due to Gram-negative bacteria, and review of patients' charts were approved by the institutional review board of the hospital. Data collection and entry Data for several variables, including demographic and clinical information, as well as the results of laboratory and imaging tests (chest radiography or computed tomography of the thorax), were collected from the medical records of patients receiving aerosolized colistin. All available results of renal function tests (creatinine, urea, creatinine clearance, urinalysis), liver function tests (serum glutamate-pyruvate transaminase, serum glutamic-oxaloacetic transaminase, alkaline phosphatase, γ-glutamyltransferase, bilirubin), creatine phosphokinase and arterial blood gases were recorded during the course of colistin treatment and at hospital discharge. Microbiological testing All causative micro-organisms were identified using routine microbiological methods. Susceptibility testing was done using both the disk diffusion method and an automated broth microdilution method (Vitek II; bioMerieux, Hazelwood, MO, USA). (The breakpoints were those defined by the National Committee for Clinical Laboratory Standards [ 13 , 14 ].) Susceptibility to colistin was tested by means of the disk diffusion method using a 10 μg colistin disk (Oxoid, Basingstoke, UK); isolates were considered sensitive if the inhibition zone was ≥ 11 mm. Intermediate sensitivity of isolated Gram-negative pathogens to antimicrobial agents was considered resistance. Multidrug-resistant was defined as resistance of the isolate to five antipseudomonal classes of antimicrobial agents (i.e. antipseudomonal penicillins, cephalosporins, carbapenems, monobactams, quinolones, colistin and aminoglycosides). An isolate was defined as colistin-only sensitive if it was resistant to all antipseudomonal agents except colistin. Definition of pneumonia Diagnosis of pneumonia required two or more serial chest radiographs with at least one of the following: new or progressive and persistent infiltrate, consolidation, cavitation, or pleural effusion. In addition, patients were required to have had fever >38°C with no other recognized cause or an abnormal white blood cell count (leucopenia [<4000 white blood cells/mm 3 ] or leucocytosis [≥ 12,000 white blood cells/mm 3 ]), and at least two of the following: new onset of purulent sputum, change in the character of sputum, increased respiratory secretions, or increased requirement for suctioning; new onset or worsening of cough, or dyspnoea or tachypnoea; rales or bronchial breath sounds; or worsening gas exchange. Pneumonia was considered to be ventilator associated (VAP) when its onset occurred 48 hours after the initiation of mechanical ventilation, and was judged not to have been incubating before the initiation of mechanical ventilation [ 15 ]. Definition of outcome The definition of positive outcome (cure or improvement) of pneumonia was based on clinical (fever defervescence, resolution or partial resolution of presenting symptoms and signs of pneumonia, decrease in suctioning requirements), radiological (decrease or disappearance of presenting findings on chest x-ray), and laboratory findings (improvement in arterial blood gases, or normalization of white blood cell count and C-reactive protein). Results From 1 October 2000 through 31 January 2004, 152 patients received treatment with intravenous colistin for infections with multidrug-resistant Gram-negative bacteria. Eight out of 152 patients were identified as having received aerosolized colistin for the management of Gram-negative nosocomial pneumonia. Table 1 describes the demographic and clinical features of these patients, including comorbidities, responsible pathogen(s) and susceptibility of the pathogen(s) to commonly tested antimicrobial agents, as well as the outcome of the infection and of the patient. The mean age of the patients was 59.6 years and most of them were male (six out of eight). All patients had been admitted to the ICU, with a mean Acute Physiology and Chronic Health Evaluation II scores on the day of ICU admission and on day 1 of aerosolized colistin administration of 14.6 and 17.1, respectively. During the preceding 3 months, three patients had been hospitalized in the same or another unit. All patients had received other antimicrobial regimens before aerosolized colistin was initiated. In addition, three patients received immunosuppressive treatment (steroids) and four received immunoglobulin therapy during their hospitalization. The responsible pathogens in the eight cases of nosocomial pneumonia were Acinetobacter baumannii (seven out of eight) and P aeruginosa (one out of eight) strains. Only in one case was a second strain isolated from the same culture specimen, and it was found to be methicillin-resistant Staphylococcus aureus . Half of the isolated pathogens were sensitive only to colistin; the rest were multidrug-resistant strains. All patients received mechanical ventilatory support for a mean of 19.4 days. Colistin was prepared for nebulization; 1 or 2 million IU colistin was diluted in 2 or 4 ml sterile normal saline 0.9%, respectively. In patients undergoing mechanical ventilation aerosolized colistin was delivered by means of the Siemens Servo Ventilator 300 (Siemens-Elma AB, Solna, Sweden). In spontaneously breathing patients colistin was administered as follows: 1,000,000 IU were added to 4 ml normal saline and the solution was nebulized with 8 l/min oxygen flow and inhaled via a face mask. This technique of administration of aerosolized medication is commonly used worldwide for the administration of bronchodilators in nebulized form. The daily dose of aerosolized colistin ranged from 1.5 to 6 million IU divided into three or four doses, and the duration of administration ranged from 3 to 32 days (mean 10.5 days). No strictly uniform dosing strategy for aerosolized colistin was applied, and differences in regimen reflect the differing approaches of the individual attending physicians. In addition, seven out of eight patients received concomitant intravenous treatment with colistin or other antimicrobial agents with activity against Gram-negative bacteria, such as β lactams, quinolones and aminoglycosides. Only one patient received aerosolized colistin as monotherapy; she had received intravenous colistin therapy before aerosolized colistin for 7 days and continued to receive the intravenous therapy after the end of aerosolized therapy (for 32 days). The pneumonia was observed to respond to treatment in seven out of eight patients who received supplemental therapy with aerosolized colistin. Four episodes of pneumonia were cured and three were improved at the end of treatment. Only one out of the eight patients who received aerosolized colistin for the treatment of multidrug-resistant Gram-negative pneumonia deteriorated and finally died. He was a 50-year-old multiple trauma patient, who was admitted to the ICU with fractures located at C4–C5, haemothorax and functional dissection of the spinal cord due to a car accident. His past medical history was noteworthy for arterial hypertension, Wolff–Parkinson–White syndrome, chronic renal insufficiency due to polycystic kidney disease and ankylosing spondylitis, for which he was receiving steroid therapy. During his prolonged hospitalization in the ICU, the patient developed pneumonia due to multidrug-resistant A baumannii , requiring intubation. His clinical condition became complicated by sepsis syndrome due to an infection caused by a colistin-only sensitive P aeruginosa strain, which was unresponsive to administered antimicrobial treatment. On day 95 of his hospitalization in the ICU, he died from septic shock and multiple organ failure. Follow-up cultures were available for five out of eight patients. In four of them the responsible pathogen was eradicated, and in one case the pathogen persisted in repeated specimen cultures; this patient died. Superinfection with Gram-positive micro-organisms or yeasts was not observed. No Gram-negative bacterium developed resistance to colistin in subsequent specimen cultures during or at the end of aerosolized treatment. Administration of aerosolized colistin was well tolerated by all patients. During treatment, all patients were closely monitored for possible respiratory adverse reactions, but none of them experienced chest tightness, bronchoconstriction, or apnoea. Only two patients, who had history of chronic obstructive pulmonary disease, received concurrent treatment with inhaled β 2 agonist. Only in the patient who died did renal function worsen (baseline serum creatinine increased by 1.4 mg/dl) during aerosolized colistin treatment. This patient, as mentioned above, had a history of polycystic kidney disease and chronic renal failure, and died from septic shock and multiple organ failure. No deterioration in renal function was observed in the other seven patients during colistin treatment. One patient had baseline serum creatinine levels of 5.4 mg/dl, and at the end of colistin treatment serum creatinine had decreased to 4.5 mg/dl. That particular patient was already receiving haemodialysis treatment before the initiation of intravenous or aerosolized colistin. Of 152 patients who received treatment with intravenous colistin for infections with multidrug-resistant Gram-negative bacteria during the period of study, 55 had received less than 72 hours of intravenous colistin and were excluded from all analyses. Medical records were not available for three patients; in addition, one patient was in the hospital during data collection. Thus, 93 patients were further analyzed. Forty-five of these patients received intravenous colistin for the treatment of nosocomial pneumonia due to Gram-negative bacteria. Survival and clinical cure rates for the infection were better, although not statistically significantly so, in patients with pneumonia who received additional aerosolized colistin than in patients who received only intravenous colistin treatment (survival: 7/8 patients [87.5%] versus 34/45 patients [75.6%], P = 0.41; clinical cure: 7/8 patients [87.5%] versus 30/45 patients [66.7%], P = 0.67). Discussion Aerosolized colistin may be an effective adjunctive intervention for the treatment of nosocomial pneumonia due to multidrug-resistant Gram-negative bacteria in patients without cystic fibrosis. Colistin and polymyxin E are old antibiotics; colistin was almost abandoned for many years because of its reported nephrotoxicity and neurotoxicity. This medication was reintroduced into clinical practice just a few years ago, and this resulted mainly from increased resistance rates among Gram-negative bacteria, especially in the ICU setting, and the absence of new and effective alternative therapeutic options [ 16 - 18 ]. The idea of using colistin or polymyxin B (which belongs to the same group of antibiotics, and has similar antimicrobial spectrum, usage indications and toxicities as colistin) in the nebulized form for the management of pneumonia due to Gram-negative bacteria is not new. In 1963, Pino and coworkers [ 19 ] used aerosolized colistin in patients with pulmonary suppurations. A few years later, Marschke and Sarauw [ 20 ] reported two cases of pneumonia due to P aeruginosa strains in patients with underlying bronchiectasis and chronic bronchitis, in which polymyxin B was given by inhalation. Both patients experienced dyspnoea due to airway obstruction. Recently, aerosolized colistin was used successfully to treat and prevent pneumonia caused by P aeruginosa in patients with human immunodeficiency syndrome and in patients with nosocomial pneumonia and tracheobronchitis [ 21 - 23 ]. There is extensive experience with administration of aerosolized colistin to patients with cystic fibrosis, in whom this type of treatment is used to prevent or treat lung infections with P aeruginosa strains. Notably, studies found that nebulized colistin reduced the number of relapses of lung infections and subsequently the decline in lung function among patients with cystic fibrosis [ 24 - 27 ]. The pharmacokinetic properties and dosing strategies of aerosolized colistin are not well defined. Whether the various forms of colistin used for inhalation therapy (e.g. dry powder formulation for inhalation, colistin solutions for nebulization) or the different types of nebulizing systems influence the effectiveness and safety of colistin remains to be determined [ 28 - 31 ]. Adverse effects of aerosolized colistin or polymyxin B are a major concern; potential adverse effects include bronchoconstriction, chest tightness and apnoea due to neuromuscular blockade. One study conducted in 58 children with cystic fibrosis who received nebulized colistin for the treatment of lung infections [ 32 ] reported that 20 of them experienced a decrease in forced expiratory volume in 1 s by greater than 10% from baseline. In addition, another study [ 33 ] found that 35 out of 46 adult patients with cystic fibrosis who also received nebulized colistin for lung infection developed chest tightness. However, treatment with inhaled β 2 agonists before the initiation of aerosolized colistin was able to prevent the development of such side effects in the respiratory system. Another significant concern regarding the use of aerosolized colistin for the treatment of nosocomial pneumonia is dissemination of multidrug-resistant bacteria through nebulizer devices [ 34 , 35 ]. However, this potential problem could be eliminated by strict use of appropriate infection control guidelines by medical and nursing hospital staff. Our study is not without limitations. It is a small case series and is of a retrospective design. In addition, there is no control group of patients receiving treatment with only intravenous antimicrobial agents. Furthermore, some of the patients also received intravenous treatment with other antimicrobial agents, which might have influenced the outcomes. Two major risks are arising from the wide use of colistin: the emergence of Gram-negative bacteria, such as P aeruginosa and A baumannii , resistant to colistin; and an increase of infections due to Gram-positive and Gram-negative pathogens, such as Proteus and Serratia spp., inherently resistant to colistin. Consequently, there is an urgent need to restrict the use of colistin use in order to minimize these risks. Conclusion Inhaled colistin may be beneficial in the treatment of nosocomial pneumonia (ventilator associated or not) due to multidrug-resistant, Gram-negative bacteria. However, the severity of these infections in the ICU setting means that treatment just with aerosolized colistin is unlikely to be sufficient. This is in contrast to therapeutic strategies employed in patients with cystic fibrosis, in which initial lung colonization with P aeruginosa strains is commonly treated with aerosolized colistin alone. Randomized controlled trials studying the possible additional benefits and risks associated with use of nebulized colistin, as an adjunct to intravenous antimicrobial treatment, in patients with pneumonia due to multidrug-resistant Gram-negative bacteria are urgently needed. Key messages • Aerosolized administration of colistin is a promising adjunctive therapy for management of patients with pneumonia (whether ventilator associated or not) due to multiresistant Gram-negative bacteria • Aerosolized colistin was safe in this group of patients. • There is an urgent need for randomized controlled trials examining the efficacy and safety of aerosolized colistin for the management of patients with nosocomial pneumonia. Abbreviations ICU = intensive care unit; VAP = ventilator-associated pneumonia. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AM and MEF conceived the study. SKK, ZM, KR and AMK collected data. All authors contributed to the writing and preparation of the manuscript.

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1065265

The N. gonorrhoeae Type IV Pilus Stimulates Mechanosensitive Pathways and Cytoprotection through a pilT-Dependent Mechanism

The Neisseria gonorrhoeae type IV pilus is a retractile appendage that can generate forces near 100 pN. We tested the hypothesis that type IV pilus retraction influences epithelial cell gene expression by exerting tension on the host membrane. Wild-type and retraction-defective bacteria altered the expression of an identical set of epithelial cell genes during attachment. Interestingly, pilus retraction, per se, did not regulate novel gene expression but, rather, enhanced the expression of a subset of the infection-regulated genes. This is accomplished through mitogen-activated protein kinase activation and at least one other undefined stress-activated pathway. These results can be reproduced by applying artificial force on the epithelial membrane, using a magnet and magnetic beads. Importantly, this retraction-mediated signaling increases the ability of the cell to withstand apoptotic signals triggered by infection. We conclude that pilus retraction stimulates mechanosensitive pathways that enhance the expression of stress-responsive genes and activate cytoprotective signaling. A model for the role of pilus retraction in influencing host cell survival is presented.

Introduction Many pathogenic and nonpathogenic bacteria produce type IV pili (Tfp), among them, Neisseria gonorrhoeae , N. meningitidis, Pseudomonas aeruginosa, Legionella pneumophila, enteropathogenic and enterohemorrhagic Escherichia coli, and Vibrio cholerae [ 1 ] . Tfp are fimbriate organelles that play a crucial role in the interaction of the bacterium with its environment, as evidenced by their requirement for motility [ 2 ], biofilm formation [ 3 , 4 ], and horizontal gene transfer [ 5 , 6 , 7 ]. These appendages also promote bacterial attachment to host cells and contribute to virulence [ 8 , 9 , 10 , 11 , 12 ]. Recent evidence has shown that the gonococcal Tfp can physically retract—a process that underlies twitching motility [ 13 ] (i.e., the ability of the bacterium to move on solid surfaces [ 14 ]). It is now generally believed that twitching motility occurs via extension, substrate tethering, and retraction of the pilus filament. Two inner membrane/cytoplasmic ATPases, PilF and PilT, take part in these activities. PilF mediates pilus assembly, as pilF mutants produce pilin subunits but are not piliated [ 15 ]. PilT is involved in pilus disassembly, as pilT mutants are piliated but cannot retract their pili [ 13 , 16 ]. Neither mutant is motile. Pilus retraction allows gonococci to form organized microbial communities on the cell surface and on synthetic substrates (S. Lee and M. S., unpublished data), via both specific and nonspecific interactions. During attachment to host cells, microcolonies stimulate the formation of cortical plaques—structures in the cell cortex containing high concentrations of transmembrane receptors, nonreceptor tyrosine kinases and their anchors, and components of the cortical cytoskeleton [ 10 , 17 ]. Though pilT mutants adhere normally to both synthetic surfaces and epithelial cells, they form disordered microcolonies, fail to induce cortical plaques, and are less invasive than their wild-type (wt) parent strain [ 17 ]. Retraction of a single gonococcal pilus can exert forces up to 80–100 pN on its substrate [ 13 , 18 ]. Forces of lesser magnitude can elongate the membrane into microvillus-like structures [ 19 , 20 ], promote cytoskeleton rearrangements and protein clustering [ 21 , 22 ], induce calcium fluxes [ 23 , 24 ], and alter gene expression [ 25 , 26 , 27 , 28 ]. Pilus retraction has therefore been speculated to induce host cell signaling by exerting mechanical tension on the membrane [ 17 ]. Indirect support for a mechanical signaling hypothesis comes from observations that pilT mutants, unlike wt piliated strains, can neither trigger cortical plaque formation [ 10 ] nor activate PI-3 kinase (S. Lee and M. S., unpublished data), a member of a mechanical stress–activated pathway. Moreover, a pilT mutant induces an attenuated calcium flux in epithelial cells, as compared to infection with wt gonococci (P. Ayala and M. S., unpublished data). Here we provide further evidence that pilus retraction acts as a mechanical stimulus by activating mechanical stress–signaling pathways that alter epithelial cell gene expression and generate a cytoprotective environment within the host cell. Results Pilus Retraction Enhances the Expression of Cell Stress/Survival Genes We used microarrays to examine the transcriptional profiles of T84 human colorectal epithelial cells infected with retraction-proficient (N400) or retraction-deficient (N400 pilT ) gonococci for 3 h. Infection with N400 or N400 pilT induced transcriptional changes in the same genes. Contrary to expectations, no genes responded uniquely to infection with either strain. Instead, infection with pilT affected the level of expression of a small subset of infection-responsive genes. To segregate the genes responding to pilus retraction, a wt to pilT fold-change expression ratio (W/P) was calculated for each infection-regulated gene. This method identified, out of approximately 300 infection-regulated genes, 69 probe sets (representing 52 genes) whose expression appeared to be enhanced by pilus retraction ( Figure 1 ). Figure 1 Infection-Regulated, Retraction-Enhanced Epithelial Cell Genes Wt and pilT values represent the mean fold-change in the transcript level of each gene in infected cells compared to uninfected cells ( n = 2). W/P values represent the degree of enhancement of gene expression resulting from pilus retraction and are the result of dividing the wt fold-change value by the pilT fold-change value from two independent experiments. The p -value for each gene represents the statistical significance of the difference in its expression level (as determined by Cyber-T analysis) between wt and pilT . The color code assigned to each gene represents its degree of response to infection as expressed by its fold-change value, W/P, and p -value. To confirm the microarray results, real-time quantitative RT-PCR was initially performed on two infection-regulated genes, DUSP5 and ADM . According to our microarray data, DUSP5 expression was enhanced by pilus retraction (W/P = 1.63), and ADM expression was not (W/P ≈ 1.0). RT-PCR results corroborated the microarray analysis, as DUSP5 transcript levels were significantly higher in N400-infected cells than N400 pilT -infected cells, whereas ADM transcript levels were similar in both sets of cells ( Figure 2 A). Ten additional genes predicted to respond to retraction and five additional genes predicted to be not affected by retraction were similarly analyzed by real-time quantitative RT-PCR ( Figure 2 B). In every case, the presumptive positives yielded W/P ratios of 1.5 or more, whereas the presumptive negatives yielded W/P ratios of approximately 1.0. Figure 2 Real-Time Quantitative RT-PCR Verification of Microarray Results and Initial Characterization of Retraction-Enhanced Genes (A) Microarray (top panels) and real-time quantitative RT-PCR (bottom panels) expression profiles of ADM and DUSP5 in uninfected cells (UI), N400-infected cells (WT), and N400 pilT -infected cells (pilT) . Microarray data are shown as box-plots ( n = 3). RT-PCR data are plotted as triplicate samples from one representative experiment. (B) Real-time quantitative RT-PCR verification of retraction-enhanced expression of selected genes. Data are expressed as average W/P (±SEM, n = 3). Genes with a W/P statistically greater than 1.0 ( p < 0.05) are denoted with an asterisk. (C) Grouping of retraction-enhanced genes according to function, based on published reports (see Table S1 ). Some genes have multiple functions and thus appear in more than one group. (D) Genes in this study that are known to be induced by environmental stress, mechanical stress, or MAPK signaling (see Table S1 ). The identification of genes whose expression is enhanced by pilus retraction raised the question of whether these genes share a common regulatory pathway or perform similar functions. The majority of genes whose expression is enhanced by retraction are involved in the cell stress response and survival ( Figure 2 C). Over half of these can be induced by environmental or other cellular stresses, and a striking number can be induced specifically by mechanical stress ( Figure 2 D). Importantly, the majority of the genes from both groups can also be induced by mitogen-activated protein kinases (MAPKs; Figure 2 D) (For literature citations, see Table S1 .) These results indicate that pilus retraction may enhance infection-induced gene expression through the MAPK pathway. ERK, JNK, and P38 MAPK Are Activated by Infection and Enhanced by Pilus Retraction The MAPK cascades are well known for their involvement in the stress response, including the response to bacterial infection. Previous studies have shown that JNK is activated in N. gonorrhoeae –infected HeLa, Chang, and phagocytic cells [ 29 , 30 ], and MAPK signaling is induced in conjunctival cells by N. meningitidis (R. Bonnah and M. S., unpublished data). To study the role of MAPK signaling in retraction-enhanced gene expression, we first determined which of these pathways are activated in infected T84 cells. Compared to resting cells ( Figure 3 A, left panel), the addition of medium alone slightly increased the levels of ERK-p, JNK-p, and P38-p ( Figure 3 A, UI), but levels of each phosphorylated kinase returned to baseline after 90 min. Infection with N400 dramatically increased the levels of all three activated kinases by 60 min post-infection ( Figure 3 A, WT). Densitometric analysis of immunoblots from two independent experiments is shown in Figure 3 B. ERK-p levels were elevated throughout the course of infection, with only a slight decrease in phosphorylation visible by 3 h post-infection. In contrast, P38-p and JNK-p levels peaked between 60 and 90 min post-infection and dropped noticeably by 3 h post-infection. Figure 3 Levels of Activated MAPK in Infected Cells and Their Involvement in Retraction-Enhanced Gene Expression (A) Representative immunoblot showing ERK-p, P38-p, and JNK-p, in uninfected cells before (0 h) and after medium change (UI), or in cells infected with N400 (WT) or N400 pilT (pilT). Total P38 protein levels in each sample served as the internal control (bottom lanes). (B and C) ERK-p JNK-p, and P38-p levels over time in cells infected with N400 and N400 pilT, respectively. Immunoblots from (A) were analyzed by densitometry, and levels of activated kinase from infected cells were normalized to that from uninfected cells (UI). Values represent mean normalized protein levels (±SEM, n = 2). Solid markers indicate a significant difference between wt and pilT -induced MAPK phosphorylation at that time point ( p < 0.05); thus, ERK-p is significant at 60, 90, and 180 min; JNK-p is significant at 60 min; and P38-p is significant at 60 and 90 min. (D) Representative immunoblot showing ERK-p, MAPKAPK2-p, and c-Jun-p in cells preincubated with vehicle (DMSO) or MAPK inhibitors and infected for 90 min with N400 (WT) or left untreated (UI). Total P38 protein levels in each sample served as the internal control (bottom lanes). (E) Real-time quantitative RT-PCR analysis of the effect of MAPK inhibitors on the expression of retraction-responsive genes. Light bars indicate cells infected with N400 in the presence of vehicle (DMSO); dark bars indicate cells infected with N400 in the presence of MAPK inhibitors. Values represent the fold-change (±SEM, n = 2) in transcript levels compared to uninfected, DMSO treated control. A significant difference in expression between the two conditions is denoted by an asterisk ( p < 0.1). We next examined MAPK phosphorylation in T84 cells infected with N400 pilT to determine whether kinase activation was influenced by pilus retraction. Low levels of all three activated MAPKs were detected in N400 pilT -infected cells only after 90 min of infection ( Figure 3 A, PT). Densitometric analysis of immunoblots from two independent experiments is shown in Figure 3 C. Although the kinetics of MAPK activation appear to be different in wt- and pilT -infected cells, a firm conclusion cannot be drawn from these results, given the delayed onset of activation and the low levels of phosphorylation of each enzyme. Taken together, these results demonstrate that infection by piliated gonococci activates all three MAPK pathways and that pilus retraction enhances this activation. MAPK Signaling Is a Mediator of Retraction-Dependent Enhancement of Gene Expression We next determined whether MAPK signaling regulates the expression of retraction-enhanced genes. T84 cells were preincubated with vehicle or MAPK inhibitors SB203588, U0126, and SP600125, and assessed for ERK, P38, and JNK activation by immunoblotting for ERK-p, MAPKAPK2-p, and c-Jun-p, respectively. MAPK inhibitors dramatically reduced the levels of all three activated kinases in both uninfected and N400-infected cells ( Figure 3 D). They also significantly reduced the transcript levels of four of the five retraction-responsive genes in N400-infected cells, as judged by real-time quantitative RT-PCR ( Figure 3 E). In contrast, the inhibitors did not affect the transcript levels of genes with a W/P of approximately 1.0. Interestingly, cyr61 expression was unaltered by MAPK inhibitors. This gene was shown by microarray (W/P = 2.15) and RT-PCR analysis (W/P = 1.86) to respond to retraction. These results implicate MAPK signaling in the regulation of some, but not all, retraction-responsive genes. They indicate that other pathways also influence the response of genes to pilus retraction. Mechanical Stress Activates MAPK Signaling and Upregulates Retraction-Responsive Genes A significant number of the retraction-responsive genes are known to be induced specifically by mechanical strain on the cell membrane. Although substantial force is generated by pilus retraction in vitro, this force has not yet been demonstrated to influence host responses to infection. To examine this issue, we determined whether artificial mechanical force on the epithelial cell membrane could mimic retraction-induced MAPK activation and retraction-enhanced gene expression. To generate mechanical stress in a manner similar to that of pilus retraction, a modified magnet-based force assay was used [ 31 ]. Magnetic beads were coated with crude pili preparations (CPPs) from piliated gonococci and added to T84 cells ( Figure 4 A). Within 30 min, small clusters of approximately two to ten beads attached to the cells, with each cell containing two to three clusters of beads (data not shown). Cell monolayers were then placed 10 mm beneath the magnet. At this distance, the magnet generates an upward force of 4 pN per bead ( Figure 4 B), or approximately 20–100 pN per cell. Figure 4 Artificial Force Triggers MAPK Phosphorylation and Induces the Expression of Retraction-Enhanced Genes (A) Representation of the magnet/magnetic bead assay. (B) Average force generated on one bead as a function of magnet distance from the culture dish. Data represent the forces calculated from four identical magnets (±SEM). All subsequent assays were performed using a magnet distance of 10 mm, which corresponds to a force of 4 pN per bead (dotted line). (C) Magnet-induced clustering of actin beneath magnetic beads. CPP-coated beads were seeded onto T84 cells and exposed to the magnet for 1 h (top panels) or left untreated (no magnet, bottom panels). Differential interference contrast images (left panels) reveal the location of the beads; phalloidin staining (middle panels) shows the presence of actin at the same site. Right panels show the two previous images merged. (D) Representative immunoblot of ERK-p, JNK-p, and P38-p in cells seeded with CPP-coated beads, BSA-coated beads, or no beads, and exposed to the magnet for 15 or 30 min. Total P38 protein levels in each sample served as the internal control (bottom panels). (E) Quantitation of ERK-p, JNK-p, and P38-p signals by densitometry from the representative immunoblot shown in (D), normalized to the no-bead control. Solid lines indicate signals from cells exposed to membrane-coated beads; dotted lines indicate signals from cells exposed to BSA-coated beads. (F) Real-time quantitative RT-PCR analysis of the transcript levels of selected genes in cells seeded with CPP beads and exposed to the magnet for 3 h. Data represent the average fold-change (±SEM, n = 2) compared to a no-magnet control. A significant difference in expression on force induction is denoted by an asterisk ( p < 0.1). T84 cells seeded with CPP-coated beads and exposed to the magnet were first examined for the presence of actin recruitment into cortical plaques (see Introduction). The clustering of actin near these beads would indicate that the magnetic force was sufficient to mimic pilus retraction forces from the bacterial microcolony. In the presence of magnetic force, actin concentrated in the cell cortex around membrane-coated beads ( Figure 4 C, top panel). In contrast, actin did not cluster with the beads in the absence of the magnet ( Figure 4 C, bottom panel). Thus, the force generated by this magnet system was sufficient to recruit actin to the site of the attached beads. We next determined whether magnetic forces applied to CPP-coated beads were sufficient to activate MAPK and alter gene expression. The levels of all three phosphokinases were slightly reduced when the magnetic field was applied to cells incubated with medium alone ( Figure 4 D, no beads). Levels of each phosphorylated kinase from bead-treated samples ( Figure 4 D, CPP) were normalized to those from the no-bead samples to account for the effect of the magnet alone on MAPK phosphorylation. Following normalization, increased levels of all three phosphokinases are evident within the short time course ( Figure 4 E, solid lines). In parallel experiments, cells were seeded with bovine serum albumin (BSA)-coated beads and exposed to the magnet. Under these conditions, less force was applied to the cells, as fewer bead clusters attached to the cells, and each cluster contained only two to three beads on average (data not shown). Again, levels of each phosphorylated kinase from bead-treated samples ( Figure 4 D, BSA beads) were normalized to those from the no-bead samples to account for the effect of the magnet alone on MAPK phosphorylation. Despite lower forces, BSA-coated beads also activated ERK, JNK, and P38 ( Figure 4 E, dashed lines). Interestingly, force-induced activation of both JNK and ERK was higher in cells treated with BSA-coated beads. This can most likely be attributed to the fact that BSA-coated beads, unlike CPP beads, induce no MAPK activation in the absence of force (data not shown). Thus when force-induced MAPK activation is calculated, the CPP-coated beads are normalized to a higher level of “background” activation than are the BSA-coated beads. The observation that force induction via both CPP- and BSA-coated beads can induce these signals strongly indicates that activation of MAPK cascades is, in part, a response to stress forces on the membrane rather than to force mediated through specific adhesin–receptor contacts between the bacterium and the host. To examine the effect of mechanical stress on gene expression changes, cells seeded with CPP-coated beads were exposed to magnetic force for 3 h, and gene expression levels were analyzed by real-time quantitative RT-PCR. Transcript levels were expressed as the ratio of signals from magnet-stimulated cells to those from cells not subjected to magnetic force. All three “enhanced” genes tested, EGR1, DTR, and DUSP5, were upregulated in cells exposed to magnetic force ( Figure 4 F). In contrast, neither ADM (W/P ≈ 1.0) nor cyr61 (which did not respond to MAPK inhibitors; see Figure 3 E) was affected by the magnet. In this and the previous experiment, magnet-induced changes were of lower magnitude than those induced by infection. The most plausible explanation for this difference is that pilus retraction from a microcolony likely generates greater force than a magnet acting on a small cluster of beads. In our magnet assay, an average force of 20–100 pN was placed on each cell. During an infection, each pilus can induce this amount of force. Thus, if there are 10–100 bacteria per microcolony, and each bacterium expressed 10 pili (a conservative estimate), pilus retraction from a single microcolony could place forces of 10 4 –10 5 pN on the cell. Nonetheless, our method of artificial force application did indeed activate all three MAPK cascades and increased the expression level of each gene examined by approximately 1.5-fold. (Note that a minimum 1.5-fold change in expression level was found to accurately identify retraction-responsive genes in the microarray experiment.) Together, these results demonstrate that retraction-enhanced MAPK activation and gene expression changes can be replicated by artificial force. Pilus Retraction Mediates Host Cell Cytoprotection Many of the retraction-responsive genes are known to protect cells from apoptosis and from a variety of cellular stresses. Moreover, prolonged ERK activation accompanied by transient JNK and P38 activation (as observed in a wt infection; see Figure 3 A and 3 B) is hypothesized to mediate cytoprotection [ 32 , 33 , 34 , 35 ]. We therefore investigated whether pilus retraction was involved in determining cell fate by assaying infected cells for cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase 8. PARP is a 116-kDa nuclear protein that mediates DNA repair in response to cell stress and is required to maintain cell viability [ 36 , 37 ]. During programmed cell death, the protein is cleaved by caspase 3 or caspase 7, a terminal step in the caspase cascade [ 38 , 39 ]. Caspase 8, however, is an initiator caspase that is upstream of caspase 3, caspase 7, and PARP, and represents an earlier event in the apoptosis cascade. Thus, increased levels of cleaved PARP or caspase 8 indicate that a cell is undergoing apoptosis. Cells infected with N400 for 6 h contained lower levels of both cleaved PARP and cleaved caspase 8 than did uninfected cells ( Figure 5 A). In contrast, N400 pilT -infected cells had higher levels of cleaved PARP and cleaved caspase 8 than did both uninfected and wt-infected cells. These results indicate that piliated gonococci that cannot retract pili induce low levels of programmed cell death in a culture. In contrast, gonococci capable of retracting their pili lower the tendency for cells to enter the apoptosis pathway. Figure 5 Pilus Retraction during Bacterial Attachment Promotes Host Cell Cytoprotection (A) Levels of cleaved PARP and cleaved caspase 8 in T84 cells infected for 6 h with N400 (WT) or N400 pilT (pilT), normalized to cleaved PARP or cleaved caspase 8 levels in uninfected cells. (B) Levels of cleaved PARP and cleaved caspase 8 in T84 cells infected with N400 (WT) or N400 pilT (pilT) or left uninfected (UI) for 4 h, then incubated with STS (1 μM) for an additional 4 h to induce apoptosis. A significant difference from uninfected cells is denoted by two asterisks ( p < 0.05). (C) Cleaved PARP and cleaved caspase 8 levels in cells exposed to magnetic force. T84 cells were seeded with CPP- or BSA-coated beads and exposed to the magnet for 2 h or were left unexposed, then incubated with STS (1 μM) for an additional 4 h away from the magnet. The cleaved PARP and cleaved caspase 8 level in cells without beads and not exposed to magnetic force is arbitrarily assigned a value of 1.0, and all other treatments are expressed relative to this value. For all experiments, cleaved protein levels were quantified by densitometry of immunoblot signals. Values represent the mean levels of cleaved target (±SEM) from two independent experiments. A significant difference from untreated cells is denoted by two asterisks ( p < 0.05) or by a single asterisk ( p < 0.1). We next determined whether this cytoprotective effect of pilus retraction was sufficient to protect cells from staurosporine (STS)-induced apoptosis. STS is a cell-permeant protein kinase inhibitor that induces apoptosis at micromolar concentrations [ 40 , 41 ]. Infection of urethral epithelium with N. gonorrhoeae was recently reported to protect these cells from STS-induced apoptosis [ 42 ]. Both N400 and N400 pilT infection protected T84 cells from STS-induced apoptosis, as compared to uninfected cells ( Figure 5 B). However, cleaved PARP and cleaved caspase 8 levels in N400 pilT -infected cells were higher than in wt-infected cells, indicating that pilus retraction enhances protection from STS-induced apoptosis. Finally, we examined whether this retraction-enhanced cytoprotection is specifically mediated by mechanical force. In the absence of force, CPP-coated beads provided moderate protection from STS-induced apoptosis, demonstrated by lower cleaved PARP and cleaved caspase 8 levels than the no-bead cell control ( Figure 5 C). This result is similar to that seen in pilT -infected cells and suggests that components in the bacterial membrane are sufficient to protect against STS-induced apoptosis. Cells seeded with CPP-coated beads and exposed to the magnetic field had still lower cleaved PARP and cleaved caspase 8 levels, consistent with data from wt-infected cells ( Figure 5 B). BSA-coated beads did not protect against STS-induced apoptosis in the absence of magnetic force. However, when force was applied to these cells, the level of cleaved PARP and cleaved caspase 8 was reduced nearly to the value observed for membrane-coated beads in the presence of the magnet. Together, these data indicate that nonspecific membrane tension is capable of protecting the host cell against apoptosis. Discussion Retraction of the N. gonorrhoeae Tfp during bacterial attachment elicits host cell signaling cascades essential for the establishment of intimate attachment and promotion of bacterial invasion [ 17 ]. We tested the hypothesis that Tfp retraction induces changes in epithelial cell gene expression during bacterial attachment. Pilus retraction, per se, did not regulate a unique set of genes. Rather, retraction enhanced the expression of a small subset of infection-regulated genes (see Figure 1 ), many of which are known to respond specifically to mechanical stress and to be induced by the MAPK cascade. We confirmed that wt bacteria activated MAPKs ERK, JNK, and P38 at a higher level than the pilT mutant. Moreover, MAPK inhibitors lowered the expression level of all but one retraction-responsive gene selected for further examination (see Figure 3 ). These results strongly indicate that MAPK signaling plays a major role in the enhancement of gene expression by pilus retraction. Importantly, artificial force placed on the cell membrane using magnets and magnetic beads can replicate the gene expression changes and MAPK activation observed using wt bacteria, indicating that pilus retraction may induce these events via mechanical force. Although the total force produced by pilus retraction within a bacterial microcolony is not known, we estimate that it is on the order of 10 4 –10 5 pN, based on 100 pN per retraction event, approximately 10 pili per bacteria, and roughly 10–100 bacteria per microcolony. In comparison, this amount of force is equivalent to that applied to integrin complexes in the periodontal ligament by a human bite [ 31 ]. Retraction forces from a microcolony could therefore be physiologically relevant. We cannot exclude the possibility that pilus retraction enhances these signaling events by mechanisms independent of membrane tension (i.e., through secondary receptor engagement or via an inherent difference in the pilus structure/composition between wt and pilT bacteria). Our data with CPP-coated beads strongly argue against these possibilities, however. The CPP preps used for bead coating were from wt cultures, and thus were identical. In addition, the magnet pulled the beads upward. This should pull the bead farther from the cell surface, making secondary receptor engagement less likely. The possibility remains, however, that pilus differences or secondary receptor engagement may act in concert with membrane tension to generate the higher levels of MAPK activation and gene expression changes seen with infection. Further research is needed to examine this possibility. Nonetheless, we are confident that force plays at least some role in the signaling events identified through this work. We have begun to assess the biological functions of enhanced gene expression and MAPK activation during gonococcal infection. ERK, JNK, and P38 play a role in determining cell survival during stress and entry into the apoptosis pathway [ 32 ]. Moreover, nearly half of the identified retraction-enhanced genes are known to be involved in cell cycle/survival signaling. We show that cells infected with wt bacteria have lower levels of cleaved PARP and cleaved caspase 8 than do uninfected and pilT -infected cells. Pilus retraction is therefore predicted to enhance the ability of the cell to withstand apoptosis-inducing signals generated by infection. Indeed, cells infected with wt bacteria withstood STS-induced apoptosis better than uninfected cells and cells infected with pilT . The effect of N. gonorrhoeae infection on cell fate has been a long-standing controversy. The neisserial porin has been reported to protect cells from apoptosis [ 43 ] as well as to induce programmed cell death [ 44 ]. These conflicting observations are likely a result of differences in experimental systems and bacterial strains. We believe that our results may clarify the issue of N. gonorrhoeae and programmed cell death, through the identification of another bacterial factor (i.e., pilus retraction) involved in such a response. A number of factors influence the ability of the cell to withstand apoptosis, including the signaling cascades that are activated and the degree and duration of the activation of these cascades [ 32 ]. They also include the virulence genes expressed by the infecting bacteria. The bacterial strains used for previous studies on N. gonorrhoeae and apoptosis differed in their piliation state and their ability to invade the host cell. Our results indicate that piliated bacteria, in the absence of pilus retraction, slightly increase the tendency of the infected cell to undergo apoptosis (see Figure 5 A). However, these bacteria are still able to moderately protect infected cells from STS-induced apoptosis, indicating that a certain level of cytoprotection is provided by other bacterial factors. In contrast, bacteria that can retract their pili, and thus presumably induce mechanical stress on the host-cell membrane, strongly mediate pro-survival signaling. The influence of mechanical stress on apoptosis has been studied in some detail. Importantly, such studies indicate that different stress patterns result in different cellular outcomes. Extended, repetitive mechanical force increases the expression of genes encoding cytoprotective heat shock proteins and lowers the number of apoptotic cells in a culture [ 45 ]. Suppression of apoptosis requires permanent membrane tension or rhythmic, pulsatile forces [ 46 ], which are thought to allow the cell to adapt to new environmental conditions. Retraction events in N. gonorrhoeae generate strong, pulsatile forces every 1–20 s [ 13 ]. The nature of the pilus retraction force may therefore be the key to counteracting infection-induced apoptosis. Our data strongly indicate that pilus retraction from a microcolony is capable of stimulating mechanoprotective signals. In light of the results presented here and elsewhere, we propose a model to explain how pilus retraction by N. gonorrhoeae influences survival signaling in the infected cell ( Figure 6 ). Initial contact between the bacterium and the epithelial cell activates MAP kinases and alters gene expression at a low level. The cell senses “stress” from the infection, the degree of which varies depending on the metabolic state of the cell and the constellation of virulence factors expressed by the infecting strain. As a result of this stress, the cell is poised to enter the apoptosis pathway. In the absence of pilus retraction and membrane tension, the low levels of activated MAP kinases may or may not be enough to counteract this stress. As the infection proceeds, microcolonies are formed. Pilus retraction from microcolonies is hypothesized to exert stress forces on the membrane, amplifying the levels of activated MAPK, enhancing the transcription of infection-induced genes, and possibly activating other as-yet-unidentified pathways. The end result is the enhanced stimulation of pro-survival pathways and an overriding of pro-apoptotic stress signals. In other words, the fate of the infected cell is decided by the type of signaling networks induced by infection and the extent of activation of these networks. Pilus retraction tips the balance in favor of cell survival. Figure 6 Model of the Role of Pilus Retraction in Promoting a Cytoprotective Environment during Gonococcal Infection of an Epithelial Cell (1) Initial contact between the bacterium and host cell activates low levels of MAPK, and transcription of infection-induced genes. This level of signaling may or may not be able to protect the cell from apoptosis; thus, the host cell “teeters” on the edge of life and death. (2) As the infection proceeds, microcolonies of gonococci are formed, and more pili are locally available to retract. (3) Pilus retraction amplifies MAPK activation, which in turn enhances the transcription of mechanical stress–induced genes. (4) Pilus retraction may also stimulate other pathways that mediate gene expression and survival signaling. Overall signaling events tip the balance in favor of cell survival. We have used a tissue culture system to study the interplay between pilus retraction, host cell signaling, and gene expression during the attachment phase of N. gonorrhoeae infection. How these interactions may affect the disease in vivo remains to be clarified. Our results make teleologic sense when the bacterial life cycle and gonococcal disease are taken into consideration. N. gonorrhoeae does not survive on fomites and has no intermediate host. Transmission depends on person-to-person spread. Simple mucosal gonorrhea infections can be mild, and inflammatory responses begin days after exposure [ 47 ]. Moreover, a significant number of infected individuals carry gonococci without overt symptoms of disease [ 47 , 48 ]. Indeed, the ability of the bacterium to survive as a species requires a relatively healthy host. Our model for pilus retraction is consistent with these considerations. Materials and Methods Reagents Antibodies to PARP, caspase 8, c-Jun, phospho-c-Jun (Ser63), P44/42 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204), phospho-MAPKAPK2 (Thr334), p38 MAPK, phospho-p38 MAPK (Thr108/Tyr182), SAPK/JNK, and phospho-SAPK/JNK (Thr183/Tyr185) were purchased from Cell Signaling Technology (Beverly, Massachusetts, United States). MAPK inhibitors SB203588, U0126, and SP600125 were purchased from Calbiochem (San Diego, California, United States) and used at a final concentration of 10 μM unless otherwise stated. STS was purchased from Cell Signaling Technology and used at a final concentration of 1 μM to induce apoptosis. Neodymium iron boron (NdFeB) magnets (Eneflux Armtek Magnetics, Bethpage, New York, United States) measured 2 in. in diameter by 1 in. thick and were grade 30 (MGOe). Cell lines, bacterial strains, and infections T84 human colonic epidermoid cells (American Type Culture Collection, Manassas, Virginia, United States) were maintained in DMEM-F-12 plus 5% heat-inactivated, filter-sterilized fetal bovine serum at 37 °C and 5% CO 2 . For all experiments, cells were seeded into 35-mm dishes and allowed to become confluent before infection. N. gonorrhoeae strains N400 and N400 pilT [ 49 ] were used for all infections and were maintained on GCB agar plus Kellogg's supplements at 37 °C and 5% CO 2 . Piliation and Opa phenotypes were monitored by colony morphology. Only piliated, Opa − bacteria were used. For infection experiments, bacteria were resuspended in GCB liquid medium and added to the epithelial cells at a multiplicity of infection of 50. RNA isolation and microarray analysis T84 cells were infected with N400 or N400 pilT or treated with GCB medium alone for 3 h. For RNA isolation, labeling, and microarray hybridization procedures, see Protocol S1 . Comparative analysis was performed using MAS 5.0 algorithms to determine fold-change values between uninfected and infected samples from the same experiment, with uninfected samples representing the baseline. Statistical analysis was performed on natural-log transformed data using Cyber-T ( http://visitor.ics.uci.edu/genex/cybert/ ). Subsequent data analysis was performed using Excel (Microsoft, Redmond, Washington, United States) and GeneSpring version 4.0 (Silicon Genetics, Redwood City, California, United States). Genes with a “presence call” p -value of less then 0.1 across all chips were eliminated from analysis, as were genes that were given a “no change” call across all samples. A gene was identified as differentially regulated if the fold-change was greater than ±1.5 in at least two out of three experiments. “Enhanced” genes were identified by calculating the ratio of the fold-change for the wt-infected cells to the fold-change for the pilT -infected cells (W/P). Gene expression was considered to be enhanced by pilus retraction if the W/P, averaged from at least two out of three individual experiments, was greater than 1.5, and the individual W/P from each experiment was greater than1.25. Real-time RT-PCR analysis One microgram of total RNA (as isolated above) was reverse-transcribed to generate cDNA, using the iScript cDNA synthesis kit (Bio-Rad, Hercules, California, United States). As a control, parallel samples were run in which reverse transcriptase was omitted from the reaction mixture. Quantitative real-time PCR was performed using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, California, United States). Amplification was carried out using TaqMan master mix (Applied Biosystems), and pre-designed TaqMan probes (Assays on Demand, Applied Biosystems) according to the manufacturer's instructions. Assay numbers are given in Table 1 . Reactions were performed in triplicate in a 20-μl volume, with the following cycle parameters: 95 °C/10 min enzyme activation, 95 °C/15 s, 60 °C/1 min for 40 cycles. Data analysis was performed using the comparative Ct method (Applied Biosystems) to determine relative expression levels. Table 1 Assays on Demand (TaqMan Probes and Primers) Used for Real-Time Quantitative RT-PCR in This Study Immunoblotting T84 cells were infected with N400 or N400 pilT or treated with GCB medium alone for specified times. Following infection, cells were lysed with 150 μl of 1× SDS lysis buffer (62.5 mM Tris-HCl [pH 6.8], 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.1% w/v bromophenol blue), scraped into Eppendorf tubes, vortexed for 15 s, and immediately stored at −20 °C. For PARP and caspase 8 assays, samples were incubated with 150 μl of cell lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP40, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na 3 VO 4 , 1 μg/ml Leupeptin) for 20 min on ice, followed by a 15-s sonication. Samples were boiled for 5 min at 100 °C, then separated by SDS 8% polyacrylamide gels and transferred onto nitrocellulose sheets. Membranes were probed with the specified antibodies following the manufacturer's protocol. CPPs and bead coating N. gonorrhoeae CPPs were generated from piliated, Opa − gonococci. Bacteria were scraped from overnight cultures (grown on plates) into HBSS and vortexed for 2 min, followed by centrifugation at 14,000 g for 5 min. Supernatants were removed, quantitated by spectrophotometric analysis, and stored at −80 °C until use. Pili preparations were assayed for the presence of pili via indirect immunofluorescence microscopy and immunoblot, using anti-pilin antibody (data not shown). Bio-Mag Plus carboxy-modified paramagnetic microspheres (Bangs Laboratories, Fishers, Indiana, United States), were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, hydrochloride (EDAC), and incubated with piliated N. gonorrhoeae CPPs or BSA as per the manufacturer's instructions. Bead coating was confirmed by immunoblotting using antibodies to BSA (ICN Biomedicals, Irvine, California, United States) and pilin (antibody SM1; data not shown). Immunofluorescence microscopy T84 cells were grown on coverslips to 50% confluency and incubated with either BSA-coated or CMP-coated magnetic beads for 15 min. Unbound beads were washed off and the magnet placed at a distance of 10 mm from the cell surface for 1 h. The medium was then aspirated, and the cells fixed for 15 min at room temperature in 4% paraformaldehyde. Cells were blocked and permeabilized in isotonic PBS containing BSA (3%, w/v) and saponin (0.02% w/v) for 1 h at room temperature, followed by staining with Alexa-Fluor 594 phalloidin (Molecular Probes, Eugene, Oregon, United States) at 1:1,000 for 30 min. Samples were rinsed extensively in PBS before mounting in Fluoromount-G (Fisher Scientific, Hampton, New Hampshire, United States). Images were obtained with a Deltavision Restoration Microscope (Applied Precision, Issaquah, Washington, United States) fitted with a Nikon (Tokyo, Japan) 60× oil-immersion objective and processed at a Silicon Graphics (Mountain View, California, United States) workstation with accompanying API software. The images were subsequently exported to Adobe Photoshop (version 7.0) and Adobe Illustrator (version 11.0) (Adobe Systems, San Jose, California, United States) for manuscript preparation. Calculation of magnetic force To quantify the amount of force that the magnet exerts per magnetic bead, the change-in-mass method [ 31 ] was used. Briefly, the mass of a known number of dry beads (0.12 g) was measured on an electronic balance in the presence and absence of the magnet. Given the mean bead diameter of 1.5 μm and the bead density of 2.5 × 10 3 kg/m 3 (Bangs Laboratories), the number of beads in this sample was calculated to be 1.2 × 10 10 . The change in mass of the beads in the presence of the magnet was entered into the equation—force = Δmass × acceleration (with acceleration being equal to gravity, or 9.81 m/s 2 )—to give a value for the force. Change-in-mass measurements were taken at varying distances from the magnet to determine force as a function of distance (see Figure 4 B). Magnetic force experiments T84 cells were grown to confluency in 35-mm culture dishes. Before assay, the cells were incubated with prewarmed, serum-free medium for 2 h. Cells were then incubated for 30 min with medium alone, or with CPP- or BSA-coated beads diluted in the same medium. Cells were then washed with fresh, serum-free medium to remove unbound beads. Magnets were placed at a distance of 10 mm from the bottom of the tissue culture dish, and the dishes were incubated for the specified time at 37 °C, 5% CO 2 . The samples were then processed for RNA isolation or SDS-PAGE, as described above. Control samples were treated in parallel but were not exposed to the magnet. Statistics Statistical analysis was performed using standard t -test analysis with SPSS version 11.0 (SPSS, Chicago, Illinois, United States) unless otherwise stated. Supporting Information Protocol S1 MIAME Checklist (42 KB DOC). Click here for additional data file. Table S1 Supplementary References (409 KB DOC). Click here for additional data file. Accession Numbers The GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession numbers for the genes and gene products discussed in this paper are ADM (D14874), cyr61 (Y11307), DTR (M60278), DUSP5 (U15932), EGR1 (X52541), PilF (U32588), and PilT (S72391).

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1065266

Gamma-Herpesvirus Latency Requires T Cell Evasion during Episome Maintenance

The gamma-herpesviruses persist as latent episomes in a dynamic lymphocyte pool. Their consequent need to express a viral episome maintenance protein presents a potential immune target. The glycine–alanine repeat of the Epstein–Barr virus episome maintenance protein, EBNA-1, limits EBNA-1 epitope presentation to CD8 + T lymphocytes (CTLs). However, CTL recognition occurs in vitro, so the significance of such evasion for viral fitness is unclear. We used the murine gamma-herpesvirus-68 (MHV-68) to define the in vivo contribution of cis -acting CTL evasion to host colonisation. Although the ORF73 episome maintenance protein of MHV-68 lacks a glycine–alanine repeat, it was equivalent to EBNA-1 in conferring limited presentation on linked epitopes. This was associated with reduced protein synthesis and reduced protein degradation. We bypassed the cis- acting evasion of ORF73 by using an internal ribosome entry site to express in trans -a CTL target from the same mRNA. This led to a severe, MHC class I–restricted and CTL-dependent reduction in viral latency. Thus, despite MHV-68 encoding at least two trans -acting CTL evasion proteins, cis- acting evasion during episome maintenance was essential for normal host colonisation.

Introduction Latent herpesviruses can persist as episomes in quiescent cells without viral protein expression. However, the gamma-herpesviruses are characteristically latent in memory lymphocytes, which intermittently divide. Viral genomes must therefore be replicated and segregated between daughter cells in step with cellular mitosis. This requires a viral episome maintenance protein, creating a potential target for the immune recognition of latently infected cells. A glycine–alanine repeat in the Epstein–Barr virus (EBV) episome maintenance protein, EBNA-1 [ 1 ], inhibits its degradation [ 2 ] and translation [ 3 ] such that EBNA-1 epitopes are poorly presented to CD8 + T lymphocytes (CTLs) [ 4 , 5 , 6 ]. However, the in vivo effectiveness of the glycine–alanine repeat and its quantitative contribution to host colonisation remain unknown. In vitro studies have suggested that abortive EBNA-1 translation products still provide sufficient epitopes for EBNA-1-specific CTLs to recognise latently infected B cells [ 7 , 8 , 9 ]. In vivo immune function is difficult to analyse directly with human viruses. However, the murid pathogen murine gamma-herpesvirus-68 (MHV-68) affords an opportunity to manipulate a gamma-herpesvirus in its natural host. MHV-68 is a gamma-2-herpesvirus [ 10 ], more closely related to the Kaposi's sarcoma–associated herpesvirus than to EBV [ 11 ], but clear functional parallels exist between all three viruses. Like EBV, MHV-68 causes an acute infectious mononucleosis-like illness, associated with a massive expansion of latently infected germinal centre B cells, and it persists in memory B cells [ 12 , 13 , 14 ]. The episome maintenance protein of gamma-2-herpesviruses is encoded by ORF73 [ 15 ]. Just as EBNA-1-deficient EBV [ 16 ] and ORF73-deficient Kaposi's sarcoma–associated herpesvirus [ 17 ] fail to maintain latency in vitro, ORF73-deficient MHV-68 has a profound latency deficit in vivo [ 18 , 19 ]. At least two MHV-68 gene products inhibit CTL recognition of latently infected cells. The M3 chemokine-binding protein [ 20 , 21 ] is abundantly secreted by lytic virus [ 22 ]. This co-exists with latent virus in infected lymphoid tissue [ 14 , 23 , 24 ]. Bystander protection by M3 [ 25 ] probably explains why M3 disruption causes mainly a reduction in MHV-68 latency amplification [ 26 , 27 ]. Protection by M3 may be quite context-dependent [ 28 ] and probably functions best in acute infection, before M3-specific immunity is established. A second MHV-68 CTL evasion protein, K3, degrades major histocompatibility complex (MHC) class I heavy chains [ 29 ] and TAP (transporter associated with antigen processing) [ 30 ]. K3 is transcribed in latency as well as in the viral lytic cycle, and again protects against CTLs during latency amplification [ 31 ]. However, K3 is not expressed in all forms of latency: it is not detectable in the MHV-68-infected S11 tumor line [ 32 ] or in persistently infected, B cell–deficient mice [ 33 ]. Notably, ORF73 disruption causes a much more profound latency deficit than does K3 disruption, implying that immune evasion by K3 is not important in every cell expressing ORF73. These restrictions on M3 and K3 function imply that further immune evasion mechanisms contribute to MHV-68 latency. Selective EBNA-1 expression occurs in normal EBV persistence [ 34 , 35 ] and is characteristic of EBV-associated Burkitt's lymphoma [ 36 ]. The gamma-2-herpesviruses presumably implement an equivalent program of selective ORF73 expression. We have used MHV-68 to determine the in vivo importance of avoiding epitope presentation during episome maintenance. We first established that the MHV-68 ORF73 is equivalent to EBNA-1 in reducing the presentation of an MHC class I–restricted epitope linked to it in cis. We then modified the ORF73 transcript to bypass this evasion, and used the mutant virus to define the consequences of epitope presentation for viral fitness. Our analysis of MHV-68 implies that if MHC class I–restricted viral epitope presentation occurs during gamma-herpesvirus episome maintenance, even a relatively small CTL response can very effectively clear latent infection. Results Limited Presentation of a CD8 + T Cell Epitope Linked in cis to ORF73 As with EBNA-1, CTL epitopes in the MHV-68 ORF73 have been hard to find, perhaps reflecting limited ORF73 entry into the MHC class I antigen-processing pathway. To determine whether ORF73 is similar to EBNA-1 in its resistance to MHC class I–restricted antigen presentation, we introduced the H2-K b -restricted SIINFEKL epitope of ovalbumin (OVA) near the ORF73 C-terminus (73-SC) or N-terminus (73-SN). L929-K b cells transfected with 73-SC or 73-SN were poorly recognised by the SIINFEKL-specific hybridoma, B3Z ( Figure 1 A), suggesting poor antigen processing. There was no evidence for ORF73 inhibiting SIINFEKL presentation from co-transfected OVA ( Figure 1 B). The apparent immune evasion therefore acted in cis rather than in trans, and it was not due to any ORF73 toxicity. Figure 1 Inhibition of MHC Class I–Restricted Epitope Presentation by Physical Linkage to ORF73 (A) The SIINFEKL epitope of OVA was introduced into ORF73 near either its N-terminus (ORF73-NC) or its C-terminus (ORF73-SC). Both ORF73 derivatives were cloned into the pcDNA3 expression vector and compared with OVA in the same vector for their capacity to stimulate the SIINFEKL-specific T cell hybridoma B3Z after transfection into L929-K b cells. After 48 h, beta-galactosidase production was assayed by cell lysis in the presence of chlorophenol-red-beta- D -galactoside and reading absorbance at 595 nm. nil, vector only. (B) L929-K b cells were co-transfected with OVA plus the plasmid indicated. C1–C4 are control plasmids, expressing MHV-68 ORFs 19, 30, 31, and 35, respectively. K3 degrades MHC class I heavy chains and m152 is a murine cytomegalovirus gene that retains MHC class I molecules in the endoplasmic reticulum. Net absorbance = A 595 with co-transfection − A 595 with untransfected cells (<0.02). (C) Hybrids of OVA and ORF73-SC were made to identify regions of ORF73 that inhibited SIINFEKL presentation. Responses are expressed as 100(A 595 with plasmid − A 595 with untransfected)/(A 595 with OVA transfection − A 595 with untransfected). nil, vector only. Mean ± standard deviation (SD) values of triplicate cultures are shown. Each graph is representative of at least three separate experiments. In at least one experiment, equal transfection efficiency was confirmed by co-transfecting a GFP expression plasmid and checking fluorescence under ultraviolet illumination. (D) ORF73 was fused to the C-terminus of the OVA coding sequence in pcDNA3. C-terminal deletions were then made as shown. Each construct was transfected into L929-H2-K b cells. The shaded area in (D–G) highlights a region of ORF73 that appeared to be important for inhibiting epitope presentation. (E) N-terminal ORF73 truncations were generated by PCR and fused in frame to amino acid 325 of OVA. Each construct was transfected into L929-H2-K b cells and assayed for SIINFEKL presentation as in (D). (F) Progressive truncations of ORF73-SN were assayed for their capacity to present the SIINFEKL epitope to B3Z cells after transfection into L929-K b cells. Selective presentation from the ORF73-SN-PstI construct was confirmed in multiple experiments, including independent plasmid preparations. (G) PCR-generated C-terminal truncations of ORF73-SN were assayed for SIINFEKL presentation after transfection of L929-H2-K b cells. Deletions across the area identified as important for inhibiting epitope presentation in (D–E) again improved epitope presentation. To identify possible contributions of the N-terminal and C-terminal regions of ORF73 to its poor epitope presentation, we made hybrids of OVA (amino acid residues 1–325) and 73-SC using a shared PstI site ( Figure 1 C). N-terminal ORF73 (amino acid residues 1–150) diminished SIINFEKL presentation from OVA relatively little, suggesting that any inhibitory segment was C-terminal of its PstI site (amino acid residues 151–314). With N-terminal OVA, SIINFEKL presentation from 73-SC remained low. A Key Region of ORF73 for cis- Acting Immune Evasion We adopted two strategies to localise an inhibitory segment C-terminal of the ORF73 PstI site. First, we fused the entire ORF73 coding sequence to the C-terminus of OVA, thereby inhibiting SIINFEKL presentation from OVA. C-terminal truncation of the fusion protein ( Figure 1 D) up to the ORF73 HinDIII site (residue 278) then had little effect, but truncation up the ORF73 KpnI site (residue 206) restored SIINFEKL presentation somewhat. N-terminal truncation ( Figure 1 E) to ORF73 residue 166 (OVA-73C) did not compromise the inhibition, but further truncation to residue 186 (OVA-73B) and then residue 206 (OVA-73A) progressively improved SIINFEKL presentation. These data were consistent with the region of ORF73 encoding approximately residues 170–220 (the shaded region in Figure 1 D– 1 G) reducing antigen presentation. We also truncated 73-SN from its C-terminus and looked for presentation of its N-terminal SIINFEKL epitope ( Figure 1 F). With SIINFEKL in this context, the same C-terminal truncations as in Figure 1 D (amino acid residues 1–204) gave no epitope presentation, presumably because SIINFEKL was less efficiently processed from 73-SN than it was from OVA. However, a further truncation up to the ORF73 PstI site (residue 150) dramatically improved SIINFEKL presentation. PCR-generated C-terminal truncations of 73-SN ( Figure 1 G) supported the idea of a region just upstream of the ORF73 KpnI site (ORF73-SN-B) limiting the presentation of SIINFEKL from 73-SN. These results were therefore consistent with those shown in Figure 1 D and 1 E. The Effects of ORF73 on the Turnover of Linked OVA The inhibition of EBNA-1 epitope presentation by its glycine–alanine repeat has been attributed principally to reduced protein synthesis and secondarily to reduced protein degradation [ 3 ]. We therefore analysed the effect of ORF73 on OVA turnover using constructs equivalent to those in Figure 1 E, except that we removed the signal sequence of OVA to avoid any protein secretion (SOVA, Figure 2 A). All constructs were cloned into pcDNA3 and transfected into 293T cells. We observed a hierarchy of SOVA/ORF73 antigen presentation ( Figure 2 B) similar to that seen with the OVA/ORF73 hybrids: amino acids 206–314 of ORF73 (SOVA-73A) reduced somewhat SIINFEKL presentation from SOVA; amino acids 166–314 (SOVA-73C) reduced it further; including an additional 40 amino acids of ORF73 (SOVA-73E) gave no additional inhibition. Figure 2 Inhibition of Epitope Presentation by ORF73 Fusion to OVA Correlates with Reduced Translation of the Fusion Protein (A) N-terminal ORF73 truncations equivalent to those in Figure 1 E were fused in frame to the C-terminus of OVA amino acids 41–325, thereby removing both the OVA signal sequence and the ORF73 nuclear localisation signal. (B) Serial dilutions of an expression plasmid containing each fusion gene were transfected into L929-H2-K b cells as in Figure 1 . SIINFEKL presentation was assayed using beta-galactosidase production from the B3Z hybridoma. (C) Equivalent transfected cells were immunoblotted with an anti-OVA rabbit serum (OVA). Fusion products are indicated by arrowheads where visible. Parallel immunoblots for neomycin phosphotransferase II (NPT), which is expressed from a different promoter of the same plasmid (pcDNA3), were used to control for transfection efficiency. The endogenous neomycin phosphotransferase II expressed by 293T cells was not visible at this exposure. One of three equivalent experiments is shown. (D) Forty-eight hours after transfection with the constructs indicated, 293T cells were pulse-labelled (P) for 30 min with 35 S-cysteine/methionine, followed by a 2-h chase (C) with excess unlabelled cysteine/methionine. OVA derivatives were then immunoprecipitated with an OVA-specific rabbit serum and resolved by SDS-PAGE. The specific bands corresponding to each fusion protein are indicated by arrowheads. The graph shows densitometry readings for each band. (E) 293T cells transfected with selected fusion proteins were labelled for a variable period (15–120 min) as indicated. OVA derivatives were then immunoprecipitated and analysed as in (D). Arrowheads show the predicted position of the relevant fusion proteins for the 120-min label samples. (F) Either SOVA-ORF73A or SOVA was transfected into 293T cells. Forty-eight hours later the cells were pulse-labelled (P) for 15 min with 35 S-cysteine/methionine, followed by a 15-min (C1), 45-min (C2), and 105-min (C3) chase with excess unlabelled cysteine/methionine. This was done in the presence or absence of 100 μM lactacystin. The graph shows densitometry readings for each specific band. Steady-state protein levels, determined by immunoblotting transfected cell lysates ( Figure 2 C), were greatest with SOVA, followed by SOVA-73A, and least with SOVA-73D-E. Parallel immunoblots for neomycin phosphotransferase II, expressed from a different promoter of the same plasmid, showed no significant variation in signal, arguing against an effect of ORF73 on cell viability or transfection efficiency. Pulse-chase metabolic labelling of transfected 293T cells and immunoprecipitation with an OVA-specific antiserum ( Figure 2 D) showed that all the ORF73/SOVA fusions were more stable than SOVA alone, and that their labelling was reduced as more ORF73 sequence was attached. The differences in protein synthesis rate ( Figure 2 D) correlated with steady-state protein levels (see Figure 1 C). Using a variable labelling window ( Figure 2 E), SOVA and SOVA-73A were detectable after a 15-min pulse, whereas SOVA-73E was hard to discern even after a 120-min pulse. The apparent stability conferred by ORF73 on SOVA ( Figure 2 D) was confirmed by further analysis of SOVA-73A, the fusion protein that labelled most efficiently ( Figure 2 F). After a 20-min pulse, most labelled SOVA was lost over the next 2 h, whereas the labelled SOVA-73A was relatively well maintained. Proteasome inhibition with lactacystin partially stabilised SOVA, but it remained less stable than SOVA-73A. Thus, the stability afforded by amino acids 206–314 of ORF73 appeared to extend beyond protection against proteasome-mediated degradation. Overall, the cis- acting immune evasion of ORF73 appeared functionally similar to that of EBNA-1, in that reduced epitope presentation was associated with reduced protein synthesis and reduced protein degradation. In contrast to EBNA-1, these functions were mediated by distinct regions of ORF73. The key region for inhibiting epitope presentation (see Figure 1 D– 1 G) corresponded to that responsible for reducing protein synthesis. An MHV-68 Mutant That Lacks cis- Acting Immune Evasion The region of ORF73 responsible for cis- acting immune evasion—that encoding amino acids 170–220—shows considerable amino acid homology to both EBNA-1 and the Kaposi's sarcoma–associated herpesvirus ORF73 [ 37 ]. Any mutagenesis was therefore likely to compromise other ORF73 functions. Also, altering this region would change the steady-state levels of ORF73, with likely toxic effects in latently infected cells. In order to keep ORF73 function intact, therefore, we bypassed the cis- acting immune evasion not by mutating the ORF73 protein, but by modifying its mRNA. Thus, we inserted an internal ribosome entry site (IRES) just downstream of the ORF73 coding region and used this to co-express either green fluorescent protein (GFP) or three tandem MHC class I–binding peptides ( Figure 3 A). Figure 3 Modification of the MHV-68 Genome to Overcome cis- Acting Immune Evasion by ORF73 (A) An IRES element was inserted just downstream of ORF73, between its stop codon and that of M11. This allowed either three tandem CD8 + T cell epitopes (EPI) or GFP to be translated from the ORF73 mRNA. (B) DNA from BAC-cloned viral genomes (BAC) or virus-infected cells (VIR) was digested with NcoI, electrophoresed, transferred to nylon membranes, and blotted with a probe corresponding to the BamHI-G genomic fragment shown in (A). The predicted bands for WT virus were 1,021 bp, 3,121 bp, and 4,630 bp. The IRES-GFP insert introduced an NcoI site such that the WT 3,121-bp band was cut into 2,975-bp and 1,466-bp fragments. The NcoI site was lost from the IRES-EPI insert, such that the WT 3,121-bp band became a 3,861-bp band. (C) BHK-21 cells were infected (0.01 PFU/cell) with WT, GFP, or EPI viruses as indicated. Plaque titres of cell cultures are shown with time after infection. (D) H2 b MEF-1 cells or L929-K b cells were left uninfected (UI) or infected for 2 h with MHV-68 expressing either OVA under a strong lytic promoter (OVA) or the SIINFEKL epitope of OVA as part of the ORF73-IRES-EPI construct (EPI). B3Z cells were then added, and 18 h later their beta-galactosidase response was assayed using chlorophenol-red-beta- D -galactoside substrate. Mean ± SD values of triplicate cultures are shown. The data are from one or two equivalent experiments. (E) A20-syndecan-1 cells were infected (20 PFU/cell) with GFP − WT virus, WT virus with an HCMV IE1 promoter-driven GFP expression cassette (HCMV IE1-GFP), or with the ORF73-IRES-GFP virus. The numbers indicate the percentage of total cells in the gated region (GFP + ). Expression from the HCMV IE1 promoter is probably limited to lytic infection, whereas ORF73 is expressed in latency. Southern blots confirmed the predicted genomic structure of the ORF73-IRES-epitope (EPI) and ORF73-IRES-GFP viruses ( Figure 3 B). Both mutants showed unimpaired growth in vitro ( Figure 3 C). Infection of H2-K b fibroblasts with the EPI virus established that its SIINFEKL epitope could be processed and presented ( Figure 3 D). MHV-68 expressing OVA from an intergenic expression cassette under the control of an ectopic viral M3 promoter (MHV-OVA), which shows high-level lytic cycle OVA production (data not shown), was tested in parallel. In murine embryonic fibroblast (MEF)-1 cells, which support MHV-68 lytic replication, MHV-OVA showed better SIINFEKL presentation than did the EPI virus. In L929 cells, which support viral entry into the lytic cycle relatively poorly [ 38 ], the EPI virus gave better SIINFEKL presentation than did MHV-OVA. These data were consistent with the EPI virus presenting SIINFEKL in latency. The GFP mutant provided further evidence that the IRES constructs were expressed in latency. Although MHV-68 is predominantly latent in B cells in vivo, it appears to infect B cells poorly in vitro. This may reflect that efficient infection by MHV-68 virions requires cell-surface glycosaminoglycans [ 39 ]. We therefore enhanced infection of the A20 B cell line by transducing it with a retroviral vector expressing the extracellular domain of syndecan-1, a major carrier of cell-surface glycosaminoglycans [ 40 ], linked to the transmembrane and cytoplasmic domains of H2-D b . This form of syndecan-1 resists proteolytic cleavage. GFP expressed from a human cytomegalovirus IE-1 promoter [ 41 ] gave green fluorescence in relatively few A20-syndecan-1 cells. Although the ORF73-IRES-GFP virus gave much weaker fluorescence, many more cells were positive ( Figure 3 E). These data were consistent with B cell infection being predominantly latent, and with gene expression from the IRES constructs in latency. Epitope Presentation during Episome Maintenance Leads to a Severe In Vivo Latency Deficit We tested the capacity of the EPI virus to replicate in vivo by intranasal infection of C57BL/6J mice ( Figure 4 ). There was no difference between wild-type (WT) and EPI viruses in lytic replication in lung epithelial cells or in seeding latent virus to the spleen ( Figure 4 A). However, by 14 d after infection, when WT virus had reached its peak latent load, the titre of EPI virus was drastically reduced ( Figure 4 B). In agreement with the reduced number of infectious centres, the EPI virus genome load was low ( Figure 4 C) and there was little virus-driven B cell activation, T cell activation, or Vbeta4 + CD8 + T cell expansion ( Figure 4 D). The GFP control virus showed no such deficit, so it was not the IRES element that compromised ORF73 function. An independently derived EPI mutant showed a similar in vivo latency deficit, and reverting the EPI mutation restored latency establishment to normal levels ( Figure 4 E). The latency deficit was therefore due specifically to the expression of a poly-epitope construct downstream of ORF73. Figure 4 Replication of the IRES-EPI Virus In Vivo (A) Six days after intranasal infection with WT or EPI viruses as indicated, infectious virus in lungs was titred by plaque assay (left panel) and infectious plus latent virus in spleens was titred by infectious centre assay (right panel). Each point shows an individual mouse. Pre-formed, infectious virus was undetectable in equivalent, freeze-thawed spleen samples, so the infectious centres represent latent virus. (B) By 14 d post-infection, infectious centre titres were much lower with the EPI virus than with WT. The GFP control virus is shown for comparison. This difference was preserved at day 19 post-infection, indicating that the EPI virus was not merely delayed in host colonisation. (C) DNA was extracted from spleens and its viral genome content quantitated by real-time PCR. Genome loads broadly reflected the infectious centre titres, indicating that the viral load was reduced rather than the efficiency of ex vivo reactivation. (D) As a further measure of host colonisation, we measured B cell activation (CD69 expression on CD19 + B cells) at 14 d post-infection and CD8 + T cell activation (loss of CD62L expression) at 19 d post-infection. We also measured the day 19 expansion of the Vbeta4 + CD8 + T cell subset that is characteristic of MHV-68-associated infectious mononucleosis. All these measures correlated closely with the viral latent load in lymphoid tissue and were markedly reduced with the EPI virus compared to WT or GFP. GFP expression was undetectable in ex vivo B cells after infection with the GFP virus (data not shown). (E) C57BL/6J mice were infected intranasally with WT virus, the EPI mutant, an independently derived EPI mutant (EPI-IND), or a revertant of the EPI virus (EPI-REV). Splenic infectious centres were then measured 13 and 17 d post-infection. The dashed line shows the lower limit of assay sensitivity. Antigen-Specific Immune Responses to the EPI Virus The EPI virus was notably controlled without a need for the massive T cell activation that characterises MHV-68- or EBV-associated infectious mononucleosis ( Figure 4 D). We measured virus-specific immune responses ( Figure 5 ) to gain some idea of what effector response might be responsible for the latency amplification deficit. At 13 d post-infection, ELISPOT assays ( Figure 5 A) showed a low CD4 + T cell response to the EPI virus compared to WT. A similar reduction in CD4 + T cell response is seen with MHV-68 specifically made to be latency deficient [ 42 ], presumably because lytic reactivation after latency amplification normally provides a large CD4 + T cell stimulus. Virus-specific serum antibody titres were marginally higher in the EPI-virus-infected mice ( Figure 5 B). CD8 + T cell responses to immunodominant MHV-68 lytic epitopes (p56 and p79) were comparable between WT and EPI viruses at day 13 post-infection ( Figure 5 A and 5 C). Thus, the EPI virus was most likely being cleared by CTLs directed against an ORF73-associated epitope. Figure 5 Antigen-Specific Immune Responses to the IRES-EPI Virus (A) CD8 + and CD4 + T cell responses were measured by interferon-gamma ELISPOT assay 13 d post-infection. The response to virus-exposed targets (VIR) is mediated by CD4 + T cells; the response to the p56, p79, and SIINFEKL (OVA) peptides is mediated by CD8 + T cells [ 62 ]. The mean number of spots with untreated targets was subtracted from the number of spots with each specific target. There was a response to the OVA peptide in the IRES-epitope construct, but not to the ASNENMETM peptide (NP). Mean ± SD values of five mice per group are shown. (B) Total and MHV-68 virion-specific serum IgG responses were measured by ELISA at 18 d post-infection. “Naive” indicates age-matched, uninfected controls. Mean ± SD absorbance values of four mouse sera per group are shown. (C) Spleen cells were stimulated for 5 h in the presence of Brefeldin A plus the peptide indicated and then stained for cell-surface CD8 and intracellular interferon-gamma. The percentage of interferon-gamma + CD8 + cells without peptide was subtracted from the value with peptide to give the specific response. Mean ± SD values of five mice per group are shown. There was no ASNENMETM-specific response to the EPI virus ( Figure 5 A and 5 C). Also, there was no evidence of an enhanced response to the p79 epitope, which was present both in the IRES-epitope construct and in its native context in ORF61 [ 43 ]. These epitopes were probably not processed from the polytope construct, since transfecting it as an expression plasmid into H2-K b - or H2-D b -expressing L929 cells stimulated the SIINFEKL-specific hybridoma B3Z but not T cell hybridomas specific for ASNENMETM or p79 (data not shown). In contrast to the lack of response to ASNENMETM, there was a clear response to the SIINFEKL epitope co-expressed with ORF73 ( Figure 5 A and 5 C), which was also presented in vitro (see Figure 3 D). By 18 d post-infection ( Figure 5 C), the CD8 + T cell response to WT infection had made its characteristic shift in immunodominance from the p56 epitope associated with epithelial infection to the p79 epitope associated with B cell infection [ 43 ]. This did not occur with the EPI virus, presumably because the number of latently infected B cells remained low. Thus, it seemed likely that SIINFEKL-specific CTLs eliminated the EPI virus. Attenuation of the EPI Virus Is H2-Type-Restricted As all of the CTL epitopes in the IRES-epitope construct were H2 b -restricted, a major prediction was that the EPI virus would not be attenuated in H2 d mice. This was found to be the case ( Figure 6 ). In H2 d BALB/c mice, the EPI virus attained infectious centre titres in the spleen equivalent to WT virus ( Figure 6 A). B cell activation ( Figure 6 B), splenomegaly ( Figure 6 C), and viral genome load ( Figure 6 D) were also normal. We further assayed latency by in situ hybridization for the expression of viral tRNA homologues in splenic germinal centres ( Figure 6 E). These are expressed at high levels in MHV-68-infected lymphoid tissue and provide an additional marker of latency establishment [ 44 ]. The EPI virus showed no viral tRNA + cells in C57BL/6J mice and normal numbers in BALB/c mice. These data supported the idea that the attenuation of the EPI virus was due to the expression of a CTL target from the ORF73 mRNA. Figure 6 Normal EPI Virus Replication in Non-H2 b Mice (A) Infectious centre titres in individual spleens were determined 14 d after intranasal infection of C57BL/6J (H2 b ) or BALB/c (H2 d ) mice with WT or EPI virus. (B) CD69 expression on splenic B cells was measured by flow cytometry 14 d post-infection. B cells from uninfected mice were less than 5% CD69 + . (C) The weights of individual spleens are shown 14 d post-infection, with spleens of age-matched, uninfected mice (UI) for comparison. (D) Viral genome loads in individual mice were determined by real-time PCR at 14 d post-infection. (E) Viral tRNA expression in infected germinal centres was visualised by in situ hybridization with a digoxigenin-labelled riboprobe specific for tRNAs 1–4. Representative follicles of at least five sections per mouse and three mice per group are shown. tRNA + follicles were abundant with WT virus and with the EPI virus in BALB/c mice, but were not seen with the EPI virus in C57BL/6J mice. (F) BALB/c or C57BL/6J mice were infected intranasally with MHV-68 expressing OVA from an intergenic expression cassette (OVA) or with WT virus. The extent of lymphoid colonisation was determined by infectious centre assay of spleens 12 and 15 d post-infection. Mean ± SEM titres of five mice per group are shown. In contrast to the EPI virus, the OVA virus showed no defect in host colonisation. In contrast to the EPI virus, MHV-OVA showed no significant attenuation in either BALB/c or C57BL/6J mice compared to WT virus ( Figure 6 F). MHV-OVA expresses the SIINFEKL epitope at high levels during lytic infection (see Figure 3 D). Thus, SIINFEKL expression during episome maintenance, when epitopes are not normally presented, was catastrophic for the virus, whereas SIINFEKL expression outside of this context, when MHV-68 does not rely on limiting epitope presentation for its survival, had little effect. Attenuation of the EPI Virus Is CD8 + T Cell–Dependent The MHC class I restriction of the EPI virus's latency deficit and its association with anti-SIINFEKL immunity implied that CD8 + T cells were eliminating latently infected cells. This was confirmed by rescuing the EPI virus with CD8 + T cell depletion ( Figure 7 ). Thus, in C57BL/6J mice treated with an anti-CD8 monoclonal antibody, the EPI virus achieved WT levels of B cell activation ( Figure 7 A), viral genome load ( Figure 7 B), and infectious centres ( Figure 7 C). The ORF73 CTL evasion (see Figure 1 ) that was bypassed in the EPI virus was therefore essential for in vivo episome maintenance. Figure 7 Rescue of the EPI Virus by CD8 + T Cell Depletion Mice were left undepleted (UD) or depleted of CD8 + T cells (CD8 − ) by an initial intravenous injection of mAb YTS169 2 d before infection, followed by intraperitoneal injections of the same antibody every 2–3 d up to the time of sampling. Infection was by intranasal inoculation of either WT or EPI viruses. (A) Depletion was 95%–99% complete as assessed by flow cytometry of spleen cells. CD69 expression on splenic B cells was measured 13 d post-infection. (B) Genome loads were measured 13 d post-infection by real-time PCR. Each point shows an individual mouse. (C) The infectious centre titres of individual mice at 13 d post-infection are shown for one of two equivalent experiments. The titres of pre-formed, infectious virus in freeze-thawed spleens were less than 5% of the infectious centre titres, so even after CD8 + T cell depletion, the infectious centre assay essentially measured latent virus. By 13 d post-infection, the lungs of both immunocompetent and CD8 + T cell–depleted mice were clear of infectious virus. (D) In situ hybridization for viral tRNA expression in splenic germinal centres is shown 13 d after infection of CD8 + T cell–depleted or undepleted mice, infected with either EPI or WT virus. Spleens of two representative mice are shown in each case. Discussion The MHV-68 ORF73 lacks the glycine–alanine repeat of EBNA-1 but still conferred poor presentation on a linked CTL epitope. Thus, despite different means, MHV-68 and EBV have arrived at a similar end of inhibiting epitope presentation during episome maintenance. Neither molecular mechanism is fully understood, but both seem to rely primarily on limiting protein synthesis. A large proportion of CTL epitopes are derived from abortive translation events [ 45 ]. Understanding the mechanism of cis- acting CTL evasion therefore means understanding the major source of abortive translation events, whether damaged RNA, ribosomal errors, or protein misfolding. Since the key evasion regions of EBNA-1 and ORF73 are located centrally, they are translated only after potential N-terminal epitopes, and so may exert their inhibitory effects prior to translation, as RNA. Our aim here was to ask what cis- acting evasion contributes to the fitness of a gamma-herpesvirus. Inserting an IRES element downstream of ORF73 allowed us to bypass cis- acting immune evasion in MHV-68. CTLs then wiped out latency. We conclude that avoiding epitope presentation during episome maintenance is fundamental to gamma-herpesvirus survival. As yet, no endogenous CTL epitopes to our knowledge have been described for the MHV-68 ORF73. This may reflect its cis- acting immune evasion in the same way that EBV infection was initially thought not to elicit EBNA-1-specific CTLs [ 46 , 47 ]. However, EBNA-1 epitopes can be presented by cross-priming [ 5 ]. It seems likely that MHV-68 will elicit ORF73-specific CTLs by a similar route. Certainly there is no lack of predicted cleavable, MHC class I–binding peptides in ORF73; for example, H2-D b , FSSTHPYTL; H2-K b , QCVTYYLL; H2-D d and H2-K d , KYQGMRRHL; and H-L d , APPSPDVDV. Thus, evasion must occur at the level of endogenous ORF73 presentation. The effectiveness of immune evasion is inevitably context-dependent. Defining its impact on host colonisation therefore requires natural thresholds of in vivo antigen presentation. The results are not always predictable. For example, MHV-68 transcribes its K3 gene in the lytic cycle as well as in latency, but a lack of K3 has no discernable impact on primary lytic infection, only on latency amplification [ 31 ]. The recognition of EBNA-1 during latency III [ 7 , 8 , 9 ] does not necessarily imply EBNA-1 recognition during latency I, when autoregulation [ 48 ] and a cell cycle dependence [ 49 ] of the Qp promoter reduce EBNA-1 transcription. Our results with MHV-68 suggest that the EBNA-1 glycine–alanine repeat is a key component of in vivo EBV persistence. Of course MHV-68 is not EBV, and it is possible that the expression of a strong MHC class I–binding peptide exaggerated somewhat the potential of ORF73-specific CTLs to control infection. However, a clear message is that cis- acting CTL evasion is an important feature of the gamma-herpesvirus lifecycle. Latency-associated trans -acting CTL evasion comes into play during the MHV-68 growth program, when rapid cell division probably raises ORF73 production above a level that can be disguised by cis- acting evasion, and additional viral gene products are expressed. This trans- acting evasion allows latency amplification to progress despite evidence of a CTL response to at least one viral growth program antigen [ 32 ]. However, trans -acting evasion alone was insufficient for even an initial amplification of MHV-68 latency. SIINFEKL production from the EPI virus did not simply compromise K3 function, since a complete loss of K3 typically gives a 1-log reduction in infectious centres with relatively little effect on the viral genome load [ 31 ], whereas the EPI virus showed a 3- to 4-log infectious centre deficit and a severely reduced genome load. cis -acting immune evasion therefore operated in a distinct setting relatively early in latency establishment. Multiple patterns of both EBV [ 50 , 51 ] and MHV-68 [ 14 ] latent gene expression occur in acutely infected lymphoid tissue. Notably, EBV implements EBNA-1-only latency even during acute infectious mononucleosis [ 35 ]. Thus, the extreme dependence of MHV-68 on cis- acting evasion probably reflects early B cell entry into “ORF73-only” latency. The rather modest SIINFEKL-specific response to the EPI virus contrasted with the massive CTL activation stimulated by WT virus. For example, in one experiment WT virus progressed from 4.7 × 10 3 infectious centres per spleen at day 10 of infection to 6.4 × 10 4 at day 14 (means of five mice), while the percentage of CD8 + T cells expressing CD69 increased from 10.7% to 20.8%. Over the same time, the EPI virus infectious centres fell from 7.3 × 10 2 to 4.6 × 10 1 , with 6.9% and 5.9%, respectively, of CD8 + T cells expressing CD69. Indeed the numerous lytic antigen-specific CTLs stimulated by EBV [ 52 ] and MHV-68 [ 43 ] infections imply an immune response failure, since latently infected B cells proliferate and progress to lytic gene expression without hindrance by latent antigen-specific CTLs. It is crucial in persistent viral infections for the immune system to attack appropriate targets. End-stage cells may stimulate large T cell responses, but the control of infection depends more on overcoming immune evasion. A major challenge in vaccinating against complex pathogens is to direct the immune system against the key, self-renewing population that maintains the parasite load. Materials and Methods Mice C57BL/6J and BALB/c mice (Harlan Olac, Bicester, United Kingdom) were kept in Cambridge University animal facilities in accordance with United Kingdom Home Office guidelines (project licence 80/1579). Mice were infected intranasally with 2 × 10 4 plaque-forming units (PFU) of MHV-68 under brief halothane anaesthesia. T cell subset depletion was by intravenous and then intraperitoneal injection of purified mAb YTS169 [ 53 ]. Cell lines BHK-21 cells, MEF-1 cells, NIH-3T3-CRE cells [ 31 ], A20 cells, L929 cells transfected with H2-K b [ 54 ], and the B3Z T cell hybridoma [55 were all grown in DMEM, supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum (complete DMEM). A20-syndecan-1 cells were made by retroviral transduction of A20 cells with a vector expressing the extracellular domain of murine syndecan-1 linked to the transmembrane and cytoplasmic domains of H2-D b , and will be described in detail elsewhere. MEFs were harvested at 13 d of gestation and were grown in complete DMEM with 50 μM 2-mercaptoethanol. Plasmids We amplified ORF73 by PCR (Hi-Fidelity PCR kit, Roche Diagnostics, Lewes, United Kingdom), including EcoRI and SalI restriction sites at its respective 5′ and 3′ ends, and cloned the product into the EcoRI and SalI sites of pSP73 (Promega, Chilworth, United Kingdom) to make pSP73-ORF73. To introduce the SIINFEKL epitope of OVA [ 56 ] near the 3′ end of ORF73, we digested pSP73-ORF73 with HinDIII and dephosphorylated it ( P. borealis alkaline phosphatase, Roche Diagnostics). Two complementary oligonucleotides (5′- AGCTAGTATAATCAACTTTGAAAAACTGCT and 5′- AGCTAGCAGTTTTTCAAAGTTGATTATACT) (Sigma-Genosys, Cambridge, United Kingdom) were then heated, annealed, phosphorylated, and ligated into the HinDIII site (T4 DNA ligase, New England Biolabs, Hitchin, United Kingdom). Thus, amino acid residues 277–283 of ORF73 (QASGTQH) were changed to QASIINFEKLLASGTQH (ORF73-SC). Oligonucleotide insertion was confirmed by DNA sequencing. To insert the SIINFEKL coding sequence in the 5′ end of ORF73, we amplified ORF73 by PCR, pairing the 5′ primer , containing a 5′ EcoRI restriction site (underlined) upstream and a HinDIII site (double-underlined) downstream of the ORF73 start codon (bold), with a 3′ primer downstream of the ORF73 BstEII site, and containing an XhoI site. This PCR product was cloned into the EcoRI and XhoI sites of pSP73. The complementary oligonucleotides 5′- AGCTAGTATAATCAACTTTGAAAAACTGAC and 5′- AGCTGTCAGTTTTTCAAAGTTGATTATACT were then inserted into the HinDIII site, changing amino acid residues 1–6 of ORF73 from MPTSPP to MQASIINFEKLTASPP (ORF73-SN). The modified 5′ end of ORF73 was then subcloned as an EcoRI/BstEII fragment into pSP73-ORF73, thereby reconstituting the gene with its 3′ HinDIII site intact. Each form of ORF73 was then cloned into the pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, California, United States). We made 3′ deletions of pcDNA3-ORF73-SN by digesting it with HinDIII or KpnI, each of which cuts within ORF73 and within the pcDNA3 polylinker 5′ of its EcoRI site. The N-terminal ORF73 fragment was then gel-purified and ligated into a new pcDNA3 vector. We generated a 3′ PstI deletion by digestion with PstI, gel purification, and ligation of the vector back to itself, since PstI cuts downstream of the pcDNA3 XhoI site. We subcloned the N-terminal 325 amino acid residues of OVA as an EcoRI/XhoI fragment from pMSCV-OVA-IRES-GFP [ 57 ] into pSP73. Hybrids of 5′ ORF73 and 3′ OVA (which contains the SIINFEKL epitope), or 5′ OVA and 3′ ORF73 (with its SIINFEKL insert), were made by cutting each at a unique internal PstI site and swapping an in-frame 3′ PstI/XhoI fragment between them. Each form of ORF73/OVA was then subcloned as an EcoRI/XhoI fragment into the EcoRI and XhoI sites of pcDNA3. To fuse the ORF73 coding sequence to the C-terminus of OVA, we PCR-cloned the N-terminal 325 amino acid residues of OVA without a stop codon into the EcoRI and XhoI sites of pcDNA3 and ligated in PCR-cloned ORF73 as an XhoI/ApaI fragment, downstream of and in frame with the OVA coding sequence. 3′ HinDIII and KpnI truncations of this construct were generated as above. We also generated 5′ ORF73 truncations as XhoI/ApaI-digested PCR products, starting at amino acid residue 126 (OVA-73E), 146 (OVA-73D), 166 (OVA-73C), 186 (OVA-73B), or 206 (OVA-73A), and fused these to OVA 1–325 in pcDNA3. We also cloned OVA lacking its signal sequence, with translation starting at its methionine residue 41 (SOVA), and made the same fusions with N-terminal ORF73 truncations A–E. Recombinant viruses The MHV-68 M11 and ORF73 coding sequences (genomic co-ordinates 103418–103933 and 104868–103924, respectively) overlap by 10 bp at their 3′ ends [ 11 ]. We therefore duplicated this overlap to generate an insertion site between them. Thus, we PCR-cloned genomic co-ordinates 131171–103933, including XhoI and SalI restriction sites at the respective 5′ and 3′ ends, and cloned the fragment into the XhoI and SalI sites of pSP73-ORF73 (see Plasmids, above) to make pSP73-ORF73-M11, with the M11 and ORF73 coding sequences each complete and separated by a SalI site. The M11/ORF73 genomic overlap (the stop codons of ORF73 on the noncoding strand and M11 on the coding strand are underlined) was thus changed to TTTATGTC GTCGACTTATGTCTGAG. We then generated a poly-epitope construct downstream of an encephalomyocarditis IRES for insertion into the SalI site. We first inserted the adenovirus E19K leader sequence as two complementary oligonucleotides (5′- AATTGACCACCATGAGGTACATGATTTTAGGCTTGCTCGCCCTTGCGGAGTCTGCAGCGCGAATTCAGATCTCTCGAGTGAT and 5′- TCGAATCACTCGAGAGATCTGAATTCGCGCTGCAGACTGCCGCAAGGGCGAGCAAGCCTAAAATCATGTACCTCATGGTGGTC) into the EcoRI and XhoI sites of pMSCV-IRES-NEO [ 57 ]. Two complementary oligonucleotides encoding the peptide sequence MTSINFVKIASNENMETMSIINFEKL (5′- AATTCCTACCACCATGACCAGTATCAACTTTGTGAAGATAGCTTCCAATGAAAACATGGAGACTATGAGTATAATCAACTTTGAAAAACTGTGAC and 5′- TCGAGTCACAGTTTTTCAAAGTTGATTATACTCATAGTCTCCATGTTTTCATTGGAAGCTATCTTCACAAAGTTGATACTGGTCATGGTGGTAG) were then inserted into the EcoRI and XhoI sites of the pMSCV-NEO-leader construct. TSINFVKI is an H2-K b -restricted epitope from the MHV-68 ORF61 [ 43 ]; ASNENMETM is an H2-D b -restricted epitope from the influenza A/PR/8/34 nucleoprotein [ 58 ]. We amplified the leader-epitope construct by PCR, using the primers 5′- CCCCCATGGCCAGGTACATGATTTTAGGCTTGCTC and 5′- CCCGTCGACTCACAGTTTTTCAAAGTTGATTATACT, thereby adding a 5′ NcoI site and replacing the 3′ XhoI site with a 3′ SalI site, and cloned it into the NcoI and SalI sites of pMSCV-IRES-GFP [ 59 ]. Thus, the GFP coding sequence downstream of the IRES was replaced by the leader-epitope construct. We used the 3′ SalI site and an XhoI site just 5′ of the IRES to excise an IRES-leader-epitope XhoI/SalI fragment and cloned it into the SalI site of pSP73-ORF73-M11. We also subcloned an XhoI/SalI IRES-GFP fragment from pMSCV-IRES-GFP into the SalI site of pSP73-ORF73-M11 to make a control virus. Each ORF73-IRES construct was then subcloned into a larger genomic fragment for recombination into the MHV-68 genome. To do this, we used a BamHI-G genomic fragment [ 10 ] (genomic co-ordinates 101653–106902), cloned into pACYC184 (New England Biolabs) lacking a BspHI site [ 60 ]. The ORF73-IRES constructs and pACYC184-BamHI-G were digested with BstEII (genomic co-ordinate 104379) and BspHI (genomic co-ordinate 103750). Because BspHI is blocked by methylation, we used plasmids derived from Dam − E. coli. Finally, the mutant BamHI-G fragments were subcloned into the BAC mutagenesis shuttle vector pST76K-SR. Rec A–mediated recombination into the MHV-68 BAC was then carried out as previously described [ 41 ]. Sequence analysis revealed that the E19K leader sequence had been mutated during cloning, destroying the NcoI site and changing the start of the coding sequence downstream of the IRES from MARYMILG to MILG. Since this change was unlikely to prevent epitope presentation, no attempt was made to correct it. The ORF73-IRES-epitope BAC was subsequently reverted using an unmutated BamHI-G clone in pST76K-SR. MHV-OVA was generated by cloning OVA cDNA into EcoRI and XhoI sites of an ORF57/ORF58 intergenic expression cassette, driven by an ecotopic MHV-68 M3 promoter [ 61 ]. This virus will be described in more detail elsewhere. All BACs were reconstituted into infectious virus by transfecting 5 μg of BAC DNA into BHK-21 cells with Fugene-6 (Roche Diagnostics). The BAC cassette was removed by serial viral passage through NIH-3T3-CRE cells. Virus stocks were grown and titred on BHK-21 cells. Virus assays Infectious virus in freeze-thawed lung and spleen homogenates was plaque assayed on MEFs. Latent plus pre-formed virus in spleens was assayed on MEFs by explant culture of single-cell suspensions [ 39 ]. Cells expressing viral tRNAs 1–4 were detected by in situ hybridization of formalin-fixed, paraffin-embedded spleen cell sections, using a digoxigenin-labelled riboprobe transcribed from pEH1.4 [ 44 ]. Hybridized probe was detected with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Boehringer Ingelheim, Bracknell, United Kingdom) according to the manufacturer's instructions. The viral genome load in individual spleens was measured by real-time PCR. DNA was extracted (Wizard genomic DNA purification kit, Promega) and a portion of the MK3 ORF (genomic co-ordinates 24832–25071) amplified by PCR from 10 ng of each sample (Rotor Gene 3000, Corbett Research, Cambridge, United Kingdom). PCR products were quantitated with Sybr green (Invitrogen) and compared with a standard curve of cloned MK3 template, serially diluted in uninfected cellular DNA and amplified in parallel. The MK3 copy number was calculated from the cycle number at which the Sybr green signal crossed a set threshold on the standard curve. Amplified products were distinguished from paired primers by melting curve analysis, and the correct size of the amplified products was confirmed by electrophoresis and staining with ethidium bromide. Southern blotting Viral DNA was isolated from infected BHK-21 cells by alkaline lysis [ 39 ], digested with NcoI, electrophoresed on a 0.8% agarose gel, and transferred to positively charged nylon membranes (Roche Diagnostics). A 32 P-dCTP-labelled probe (APBiotech, Amersham, United Kingdom) was generated from the BamHI-G genomic fragment by random primer extension (Nonaprimer kit, Qbiogene, Bingham, United Kingdom) according to the manufacturer's instructions. Membranes were hybridized with probe (65 °C, 18 h), washed to a stringency of 0.2× SSC with 0.1% SDS, and exposed to X-ray film. Metabolic labelling and immunoprecipitation Cells were metabolically pulse-labelled with 35 S-cysteine/methionine (APBiotech) and chased with 1 mM unlabelled cysteine and methionine [ 29 ]. Labelled cells were lysed on ice for 30 min in 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM PMSF, plus Complete protease inhibitors (Roche Diagnostics). Cell debris and nuclei were removed by centrifugation (13,000 × g, 15 min). Lysates were precleared with rabbit anti-actin whole serum and formalin-fixed S. aureus (Sigma Chemical, Poole, United Kingdom), and then again with protein A-sepharose. OVA was precipitated with rabbit anti-OVA serum (Abcam, Cambridge, United Kingdom) followed by protein A-sepharose. Beads were washed five times in 1% Triton X-100 buffer. Samples were dissociated (95 °C, 2 min) in Laemmli's buffer prior to SDS-PAGE. Gels were fixed, dried, and exposed to X-ray film. Immunoblotting Cells were lysed as for immune precipitations (above). Post-nuclear lysates were denatured (95 °C, 2 min) in Laemmli's buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Membranes were blocked in PBS/0.1% Tween-20/10% non-milk fat and probed with rabbit anti-OVA serum or rabbit anti-neomycin phosphotransferase II serum (Upstate, Milton Keynes, United Kingdom), followed by horseradish-peroxidase-coupled donkey anti-rabbit IgG pAb (APBiotech) and ECL substrate development. Antigen presentation assays L929-K b cells (1–2 × 10 5 /well in 24-well plates) were transfected with 1 μg of plasmid using Fugene-6. Forty-eight hours later, B3Z cells (5 × 10 5 ) were added to each well. B3Z is an H2-K b -restricted, SIINFEKL-specific T cell hybridoma that produces beta-galactosidase in response to T cell receptor ligation [ 55 ]. For virus infections, L929-K b cells or MEF-1 cells (5 × 10 5 /well) were infected for 2 h and washed once before adding B3Z T cell hybridoma cells (5 × 10 5 /well). After a further 18 h, the cells were washed once in PBS and lysed in PBS/5 mM MgCl 2 /1% NP-40/0.15 μM chlorophenol-red-beta- D -galactoside (Merck Biosciences, Nottingham, United Kingdom) to assay beta-galactosidase activity. After 2–4 h at 37 °C, the absorbance at 595 nm was read on a Bio-Rad (Hercules, California, United States) Benchmark microplate reader. ELISA and ELISPOT assays For IFN-γ ELISPOT assays [ 62 ], duplicate dilutions of effector cells were incubated with 3 × 10 5 naive irradiated syngeneic spleen cells in nitrocellulose-bottomed 96-well plates (Millipore Corporation, Bedford, Massachusetts, United States) coated with rat anti-mouse IFN-γ mAb (BD-Pharmingen, San Diego, California, United States). The naive spleen cells were either (1) untreated, (2) pulsed with 1 μM AGPHNDMEI (p56), 1 μM TSINFVKI (p79), 1 μM ASNENMETM, or 1 μM SIINFEKL peptide, or (3) infected with WT MHV-68 (2 PFU/cell). After 48 h culture at 37 °C in complete RPMI/50 μM 2-mercaptoethanol/10 U/ml human recombinant IL-2, captured IFN-γ was detected with a further, biotinylated rat anti-mouse IFN-γ mAb (BD-Pharmingen), followed by streptavidin-alkaline phosphatase (Dako Cytomation, Ely, United Kingdom) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate. Virus-specific and total serum IgG levels were measured by ELISA. Maxisorp ELISA plates (Nalge Nunc, Rochester, New York, United States) were coated overnight with either affinity-purified goat anti-mouse IgG sera (Sigma Chemical) or 0.05% Triton X-100–disrupted MHV-68 [ 63 ]. After incubation with 2-fold serum dilutions, bound murine IgG was detected with alkaline phosphatase-conjugated goat anti-mouse IgG–Fcγ serum and nitrophenylphosphate substrate (Sigma Chemical). Absorbance was read at 405 nm. Flow cytometry A20-syndecan-1 cells infected with GFP + viruses were trysinized, washed in PBS, and analysed directly for green channel fluorescence. Spleens were disrupted into single-cell suspensions, washed in PBS/0.1% BSA/0.01% azide, and incubated for 15 min on ice with 5% mouse serum/5% rat serum and anti-CD16/32 mAb. Specific staining (1 h, 4 °C) was with fluorescein-isothiocyanate-coupled anti-CD69 and phycoerythrin-coupled anti-CD19 (BD-Pharmingen), or tricolour-coupled anti-CD8 (Caltag Laboratories, Burlingame, California, United States), Fluorescein-isothiocyanate-coupled anti–T cell receptor Vbeta4, and phycoerythrin-coupled anti-CD62L (BD-Pharmingen). For intracellular cytokine staining, spleen cells (5 × 10 5 –1 × 10 6 in 200 μl of complete RPMI/50 μM 2-mercaptoethanol/10 U/ml human recombinant IL-2/10 μg/ml Brefeldin A) were stimulated (5 h, 37 °C) with 1 μM ASNENMETM, 1 μM AGPHNDMEI, 1 μM TSINFVKI, or 1 μM SIINFEKL peptides, or left without peptide. All cells were then washed in PBS/10 μg/ml Brefeldin A, blocked with anti-CD16/32, stained with tricolour-conjugated anti-CD8 plus fluorescein-isothiocyanate-conjugated anti-I-A b (1 h, 4 °C), washed twice, fixed in 2% paraformaldehyde (30 min, 4 °C), washed once, permeabilized with 0.5% saponin, washed once, stained with phycoerythrin-coupled anti-interferon-gamma (BD-Pharmingen), and washed twice. All cells were analysed on a FACS Calibur using Cellquest software (Becton-Dickinson, Oxford, United Kingdom). I-A b staining was used to exclude B cells and myeloid cells. Data were graphed with FCSPress v1.3 ( www.fcspress.com ).

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1065267

Neutralizing Aptamers from Whole-Cell SELEX Inhibit the RET Receptor Tyrosine Kinase

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance.

Introduction The identification of tumor-specific molecular markers is a powerful tool in cancer diagnostics, and the targeting of tumor-specific pathways is the best hope for developing nontoxic and efficient anticancer therapies. Targeting of cancer cells relies on the development of molecular beacons, suited for in vivo applications, that are endowed with the required affinity, specificity, and favorable pharmacokinetic properties. With the systematic evolution of ligands by exponential enrichment (SELEX) technology [ 1 , 2 ], specific macromolecular ligands—aptamers—can be generated by screening very large pools of oligonucleotides containing regions of random base composition with reiterated cycles of enrichment and amplification. At each cycle, the individual oligonucleotides with affinity for the desired target are kept, those with affinity for the sham target are rejected, and the population is enriched in oligonucleotides that distinguish between sham and real target. Aptamers that recognize a wide variety of targets, from small molecules to proteins and nucleic acids, and from cultured cells to whole organisms, have been described [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. These oligonucleotides generally meet the requirements for in vivo diagnostic and/or therapeutic applications: Besides their good specificity and affinity, they are poorly immunogenic, and the SELEX technology can now accept chemically modified nucleotides for improved stability in biological fluids [ 11 ]. Conspicuously, less than fifteen years after the first applications of the technique, several lead compounds, including an anti-vascular endothelial growth factor aptamer [ 12 ], are currently under clinical trials [ 13 ]. Receptor tyrosine kinases (RTKs) are involved in a variety of signaling processes that regulate cell growth and proliferation and in several cancers [ 14 ]. RTKs are privileged targets for cancer therapy, which is underscored by the promising outcome of clinical trials with small molecules or antibody inhibitors [ 14 ]. In the present study, we validated a general strategy to target transmembrane receptors by SELEX. The RET (rearranged during transfection) RTK is physiologically stimulated by any member of the glial cell line-derived neurotrophic factor (GDNF) family [ 15 , 16 ]. Germline mutations in the RET gene are responsible for constitutive activation of the receptor and for inheritance of multiple endocrine neoplasia (MEN) type 2A and 2B syndromes and of familial medullary thyroid carcinoma [ 17 , 18 , 19 , 20 ]. Mutations in the extracellular domain of RET, responsible for MEN2A syndrome, lead to constitutive dimerization of two mutated RET molecules. Conversely, a single point mutation, within the RET catalytic domain, that causes the MEN2B syndrome, involves an intramolecular mechanism to convert RET into a dominant transforming gene. Therefore, RET constitutes a model system of choice [ 20 ], in that the transforming mutations located in the extracellular domain simplify the issue of intracellular accessibility for a molecule targeting the receptor mutated in the extracellular domain (in its monomeric or dimeric form) and might provide alternative models (e.g., RET with mutations of the 2B kind) for controls or to elucidate the mode of target recognition. Here we adopted a whole-cell SELEX strategy to target RET in a complex environment that is expected to expose a native protein to the selection procedure, thus best mimicking in vivo conditions. We obtained aptamers that not only recognize the extracellular domain of RET, but also block RET downstream signaling and subsequent molecular and cellular events. The fact that aptamers with antioncogenic activity were isolated in the absence of a specific selective pressure suggests that our method could be used to identify active macromolecules with potential therapeutic interest against other transmembrane receptors. Results A library of 2′-fluoropyrimidine (2′F-Py), nuclease-resistant RNAs was subjected to a differential SELEX protocol against intact cells expressing different forms of the human RET oncogene ( Figure 1 ). For the selection step, PC12 cells were used that express the human RET C634Y mutant receptor (PC12/MEN2A). RET C634Y is mutated in the extracellular domain and forms spontaneously active homodimers on the cell surface, which induces biochemical and morphological changes that mirror the RET-dependent human pheochromocytoma phenotype of MEN2 syndromes [ 21 ]. The counterselection necessary to avoid selecting for aptamers that nonspecifically recognized the cell surface included a first step against parental PC12 cells in order to eliminate nonspecific binders of the PC12 cell surface, followed by a second counterselection step against PC12/MEN2B cells that expressed an allele of RET (RET M918T ) mutated in the intracellular tyrosine kinase domain. PC12/MEN2B and PC12/MEN2A cells have a similar morphology, but the extracellular domain of the RET M918T receptor is identical to the wild type and, in the absence of the ligand and co-receptor, remains monomeric. This step was originally aimed at selecting aptamers that recognize specifically the dimeric form of the extracellular domain. 10.1371/journal.pbio.0030123.g001 Figure 1 Schematic Protocol for the Selection of PC12/MEN2A Cell-Specific Aptamers A pool of 2′F-Py RNAs was incubated with suspended parental PC12 cells (Counterselection 1). Unbound sequences were recovered by centrifugation and incubated with adherent PC12/MEN2B cells (Counterselection 2). Unbound sequences in the supernatant were recovered and incubated with adherent PC12/MEN2A cells for the selection step (Selection). Unbound sequences were discarded by several washings, and bound sequences were recovered by phenol extraction. Sequences enriched by the selection step were amplified by RT-PCR and in vitro transcription before a new cycle of selection. After 15 rounds of selection, the pool of remaining sequences bound PC12/MEN 2A cells in a saturable manner with an apparent Kd approximating 100 nM. From this pool, 67 sequences were cloned and analyzed. Two individual sequences (D14 and D12) dominated the selection and constituted together more than 50% of the clones, four other sequences represented together 25% of the clones, and eight sequences were present only once. As is often the case for a selection against a complex target [ 7 , 22 ] (and in contrast to in vitro SELEX on purified proteins) we found almost no similarity among sequences, except for clones D24 and D4, which shared common sequence motifs and structure prediction ( Figure 2 A). 10.1371/journal.pbio.0030123.g002 Figure 2 Predicted Structure and Association Constants of D4 and D24 (A) Comparison of a secondary structure prediction for the D4 and D24 aptamers . Structures were predicted using MFOLD software version 3.1 (available at http://www.bioinfo.rpi.edu/applications/mfold/ ). (B) Binding curve of the D4 aptamer on PC12/MEN2A. D4 was 32 P-radiolabeled and incubated at different concentrations on cell monolayers. The background binding value for a D4 scrambled sequence is subtracted from every data point. Scatchard analysis (inset) was used for the evaluation of the binding constant. (C) Binding of the 32 P-labeled D4 aptamer to several cell lines expressing (or not) human RET. Binding was performed on the cell lines indicated in the same condition at 50 nM, and the results are expressed relative to the background binding detected with the starting pool of sequences used for selection. Expression of RET could not be detected by Western blot in HeLa, NBTII, PC12wt and NIH3T3 cells, whereas PC12/MEN2A and NIH3T3/MEN2A express RET C634Y and PC12/MEN2B and NIH3T3/MEN2B express RET M918T . We assessed binding to PC12/MEN2A cells of all individual aptamers that were found more than once and also of some unique sequences (including D4 and D24). Several sequences bound PC12/MEN2A cells with apparent Kd values ranging from 30 to 70 nM ( Figure 2 B and unpublished data), but not parental PC12, rat-derived bladder carcinoma (NBTII), or human cervical carcinoma (HeLa) cells ( Figure 2 C and unpublished data). As a first attempt to deconvolute the complex pool of winning aptamers, we first produced a recombinant fragment of RET, EC-RET C634Y [ 23 ], but all attempts to identify in the winning pool aptamers binding to EC-RET C634Y were fruitless. Likewise, SELEX against this purified EC-RET C634Y protein gave rise to aptamers unable to recognize the PC12/MEN2A cells, suggesting that they did not bind to the RET protein present in its native conformation on the cell surface. Consequently, we screened the winning pool of aptamers for the ability to interfere with the biological activity of RET. To this end, we used an in vitro cell system in which we assessed the capability of each aptamer to inhibit RET C634Y autophosphorylation and receptor-dependent downstream signaling. Mutant RET C634Y , expressed in PC12/MEN2A cells, forms homodimers on the cell surface that cause constitutive activation of its tyrosine kinase activity [ 24 ] and induce several downstream signaling cascades, including the activation of extracellular signal-regulated protein kinase (ERK) [ 25 ]. As previously reported [ 25 ], levels of phosphorylated RET and ERK were constitutively high in untreated PC12/MEN2A cells due to the presence of the active RET C634Y allele. Surprisingly, some of the tested aptamers inhibited RET C634Y and ERK phosphorylation, compared to the control starting pool and to the other aptamers ( Figure 3 A and unpublished data). In all experiments, inhibition of phosphorylation was more rapid and quantitative for ERK than for RET C634Y . We believe that this is due to a different sensitivity to changes in RET tyrosine kinase activity of the two processes and/or to differences in the half-lives of the phosphorylated forms of the two proteins [ 26 ]. In a dose-response experiment ( Figure 3 B, left panel), the best inhibitor, D4, was effective at a concentration of 200 nM to inhibit RET C634Y autophosphorylation up to 70% and to drastically reduce ERK phosphorylation. Time-activity studies showed that the treatment of PC12/MEN2A cells at 200 nM for 1 h was sufficient to significantly inhibit RET C634Y autophosphorylation and to drastically reduce ERK phosphorylation ( Figure 3 B, right panel). 10.1371/journal.pbio.0030123.g003 Figure 3 Effect of Selected Aptamers on RET C634Y Activity (A) PC12/MEN2A cells were either left untreated or treated for 16 h with 150 nM of the indicated RNA aptamer, or the starting RNA pool (pool). Cell lysates were immunoblotted with anti-(phospho)-ERK (pErk), then stripped and reprobed with anti-ERK (Erk) to confirm equal loading. Values below the blots indicate signal levels relative to untreated controls. (B) PC12 / MEN2A cells were treated for 1 h with increasing amounts of D4 (left blots) or with 200 nM D4 for the indicated incubation times (right blots). Cell lysates were immunoblotted with anti-(Tyr-phosphorylated)-RET (pRet) or anti-(phospho)-ERK (pErk) antibodies, as indicated. To confirm equal loading the filters were stripped and reprobed with anti-RET (Ret) or anti-ERK (Erk) antibodies, respectively. In (A) and (B), “C” indicates mock-treated cells. Quantitations were done on the sum of the two RET- or ERK- specific bands, and values are expressed relative to the control, arbitrarily set to 1. Standard deviations are indicated ( n = 4). Comparison of the predicted structures of D4 and of the related clone D24 ( Figure 2 A) suggests that a conserved stem-internal loop-stem is crucial for binding. Consistently, we found that replacing the apical loop with a stable tetraloop (UUGC) or deleting nucleotides not included in the conserved structure did not significantly affect binding of D4 to PC12/MEN2A cells (unpublished data). However, only the full-length D4 inhibits RET C634Y signaling, demonstrating that binding is necessary but not sufficient for inhibition. A 2′F-Py RNA oligonucleotide of identical composition but with a scrambled sequence (D4Sc) was ineffective for both binding and inhibition. The D4 aptamer bound to PC12/MEN2A with an estimated apparent Kd of 35 ± 3 nM ( Figure 2 B), but also to PC12/MEN2B cells ( Figure 2 C and unpublished data), suggesting that one of the counterselection steps employed in the SELEX procedure was ineffective in this case. The D4 aptamer bound to transfected NIH3T3 cells expressing at similar levels the two mutant forms (RET C634Y and RET M918T ) of the RET receptor (NIH/MEN2A and NIH/MEN2B, respectively [ Figure 2 C; see also below]). Binding was dependent on expression of human RET, as D4 did not recognize parental untransfected PC12, NIH3T3 cells, or other cell lines, including rat NBTII, human HeLa cells, and mouse MN1 ( Figure 2 C and unpublished data). Interestingly, the latter, a mouse motor neuron-neuroblastoma fusion cell line, expresses the mouse RET wt , suggesting some species-specificity in RET recognition by D4. Finally, D4 bound a human neuroblastoma cell line (SK-N-BE) that naturally expresses endogenous RET (L. Cerchia et al., personal communication). Consistently with what was observed for the pool of winning aptamers, D4 was unable to bind the purified EC-RET C634Y protein (unpublished data), thus supporting the specificity for the membrane-bound RET. We next determined whether D4 could inhibit wild-type RET. Cells from a PC12-derived cell line expressing the human wild-type RET (PC12/wt) were stimulated with a mixture containing GDNF and soluble GDNF family receptor α1 (GFRα1), and either treated with the D4 aptamer or with the starting pool of 2′F-Py RNA as a negative control. As shown in Figure 4 A, the D4 aptamer, but not the control RNA pool, strongly inhibited GDNF-induced phosphorylation of RET (left panel) and of the downstream effector ERK (middle panel). A similar inhibitory effect was observed in PC12-α1/wt cells, a PC12-derived cell line that stably expresses both human RET and GFRα1 (unpublished data). In contrast, D4 was inactive in inhibiting the signaling triggered by the unrelated nerve growth factor (NGF) receptor tyrosine kinase TrkA, thus indicating that D4-induced inhibition of ERK phosphorylation was specific for RET intracellular signaling ( Figure 4 A, right pane) 10.1371/journal.pbio.0030123.g004 Figure 4 D4 Aptamer Inhibits RET wt but Not RET M918T Activity (A) PC12/wt cells were treated for 10 min with GDNF (50 ng/ml) and soluble GFRα1 (1.6 nM), or 5 min with NGF (100 ng/ml), together with 200 nM of either the D4 aptamer or the starting RNA pool. “C*” indicates cells treated with GDNF and GFRα1 in the absence of aptamer. (B) PC12/MEN2B cells were starved for 6 h and then treated for 1 h with 200 nM D4 or the starting RNA pool. Cell lysates were immunoblotted with anti-(Tyr-phosphorylated)-RET or anti-(phospho)-ERK antibodies, as indicated (see Figure 3 legend). In (A) and (B), “C” indicates mock-treated cells. Quantitations were done as in Figure 3 , and relative abundances are expressed relative to controls, arbitrarily set to 1. Standard deviations are indicated ( n = 4). Although the D4 aptamer binds PC12/MEN2B cells, treating these cells with 200 nM D4 for 1 h ( Figure 4 B) or longer, or at higher D4 concentrations (unpublished data), did not interfere with signaling due to the monomeric RET M918T . This further confirms that inhibition of ERK phosphorylation is not a nonspecific effect of exposing the cells to the D4 aptamer. The kinase and the biological activities of RET M918T , although constitutive, are responsive to GDNF stimulation in the presence of GFRα1 [ 27 , 28 ]. Similarly to the inhibition of RET wt activity, the treatment of PC12/MEN2B cells by D4 abolished the GDNF-dependent overstimulation of RET and ERK phosphorylation (unpublished data). These data strongly suggest that D4 inhibits exclusively the dimerization-dependent RET activation. We then searched for phenotypic effects of D4 on RET-dependent cell differentiation and transformation. First we measured neurite outgrowth in PC12-α1/wt cells following GDNF stimulation. As shown in Figure 5 , cells extended long neurite-like processes in response to a 48-h exposure to GDNF ( Figure 5 B) with respect to the nonstimulated control cells ( Figure 5 A). Treatment of the cells with the D4 aptamer ( Figure 5 C), but not with the D4Sc scrambled control ( Figure 5 D), significantly decreased the proportion of neurite outgrowth ( Figure 5 E). To biochemically monitor differentiation, we determined the levels of the nerve growth factor-inducible protein (VGF) in cell extracts following 48 h of treatment. VGF is an early gene that is rapidly induced by both NGF and GDNF in PC12 cells [ 29 ]. As expected, in GDNF-treated cells, VGF expression was stimulated and, consistent with the phenotypic effects reported above, treatment with D4, but not with D4Sc, kept the VGF levels close to basal ( Figure 5 F). 10.1371/journal.pbio.0030123.g005 Figure 5 D4 Aptamer Inhibits the GDNF-Induced Differentiation of PC12-α1/wt Cells Cells were either left unstimulated (A), stimulated with GDNF (B), or with GDNF together with D4 or D4Sc (C and D, respectively). Following 48 h of GDNF treatment, the percentage of neurite outgrowth was calculated. The data represent the average of three independent experiments and are expressed as percentage of neurite-bearing cells/total cells analyzed (E). Following 48 h of treatment, cells were lysed and proteins immunoblotted with anti-VGF antibodies. Equal loading was confirmed by immunoblotting with anti-ERK antibodies as indicated (F). Upon expression of either RET C634Y or RET M918T , NIH3T3 cells show drastic changes in their morphology [ 24 ]. We treated NIH/MEN2A and NIH/MEN2B cells stably expressing the RET mutants with D4 for 72 h, and analyzed the morphological changes induced by the aptamer. As shown in Figure 6 , NIH/MEN2A and NIH/MEN2B cells have a spindle shape, long protrusions, and a highly refractive appearance ( Figure 6 B and 6 E, respectively). As expected, D4-treated NIH/MEN2A cells ( Figure 6 C) reverted to a flat and polygonal morphology similar to the parental NIH3T3, whereas no morphological changes were observed in NIH/MEN2B ( Figure 6 F), which is consistent with the notion that constitutive signaling from RET C634Y , but not from RET M918 , is inhibited by D4. On the other hand, treatment with D4Sc had no effects on any cell line ( Figure 6 D and unpublished data). 10.1371/journal.pbio.0030123.g006 Figure 6 D4 Aptamer Reverts the Transformed Morphology of NIH/MEN2A Cells NIH3T3-derived cell lines were either left untreated (A, B, and E) or treated with D4 (C and F) or D4Sc (D), and the cells were maintained in culture for 72 h. Each experiment was repeated a minimum of three times. Discussion RTKs are involved in a variety of signaling pathways that affect cell growth and differentiation. Targeting specifically RTKs holds potential for dissecting the molecular mechanisms of receptor function, but also for diagnosis and therapeutics of cancer [ 14 ]. Here we employed a modified SELEX procedure to target the RET RTK, and we obtained nuclease-resistant RNA ligands capable of binding and inhibiting the protein on the cell surface. Aptamers against recombinant heregulin 3 (HER3) RTK have been recently isolated and shown to inhibit the heregulin-induced activation of the HER3/HER2 dimer [ 30 ]. However, finding the most efficient binders and inhibitors is likely to generally rely on the recognition of the target protein in its native state. In the case of transmembrane receptors, whole-cell SELEX offers the advantage of selecting molecules capable of recognizing the target protein in its natural glycosylation state and presented in its physiological environment. An important drawback of this strategy is the lack of knowledge of the identity and abundance of the effective targets and the possibility that unwanted aptamers may dominate the selection, preventing the emergence of the molecules of interest. However, the abundance of the target protein and an appropriate selection scheme might provide sufficient selective pressure to favor the wanted aptamers [ 10 ]. The D4 aptamer binds to different cell types, provided that human RET is expressed on the cell surface, and specifically inhibits both RET and ERK phosphorylation, strongly suggesting that RET is the bona fide target of D4. Interestingly, aptamers isolated by whole-cell SELEX were unable to bind purified EC-RET C634Y and, conversely, aptamers coming from the selection with purified EC-RET C634Y were unable to bind the membrane-bound RET. Thus, it is likely that D4 binding is dependent on the association of RET with the cellular membrane, which might reflect changes in the receptor's conformation/modification state or, alternatively, might imply unidentified molecular components interacting with RET at the cell surface. This latter possibility is supported by a recent report demonstrating that the presence of heparan sulfate glycosaminoglycan on the cell surface is required for RET-dependent GDNF intracellular signaling [ 31 ]. Our interpretation of the D4 aptamer's mode of action relies upon three observations: (1) D4 binds with similar affinities to cells expressing RET in a monomeric or dimeric form; (2) D4 inhibits dimerization-dependent RET activation, as a consequence either of GDNF stimulation of RET wt or RET M918T or of constitutive dimerization of the RET C634Y mutant; and (3) D4 does not inhibit a monomeric form of RET that is constitutively activated by a mutation in the intracellular kinase domain (RET M918T ). These results taken together are compatible with the notion that D4 acts by interfering with the formation of a stable, active RET dimer, regardless of whether dimerization is caused by the formation of the RET/GDNF/GFRα1 complex or by the direct interaction of two mutated RET C634Y proteins. This might occur either by D4 binding to monomeric RET, which would impede subsequent formation of the dimer, or by binding directly to the dimer. Differential whole-cell SELEX strategies (this work; see also [ 5 , 7 , 8 , 10 ]) can be employed to identify new markers on the surface of a given cell type, define the specificity of a cellular state, and/or allow in vivo targeting for diagnostic and therapeutic applications. The identification of lead compounds by reiterated affinity selection on living cells appears crucial when the molecular target is a membrane-bound or large transmembrane protein for which the conformation is frequently dictated by the interaction with other molecules, including membrane constituents [ 31 ]. Given that several of these proteins, as transmembrane receptors, integrins, and adhesion molecules, are involved in cell proliferation, apoptosis, and differentiation, aptamers for these targets could be promising prognostic tools in human therapy for widespread, devastating diseases such as cancer and neurodegeneration. Materials and Methods Cell culture and immunoblot analysis Growth conditions for PC12 cells and derived cell lines were previously described [ 32 ]. NIH/MEN2A and NIH/MEN2B cells were obtained from NIH3T3 cells stably transfected with vectors expressing human RET C634Y and RET M918T . To assess the effects of aptamers on RET activity, cells (160,000 cells per 3.5-cm plate) were serum-starved for 2 h and then treated with the indicated amount of RNA aptamers or the starting RNA pool after a short denaturation-renaturation step. When indicated, 2.5S NGF (Upstate Biotechnology, Lake Placid), GDNF (Promega), or recombinant rat GFRα1-Fc chimera (R&D Systems, Minneapolis, Minnesota, United States) were added to the culture medium. Cell extracts and immunoblotting analysis were performed as described [ 23 ]. The primary antibodies used were anti-RET (C-19), anti-VGF (R-15), and anti-ERK1 (C-16) (all three, Santa Cruz Biotechnology, Santa Cruz, California, United States); and anti-(Tyr-phosphorylated) RET and anti-phospho-44/42 MAP kinase (also indicated as anti-[phospho]-ERK) monoclonal antibodies (E10) (both from Cell Signaling, Beverly, Massachusetts, United States). Four independent experiments were performed. Cell transformation and neurite outgrowth bioassay PC12-α1/wt or NIH3T3 cells were plated at equal density on 12-well culture plates. Aptamers were added at 3 μM final concentration to the growth medium. To ensure the continuous presence of a concentration of at least 200 nM, this treatment was renewed every 24 h, which takes into account the half-life of the D4 aptamer in 10% serum (approximately 6 h, unpublished data). At least 15 random fields were photographed every 24 h with a phase-contrast light microscope. To evaluate the effects of D4 on cell differentiation, cells were pretreated for 6 h with 400 nM D4 or D4Sc and then incubated with 50 ng/ml GDNF together with 3 μM of the appropriate aptamer (see above). At 24 and 48 h of GDNF stimulation, 50 cells per frame were counted and scored as having neurites or not. A neurite was operationally defined as a process outgrowth with a length more than twice the diameter of cell body. Ex vivo SELEX The SELEX cycle was performed essentially as described [ 33 ]. Transcription was performed in the presence of 1 mM 2′F-Py and a mutant form of T7 RNA polymerase (T7 Y639F , kind gift of R. Souza) [ 11 ] was used to improve yields. 2′F-Py RNAs were used because of their increased resistance to degradation by seric nucleases. The complexity of the starting pool was roughly 10 14 . 2′F-Py RNAs (1–5 nmol) were heated at 85 °C for 5 min in 3 ml of RPMI 1640, snap-cooled on ice for 2 min, and allowed to warm up to 37 °C before incubation with the cells. Two counterselection steps were performed per cycle. To avoid selecting for aptamers nonspecifically recognizing the cell surface, the pool was first incubated for 30 min at 37 °C with 10 7 PC12 cells, and unbound sequences were recovered by centrifugation. These were subsequently incubated with 10 7 adherent PC12/MEN2B cells, expressing a human RET receptor mutated in the intracellular domain (RET M918T ), and unbound sequences were recovered for the selection phase. This step was meant to select sequences recognizing specifically the human RET receptor mutated in the extracellular domain (RET C634Y ) expressed on PC12/MEN2A cells. The recovered sequences were incubated with 10 7 adherent PC12/MEN2A cells for 30 min at 37 °C in the presence of nonspecific competitor RNA (total yeast RNA) and recovered after several washings with 5 ml of RPMI by total RNA extraction (Extract-All, Eurobio, Les Ulis, France). During the selection process, we progressively increased the selective pressure by increasing the number of washings (from one for the first cycle up to five for the last three cycles) and the amount of nonspecific RNA competitor (100 μg/ml in the last three cycles), and by decreasing the incubation time (from 30 to 15 min from round 5) and the number of cells exposed to the aptamers (5 × 10 6 in the last three cycles). To follow the evolution of the pool we monitored the appearance of four-base restriction sites in the population, which reveals the emergence of distinct families in the population [ 34 ]. After 15 rounds of selection, sequences were cloned with TOPO-TA cloning kit (Invitrogen, Carlsbad, California, United States) and analyzed. Binding experiments Binding of individual aptamers (or the starting pool as a control) to PC12 cells and derivatives was performed in 24-well plates in triplicate with 5′- 32 P-labeled RNA. 10 5 cells per well were incubated with various concentrations of individual aptamers in 200 μl of RPMI for 10 min at 37 °C in the presence of 100 μg/ml polyinosine as a nonspecific competitor. After extensive washings (5 × 500 μl of RPMI), bound sequences were recovered in 350 μl of SDS 0.6%, and the amount of radioactivity recovered was normalized to the number of cells by measuring the protein content of each well. Binding of individual sequences to different cell lines was performed in the same condition at 50 nM only. For the binding curve of D4 to PC12/MEN2A cells (see Figure 2 B), nonspecific binding was assessed using a 5′- 32 P-labeled naive pool of 2′F-RNAs (i.e., the starting pool of the selection), and the background values obtained were subtracted from the values obtained with the D4 aptamer. Apparent Kd values for each aptamers were determined by Scatchard analysis according to the equation [bound aptamer]/[aptamer] = −(1/Kd) × [bound aptamer] + ([T] tot /Kd) where [T] tot represents the total target concentration. Supporting Information Accession Numbers The Swiss-Prot ( http://www.ebi.ac.uk/swissprot/ ) accession numbers for the proteins discussed in this paper are ERK (P27361), GDNF (P39905), GFRα1 (P56159), NGF (P01138), RET RTK (P07949), TrkA (P04629), and VGF (P20156).

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1065268

The Bacteria's Guide to Survival

From The Worst Case Scenario Survival Handbook —with handy entries like “How to escape from killer bees” and “How to escape from quicksand”—to The Zombie Survival Guide: Complete Protection from the Living Dead , survival guides are one of the latest publishing fads. If there was a market for it, a survival guide for bacteria might include topics like “How to use your pili to keep your host from going apoptotic.” A host's cells can respond to a bacterial infection with apoptosis, or programmed cell death. For bacteria that pass directly from host to host, this can pose a problem. If the bacteria are highly virulent and induce too much cell death, they could take down their host before they're able to jump ship, thus hurting the bacteria's chances of survival in the long run. Earlier studies suggested that bacteria can use their pili, finger-like appendages that many bear on their surface, to pull on a host's cell membranes and thus influence the cell's behavior. But these studies, which looked at mutant bacteria that could not retract their pili, did not examine the matter of how the bacteria coax their hosts to stay alive. Now, in PLoS Biology , a group of researchers present more direct evidence that bacteria can induce changes in hosts' gene expression—and possibly keep the host cells alive longer—through tiny tugs on cell membranes. The study, led by Magdalene So, examined gene activity in human epithelial cells infected with Neisseria gonorrhoeae , the bacteria responsible for the sexually transmitted disease gonorrhea. By comparing cells infected with normal N. gonorrhoeae to those infected with a mutant strain with defective pili, the researchers found a subset of 52 host genes that had higher activity when the host was infected with the normal bacteria, suggesting that the pulls of the pili were responsible. They also ran a key control experiment with an artificial mechanical pull on the host cell membrane. By coating magnetic beads with a preparation of bacterial pili, the beads attached themselves to the cell membranes. Then, in the presence of a magnetic field, the beads tugged on the cell membrane, approximating the effects on gene expression during infection with normal bacteria. Thus, the mechanical tugs seem responsible for triggering a signaling cascade in the host cells, which ultimately affects the host's gene expression. Many of the genes that increased in activity due to the tugs were already known to regulate apoptosis and cellular response to stress, including mechanical strain on the membrane. Also, a majority of these genes were known to be induced by a family of proteins called mitogen-activated protein kinases, or MAPKs. The researchers showed that blocking MAPKs reduced the activity of several of the genes that are usually enhanced by infection with the normal bacteria. Also, they found that cells infected with the bacteria tended to survive treatment with staurosporine, a chemical that normally induces apoptosis. Overall, the group's findings support previous speculations that some bacteria influence gene expression and the fate of cells in their hosts by tugging on the host cells' membranes with their pili. For bacteria like N. gonorrhoeae that pass directly from host to host, the researchers argue, it would be in a bacterium's interest to help keep its host alive. And bacteria appear to do this with the help of their pili.

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1065269

A Small RNA That Neutralizes a Protein Linked to Tumor Development

For most of human history, cancer has been incurable. But with the invention of anesthesia in the mid-19th century, surgeons were able to remove some forms of cancer surgically. Radiotherapy arrived next, soon after the discovery of X rays in 1896. Chemotherapy, now a mainstay of cancer treatment, did not arrive until the mid-1940s, when nitrogen mustard, an alkylating agent related to the mustard gas used in the two World Wars, was developed as an anticancer agent. Unfortunately, although cancer cells are hypersensitive to the effects of alkylating agents—molecules that introduce lethal changes into the cell's DNA—normal cells are also targeted by them, although less damage is caused because normal cells typically divide slower than cancer cells. Using chemotherapy based on alkylating agents to treat cancer is like using a sledgehammer to crack a nut. But with improved knowledge about how cancer cells differ from normal cells, chemotherapeutics are now being designed that hit only cancer cells. Many of these new chemotherapeutics target protein receptors called tyrosine kinases. These receptors, which sit on the cell surface, normally stimulate intracellular pathways that control proliferation and other cellular functions in response to growth factors. In tumors, these receptors often have mutations that allow them to become active without growth factor binding, which results in the uncontrolled proliferation that is characteristic of cancer cells. For instance, mutations in the RET receptor tyrosine kinase are responsible for multiple endocrine neoplasia (MEN) type 2 syndromes. Whereas external stimulation by a growth factor is normally needed before two RET molecules can bind together (a process called dimerization) to activate intracellular signaling cascades, in MEN type 2A, a mutation in the RET receptor tyrosine kinase provokes (or induces) dimerization without external stimulation. In recent years, several proteins and various small synthetic chemicals have been designed that specifically inhibit the activity of mutated receptor tyrosine kinases and show anticancer activity. Domenico Libri and colleagues are now working on another class of molecules, called aptamers, that have potential as anticancer drugs. Aptamers—single-stranded nucleic acid molecules that are 50–100 bases long and can be selected for their ability to bind directly and tightly to specific proteins—are less likely to be targeted and destroyed by the body's natural defenses than some other types of potential therapeutic molecules. To find an aptamer able to recognize the RET receptor kinase within a cellular membrane environment, the researchers used whole-cell SELEX (systematic evolution of ligands by exponential enrichment), a process in which large pools of oligonucleotides are enriched for molecules that can distinguish between a real and sham target. First, they incubated a large pool of RNAs with PC12 cells, a rat cell line not expressing RET, to remove sequences binding non-specifically to the PC12 cell surface. Unbound sequences were recovered and applied to PC12 cells expressing human RET with the MEN type 2A mutation that causes dimerization. This time, bound sequences were retained, and the whole selection process was repeated another 14 times to select for aptamers that recognize the dimeric form of the RET extracellular domain. A newly synthesized molecule, D4, inhibits cellular differentiation Of the 67 sequences pulled out of the final pool of RNAs, the researchers found one sequence, D4, that not only bound the extracellular domain of RET but also blocked RET downstream signaling events and subsequent cellular and molecular changes. The researchers suggest that D4 blocks the dimerization-dependent activation of RET—whether it's induced by its physiological signaling molecule or by an activating mutation—and suggest that their method can be used to identify macromolecules with potential therapeutic effects against other transmembrane receptors involved in tumorigenesis, particularly since the whole-cell SELEX approach should efficiently select aptamers that recognize these receptors as they are found on the surface of tumor cells.

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1065270

How a Latent Virus Eludes Immune Defenses

For a virus to survive, it must elude the ever vigilant immune sentinels of its host. A latent virus can escape immune detection if it resides in nondividing cells and doesn't produce any proteins. No viral proteins means no red flags for immune cells. If the virus targets one of the many cell types that rarely divide, it's relatively safe while latent. But some viruses, like the gamma-herpesvirus, infect B cells of the immune system, which occasionally divide. The gamma-herpesvirus genome persists as circular pieces of DNA called episomes. When an infected B cell divides, the latent gamma-herpes virus episome must replicate and segregate into daughter cells along with the cell's genome. Viral replication and segregation requires the services of a protein called the episome maintenance protein—a potentially recognizable target for immune cells. Gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma–associated herpesvirus (KSHV), can induce uncontrolled lymphocyte (immune cell) proliferation and result in lymphoma, Hodgkin's disease, and Kaposi's sarcoma. These diseases arise from the persistent latent infections that take hold after initial infections are controlled by immune defenses. The episome maintenance protein produced by EBV, called EBNA-1, harbors an amino acid element in its epitope—the region that binds to a T cell and triggers an immune response—that helps the viral protein evade the killer T cells that could destroy it. Lab studies show that the amino acid element limits EBNA-1's interaction with T cells by inhibiting synthesis and, to a lesser degree, degradation of the protein. How this evasive action works or helps the virus in a living organism is not entirely clear. But if T cells aren't presented with bits of viral protein, they have no way of knowing the virus is present. In a new study, Neil Bennett, Janet May, and Philip Stevenson explore this question by studying virus–host interactions in mice infected with the murine gamma-herpesvirus-68 (MHV-68). Though MHV-68 infects mice, it behaves similarly to EBV and KSHV infections in humans, producing an acute mononucleosis-like illness and a pervasive pool of latently infected B cells. The episome maintenance protein in MHV-68 and KSHV is called ORF73. None of the viruses can maintain latent infections with deficient episome maintenance proteins. Stevenson and colleagues first demonstrated that ORF73 limits T cell recognition and then identified a key region responsible for immune evasion by modifying different regions of the viral protein. In the next round of experiments, the authors asked how the viral protein manages this feat. They discovered that ORF73 limits T cell recognition much like EBNA-1 does, by reducing synthesis and degradation of the protein. One region strongly associated with inhibiting epitope presentation to killer T cells corresponded to reduced protein synthesis. When the authors modified the ORF73 transcript to circumvent T cell evasion, the T cells “wiped out” latent virus. These results indicate that avoiding epitope presentation during episome maintenance is key to the virus's survival. MHV-68 virions emerging from infected cells Interestingly, the MHV-68 episome maintenance protein mediates immune evasion even though it lacks the amino acid element that does the job for EBV. Future studies will have to determine the responsible MHV-68 epitope and the mechanisms that engineer immune avoidance. Since a majority of epitopes that killer T cells recognize come from aborted translation events, it may be that evasive action is taken at the RNA transcript stage, before RNA is translated into protein. Evading killer T cells, the authors argue, is key to the survival of the gamma-herpesvirus. By figuring out just how evasion occurs, scientists can identify a promising target for controlling infection.

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1065312

Microcirculation abnormalities in patients with fibromyalgia – measured by capillary microscopy and laser fluxmetry

This unblinded preliminary case-control study was done to demonstrate functional and structural changes in the microcirculation of patients with primary fibromyalgia (FM). We studied 10 women (54.0 ± 3.7 years of age) with FM diagnosed in accordance with the classification criteria of the American College of Rheumatology, and controls in three groups (n = 10 in each group) – age-matched women who were healthy or who had rheumatoid arthritis or systemic scleroderma (SSc). All 40 subjects were tested within a 5-week period by the same investigators, using two noninvasive methods, laser fluxmetry and capillary microscopy. The FM patients were compared with the healthy controls (negative controls) and with rheumatoid arthritis patients and SSc patients (positive controls). FM patients had fewer capillaries in the nail fold (P < 0.001) and significantly more capillary dilatations (P < 0.05) and irregular formations (P < 0.01) than the healthy controls. Interestingly, the peripheral blood flow in FM patients was much less (P < 0.001) than in healthy controls but did not differ from that of SSc patients (P = 0.73). The data suggest that functional disturbances of microcirculation are present in FM patients and that morphological abnormalities may also influence their microcirculation.

Introduction Vasospastic symptoms occur in about 30% of patients with primary fibromyalgia (FM) [ 1 ]. These patients present with Raynaud's phenomenon and intolerance to cold [ 2 ]. Frodin and colleagues, using nailfold capillaroscopy in FM patients, found slight morphological changes, such as moderate enlargement of capillary loops and variations in calibre [ 3 ]. Jeschonneck and colleagues showed decreased microcirculatory blood flow above tender points in FM patients [ 4 ]. About 60% to 90% of systemic scleroderma (SSc) patients have Raynaud's phenomenon [ 5 ]. In patients with SSc other workers, using videomicroscopy with sodium fluorescein, have found typical changes of the nailfold capillaries, characterised by reduced capillary density, giant capillaries, avascular fields, microhaemorrhages, and disturbance of diffusion [ 6 ]. Furthermore, in rheumatoid arthritis (RA), peripheral malperfusion and vasculitis occur [ 7 ], resulting in skin ulcers, neuropathy, necrosis, or gangrene. Our aim in this preliminary study was to investigate capillary abnormalities and blood flow by two independent objective methods, capillary microscopy and laser Doppler fluxmetry, to obtain evidence of disturbed microcirculation in FM patients. Materials and methods The study group consisted of 10 women (54.0 ± 3.7 years of age) from the Outpatient Department in the Department of Rheumatology, University Hospital, Zurich, with primary FM classified in accordance with the criteria of the American College of Rheumatology [ 8 ]. The controls were three groups ( n = 10 in each group) of age-matched women who were healthy (negative controls) or who had symptomatic RA or SSc (positive controls). The subjects were studied using laser Doppler fluxmetry and capillary microscopy. None of the subjects had vasculitis. The RA patients did not have Raynaud's phenomenon and only 2 of the 10 had hypertension as a possible risk factor for small-vessel disease. Although the SSc patients did not have vasculitis, all except one presented with a typical Raynaud's syndrome, with a mean Medsger score [ 9 ] of 2.2 ± 1.03. This score describes how advanced the disease is (Table 1 ); a low scores (0 or 1) indicates no or mild SSc, and a high score (4) indicates the end stage. Smokers and patients treated with nitrate or Ca 2+ -channel blockers were excluded from the study. The study was approved by the local ethical committee of the University Hospital, Zurich. Capillary microscopy The morphology of nailfold capillaries has been studied by intravital capillaroscopy (Leica, Glattbrugg, Switzerland) at a magnification of 50× [ 10 ]. The room temperature was maintained between 22°C and 24°C. Patients were examined in a sitting position after a resting time of at least 20 minutes. The capillaries were evaluated in accordance with the criteria of the German Association of Angiology [ 11 ]. The following changes were analysed: density of capillaries (normal 7–16 capillaries per millimetre), microhaemorrhages, dilatation of capillaries, giant capillaries, and 'irregular formations' (that is, instances where capillaries were arranged in clusters with gaps in between). A dilatation was considered to be present when the arteriolar limb of the capillary loop was thicker than 50 µm and the venous limb was thicker than 20 µm, and giant capillaries were defined as those having an apex diameter of over 50 µm [ 11 ]. Laser Doppler fluxmetry Skin blood flow was measured in supine subjects at the lateral epicondyle (typical FM tender point [ 8 ]), in fingertips II and III (that is, of the forefinger and middle finger) and in the lower arm (control point) using the laser Doppler technique (PeriFlux PF3; Perimed, Järfälla, Sweden), as described elsewhere [ 12 ]. A blood-pressure cuff was positioned on the upper arm and standard laser Doppler probes for skin blood flow measurements were attached to the epicondyle, fingertips II and III, and the lower arm. The resting flow was recorded 5 minutes before the cuff pressure was inflated to a suprasystolic level for 3 minutes. After release of the cuff pressure, reactive hyperaemia was recorded at the four defined areas. The time to peak flow and type of peak were evaluated in each group [ 13 ]. Peak flow corresponds to the highest flow value after release of the cuff. Four types of reactive hyperaemia were identified [ 13 ] (Fig. 1 ). In type A, the first peak is within 23 seconds after cuff release and is followed by a second, smaller, wave. In type B, the amplitude of the second wave is greater than that of the first; the first peak is characterised by a fast dilatation of the myogen-activated arterioles and small arteries with a concomitant increase of the vessel tonus. Both types A and B are biphasic and are classified as 'normal', because so far they have been predominantly found in healthy subjects [ 13 ]. They also show the same characteristics in therapy and there is so far no proof that one type predisposes to a certain illness. Type C is monophasic; the fast initial component of the muscular reaction is absent. In type D, postocclusive reactive hyperaemia is missing. Types C and D are pathological reactions. The Mann–Whitney U test was used for statistical comparison of the groups. P values < 0.05 were considered to be statistically significant. Means ± standard deviations are given. Results Capillary microscopy The density of capillaries per millimetre in FM patients (9.92 ± 0.19) was significantly lower than in controls (11.31 ± 0.34) ( P < 0.001) but still within the normal range. Four or more capillary dilatations were detected in 2 of the 10 FM patients (Fig. 2 ), and one to three dilatations per nail fold were detected in 6 of the10 FM patients. No microhaemorrhages, giant capillaries, or avascular fields were detected in FM patients. The number of capillaries in patients with SSc (6.21 ± 1.03) was significantly lower than in healthy controls (Fig. 3 ) and significantly more microhaemorrhages were found in SSc patients (8 of 10) than in controls or in FM or RA patients (P < 0.01). Giant capillaries were detected only in SSc patients. Laser Doppler fluxmetry The time to peak blood flow at the lateral epicondyle was significantly longer in FM (7 ± 0.5 s) and SSc (7 ± 0.91 s) patients than in healthy controls (4 ± 0.34 s) (Fig. 4 ). In SSc patients, the time to the peak in the second finger (7.5 ± 1.22 s) was significantly longer than in FM patients (5 ± 0.27 s) and healthy controls (4.5 ± 0.58 s) and also in the third finger (7.5 ± 0.67 s, 5 ± 0.3 s, and 4.5 ± 0.17 s, respectively) (Fig. 4 ). In RA patients, the time to the peak in the second finger (7 ± 1.15 s) was significantly longer than in FM patients (5 ± 0.27 s) and healthy controls (4.5 ± 0.58 s). In the lateral epicondyle, both FM and SSc patients had longer times to peak than the healthy controls (Fig. 4 ). All of the FM patients showed a type-B hyperaemic response in the lower-arm and epicondyle measurements (Fig. 5 ). The monophasic, type-C response was seen at the lateral epicondyle in 6 of 10 RA patients and 6 of 10 SSc patients. One patient with SSc and one with RA showed no postocclusive reaction (type D) either in the lower arm or at the lateral epicondyle. In measurements made in the fingers, FM patients showed the postocclusive, type-B response in fingers II and III (Fig. 6a,6b ), except for a type-A response in finger II in one patient. This was significantly different from the response in healthy controls ( P < 0.01). The monophasic, type-C response was found in some patients with SSc and RA. In one patient with SSc, a type-D response was observed in all four fingers. Discussion Patients with FM and SSc present functional as well as morphological changes in microcirculation, but the diseases are distinguishable by the severity of morphological pathologies of capillaries in SSc. Specific capillary abnormalities are present in patients with SSc and have a high predictive value [ 5 ]. These changes and the irregular formations in SSc may be due to microinfarcts [ 14 ]. Our results show that the density of capillaries in patients with FM is still normal but lower than in healthy controls. In an earlier study, however, it was reported that the number of capillaries is decreased [ 15 ]. Morphological abnormalities and vascular malfunction (spasms) therefore have to be discussed as possible reasons for this decreased number of capillaries. In our study, the main finding is a longer time to peak flow (reactive hyperaemia) after occlusion in FM patients than in the healthy controls. An earlier study [ 13 ] showed that 80% of healthy persons have a biphasic type of reactive hyperaemia, such as type A or B (see Fig. 1 ). Apart from one type-A response in the second fingertip, all of the patients with FM whom we studied were recorded as having a type-B response. This type of response is classified as 'normal', but it lacks the first, fast, myogen-activated peak. The explanation may be that FM patients have a reduced primary muscular vessel reaction whereas – and this is important – the second wave is normal. The small number of patients in this preliminary study may be a limitation in that we may have coincidentally recorded 10 FM patients with a type-B response to occlusion of the blood flow. Further studies with more patients are needed to confirm this finding. The missing fast component of reactive hyperaemia in our FM population is presumably due to a higher sympathetic tonus, resulting in increased vasoconstriction; this increased vasoconstriction would explain both the significantly increased time to peak, especially at the lateral epicondyle – which is a tender point in FM – and the reduced density of vessels. Earlier workers [ 4 ] advanced the idea that psychological and physical situations of stress might have an impact on this system. Local ischaemia, which may result from these proposed mechanisms in more advanced stages of the disease, could be a possible explanation for Raynaud's phenomenon in a fraction of FM patients. This local ischaemia results in an influence on spinal and supraspinal structures with sympathetic and motor efferences. SSc patients showed also a prolonged time to peak flow at all points studied. It is well known that endothelial changes are present in SSc [ 14 ]. Both morphological and symptomatic disturbances in microcirculation can occur in FM but occur most often in SSc [ 16 ]. Functional changes have already been observed in FM, SSc, and RA [ 7 , 17 ]. However, the changes in FM support the hypothesis of increased sympathetic activity, and hence a functional hyperexcitability of the sympathetic nervous system [ 4 ]. Conclusion We have shown that functional changes of the microcirculation are present in patients with FM and this finding may be important for new treatment options in FM. A possible therapeutic strategy could be selective suppression of the sympathetic tone or the undertaking of symptomatic measures to activate the microcirculation, such as active and passive physical methods. Because the unblinded design was a weakness of this preliminary study, blinded studies with more patients are needed to confirm our findings. Abbreviations FM = primary fibromyalgia; RA = rheumatoid arthritis; SSc = systemic scleroderma. Competing interests The author(s) declare that they have no competing interests. Authors' contributions SM recorded measurements for all subjects and performed the capillary microscopy and laser fluxmetry. AF helped with the capillary microscopy. BA-V helped with the laser fluxmetry. UKF and RK discussed the methods and the results of the measurements. DU gave advice with respect to the study design and manuscript. HS developed the study and supervised the work of SM. All authors read and approved the final manuscript.

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1065313

Rheumatoid arthritis synovium contains plasmacytoid dendritic cells

We have previously described enrichment of antigen-presenting HLA-DR + nuclear RelB + dendritic cells (DCs) in rheumatoid arthritis (RA) synovium. CD123 + HLA-DR + plasmacytoid DCs (pDCs) and their precursors have been identified in human peripheral blood (PB), lymphoid tissue, and some inflamed tissues. We hypothesized recruitment of pDCs into the inflamed RA synovial environment and their contribution as antigen-presenting cells (APCs) and inflammatory cells in RA. CD11c + myeloid DCs and CD123 + pDCs were compared in normal and RA PB, synovial fluid (SF), and synovial tissue by flow cytometry, immunohistochemistry, and electron microscopy and were sorted for functional studies. Nuclear RelB - CD123 + DCs were located in perivascular regions of RA, in a similar frequency to nuclear RelB + CD123 - DCs, but not normal synovial tissue sublining. Apart from higher expression of HLA-DR, the numbers and phenotypes of SF pDCs were similar to those of normal PB pDCs. While the APC function of PB pDCs was less efficient than that of PB myeloid DCs, RA SF pDCs efficiently activated resting allogeneic PB T cells, and high levels of IFN-γ, IL-10, and tumor necrosis factor α were produced in response to incubation of allogeneic T cells with either type of SF DCs. Thus, pDCs are recruited to RA synovial tissue and comprise an APC population distinct from the previously described nuclear RelB + synovial DCs. pDCs may contribute significantly to the local inflammatory environment.

Introduction Plasmacytoid dendritic cells (pDCs) are a distinct population of antigen-presenting cells (APCs) with the capacity for potent antigen-presenting function and production of large amounts of cytokines, including tumor necrosis factor (TNF)-α and IFN-α. Human pDCs can be identified by cell-surface expression of MHC molecules, the α-chain of the IL-3 receptor (CD123), and the presence of blood dendritic-cell (DC) antigens known as BDCA2 and BDCA4 in a proportion of cells [ 1 ]. In comparison with CD11c + myeloid DCs, pDCs display a distinct set of chemokine and Toll-like receptors [ 2 - 4 ]. In response to viruses and CpG DNA, pDCs become activated to produce IFN-α and their APC function is enhanced [ 5 - 8 ]. While pDCs were first demonstrated in the T-cell areas of lymph nodes [ 5 , 9 ], precursors of this DC population have been isolated from several sources, including normal peripheral blood (PB), thymus, fetal liver, and bone marrow [ 10 ]. Although they do not reside in normal peripheral tissues, pDCs have been shown to infiltrate certain inflamed tissues and tumor sites, including the skin in psoriasis and lupus, the cerebrospinal fluid in multiple sclerosis, and melanoma and ovarian carcinoma [ 11 - 15 ]. While pDCs play an important effector role in viral disease, being the major producers of IFN-α and having a primary role in innate immunity, there is also evidence that they may play an immunoregulatory role, through the induction of Th2 (T helper 2)-type cytokines [ 9 , 16 - 18 ]. The synovial autoimmune reaction of rheumatoid arthritis (RA) is characterized by lymphocyte, macrophage, and DC infiltration that can progress to the development of lymphoid tissue in established disease [ 19 - 21 ]. DCs are likely to contribute to the formation and maintenance of such organized lymphoid tissue and antigen presentation in RA and other autoimmune lesions [ 22 - 24 ]. We have previously shown that the effector site in RA synovial tissue is enriched in differentiated myeloid DCs, which express CD33, CD11c, MHC and costimulatory molecules, and nuclear RelB [ 21 , 25 ]. Translocation of RelB to the nucleus of myeloid DCs is associated with APC function, particularly through increased expression of MHC molecules CD86 and CD40 [ 26 ]. The proinflammatory cytokines TNF-α and IL-1β are key contributors to the inflammatory cytokine cascade in RA [ 27 , 28 ]. This relates to a number of actions, but activation of the endothelium by TNF-α is particularly important in cellular recruitment to the synovium [ 29 - 31 ]. Since RA is characterized by endothelial activation, leukocyte recruitment, and the development of high endothelial venules, we hypothesized that pDCs would be enriched in inflamed but not normal synovium. Since the functional role of pDCs in disease pathogenesis is only partly understood, we also wished to address whether these cells represent a population distinct from the described nuclear RelB + synovial DCs, and whether they may contribute as APCs or inflammatory cells in RA [ 21 ]. Materials and methods Patients and controls Thirty patients who fulfilled the American College of Rheumatology criteria for RA were included [ 32 ]. Of these, 10 provided synovial fluid (SF) samples and 27 provided PB samples. Of the 30 patients, 80% were seropositive, 62% were female, and 73% were taking at least one disease-modifying antirheumatic drug or low-dose prednisone or both. Synovial tissue was obtained at arthroscopy from seven patients with RA, of whom three were untreated and four were taking at least one disease-modifying antirheumatic drug and low-dose prednisone. The duration of disease ranged from 0.5 to 18 years. In addition, we studied synovial tissue from four healthy individuals with nonspecific knee pain undergoing arthroscopy, one patient who had had psoriatic arthritis for 8 years, and one patient who had had ankylosing spondylitis for 30 years. Each patient with spondyloarthropathy was taking sulfasalazine. No patient in the study was taking biologics. Synovial tissue was provided by Dr Malcolm Smith (Repatriation Hospital, Adelaide, Australia). PB buffy coats prepared from 30 healthy donors were obtained from the Red Cross Blood Transfusion Service (Brisbane, QLD, Australia). The study was approved by the Research Ethics Committee of the Princess Alexandra Hospital. Culture medium and cell isolation All cells were cultured in RPMI 1640 (Gibco, Life Technologies, Mulgrave, VIC, Australia) supplemented with 10% FCS (CSL Ltd, Parkville, VIC, Australia), 0.3 mg/ml L-glutamine (Trace Biosciences, Castle Hill, NSW, Australia), 0.12 mg/ml benzylpenicillin (CSL), and 10 μg/ml gentamicin (Delta West, Pharmacia and Upjohn, Spring Hill, QLD, Australia). The monoclonal antibodies used in this study include FITC, phycoerythrin (PE), and purified anti-CD11c, CD14-PerCP, PE, biotinylated and purified anti-CD123, CD86-FITC (all from BD Pharmingen, San Diego, CA, USA), BDCA2-FITC (Miltenyi Biotech, San Francisco, CA, USA), HLA-DR-biotin (Coulter Immunotech, Fullerton, CA, USA), CD40-FITC (Biolegend, San Diego, CA, USA), CD80-FITC (Cymbus Biotech, Chandlers Ford, Hants, UK), CD68 (Kp-1, DAKO, Carpinteria, CA, USA), RelB (C-19, Santa Cruz Biotech, Santa Cruz, CA, USA), and biotinylated Ulex europaeus agglutinin I (Vector Laboratories, Burlingame, CA, USA). Mononuclear cells were prepared from normal or RA PB or RA SF by density gradient centrifugation over Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) as described elsewhere [ 33 ]. T cells were purified from PB mononuclear cells by passing the cells over a nylon wool column, followed by immunomagnetic depletion of remaining monocytes, DCs, B cells, and NK (natural killer) cells using monoclonal antibodies against CD14, CD16, CD19, CD56, and HLA-DR (all from BD Pharmingen), followed by goat antimouse immunoglobulin magnetic beads, then passage through a strong magnetic field (MACS, Miltenyi Biotech), and collection of the unbound fraction. On analysis by flow cytometry, the unbound fraction routinely contained 95–98% CD3 + T cells. DC-enriched non-T cells were produced by immunomagnetic depletion of T, B, and NK cells from non-T cells, by incubation with monoclonal antibodies against CD19, CD16, CD56, and CD3. Flow cytometric analysis and selection of cells by cell sorting To enumerate CD123 + and CD11c + subsets of DCs, mononuclear cells from normal PB or RA SF were stained for four-colour flow cytometry as described elsewhere [ 33 ], using monoclonal antibodies against CD14-PECy5, CD11c-FITC, CD123-PE, and HLA-DR-APC. Live CD14 - HLA-DR + mononuclear cells were gated for analysis. Subset percentages are expressed as percentage of total mononuclear cells. Listmode data were analyzed using Winlist 2.0 software (Verity Software House, Topsham, ME, USA). For sorting, PB or SF DC-enriched non-T cells stained with the same four markers were sorted using the Moflo flow cytometer (DAKO), gating on CD14 - HLA-DR + and either CD123 + CD11c - or CD11c + CD123 - cells, respectively. For phenotypic analysis, mononuclear cells from PB or SF were stained with CD14-PECy5, CD123-PE or CD11c-PE, HLA-DR-APC, and either a fourth monoclonal antibody or isotype control monoclonal antibody conjugated with FITC. DCs were gated as described above. Electron microscopy Electron microscopy of freshly sorted cells was carried out as described elsewhere [ 5 ]. After fixation in 2.5% glutaraldehyde in phosphate-buffered saline, the cells were post-fixed with an aqueous solution of 1% OsO 4 containing 1.5% K 4 Fe(CN) 6 . Subsequently, the specimens were dehydrated in an alcohol series and embedded into epon. Ultrathin sections (50 nm) were contrasted with lead citrate and uranyl acetate and studied with a CM100 electron microscope (Philips, Eindhoven, The Netherlands). Mixed lymphocyte reactions and cytokine analysis Various numbers of sorted PB or RA SF DCs were incubated with 10 5 allogeneic PB T cells in triplicate wells for 5 days, as described elsewhere [ 33 ]. Supernatants were removed from some cultures and [ 3 H]thymidine (1 μCi/well, ICN Biochemicals) was added to the remainder for the final 18 h. Cells were harvested onto glass-fiber filter mats and the incorporation of [ 3 H]thymidine was determined by liquid scintillation spectroscopy (Packard Topcount, Packard Instrument Co, Meriden, CT, USA). IFN-γ, IL-10, and TNF-α were measured in supernatants by ELISA using OptEIA ELISA kits (BD Pharmingen). Immunohistochemistry Frozen or paraffin-embedded sections of synovial tissue from patients with untreated active RA were obtained by arthroscopic biopsy and supplied by Malcolm Smith (Repatriation Hospital, Adelaide, Australia). Normal synovial tissue was obtained at arthroscopy from patients undergoing arthroscopy for nonspecific knee pain and in whom no abnormality was found. After fixation with acetone, sections were stained with anti-CD11c or anti-CD123 using an immunoperoxidase technique, and revealed with diaminobenzidine (brown). Frozen sections were double-stained with U. europaeus agglutinin I (Ulex), a lectin that specifically binds endothelial cells (fast red), and anti-CD123 (brown), using a double, immunoperoxidase–immunoalkaline phosphatase technique as described elsewhere [ 34 ]. Formalin-fixed paraffin-embedded sections were antigen-retrieved in 10 mM citrate buffer at pH6 in an autoclave, then stained with anti-CD123 (diaminobenzidine, brown) alone, or in combination with anti-RelB (BCIP, DAKO, purple). Sections were counterstained with hematoxylin except when they had been double-stained for CD123 and RelB and were photographed using a transmitted-light microscope (Leitz Diaplan, Leica, Germany). To quantitate infiltration by CD123 + DCs, the number of CD123 + Ulex - cells was counted in sections double-stained with CD123 and Ulex. Cells were counted in each of the entire sections from three patients and three normal controls at high power, and for each biopsy this number was corrected for the area of the section to obtain the number per mm 2 . To quantitate infiltration by CD11c + cells, the number of these cells was counted in three high-power fields of the synovial sublining in sections from three patients and three normal controls. Statistical analysis Differences were analyzed using unpaired Student's t -tests. Results CD123 + nuclear RelB - DCs are located in perivascular regions of RA synovial tissue We have previously shown that synovial tissue in RA and spondyloarthropathy is enriched in differentiated myeloid DCs that express CD33, CD11c, MHC class II, costimulatory molecules, and nuclear RelB [ 21 , 25 ]. Translocation of RelB to the nucleus is associated with maturation and APC function of myeloid DCs [ 26 ]. These nuclear RelB + DCs are absent in normal synovial tissue and are rare in RA SF [ 21 , 23 ]. To determine whether RA synovial tissue was infiltrated by CD123 + pDCs in addition to CD11c + myeloid cells, frozen synovial tissue sections, either normal or from patients with RA or spondyloarthropathy, were stained with CD11c or CD123. CD11c + cells were found both in the lining layer and adjacent to vessels in the sublining of normal synovial tissue. In contrast, CD123 only stained endothelial cells in the normal tissue (Fig. 1a,1b ). In RA synovial tissue, CD11c again stained cells adjacent to vessels, now within lymphoid aggregates in the sublining. A population of CD123 + cells with dendritic appearance was also stained adjacent to CD123 + blood vessels in RA (Fig. 1c,1d ,). Cells expressing TNF-α in RA synovial tissue were found in a similar location in serial sections (data not shown), as demonstrated previously [ 35 ]. To confirm the perivascular CD123 + cells in synovial tissue, normal and RA synovial tissue were double-stained with the endothelial cell marker Ulex agglutinin (red) and with CD123 (brown). Whereas all CD123 + structures in normal synovial tissue colocalized with Ulex agglutinin (orange), single-stained CD123 + cells (brown) were located in perivascular lymphoid aggregates and within the lumen of occasional blood vessels in RA synovial tissue (Fig. 1e,1f ). These CD123 + cells are similar in appearance to those previously demonstrated as CD123 + pDCs in human tonsil, in that they are smaller than CD11c + myeloid DCs, with shorter dendritic processes, and cell clusters gave the appearance of locally proliferating cells (Fig. 1g ) [ 5 , 36 ]. While some macrophages can express CD123, there was no colocalization in synovial tissue of CD123 and CD68 (data not shown). However, aside from the dendritic morphology, we cannot exclude that some of the CD123 + cells stained are mast cells [ 37 ]. To determine whether CD123 + cells in synovial tissue were also nuclear RelB + , formalin-fixed tissue was double-stained for RelB and CD123 without hematoxylin counterstaining. No CD123 + cells had translocated RelB to the nucleus, although some expressed cytoplasmic RelB (Fig. 1h,1i ). In contrast, nuclear staining of RelB was evident in adjacent CD123 - cells (Fig. 1h , arrows). All patients with RA showed similar infiltration by pDCs and no differences in the cell numbers or location were noted between patients with RA or spondyloarthropathy (data not shown). We quantitated pDCs in normal or RA synovial tissue by counting CD123 + Ulex - cells in synovial tissue sections from patients with RA or normal controls stained with CD123 and Ulex as shown in Fig. 1 . Whereas no pDCs infiltrated the normal tissue, approximately 22 pDCs per mm 2 were identified within the RA tissue (Fig. 2 ). This number is similar to the number of nuclear RelB + differentiated DCs identified previously in RA synovial tissue [ 38 ]. In contrast, CD11c + cells infiltrated both normal and RA synovial tissue, with significantly larger numbers in RA ( P < 0.05) (Fig. 2 ). We conclude that the CD123 + cell population is most likely a pDC population that infiltrates RA and spondyloarthropathy but not normal synovial tissue and that it is distinct from the described nuclear RelB + DCs [ 21 , 36 , 39 ]. CD11c + cells comprise immature and differentiated myeloid DCs as well as monocytes [ 1 , 34 ]. Differentiated nuclear RelB + DCs are found within the CD11c + DC population in RA and other inflammatory arthritides but not in normal synovial tissue [ 21 ]. CD11c + and CD123 + DCs in RA SF Workers in our laboratory have previously shown that RA SF is enriched in CD11c + CD33 bright CD14 - myeloid DCs with efficient APC function [ 25 , 40 ]. However, when freshly isolated, only a small proportion of SF CD33 bright CD14 - DCs have translocated RelB to the nucleus. RA and normal PB mononuclear cells contain similar proportions of CD33 bright CD14 - DCs [ 25 ]. To examine plasmacytoid and myeloid DCs in parallel, we compared RA SF with RA and healthy, control PB for the proportion of CD123 + and CD11c + HLA-DR + CD14 - DCs. After purification of mononuclear cells from either normal or RA PB or RA SF by gradient centrifugation, cells were stained with CD123-PE, CD11c-FITC, CD14-PECy5, and HLA-DR-APC. Polymorphonuclear cells were excluded on the basis of forward and side light-scatter. Since basophils and monocytes can also express CD123, potential CD123 + non-DCs were excluded by gating CD14 - HLA-DR + cells [ 10 ]. By four-color analysis, CD14 - HLA-DR + CD123 + and CD11c + DC populations could be distinguished (Fig. 3 ). The percentages of CD123 + CD11c - pDCs in RA PB and normal PB were low and did not differ from each other. This observation contrasts with the reduction in pDCs observed in blood from patients with systemic lupus erythematosus [ 41 ]. CD11c + CD123 - myeloid DCs were more common than CD123 + DCs in patient and control blood ( P < 0.005), in keeping with previous studies of normal PB [ 1 ]. RA SF contained a significantly greater percentage of CD11c + DCs than normal or RA PB ( P < 0.005) – in accord with previous studies using the markers CD33 and CD14 [ 40 ]. The proportion of CD11c+ DCs in RA SF was higher than that of RA SF CD123+ DCs ( P < 0.05). Although the difference was small, the percentage of CD123 + DCs in RA SF was higher than in RA or control PB ( P < 0.05). The data show that CD123 + DCs are present in RA SF, and that the ratio of CD11c + to CD123 + DCs is similar in RA SF to that in normal or RA PB (approximately 10:1). In RA synovial tissue, mature myeloid nuclear RelB + and CD123 + DCs have infiltrated perivascular lymphoid aggregates in similar numbers. Previously, similar numbers of immature and mature myeloid DCs were identified in RA synovial tissue [ 42 ]. Thus pDCs make up about 30% of DCs within RA synovial tissue. The present and previously published data, taken together, show that both pDCs and myeloid DCs are recruited to RA synovium, with an enrichment of pDCs in synovial tissue relative to blood or SF. CD123 + PB DCs are immature whereas SF pDCs show signs of activation In normal PB, pDCs circulate as precursors with the potential for recruitment into tissues in response to chemokines [ 2 , 43 ]. These precursors exhibit a characteristic plasmacytoid morphology on electron microscopy, a cell-surface phenotype characterized by expression of the BDCA2 antigen, by low levels of costimulatory molecule expression, and by the potential for IFN-α production in response to viral or immunostimulatory CpG DNA motifs [ 1 ]. We therefore analyzed the characteristics of sorted RA SF CD123 + DCs and compared them with control PB CD123 + DCs. On electron microscopic examination, freshly sorted PB and SF CD123 + DCs appeared similar, with a smooth surface and abundant rough endoplasmic reticulum in the cytoplasm. The nucleus was nonlobulated and abundant in euchromatin and contained a distinct nucleolus (Fig. 4 ). CD11c + PB DCs were morphologically distinct from the CD123 + pDCs, with a lobulated nucleus and some phagocytic vesicles. CD11c + DCs from SF showed more membrane ruffling and phagocytic activity than those from PB (Fig. 4 ). Thus SF CD123 + DCs morphologically resemble CD123 + DCs in PB, whereas CD11c + SF DCs display a greater level of ruffling and phagocytic activity, consistent with their enhanced level of activation, than CD11c + circulating precursors [ 21 ]. On four-color flow cytometric analysis, gated RA SF CD14 - HLA-DR + CD123 + DCs expressed low levels of CD40, CD80, and CD86. All or the majority of SF CD123 + DCs expressed the BDCA2 marker of immature pDC precursors [ 1 ]. This cell-surface phenotype closely resembles that of control PB CD123 + DC precursors, although BDCA2 was consistently expressed at high levels only by a subset of CD123 + HLA-DR + cells in PB (Fig. 4b ). No PB or SF cells expressed the DC differentiation marker CD83 (data not shown). However, SF CD123 + and CD11c + DCs expressed higher levels of cell-surface HLA-DR than the corresponding cells in PB, suggesting some cellular activation within the SF environment [ 5 , 10 , 44 ]. Thus CD123 + pDCs comprise a small proportion of RA SF mononuclear cells, which are predominantly immature but show some evidence of activation in situ . These observations regarding phenotype and PB and SF numbers are consistent with findings in two recent studies [ 39 , 45 ]. CD123 + and CD11c + SF DCs are efficient APCs We have previously shown that freshly isolated CD33 bright CD14 - CD11c + SF DCs efficiently stimulate resting T cells in allogeneic mixed lymphocyte reactions [ 21 ]. In contrast, whereas freshly isolated CD11c + PB DCs are efficient APCs in mixed lymphocyte reactions, CD123 + PB DCs usually require prior activation in the presence of IL-3 and CD154 for acquisition of APC function in this assay. To analyse the functional capability of RA SF DCs, CD11c + and CD123 + DCs were sorted from either normal PB or RA SF and incubated with freshly isolated normal allogeneic PB T cells. Freshly isolated PB CD11c + but not CD123 + DCs efficiently stimulated allogeneic T-cell proliferation and IFN-γ and IL-10 production in mixed lymphocyte reactions. Addition of IL-3 made no difference to the T-cell proliferation in response to CD123 + DCs (data not shown), suggesting that death of the APCs was not responsible. In contrast, both freshly isolated CD11c + and CD123 + SF DCs efficiently stimulated proliferation and IFN-γ and IL-10 production by resting normal allogeneic T cells (Fig. 5 ). A recent study demonstrated the capacity of RA SF to inhibit pDC differentiation in vitro [ 39 ]. The current studies are consistent, in that SF pDCs showed only some evidence of activation in situ , but once incubated in mixed lymphocyte reactions in the absence of SF they displayed enhanced APC function relative to that of PB pDCs. Whereas stimulation of mixed lymphocyte reactions either by CD11c + or by CD123 + PB DCs resulted in little TNF-α production, stimulation by either of these DCs from RA SF resulted in high levels of TNF-α secretion (Fig. 5 ). The data indicate that pDCs have the capacity for enhanced APC function relative to PB pDCs once removed from the RA SF environment. Furthermore, at the time of antigen presentation by SF DCs to T cells, production of a number of cytokines by either T cells or DCs may be stimulated, including TNF-α, and this appears to be a characteristic of RA synovial DCs rather than the subtype of stimulating DCs. Discussion Ongoing inflammation in RA involves positive feedback loops between activated T cells, B cells, DCs, macrophages, and their products, with destructive consequences for parenchymal cells. Clinical and animal data indicate that effector-site DCs play an important proinflammatory role in the perpetuation of autoimmune disease and contribute to the lymph-node-like organization of that tissue [ 22 , 46 ]. This role may be effected by local antigen presentation to CD4 + and CD8 + effector cells, but DC cytokine and chemokine secretion are also important [ 47 , 48 ]. TNF-α and IL-1β are important downstream proinflammatory and destructive cytokines in RA for somatic cells, whose release is promoted by activation of macrophages. IL-10 is highly expressed in RA, and IFN-γ is an important T-cell effector cytokine [ 49 , 50 ]. In the current studies, we show that, in addition to the previously described population of nuclear RelB + DCs, a further population of nuclear RelB - CD123 + pDCs is located in perivascular regions of RA but not normal synovial tissue sublining. Moreover, pDCs were located within blood vessels, and both DC populations were observed in perivascular areas in which cells producing TNF-α were colocated [ 35 ]. Adherence of CD123 + and CD11c + DCs to TNF-α-activated endothelium was higher than to resting endothelium in vitro (data not shown). TNF-α plays an important role in the recruitment of other leukocytes to RA synovial tissue [ 29 ], and this most likely pertains to the recruitment of pDCs to RA but not normal synovial tissue through expression of adhesion molecules such as intercellular adhesion molecule (ICAM)-1, CD62-E, and CD62-P and interaction with their ligands on pDCs [ 9 , 11 , 51 - 54 ]. Furthermore, TNF-α up-regulates synthesis of chemokines by endothelial cells [ 55 ]. During experimentally elicited allergic rhinitis, CD123 + HLA-DR + pDCs have been shown to be recruited to human nasal mucosa [ 11 ]. The gene for MxA is specifically induced by IFN-α and therefore identifies a population of activated pDCs. In contrast, BDCA2 is a marker of immature pDCs. MxA + pDCs have previously been demonstrated in involved lupus skin and inflamed tonsil [ 13 ]. In RA synovial tissue, BDCA2 was shown to stain fewer cells than CD123 or MxA, suggesting differentiation in situ of a large proportion of pDCs into cells with a capacity for production of IFN-α and other cytokines. Together, the current and previous studies demonstrate recruitment of pDCs to normal lymphoid organs as well as inflammatory sites, with local differentiation, but no recruitment to normal peripheral tissues. In contrast, CD11c + myeloid precursors populate normal resting tissues, as shown here, but additional CD11c + myeloid cellular recruitment takes place at inflammatory sites, where RelB nuclear translocation takes place [ 21 , 56 ]. We have previously shown that, like synovial pDCs, CD123 + DCs in the T-cell area of human tonsil are also nuclear RelB - [ 23 ]. The data suggest either that activation of pDCs is not associated with nuclear translocation and transcriptional activity of RelB or that conditions in tonsil and synovium do not induce sufficient RelB translocation for detection by immunohistochemistry [ 26 , 57 ]. As preliminary studies in vitro demonstrate induction of RelB in PB pDCs after stimulation with lipopolysaccharide and CpG, and reduced production of IFN-α by pDCs in RelB-deficient mice, it is likely that RelB activation does accompany pDC activation. However, RelB translocation might be quantitatively reduced or RelB might be more rapidly degraded in the nucleus of pDCs than of myeloid DCs in inflamed tissues [ 58 ]. Of relevance to the RA inflammatory lesion, stimulation of blood pDC precursors with signals including CD154, influenza virus, or CpG oligonucleotides induces production of large amounts of cytokines, including IFN-α, IFN-β, and TNF-α; induction of DC differentiation; and stimulation of APC function [ 3 , 39 , 44 , 59 ]. Although inhibitory effects of SF on DC function, and thus on T-cell proliferation and cytokine production, are confirmed here [ 21 , 39 , 60 ], factors in the RA SF environment, such as IL-3 and CD154, may be sufficient to precondition the SF pDCs for efficient APC function ex vivo [ 36 , 44 ]. IFN-γ, IL-10, and TNF-α were produced in mixed lymphocyte reactions stimulated by myeloid or pDCs derived from SF but not PB, potentially by DCs or by T cells or both. In the tissue, as a result of antigen presentation by myeloid DCs or pDCs, key effector cytokines may be produced in perivascular areas in RA, located strategically close to endothelial cells, as well as incoming leukocytes. It is not known whether pDCs are capable, like myeloid DCs, of migration from synovial tissue to draining lymph nodes. However, it seems probable that pDCs conditioned by local IL-3 and CD154, or even viral or bacterial products transported to the synovium, predominantly play local proinflammatory and antigen-presenting roles, through secretion of cytokines such as IFN-α and possibly TNF-α [ 61 , 62 ]. Conclusion pDCs are recruited to RA synovial tissue and comprise an APC population distinct from the previously described nuclear RelB + synovial DCs. The APC function of pDCs is greater in SF than in PB. Activated pDCs and interacting T cells may contribute significantly to the inflammatory environment in RA. Abbreviations APC = antigen-presenting cell; DC = dendritic cell; ELISA = enzyme-linked immunosorbent assay; FCS = fetal calf serum; FITC = fluorescein isothiocyanate; IFN = interferon; IL = interleukin; NK = natural killer; PB = peripheral blood; pDC = plasmacytoid DC; PE = phycoerythrin; RA = rheumatoid arthritis; SF = synovial fluid; TNF = tumor necrosis factor. Competing interests The author(s) declare that they have no competing interests. Authors' contributions LC, RT, LF, and PP conceived the experiments and LC, AB, LS, JP, and LF carried them out. LC, RT, and LF wrote the manuscript.

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1065314

A2B adenosine receptor activity is reduced in neutrophils from patients with systemic sclerosis

We conducted the present study to investigate protein expression and functioning of A 2A and A 2B adenosine receptors (ARs) in neutrophils of patients affected by systemic sclerosis (SSc). The presence of A 2A and A 2B ARs was assessed by immunoblotting using specific antibodies. Equilibrium A 2A and A 2B ARs binding parameters were evaluated by radioligand binding assay. Functional studies were conducted to investigate coupling of the A 2B AR to the adenylyl cyclase pathway. This is the first report of the use of Western blot analysis to confirm the presence of A 2A and A 2B ARs in human neutrophils. No significant changes in A 2A AR binding parameters or expression levels were detected between SSc patients and healthy control individuals. A significant decrease (65%) in the maximum density of A 2B AR binding sites occurred in SSc neutrophils, whereas no changes in the affinity constant values were found. Moreover, a decrease in A 2B AR mediated adenylyl cyclase activity was observed in patients with SSc. Our findings demonstrate the occurrence of selective alterations in A 2B AR density and signalling in SSc.

Introduction Systemic sclerosis (SSc), also known as scleroderma, is a connective tissue disease of unknown aetiology. Possibly an autoimmune disorder, it is accompanied in the vast majority of cases by the presence of antinuclear antibodies [ 1 ]. SSc may affect virtually any organ of the body, including skin, gastrointestinal tract, lungs, heart, kidneys, and musculoskeletal system. Altered connective tissue metabolism can cause either localized or diffuse thickening of the skin, while inflammation is associated with endothelial damage. Clinically, microvascular disturbance, teleangiectasia, Raynaud's phenomenon, polyarthralgia and polyarthritis, as well as oesophageal hypomobility, visceral muscolaris mucosa damage and pulmonary fibrosis, have been described [ 2 ]. The mechanisms leading to endothelial damage, inflammation and fibrosis are unclear. Reactive oxygen species in neutrophils may increase the extent of inflammation and fibrosis during the respiratory burst and could be involved in endothelial damage [ 3 ]. The endothelial cells of microvessels are deficient in the synthesis of catalase, which provides natural defence against superoxide damage, and appear to be particularly susceptible to superoxide injury during reperfusion [ 4 ]. Adenosine is an important endogenous regulator of neutrophil functioning. It is released intracellularly and modulates neutrophil activity by interacting with specific surface receptors [ 5 ]. Distinct adenosine receptor (AR) subtypes A 1 , A 2A , A 2B and A 3 have been identified and their functions characterized in neutrophils. Specifically, activation of A 1 ARs enhances chemotaxis, phagocytosis and adherence [ 6 , 7 ]; A 2A ARs inhibit reactive oxygen species generation, phagocytosis and adherence [ 8 - 10 ]; and A 2A and A 3 ARs inhibit neutrophil degranulation [ 11 - 14 ]. Adenosine has been shown to prevent the release of vascular endothelial growth factor from neutrophils via A 2B AR activation [ 15 ]. Because activation of ARs reduces both immune and inflammatory responses, adenosine release has been hypothesized to be a possible mechanism of cell self-protection from activated neutrophils [ 5 ]. An increase in adenosine deaminase activity has been described in patients with SSc, suggesting an alteration in adenosine control mechanisms in this disease [ 16 , 17 ]. In the present study we analyzed A 2A and A 2B AR subtypes in neutrophils from patients affected by SSc by means of expression analysis, radioligand binding assays and functional studies. Methods Chemicals and reagents Bacitracine, benzamidine, trypsin inhibitor, sodium orthovanadate, Nonidet P-40, SDS, phenylsulfonylfluoride, aprotinin and adenosine deaminase (ADA) were purchased from Sigma (St. Louis, MO, USA). Unlabelled AR agonists/antagonists and the anti-β-actin antibody were supplied by RBI/Sigma (St. Louis, MO, USA). [ 3 H]CGS 21680 (CGS 21680 = [2-p-(2-carbowyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine), [ 3 H]NECA (NECA = 5'-N-ethylcarboxamidoadenosine), and [ 32 P]α-ATP were supplied by NEN Life Sciences (Köln, Germany). Electrophoresis reagents were purchased from BioRad (Munchen, Germany). A 2A AR and A 2B AR antibodies were supplied by Alpha Diagnostic (San Antonio, TX, USA). All other chemicals were from standard commercial sources. Patients Twenty-six patients affected by SSc were included in the study (22 women and 4 men; mean age ± standard deviation 53.0 ± 11.3 years). They all fulfilled standard criteria of the American College of Rheumatology for SSc. Sixteen patients were anticentromere antibody positive and four were SCL-70 positive. Limited symptoms of disease, involving skin thickness alterations to the face, hands and feet, were present in 18 patients (mean disease duration <5 years, skin score range [according to the modified Rodnan total skin thickness score] 10–21). Diffuse symptoms with more extensive skin involvement were present in eight patients (mean disease duration <5 years, total skin thickness score range 27–30). The activity score [ 18 ] varied between 0.5 and 3.5 and the severity score [ 19 ] between 2 and 6. The erythrocyte sedimentation rate was 24 ± 23 mm/hour (mean ± standard deviation). Control samples were obtained from 26 healthy volunteers, who were similar to the patients included in the study in terms of sex distribution and age (20 women and 6 men; mean age ± standard deviation 49.0 ± 9.2 years). Informed consent to participate in the study was obtained from all individuals. Sample collection and neutrophil preparation Venous blood (20 ml) was drawn between 08:00 and 09:00 a.m. from fasting individuals by antecubital venipuncture, collected in heparinized (10 IU/L) plastic tubes and processed immediately. Neutrophils were isolated following the Boyum method [ 20 ] with some modifications. Western blot analysis Neutrophils were lysed in RIPA buffer (150 mmol/l NaCl, 50 mmol/l Tris-HCl, pH 8, 0.5% sodium deoxhycolate, 1% Nonidet P-40, 1 mmol/l phenylsulfonylfluoride, 10 μg/ml aprotinin, 100 μmol/l sodium orthovanadate) for 1 hour at 4°C. After centrifugation at 15,000 g for 30 min, soluble fractions were assayed for protein content using BioRad protein assay. Equivalent amounts of proteins (50 μg/sample) were analyzed by SDS-PAGE, using 10% (weight/vol) polyacrylamide resolving gels. Protein bands were transferred to nitrocellulose and probed with 0.1 μg/ml rabbit anti-human A 2A AR or A 2B AR antibodies. A 2A AR antibody is an affinity-purified rabbit polyclonal antibody raised against a peptide mapping to the carboxyl-terminus of A 2A AR. It specifically reacts with human, bovine, rat and pig A 2A receptors and does not cross-react with A 1 , A 2B , or A 3 AR subtypes. A 2B AR antibody is an affinity-purified rabbit polyclonal antibody raised against a region that corresponds to the second extracellular domain of A 2B AR of human origin. After washing, membranes were incubated with anti-rabbit secondary antibody conjugated to horseradish peroxidase for 2 hours at room temperature, and bands were visualized by chemiluminescence, in accordance with the manufacturer's instructions (Sigma-Aldrich). Membranes were re-probed with an anti-β-actin antibody for normalization. Binding assay For membrane preparation, cells were washed twice with 10 mmol/l Tris-HCl buffer, pH 7.4, containing 10 mmol/l MgCl 2 , in the presence of protease inhibitors (200 μg/ml bacitracine, 160 μg/ml benzamidine, 20 μg/ml trypsin inhibitor [T1]) and centrifuged at 48,000 g for 15 min at 4°C. Pellets were diluted in 20 volumes of T1 buffer, treated with ADA (2 IU/ml) for 60 min at 37°C to remove endogenous adenosine, and washed twice with 50 mmol/l Tris-HCl buffer, pH 7.4, containing 10 mmol/l MgCl 2 (T2). A 2A AR binding assay was performed by using a specific radiolabelled A 2A AR agonist, namely [ 3 H]CGS 21680 . Aliquots of neutrophil membranes (0.2–0.3 mg protein) were incubated with different [ 3 H]CGS 21680 concentrations (5–30 nmol/l) in a final volume of 250 μl of T2 buffer. Nonspecific binding was determined in the presence of 100 μmol/l NECA. After 90 min incubation at 25°C, the binding reaction was terminated by vacuum filtration through Whatman GF/C glass fibre filters (Whatman, Maidstone, UK), accompanied by three washes with ice-cold T2 buffer (4 ml). A 2A AR specificity was evaluated through competition experiments, using different AR ligands. A 2B AR binding assay was performed using 20 nmol/l [ 3 H]NECA in the presence of 50 nmol/l cyclopentyladenosine (CPA) and 100 nmol/l SCH 58261 (SCH 58261 = 5-amino-7-[phenylethyl]-2-[2-furyl]-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine) to prevent [ 3 H]NECA binding to A 1 and A 2A ARs, respectively [ 21 ]. Scatchard analysis was performed on competition experiments carried out in the presence of unlabelled NECA at concentrations ranging from 50 nmol/l to 2 mmol/l. Aliquots of neutrophil membranes (0.2–0.4 mg proteins) were incubated in a final volume of 250 μl T2 buffer. Nonspecific binding was evaluated in the presence of 100 μmol/l NECA. After 90 min incubation at 0°C, the reaction was terminated either by vacuum filtration through Whatman GF/C glass fibre filters, accompanied by three washes with ice-cold T2 buffer (4 ml), or by centrifugation at 2900 g for 15 min at 4°C. A 2B AR specificity was evaluated through competition experiments, using different AR ligands. Adenylyl cyclase assay Neutrophils were homogenized in buffer solution containing 10 mmol/l Hepes, 1 mmol/l EGTA and 10 mmol/l NaCl 2 , and then centrifuged at 46,500 g for 20 min at 4°C. Pellets were resuspended in 10 volumes of 10 mmol/l Hepes, containing protease inhibitors (200 μg/ml bacitracine and 160 μg/ml benzamidine), incubated for 30 min at 30°C with 2 U/ml ADA, and centrifuged. Adenylyl cyclase (AC) activity was measured as described by Salomon [ 22 ] and Johnson and Salomon [ 23 ], with some modifications. NECA-mediated stimulation of AC activity was assessed by incubating aliquots of membranes with increasing NECA concentrations from 0.01 nmol/l to 10 μmol/l. The reaction was started by adding membrane aliquots (10–50 μg proteins/tube), conducted for 15 min at 24°C, and then stopped by transferring samples on ice and adding 500 μl ice-cold stop solution (120 mmol/l zinc acetate, 144 mmol/l Na 2 CO 3 ). The stop solution contained [ 3 H]cAMP (10,000–15,000 cpm/sample) to monitor column recovery. Newly formed ZnCO 3 allowed precipitation of residual ATP, discarded through centrifugation at 2700 g for 8 min. Supernatants containing both [ 32 P]α-cAMP and [ 3 H]cAMP were further purified by double-step Dowex-Alumina chromatography and counted by means of a β-counter (Packard Tricarb 1600; Perkin Elmer, Wellesley, MA, USA). To evaluate A 2B AR mediated cAMP accumulation, the reaction was carried out in the presence of selective A 2A antagonist SCH 58261 at a concentration (100 nmol/l) able to block A 2A receptors completely [ 21 ]. Data and statistical analysis Affinity constant values (Kd) and maximum number of binding sites (B max ) were calculated using the nonlinear multipurpose curve-fitting computer program Graph-Pad Prism The 50% inhibitory concentration values were calculated using the same program and converted to Ki values through the Cheng and Prusoff equation. A GS-670-BIO-RAD imaging densitometer was used for semiquantitative analysis of immunoblots. Partial F test ( P < 0.01) was used to determine binding data with the best fit to a one-site or two-site model. Differences in binding parameters between SSc patients and control individuals were evaluated by one-way analysis of variance. Results In both control and SSc neutrophils, Western blot analysis identified two specific immunoreactive bands of 45 kDa and 50 kDa, corresponding to A 2A and A 2B ARs, respectively (Fig. 1 ). This confirmed the presence of both AR subtypes in human neutrophils. To characterize ARs, binding assays were conducted in neutrophil membrane fractions. SSc patients were randomly divided into two subgroups in order to obtain large amounts of protein, as required by the experiments. The selective A 2A AR agonist [ 3 H]CGS 21680 identified a homogenous population of binding sites in control individuals. Kd and B max values were 25 ± 1.3 nmol/l and 35 ± 2.4 fmol/mg protein, respectively (Fig. 2 ). Competition experiments using [ 3 H]CGS 21680 in combination with a variety of A 2A ligands revealed a pharmacological profile typical for A 2A ARs (R-PIA [R-N6-phenylisopropyladenosine] > teofilline > NECA > SCH 58261 ; data not shown). Scatchard analysis for SSc neutrophils revealed no significant differences in Kd and B max between patients (mean values: Kd = 23 ± 1.8 nmol/l, B max = 40 ± 3.2 fmol/mg protein) and healthy control individuals ( P > 0.05; Fig. 2 ), suggesting that no alteration in A 2A binding sites occurs in SSc. In agreement with this, densitometric analysis of immunoblots showed no significant changes in A 2A AR immunoreactive bands in SSc neutrophils relative to controls (optical density: 0.11 ± 0.03 for patients versus 0.15 ± 0.02 for controls). A 2B AR binding sites were identified using [ 3 H]NECA as radioligand in the presence of 50 nmol/l CPA and 100 nmol/l SCH 58261 , to prevent nonspecific binding to A 1 and A 2A AR subtypes. We performed competition experiments using a wide range (50 nmol/l to 2 mmol/l) of [ 3 H]NECA concentrations to allow the identification of A 2B AR low-affinity binding sites. Data analysis revealed that the one-site model produced a significantly better fit than the two-site model ( P < 0.05), both in control and SSc neutrophils. In our experimental conditions, control neutrophils exhibited the presence of low-affinity binding sites with Kd and B max values of 476 ± 34 nmol/l and 3696 ± 210 fmol/mg, respectively (Fig. 3 ). Competition experiments using [ 3 H]NECA in combination with a variety of AR ligands revealed a pharmacological profile typical for A 2B ARs (R-PIA > teofilline > SCH 58261 = MRS1220 > DPCPX > 2Cl-adenosine > NECA > MRS1706; Table 1 ). Scatchard analysis for SSc neutrophils showed no significant differences in Kd and B max between the two subgroups of patients. However, a significant alteration in B max was found relative to controls, whereas Kd values remained unaltered. Overall, mean values for Kd and B max in SSc were 469 ± 35 nmol/l and 1292 ± 98 fmol/mg protein, respectively ( P < 0.05; Fig. 3 ). Moreover, experiments conducted in individual patients using a concentration of NECA of 500 nmol/l showed similar specific binding values (expressed as fmol/mg protein), confirming the homogeneity of A 2B AR sites between SSc subgroups (Fig. 4 ). The alteration in A 2B AR levels in SSc patients was confirmed by immunoblotting assay. Densitometric analysis of immunoreactive bands showed a reduction in A 2B expression in SSc patients (optical density 0.22 ± 0.04) as compared with controls (optical density 0.40 ± 0.06; P < 0.05; Fig. 1 ). Functional coupling of A 2B ARs to stimulatory G proteins in neutrophil membranes was assessed by evaluating the effects of the agonist NECA (in the presence of 100 nmol/l SCH 58261 ) on AC activity. NECA stimulated AC activity in a concentration dependent manner. Dose-response curves revealed significant differences between SSc patients (EC 50 = 373 ± 26 nmol/l; E max = 35 ± 2.9%) and controls (EC 50 = 165 ± 9.3 nmol/l; E max = 43 ± 3.2%), suggesting an alteration in A 2B AR responsiveness in SSc (Fig. 5 ). Discussion In the present study we analyzed A 2A and A 2B AR subtypes in neutrophils of patients affected by SSc, by means of Western blot, radioligand binding techniques and functional studies. This is the first report of use of Western blot analysis to confirm the presence of A 2A and A 2B ARs in human neutrophils. A 2A and A 2B AR equilibrium binding parameters were measured using radioligand binding assays. Scatchard analysis of [ 3 H]CGS 21680 saturation binding to A 2A AR showed no significant difference in B max or Kd between SSc neutrophils and controls, suggesting that the A 2A AR subtype remained unaltered in SSc. Conversely, when A 2B AR was analyzed a reduction in B max (65%) was observed, with no significant change in Kd values. A 2B ARs are known to be low-affinity adenosine binding sites. Competition experiments using a variety of A 2B AR agonists and antagonists revealed a pharmacological profile typical of A 2B ARs, which is consistent with studies conducted in transfected cell models. Our findings represent the first characterization of A 2B ARs in neutrophils with binding experiments. In order to analyze a population of nonhomogenous patients and to evaluate the impact of the disease on A 2 ARs, SSc patients were randomly divided into two subgroups. No difference was found when the two groups were compared, suggesting that different degrees of disease severity and activity had no impact on the assays, but that the disease per se is required to modulate levels and functioning of A 2B receptors. Functional studies were performed to investigate whether the decrease in level of A 2B ARs was accompanied by alterations in receptor responsiveness. An evaluation of the ability of NECA to increase AC activity revealed functional coupling of A 2B receptors to G proteins. In SSc patients a significant reduction (by more than 50%) in NECA potency was observed, without any effect on agonist efficacy. Our findings suggest that a selective reduction in A 2B AR levels and responsiveness occurred in SSc. Alterations in the expression and functionality of A 2B ARs (low-affinity ARs) in patients with SSc may be responsible for the increase in free oxygen radicals, and consequent oxidative damage, that characterizes SSc. This would account for impaired control of hypoxic and inflammatory processes. In neutrophils it has long been known that adenosine and its analogues inhibit O 2 - generation, phagocytosis and cell adherence by occupying specific A 2 ARs. Because hypoxia, ischaemia and inflammation can stimulate adenosine production, A 2 AR regulation has been postulated to be a self-protective mechanism for cells from activated neutrophils [ 24 ]. Eltzschig and coworkers [ 25 ] reported that A 2B ARs are selectively upregulated in endothelial cells by hypoxia (more than fivefold increase in mRNA), which is associated with ATP hydrolysis and release of adenosine. Taken together, these findings show some coordination between AR transcription and nucleoside signalling at the vascular interface during hypoxia. We might speculate that chronic inflammatory conditions in SSc patients impaired regulatory mechanisms mediated by the anti-inflammatory effects of adenosine via A 2B AR activation. In addition, it was reported by Visser and coworkers [ 26 ] that increases in cAMP in activated neutrophils play an anti-inflammatory role. The reduced activation of cAMP we observed in SSc patients might be correlated with the inability of these patients to control the inflammatory process. It was no surprise to find an alteration in adenosinergic system responsiveness in SSc. In fact, adenosine produces a constellation of responses, including anti-inflammatory actions and vasodilatation, mediated through interactions with high-affinity receptor subtype A 2A and low-affinity receptor subtype A 2B . Moreover, in SSc and related disorders, alterations in adenosine metabolism have been suggested. Indeed, purine analogue 2-chlorodeoxyadenosine, which is utilized for the treatment of such chronic disorders [ 27 , 28 ], appears to reduce the number of abnormal fibroblasts. A 2B ARs were initially thought to be of lesser physiological relevance because of their relatively low affinity for adenosine, and it was only recently that important functions attributable to A 2B ARs were discovered. A pivotal role for them was postulated in inflammatory pathological conditions, when adenosine is released at high levels (up to the micromolar range). In light of our findings, a closer examination of A 2B AR functions may be valuable because of the potential therapeutic importance of these receptors as targets for treatment with selective agents. Conclusion Our findings demonstrated a reduction in A 2 low-affinity (A 2B ) AR density and functioning in neutrophils of patients affected by SSc, suggesting an alteration in adenosinergic system responsiveness. This reduction could relate to the increased production of free oxygen radicals and consequent oxidative damage that characterize SSc, highlighting an impairment in the ability of neutrophils to control hypoxia and inflammation. No differences between two randomly selected subgroups of SSc patients were found, thus suggesting that different degrees of disease severity and activity had no impact on the degree of A 2B AR reduction. Consequently, the functional status of A 2B ARs may be considered a marker of the disease, making it worthwhile to characterize a larger cohort of patients, including their closest relatives and patients with early SSc. Abbreviations AC = adenylyl cyclase; ADA = adenosine deaminase; AR = adenosine receptor; B max = maximum number of binding sites; CGS 21680 = (2-p-[2-carbowyethyl]pheylethylamino)-5'N-ethylcarboxamidoadenosine; CPA = cyclopentyladenosine; Kd = affinity constant; NECA = 5'-N-ethylcarboxamidoadenosine; R-PIA = R-N6-phenylisopropyladenosine; SCH 58261 = 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c)pyrimidine; SSc = systemic sclerosis. Competing interests The author(s) declare that they have no competing interests. Authors' contributions LB organized the study design and recruited the patients. LT carried out the binding experiments and statistical analysis. AR participated in the immunoblotting experiments and helped to draft the manuscript. FdF participated in the collection of human samples. AL participated in the coordination of the study and helped with problem solving. SB participated in the coordination of the study and in planning the manuscript. CM participated in the coordination of the study and designed the AC assay. All authors read and approved the final manuscript.

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1065315

Gene expression profiling in murine autoimmune arthritis during the initiation and progression of joint inflammation

We present here an extensive study of differential gene expression in the initiation, acute and chronic phases of murine autoimmune arthritis with the use of high-density oligonucleotide arrays interrogating the entire mouse genome. Arthritis was induced in severe combined immunodeficient mice by using adoptive transfer of lymphocytes from proteoglycan-immunized arthritic BALB/c mice. In this unique system only proteoglycan-specific lymphocytes are transferred from arthritic mice into syngeneic immunodeficient recipients that lack adaptive immunity but have intact innate immunity on an identical (BALB/c) genetic background. Differential gene expression in response to donor lymphocytes that migrated into the joint can therefore be monitored in a precisely timed manner, even before the onset of inflammation. The initiation phase of adoptively transferred disease (several days before the onset of joint swelling) was characterized by differential expression of 37 genes, mostly related to chemokines, interferon-γ and tumor necrosis factor-α signaling, and T cell functions. These were designated early arthritis 'signature' genes because they could distinguish between the naive and the pre-arthritic state. Acute joint inflammation was characterized by at least twofold overexpression of 256 genes and the downregulation of 21 genes, whereas in chronic arthritis a total of 418 genes with an equal proportion of upregulated and downregulated transcripts were expressed differentially. Hierarchical clustering and functional classification of inflammation-related and arthritis-related genes indicated that the most common biological activities were represented by genes encoding interleukins, chemokine receptors and ligands, and by those involved in antigen recognition and processing.

Introduction The completion of the human and mouse genome sequencing programs and the subsequent annotation of previously unidentified genes have opened a new epoch in biology and biomedical sciences. The genetic information greatly facilitated the discovery of novel disease-related genes and the mapping of signature genes for early diagnosis. More specifically, polynucleotide or oligonucleotide arrays have been applied in both human and experimentally induced disease conditions to determine characteristic expression patterns of signature genes. In an inflammatory disease such as rheumatoid arthritis (RA), the gene expression profile is extremely complex owing to the diversity of cell types involved in the pathology and the polygenic character of the autoimmune disease [ 1 - 5 ]. The overall picture of molecular interactions in an inflamed joint, deduced from gene expression studies in both RA and its corresponding animal models, involves proteins participating in immunity, inflammation, apoptosis, proliferation, cellular transformation and cell differentiation, and other processes [ 3 - 8 ]. Several studies analyzed the patterns of gene expression in peripheral blood or synovial fluid mononuclear cells, and in the inflamed synovium of human patients [ 1 , 3 - 5 , 7 , 9 - 11 ]. However, the genetic heterogeneity of the human population is a serious obstacle to the correct interpretation of data in gene expression studies. Animal models of RA can facilitate the interpretation of genome-wide gene expression by providing genetic and clinical homogeneity, and an opportunity to monitor the onset and progression of the disease [ 12 - 20 ]. DNA microarray technology was successfully applied to inflamed paws of mice or rats systemically immunized with arthritogenic compounds to induce arthritis [ 6 , 21 - 23 ]. Despite the usefulness of the information provided by these studies, the early gene expression events at the site of inflammation (joint and synovium) and the mechanisms of disease initiation remain unknown. Systemic immunization of genetically susceptible BALB/c mice with human cartilage proteoglycan aggrecan (PG) induces PG-specific immune responses that then trigger inflammation in peripheral joints [ 13 , 19 ]. PG-induced arthritis (PGIA) is a murine model which bears many similarities to RA as indicated by clinical assessments, radiographic analyses, various laboratory and functional tests, and by histopathologic studies of diarthrodial joints [ 13 , 19 , 24 , 25 ]. Moreover, genome-wide screening studies identified multiple genomic loci in PGIA [ 20 , 26 - 29 ] that are syntenic with those described in RA [ 25 ]. Both RA and PGIA are polygenic autoimmune diseases with a major permissive role of the MHC, although non-MHC genes account for a significant portion of the genetic susceptibility. PGIA can be successfully transferred into naive BALB/c or syngeneic severe combined immunodeficient (SCID) mice either with unseparated spleen cells or with antigen (PG)-stimulated T lymphocytes from arthritic donor BALB/c mice [ 30 - 32 ]. In the present study, we adoptively transferred the disease (PGIA) into syngeneic BALB/c SCID mice lacking functional T and B cells. SCID mice carry a natural mutation that prevents the V(D)J recombination in B and T lymphocytes, resulting in a failure to generate functional immunoglobulins and T cell receptors [ 33 , 34 ]. Consequently, adoptively transferred arthritis in BALB/c SCID mice is an ideal model in which activated lymphocytes of arthritic donor BALB/c mice migrate and interact with the intact innate immunity environment in the joints of BALB/c SCID mice. The gene expression profiles in normal, pre-arthritic and arthritic joints of the recipient BALB/c SCID mice were determined by using DNA microarray technology (Affymetrix). Although a significant number of genes were differentially expressed in joints with acute and chronic arthritis, in this study we focused on early genes whose expression occurred before the onset of clinical symptoms. Methods Animals, antigen and immunization The use of human cartilage from joint replacement surgeries for antigen isolation was approved by the Institutional Review Board, and all animal experiments were approved by the Institutional Animal Care and Use Committee. Female BALB/c mice at the age of 24–26 weeks (National Cancer Institute, Kingston Colony, New York, USA) were injected intraperitoneally with 100 μg of cartilage PG (measured as protein) emulsified in dimethyldioctadecylammonium bromide (DDA) adjuvant (Sigma-Aldrich, St Louis, Missouri, USA). The use of adjuvant DDA allowed us to avoid the harmful effects of oil and bacterial proteins present in Freund's adjuvants [ 35 , 36 ]. Booster injections of the same doses of PG with DDA were given on days 21 and 42. BALB/c mice develop swelling and redness of one or more limbs 7–10 days after the second or third injection with PG in adjuvant [ 25 ]. Arthritis was assessed daily, and inflammation was scored from grade 0 to grade 4 for each paw [ 13 , 36 , 37 ]. Female SCID mice of the BALB/c background (NCI/NCrC.B-17-scid/scid; henceforth BALB/c SCID ) were used for adoptive cell transfer. BALB/c SCID mice were purchased from the National Cancer Institute and maintained under germ-free conditions. Stimulation of lymphocytes in vitro , and adoptive transfer of arthritis To ensure uniformity and reproducibility of disease transfer, donor spleen cells were isolated from arthritic BALB/c mice within 1–2 weeks after the onset of inflammation. At least two paws of donor BALB/c mice were arthritic, and the cumulative inflammation score (for four paws) was in the range 5–8. Spleen cells of arthritic BALB/c mice were collected and cultured in six-well plates (2.5 × 10 6 cells/ml) with cartilage PG (50 μg/ml) for 4 days in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (HyClone Laboratories, Logan, Utah, USA). After stimulation in vitro for 4 days with cartilage PG, non-adherent cells were collected, and live cells (lymphocytes) were separated on Lympholyte-M (Cedarlane, Ontario, Canada). Finally, 2 × 10 7 lymphocytes were injected intraperitoneally on days 0 and 7 into recipient BALB/c SCID mice as described [ 32 ]. A standard scoring system used for primary arthritis was applied to the assessment of disease severity in BALB/c SCID mice [ 24 , 37 ]. Typically, one to four paws became inflamed simultaneously 3–5 days after the second cell transfer, and the rest of the peripheral joints became inflamed within 2–4 days after the onset of the first symptoms. BALB/c SCID mice were scored twice daily, and were killed as soon as the inflamed paw reached an individual arthritis score of 2, but not later than 24 hours after the onset of arthritis. This paw was designated as acute arthritic (AA), and contralateral or ipsilateral paws that were not inflamed at that time were used as pre-arthritic (PA) samples. The PA joints did not show evidence of inflammation on histopathological examination, although thickening of the synovial lining in small joints was observed occasionally (data not shown). Several arthritic BALB/c SCID mice were scored daily and were killed 8–10 days after disease onset. These joint samples represented subacute-chronically arthritic (CA) samples. In addition to PA, AA and CA experimental conditions, paws of naive non-immunized BALB/c SCID mice were used as 'absolutely negative' (control naive; AN) samples for RNA isolation and subsequent hybridization. Each sample represented RNA pooled from four paws of two mice. Probe preparation Synthesis and biotinylation of cRNA and hybridization were performed in accordance with the manufacturer's instructions (Affymetrix, Santa Clara, California, USA). In brief, total RNA was isolated from normal or inflamed paws of mice by using TRIzol reagent (Invitrogen, Gaithersburg, Maryland, USA) with additional purification on RNeasy columns (Qiagen, Valencia, California, USA). RNA quality was confirmed by spectrophotometry and electrophoresis on formaldehyde gels [ 38 ]. Double-stranded complementary DNA was synthesized with the T7-dT24 primer incorporating a T7 RNA polymerase promoter. Biotinylated cRNA was prepared with the Enzo BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Inc., Farmingdale, New York, USA) and hybridized to the murine genome Affymetrix U74v2 chip set, which included three DNA chips, MG_U74Av2, MG_U74Bv2 and MG_U74Cv2, interrogating more than 36,000 genes that represented essentially the entire mouse genome [ 39 - 42 ]. Fluorescent hybridization signals were developed with phycoerythrin-conjugated streptavidin and were further enhanced with fluorescently labeled anti-streptavidin antibodies. DNA chips were scanned to obtain quantitative gene expression levels. DNA chip hybridization, Fluidics Station operations, scanning, and preliminary data management were performed in accordance with Affymetrix protocols as described previously [ 43 , 44 ]. Microarray analysis Fluorescent intensity data from Affymetrix Microarray Suite version 5 were exported as CEL files and imported into DNA-Chip Analyzer version 1.3 [ 45 ]. Data were normalized, and expression values, based on the perfect match/mismatch (PM/MM) model, were calculated for each DNA chip. All chips were examined for the image spikes, chip and gene outliers. Exported expression values for each DNA chip were combined into a single file (three chips × four experimental conditions × three to five replicates), and imported back to DNA-Chip Analyzer; the resulting data were normalized by using an array with median probe intensity. For the pairwise comparison of experimental conditions, signals were filtered by using several criteria. Gene expression was considered above the background if it showed the signal on most chips (more than 50%; that is, for three replicates, the gene should be detectable on at least two chips; for five replicates, the gene should be present on at least three DNA chips). Fold changes for gene expression were calculated when any of three following criteria were met: (1) the gene was present in the experimental condition but absent in the basal condition; (2) the gene was present in the basal condition but absent in the experimental condition; (3) the gene was present in both basal condition and experimental conditions. Student's t -test was used to determine the statistical significance of the difference in gene expression between basal and experimental conditions ( P < 0.05 was taken as significant). An additional cut-off threshold of twofold change in gene expression (either upregulation or downregulation) was used to characterize a gene as being differentially regulated (for example, a negative twofold value corresponded to a twofold downregulation). The Fisher exact test (implemented by us in Visual Basic code for MS Excel 2000) and the Mann–Whitney U -test (SPSS, Chicago, Illinois, USA) were used to verify non-paired Student's t -test calculations of the probability of gene expression differences in pairwise comparisons. Finally, the false discovery rate was established with 500 permutations for each pairwise comparison to estimate the proportion of false-positive genes. To characterize gene expression patterns, hierarchical gene clustering was performed with a DNA-Chip Analyzer program [ 45 , 46 ]. The algorithm was based on the distance between two genes defined as 1 - r , where r is the Pearson correlation coefficient between the standardized expression values of the two genes across the samples used. To characterize functional relationships between differentially expressed genes, Gene Ontology terms classification [ 47 ], incorporated in DNA-Chip Analyzer, was performed [ 48 ]. The significance level for a functional cluster was set at P < 0.05, and the minimum size of a cluster was three genes. Venn diagram calculations were performed in Visual Basic code for MS Excel 2000 to analyze overlapping of sets of genes differentially expressed in the samples at different phases of arthritis. Results The major goal of the present study was to find and characterize early signature genes whose expressions were different (at least twofold change in the threshold level) and statistically significant ( P < 0.05) between experimental groups at different phases of joint inflammation. The induction of arthritis in BALB/c SCID mice was a multi-step process. First, donor BALB/c mice were immunized with cartilage PG to induce arthritis. Second, spleen cells from acutely arthritic (AA) donor mice were stimulated in vitro with cartilage PG, and live lymphocytes were isolated on a Lympholyte-M density gradient. Third, these antigen-stimulated donor lymphocytes were injected into BALB/c SCID mice. For gene expression profiling during the time course of the adoptively transferred arthritis, RNA was isolated from pre-arthritic paws (PA) and diseased paws (AA and CA) (Table 1 ). In addition, RNA was isolated from normal paws of naive BALB/c SCID mice and served as a baseline non-arthritic control condition (AN). Three pairwise comparisons were performed: PA versus AN, AA versus AN, and CA versus AN (hereafter denoted as PA/AN, AA/AN and CA/AN). Each experimental condition was reproduced three to five times (RNA isolation, probe preparation, and independent hybridizations), and each replicate contained RNA samples pooled from a total of four paws of two arthritic animals. When the number of replicates is low and the distribution of data in the general population is basically unknown, the applicability of Student's t -test is questionable. We therefore analyzed data by using both Student's t -test and the Fisher exact test, in which the first approach requires normal data distribution, whereas the second test does not have this requirement [ 45 , 49 , 50 ]. Setting the significance level for the difference between groups at P < 0.05 and no threshold for the fold change in expression, 1805 genes passed the Fisher exact test and 1752 genes passed the DNA-Chip Analyzer Student's t -test [ 45 ] for the PA/AN comparison. In AA/AN pairwise comparisons, 3676 genes passed the Fisher exact test and 3305 genes passed Student's t -test. Concluding that Student's t -test provided similar results and was even more conservative than the Fisher exact test, we employed the former for all further analyses. Effect of the numbers of replicates on data variability Being aware of the importance of data reproducibility, we determined the optimal number of arrays to be included in experimental design by monitoring the convergence of variance for gene expression signals in five replicates representing the condition AA. For each replicate, we pooled equal amounts of quality-controlled RNA samples, isolated from two inflamed paws of two BALB/c SCID mice that had been identically treated (in terms of the number of donor cells and antigen stimulation) and had similar disease onset and severity. A total of five replicates represented 20 paws of 10 arthritic mice. We used the coefficient of variation (CV) to measure data variability. The CV for each gene on the chip and the mean CV for the entire probe set were calculated. Mean CV reached a plateau when the number of replicates increased beyond three (Fig. 1 , experimental condition AA) and there was no significant change afterwards. Therefore, for all other experimental conditions, we used three replicates representing three independent hybridization experiments of three RNA samples isolated from six paws. Mean CV after sampling of the three repeats ranged between 0.21 and 0.25 for all experimental conditions. Arthritis 'signature' genes in pre-inflamed joints Paws of naive BALB/c SCID mice and still non-inflamed (PA) paws were clinically normal with no sign of inflammation, and comparison of these two experimental conditions (PA/AN) identified a relatively small number of differentially expressed genes. Only 37 of the 36,000 screened genes were differentially expressed (that is, showed greater than a ± twofold change relative to threshold level), of which 11 genes were over the ± threefold threshold, and seven genes changed beyond ± fivefold (Fig. 2 ). The seven genes with the most significant change in expression levels encoded chemokine CC motif receptor 5 ( Ccr5 ), chemokine CXC motif ligand 1 ( Cxcl1 ), interferon-γ-inducible protein ( Ifi47 ), membrane-spanning 4-domains subfamily A member 6C ( Ms4a6c ), tumor necrosis factor-α-induced protein 6 ( Tnfip6 ), T cell receptor β variable 13 ( Tcrbv13 ), and Terf1-interacting nuclear factor2 ( Tinf2 ) (Table 2 ). Although the upregulation of Tcrbv13 , Tgtp and interferon-induced genes might indicate the appearance of antigen-specific T cells in the synovium (Table 2 ), the significant upregulation of Tnfip6 suggests the activation of an anti-inflammatory cascade [ 51 ]. Thus, gene expression related to pro-inflammatory and anti-inflammatory events can be detected even before the migration of inflammatory leukocytes into the joints. To characterize major biological functions in context with the initiation phase of the disease, we assigned the 37 early genes (Table 2 , Additional file 1 ) to separate groups according to the corresponding protein functions and Gene Ontology classification [ 47 , 48 ]. We found that differentially expressed genes in PA joints were related to immune responses, chemokine activity (including chemotaxis), cell adhesion, proteolysis regulation, inflammation and wounding, cytokines, and cytoskeletal activity (Fig. 3 , yellow circles). All clustered genes were upregulated at the pre-inflamed phase of arthritis. Gene expression profile in acute and chronic arthritis To monitor the progression of disease, we analyzed genes that were differentially expressed in paws with acute and chronic joint inflammation. Both AA and CA experimental conditions were associated with the activity of a large number of genes: 256 genes were upregulated and 21 were downregulated in acute arthritis (AA/AN comparison), and 201 genes were upregulated and 217 were downregulated in chronic inflammation (CA/AN) (Fig. 2 , Additional files 2 and 3 ). A Venn diagram summarizes the relationships between gene sets that were differentially expressed at different phases of the disease. Only 15 genes were differentially expressed in all three phases of the disease (PA, AA, and CA), 25 genes were differentially expressed both at the PA phase and during acute inflammation, 127 genes were active both in acute and chronic phases, and 17 transcripts shared a common expression pattern in pre-inflamed and chronically inflamed joints (Fig. 2 ). Using Gene Ontology terms for the functional classification of genes differentially expressed in acute and chronic arthritis [ 47 ], dozens of cell signaling pathways and gene clusters were identified. By further filtering of functional clusters, and by combining clusters encoding proteins with similar functions, we found that the acute and chronic phases of the disease can be comprehensively described by the differential expression of 15 macro-clusters (Fig. 3 ). Six clusters were found in all three phases of inflammation; they were related to immune response, chemokine activity, cytokines, inflammation and wounding, cell adhesion, and proteolysis regulation. The most abundantly represented genes in inflamed joints were those involved in immune responses: 51 genes in AA and 25 genes in CA. These genes were upregulated as much as 31-fold (group average) in acute arthritis and 15-fold in chronic arthritis (Fig. 3 ). Cytokine and chemokine genes demonstrated the highest overexpression levels: about 64-fold in acute and 28-fold in chronic arthritis, where both groups included more than a dozen genes. Proteolysis-regulating genes (proteases and their inhibitors) were highly represented at the acute phase (45 genes), but were less abundant in chronic arthritis (19 genes). Extracellular matrix-related genes, mostly relevant to tissue repair and healing, were more abundant in chronic than acute disease. Some functional clusters were phase-specific, such as lysosome, antigen presentation, scavenger receptors, immunoglobulin binding, and complement cascade; these genes were preferentially expressed in acute joint inflammation. Suppression of genes related to the respiratory chain complex was specific to chronic inflammation (Fig. 3 ). Hierarchical clustering of arthritis phase-specific genes To identify genes whose expression might be specific for the actual phase of arthritis, and to combine transcripts by the pattern of their expression through all disease phases, we applied a hierarchical clustering technique [ 46 ]. Genes that were specific for pairwise comparisons (PA/AN, AA/AN, and CA/AN) were combined into one single file (excluding redundant genes); the merged set included 507 genes. Hierarchical clustering was performed for all experimental conditions studied (AN, PA, AA, and CA), and four major gene clusters were identified, each with a distinct expression pattern (Fig. 4 , clusters I–IV). Using further classification analysis with Gene Ontology terms, to examine the functions of genes inside each cluster, we identified genes encoding proteins whose biological functions were the most relevant to arthritis development and progression. Cluster I contained genes with major functions in collagen turnover and tissue repair; the expression of these genes reached a peak in chronically inflamed joints. Cluster II was the largest cluster including about half of all phase-specific genes (Fig. 4 ). The cluster included genes with roles in immune, inflammatory and stress responses, extracellular matrix formation, cell growth, and receptor activity. The expression of cluster II genes reached a peak at the acute phase of joint inflammation. Transcription of genes in clusters III and IV gradually decreased during disease progression (Fig. 4 ). These genes were mostly related to cytoskeleton remodeling, the formation of cell junctions, and the production of structural molecules such as desmin, β-3 laminin, envoplakin, and dystonin (for a detailed gene list see Additional files 1 , 2 , 3 ). Genes associated with early arthritis (Table 2 ) were found in clusters III and IV, further underlining the importance of cell adhesion and cytoskeleton remodeling during the initiation phase of arthritis. Expression patterns of early arthritis genes Hierarchical clustering of a large number of phase-specific genes ( n = 507) (Fig. 4 ) obscured the expression pattern of a relatively small number ( n = 37) of early arthritis genes (Table 2 ). A separate hierarchical clustering was therefore performed for these 37 early genes, and the levels of expression were monitored at later phases of the disease. Six distinct expression patterns were identified (Fig. 5 , clusters A–F) using this approach. Clusters A–D contained early arthritis genes whose transcription increased as the disease progressed, reaching a peak in the pre-inflamed joint or during inflammation. Cluster A included genes that coded for variable parts of the T cell receptor, together with genes related to cytoskeleton reorganization such as Rho interacting protein 3, myosin, and β-actin (reviewed in [ 52 , 53 ]). Cluster A genes were at the peak of their expression in the PA joint. However, most early arthritis genes in clusters C and D showed an expression peak later, at the acute phase of inflammation (Fig. 5 ), and encoded chemokine receptors ( Ccr2 and Ccr5 ) and chemokine ligands ( Cxcl1 , Ccl2 , Ccl7 , and Ccl9 ). Clusters C and D also included interferon-activating genes Ifi203 , Ifi47 , and Ifigtp , and cell differentiation antigens such as CD48 and CD53. Hierarchical clusters E and F contained four genes whose expression was downregulated in the pre-inflamed joint but returned to a 'normal' level (as expressed in naive paws) during arthritis progression. Clusters E and F included genes encoding Terf1-interacting nuclear factor 2, tissue inhibitor of metalloproteinase 1, makorin, and DNA clone 4833424O15 with unknown function (Table 2 and Fig. 5 ). Discussion This study describes genome-wide gene activity taking place in mouse joints during three major phases of autoimmune arthritis: initiation, acute inflammation, and chronic inflammation. Spleen cells from PG-immunized arthritic BALB/c mice were used to transfer the disease into non-immunized syngeneic SCID mice [ 30 , 32 ]. This adoptive transfer system minimized the individual differences that are typical in primary arthritis (induced by systemic immunization), and also excluded antigen-independent stimulation of the immune system by the adjuvant. Additional benefits of the cell transfer included a decrease in the time needed for arthritis development, and uniformity and synchronization of joint inflammation in recipient mice [ 32 ]. Two major criteria were used to select genes that might be important for arthritis development: (1) significant differences in expression levels between experimental groups and (2) the fold change in expression levels. When only the first criterion was applied, genome-wide analysis identified a large number of genes whose expression was significantly ( P < 0.05) different between any pair of the experimental conditions compared. Irrespective of the statistics used (either unpaired Student's t -test, the Fisher exact test or the Mann–Whitney U -test), the number of differentially expressed genes was found to represent about 5–10% of the entire mouse genome. We further 'filtered' these genes by using a cut-off threshold set at twofold change of expression, because this threshold could reflect a physiologically important change in gene activity, and a twofold change exceeded the average CV for all pairwise comparisons. Decreasing the number of 'false positive' genes by application of these two filtering procedures proved to be an effective technique for the identification of genes that are likely to be involved in arthritis development. The present study indicates that the number of differentially expressed genes increases with the progression of the disease. At the initiation phase, when no clinical symptoms of inflammation were yet detected, only 37 genes were upregulated or downregulated. However, a differential expression of 277 genes was observed at the acute phase, and chronic inflammation was characterized by the differential activity of 418 genes. Interestingly, most early arthritis signature genes (27 of 37) remained upregulated or downregulated in inflamed joints (Fig. 2 ). A different set of genes was also involved in acute inflammation. At the chronic phase, less than half of AA-specific genes (127 of 277) were differentially expressed, and another half was CA-specific. A very limited number of transcripts ( n = 15) remained upregulated or downregulated in all three phases of arthritis. Activated T cells must be present in the peripheral blood of recipient BALB/c SCID mice after the transfer, but donor lymphocytes can be detected in joints as early as 3–5 days after the second transfer [ 32 ]. In earlier studies [ 31 ], and in control experiments (data not shown), using fluorescein-labeled or isotope-labeled donor lymphocytes, only very few cells were found in joints during the first week of transfer, and a second cell transfer was needed to induce a significant influx of lymphocytes into the joints and cause subsequent inflammation. In this study, we detected overexpression of a T cell-specific GTPase ( Tgtp ) and T cell receptor β ( Tcrbv13 ) in still non-inflamed (pre-arthritic) paws of recipient BALB/c SCID mice as early as 3–5 days after the second injection, indicating the presence of donor BALB/c lymphocytes. Thus, the initiation and development of arthritis in adoptively transferred PGIA must depend on cooperation between adaptive immunity cells (represented by donor BALB/c lymphocytes) and cells of innate immunity (represented by non-lymphoid cells in the recipient BALB/c SCID mice). Analysis of the cellular and tissue specificity of gene expression, using public gene expression databases [ 54 - 56 ], indicated that genes encoding CD48 ( Cd48 ), membrane-spanning 4A6B and 4A6C ( Ms4a6b and Ms4a6c ), epidermal growth factor-like receptor-like protein 1 ( Emr1 ), and interferon-induced 47 kDa protein ( Ifi47 ) were most probably originating from donor lymphoid cells, whereas other early arthritis genes (Table 2 ) were related to the activation of the innate immune system (represented by macrophages, dendritic cells, and cells of myeloid lineage) of recipient BALB/c SCID mice. Transcriptional control of gene activity is only one component of the complex cellular regulatory pathways. In other words, the functional activity of a protein depends on several factors such as interaction with other proteins, phosphorylation/dephosphorylation, subcellular compartmentalization, and other post-translational modifications. All of these factors might be involved in the regulation of interactions between the donor lymphocytes and the synovial/joint cells of recipient mice that lack an adaptive immune system. The list of genes we present in this study is rather short; that is, it includes only genes profoundly affected during arthritis initiation and progression at the level of transcription. Genes and proteins that are under subtle regulatory pressure, or are controlled by non-genetic mechanisms such as protein phosphorylation and other post-translational events, could not be detected and analyzed in this study. The development of new proteomics assays, and the synthesis of existing knowledge in cellular signaling pathways with information provided by gene expression studies, will be necessary to build up a complete arthritis-related regulatory network and to unravel the mechanisms involved in the development and progression of autoimmune arthritis. Conclusions The development and progression of a complex polygenic autoimmune disease such as RA are controlled by hundreds or thousands of genes, in addition to the MHC. Despite the relatively high incidence of RA in the human population, only a few studies have applied gene array methods to the monitoring of disease progression and efficacy of treatment, or to predicting the prognosis of the disease. The major obstacles in the human studies are the relatively late diagnosis of RA, the large variety of cell types (cells of the immune system and of synovial joints) involved in autoimmune arthritic processes, and the extreme genetic heterogeneity of the human population. The present study applied an adoptively transferred murine model of RA and a microarray approach to detect differentially expressed, disease-related signature genes in PA (still non-inflamed) joints, days before the clinical symptoms or histopathological abnormalities of joint inflammation could be observed. However, the detection of early arthritis signature genes in joints can be done only in an experimental system in which particular joints have already been affected before the inflammatory symptoms can be identified. To make this experimental system uniform, that is, to exclude individual variations, we adoptively transferred antigen (PG)-specific lymphocytes (representing cells of adaptive immunity) from primarily arthritic mice into syngeneic SCID mice, which lack an adaptive immune system. In this highly synchronized and uniform system we were able to detect differentially expressed genes in still non-inflamed paws of arthritis-'prone' animals. We identified a relatively small number of mostly upregulated early arthritis signature genes (known to be involved in arthritic processes and/or autoimmunity), some of which were expressed at even higher levels in the acute phase of arthritis. These early arthritis signature genes, originating from donor cells, indicated the involvement of adaptive immunity, whereas the innate immunity genes were differentially expressed by cells of the recipients. The early signature genes, together with those that were differentially expressed in the acute (277 genes) and chronic (418 genes) phase of arthritis, are listed in the Additional files. Although many of these differentially expressed genes, detected either in the acute phase or during the progression of the disease, have been implicated in inflammation or autoimmunity, the list contains a significant number of differentially expressed genes whose function, or association with arthritis, is unknown at present. Abbreviations AA = acutely arthritic; AN = absolutely negative (control naive); CA = chronically arthritic; CV = coefficient of variation; DDA = dimethyldioctadecylammonium bromide; PA = pre-arthritic; PG = cartilage proteoglycan aggrecan; PGIA = PG-induced arthritis; RA = rheumatoid arthritis; SCID = severe combined immunodeficient. Competing interests The author(s) declare that they have no competing interests. Authors' contributions VAA performed essentially all statistical analyses and put together the draft version of the results and figures. CV isolated all RNA samples, prepared biotinylated samples and was involved in Affymetrix hybridization experiments; he also performed preliminary clustering experiments with GeneSpring version 6.2 (not included in this paper). AH performed all in vitro stimulation and adoptive transfer experiments, and assessed arthritis three or four times a day together with KM, who was also involved in all phases of the experimental processes and in the finalization of the manuscript. EGB controlled Affymetrix hybridization and scanning experiments, managed preliminary data analysis and finalized the manuscript. TTG designed experiments, controlled all experimental steps, data analysis, and finalized the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional File 1 A table (Excel file) that lists all information about the 37 genes differentially expressed (more than twofold level) in pre-arthritic joints/paws, when compared with the same genes expressed in normal (naive) joints/paws of BALB/c SCID mice (PA/AN comparison). The pre-arthritic (still non-inflamed) joints were collected within 24–48 hours after the onset of inflammatory symptoms in BALB/c SCID mice with adoptively transferred PGIA. Acutely inflamed paws from these arthritic BALB/c SCID mice were used as acute arthritic (AA) samples (the list of gene expression profiles is provided in Additional file 2 .) Click here for file Additional File 2 This file contains information about 256 upregulated and 21 downregulated genes in acutely arthritic joints/paws in five independent hybridization experiments. All genes with twofold or higher expression levels are listed. Click here for file Additional File 3 This file includes 201 upregulated and 217 downregulated genes in subacute/chronic phase of arthritis (8–12 days after onset) with the corresponding information. The gene expression levels (twofold or higher) are compared with those expressed in normal joints of naive (absolutely negative) BALB/c SCID mice. Click here for file

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1065316

Chemokine receptors in the rheumatoid synovium: upregulation of CXCR5

In patients with rheumatoid arthritis (RA), chemokine and chemokine receptor interactions play a central role in the recruitment of leukocytes into inflamed joints. This study was undertaken to characterize the expression of chemokine receptors in the synovial tissue of RA and non-RA patients. RA synovia ( n = 8) were obtained from knee joint replacement operations and control non-RA synovia ( n = 9) were obtained from arthroscopic knee biopsies sampled from patients with recent meniscal or articular cartilage damage or degeneration. The mRNA expression of chemokine receptors and their ligands was determined using gene microarrays and PCR. The protein expression of these genes was demonstrated by single-label and double-label immunohistochemistry. Microarray analysis showed the mRNA for CXCR5 to be more abundant in RA than non-RA synovial tissue, and of the chemokine receptors studied CXCR5 showed the greatest upregulation. PCR experiments confirmed the differential expression of CXCR5 . By immunohistochemistry we were able to detect CXCR5 in all RA and non-RA samples. In the RA samples the presence of CXCR5 was observed on B cells and T cells in the infiltrates but also on macrophages and endothelial cells. In the non-RA samples the presence of CXCR5 was limited to macrophages and endothelial cells. CXCR5 expression in synovial fluid macrophages and peripheral blood monocytes from RA patients was confirmed by PCR. The present study shows that CXCR5 is upregulated in RA synovial tissue and is expressed in a variety of cell types. This receptor may be involved in the recruitment and positioning of B cells, T cells and monocytes/macrophages in the RA synovium. More importantly, the increased level of CXCR5, a homeostatic chemokine receptor, in the RA synovium suggests that non-inflammatory receptor–ligand pairs might play an important role in the pathogenesis of RA.

Introduction Rheumatoid arthritis (RA) is a chronic inflammatory condition that affects multiple joints, and it results in the accumulation of leukocytes within the synovial tissue (ST) and synovial fluid (SF). The inflammatory infiltrate consists predominantly of B lymphocytes, T lymphocytes and macrophages in the ST, whereas neutrophils are mainly found in the SF. The lymphocyte infiltration is organized in lymphoid-like microstructures in just under 50% of the RA patients; however, the patients present germinal centre reactions in only 20% of cases [ 1 ]. The pathogenesis of the RA is still largely unknown but leukocytes and their products play an important role in the development of inflammation, joint destruction and pain [ 2 , 3 ]. The attraction of leukocytes into the joints is controlled by chemokines, a family of small chemotactic cytokine-like molecules that act as potent mediators of inflammation [ 4 ]. Chemokine activity is dependent on the presence of and interaction with chemokine receptors on the leukocyte surface. Indeed, chemokines and their receptors are involved together in the development and perpetuation of inflammation [ 5 ]. In vitro and in vivo experiments have indicated that blocking chemokines or their receptors could potentially provide an effective treatment of inflammatory diseases [ 5 , 6 ]. The 19 receptors so far identified belong to a super-family of G-protein-coupled receptors with seven transmembrane domains [ 7 ]. Chemokine receptors have a regulatory effect on the maturation and traffic of leukocytes, and they are implicated in several disease states [ 8 ]. There have been several reports on chemokine receptor expression on T cells from RA ST, RA SF and RA peripheral blood (PB) [ 9 - 13 ]. The expression of some chemokine receptors on monocytes/macrophages, dendritic cells and neutrophils has also been reported [ 14 - 17 ], and the importance of the role of chemokine receptors in RA is emerging [ 18 , 19 ]. CXCR5 is a chemokine receptor highly expressed in recirculating B cells, in subsets of CD4 + and CD8 + T cells and monocytes [ 20 , 21 ]. It also has been identified on B-cell infiltrates in Sjogren's syndrome [ 22 , 23 ]. CXCR5 is involved in the immune-system homeostasis and in lymphoid organogenesis [ 24 ]. Several morphological and functional studies suggest that lymphoid neogenesis takes place in RA [ 1 , 25 , 26 ]. Furthermore, an important disturbance of follicle and germinal centre formation in the spleen and Peyer's patches is observed in CXCR5-deficient mice [ 27 ]. CXCL13, the unique ligand of CXCR5, is also involved in follicular homing, as observed in CXCL13-deficient mice [ 28 ]. In view of the role of chemokine receptors in leukocyte traffic, the aim of the present study was to compare their expression in inflamed and non-inflamed tissue to shed light on which chemokine receptors may be involved in the recruitment and retention of leukocytes in ST. We examined chemokine receptor expression in ST taken from RA and non-RA patients using microarray technology, RT-PCR and immunohistochemistry. The microarray and RT-PCR experiments demonstrated the differential expression of CXCR5 , and immunohistochemistry showed that this receptor is expressed in B-cell and T-cell infiltrates, on macrophages and blood vessels. Our study identifies CXCR5 as a potentially interesting therapeutic target in RA and points to the use of antagonists to this receptor as a treatment strategy in the disease. Materials and methods Tissue and cell source Tissue samples were obtained from patients with RA ( n = 8) who fulfilled the American Rheumatism Association criteria for RA (Table 1 ). The patients' mean age was 59 ± 14.8 years with a male to female ratio of 1:8. The disease duration of six out of eight RA patients was over 10 years. ST was taken from these subjects at the time of total knee replacement. Non-RA patients ( n = 9) had knee joint symptoms for suspected articular cartilage or meniscal damage (Table 1 ). Their mean age was 47.6 ± 6.8 years with a male to female ratio of 8:1. Except for one patient, the non-RA patients had knee complaints for 1 year or less. ST biopsies were obtained from these patients at the time of arthroscopy. All samples were taken with informed consent and ethical approval. The ST samples were taken from the suprapatellar pouch and the medial gutter, which is reported to provide representative sampling of synovial membrane pathology [ 29 ]. Synovia were cut into approximately 4 mm 3 pieces and were either snap frozen in isopentane and stored in liquid nitrogen or formalin fixed and paraffin embedded. Monocytes/macrophages were isolated from the PB and SF of another four RA patients (Table 1 ). In brief, the blood and hyaluronidase-treated SF were centrifuged over a ficoll cushion (Amersham Biosciences, Chalfont St Giles, UK). The macrophages were isolated from the buffy coat layer (lymphocytes, macrophages) by adherence onto a glass dish. RNA extraction Total RNA was extracted from frozen blocks of synovia or from isolated monocytes/macrophages using TRIreagent solution (Sigma, Poole, UK) according to the manufacturer's recommendation. The quantity recovered was determined by spectrophotometry and the integrity was assessed by gel electrophoresis. For microarray experiments, equal quantities (7 μg) of RNA from each RA or non-RA patient were pooled and the messenger RNA was extracted using the mRNA GeneElute Kit (Sigma). The quantity recovered was determined by fluorometry using SYBR Green II (Molecular Probes, Leiden, The Netherlands). RNA had to be pooled since only small biopsies could be obtained from non-RA patients. Microarray technology The panorama human cytokine gene array (Sigma-Genosys, Pampisford, UK) was used. This array contains 375 different cDNAs including 16 chemokine receptors and 33 chemokines, each printed in duplicate on nylon membranes. The probe labelling and hybridization were carried out according to the manufacturer's instructions. Briefly, 33 P-radiolabelled cDNA probes were prepared from 0.5 μg mRNA (see earlier) using human cytokine cDNA labelling primers (Sigma-Genosys) and AMV reverse transcriptase at 42°C, and were purified on a Sephadex ® G-25 spin column (Sigma-Genosys). The arrays were hybridized for 17–18 hours at 65°C, washed and subjected to autoradiography for various lengths of time using Kodak BioMax MR X-ray film. The intensity of hybridization signals was quantified using the ArrayVision, version 6.0, software (Imaging Research Inc., Haverhill, UK). The intensity of each spot was corrected for background levels using the 'corners between spots' (set to 3 pixels) with or without 'segmentation' protocols, and were normalized for differences in labelling using the average values of seven housekeeping genes: β 2 -microglobulin , β-actin , cyclophilin A , glyceraldehyde-3-phosphate dehydrogenase , HLA-A 0201 heavy chain , human hypoxanthine phosphoribosyl transferase , and α-tubulin . The remaining two housekeeping genes, L19 and transferrin R , were excluded because of signal saturation and differential expression, respectively. The software performs the normalization automatically. Reverse transcription-polymerase chain reaction Total RNA aliquots from individual patients were reverse transcribed using oligo(dT 18 ) (MWG Biotech, Ebersberg, Germany) and MMLV reverse transcriptase (Promega, Southampton, UK) at 37°C for 1 hour. The reactions were terminated at 70°C for 10 min and were diluted to 80 μl with H 2 O. For two of the non-RA patients no more RNA was available for RT-PCR following microarray analysis. The PCR reactions were normalized against the ribosomal RNA L27 using specific primers (MWG Biotech) (Table 2 ). Appropriate cDNA dilutions were used subsequently for the RT-PCR reactions using specific primers for CXCR5 (MWG Biotech) (Table 2 ). Specific primers were designed from the published sequences. The number of cycles and the annealing temperature were optimized for each primer pair. The RT-PCR conditions were one cycle at 94°C for 3 min, 57°C for 1 min and 72°C for 1 min, X cycles at 94°C for 1 min, 57°C for 1 min and 72°C for 1 min, and one cycle at 94°C for 1 min, 57°C for 1 min and 72°C for 10 min. X equals 34 cycles for CXCR5 and 24 cycles for L27 . Immunohistochemistry for CXCR5 The ST from the patients that had been examined at the transcription level was also available for protein expression analysis. Paraffin embedded sections were cut 4 μm thick on 3-aminopropyltriethoxysilane-coated slides. Sections were deparaffinized and rehydrated before blocking endogenous peroxidase activity with H 2 O 2 (0.3%) in methanol. The slides were rinsed with PBS and incubated with normal serum (1:67 in PBS) for 10 min before applying anti-human CXCR5 monoclonal antibody (1:666; R&D, Abingdon, UK) and the respective IgG control (Dako, Ely, UK). The sections were rinsed with PBS and incubated with biotinylated secondary antibody. The antibody binding was detected using reagents in the Vectastain ABC Elite kit (Vector, Peterborough, UK) and the chromogen 3,3'-diaminobenzidine (DAB) (Vector). Sections were rinsed and counter stained in Mayer's haematoxylin. B cells and macrophages were localized using anti-human CD20 antibodies (1:100; Dako) and CD68 antibodies (clone PG-M1, 1:75; Dako), respectively. CD20 required antigen demasking by 15 min microwaving in citrate buffer (pH 6.0), but H 2 O 2 treatment was not necessary. CD68 antigen was demasked using 0.05% pronase in Tris-buffered saline (pH 7.2) for 10 min. Double immunohistochemistry was performed with anti-human CD3 rabbit monoclonal antibodies (Labvision) and CXCR5 antibodies. The slides were deparaffinized, rehydrated and microwaved for 15 min in citrate buffer pH 6.0 before being treated with H 2 O 2 in methanol. The slides were incubated with 2.5% normal swine serum for 20 min before applying CD3 diluted 1:60 in 2.5% serum for 30 min. The sections were rinsed with PBS and were incubated with swine anti-rabbit antibody linked to alkaline phosphatase (1:40; Dako). CD3 binding was detected using Vector Red substrate (Vector). Sections were rinsed and were either counter stained in Methyl Green (Vector) or subjected to a second round of immunohistochemistry. CXCR5 was used as for single immunohistochemistry except that blocking and antibody dilutions were made in 2.5% normal horse serum and CXCR5 was revealed with DAB-Nickel (Vector). No counter stain was performed for double immunohistochemistry sections. Results Patients and tissue selection Synovia were obtained from knee joints as this allowed the use of arthroscopic samples of non-RA (normal) as controls instead of osteoarthritic tissue, which can show more enhanced inflammatory changes. The histology of H&E-stained RA synovial sections demonstrated classic signs of inflammation. Mononuclear cell infiltrates were visible in seven out of eight patients and consisted of aggregate structures; one of these seven patients also contained more germinal-like centre structures. In addition one patient revealed a diffuse infiltration. The synovium of the non-RA patients showed minimal signs of inflammation. In eight out of nine patients no mononuclear infiltrates were observed, and in one case only a small infiltrate was seen. No thickening of the intima was observed in the non-RA compared with the RA samples. Microarray analysis of chemokine receptor expression To allow rapid preliminary screening of a large number of chemokines and their receptors in RA ST and non-RA ST, chemokine expression was investigated using microarrays. A pair of human cytokine microarrays including 16 chemokine receptors and 33 chemokines was hybridized with labelled cDNA probes prepared from mRNAs obtained from RA and non-RA pools of synovial RNA. Figure 1 shows the results of hybridization of the RA and non-RA probes to the array membranes. To reduce the bias that could be introduced during the quantification, arrays showing very similar signals for the housekeeping genes were chosen and only non-saturated and non-regulated signals/genes were used for normalization. The intensity of each spot was corrected for background levels. The analysis step was repeated eight times for each pair of autoradiogram. Of the 16 chemokine receptors present, the expression of 12 chemokine receptors was visible on the RA microarrays. These were CCR1 , CCR2a , CCR5 , CCR7 , CCR9 , CX3CR1 , CXCR1 , CXCR2 , CXCR4 , CXCR5 , CXCR6 ( STRL33 ) and Bob (Table 3 ). Expression of the same receptors could be observed on the non-RA membranes with the exception of Bob , CCR7 and CCR9 . Bob / GPR15 is an orphan receptor that is a coreceptor for human and simian immunodeficiency viruses, and its expression in the RA synovium is a novel observation that might be worthy of further investigation. The detection of CCR7 and CCR9 in RA was only possible after extended exposure times, but at the time points used for quantification no regulation was demonstrated. Four chemokine receptors ( CCR2b , CCR3 , CCR4 and CCR6 ) could not be detected in RA samples or non-RA samples under our conditions. The most obvious differences between RA samples and non-RA samples were for the chemokine receptors CXCR5 and CXCR2 , and to a lesser extent CXCR4 , which gave stronger signals in RA samples (Fig. 1 ). In order to quantify the differential expression of these receptors the densities of autoradiographic spots were measured using ArrayVision software (Table 3 ). The criteria we set for a gene to be considered as upregulated or downregulated were a RA/non-RA ratio higher than 3 or lower than 0.3, respectively, and a 95% confidence interval below 10% (criteria as [ 30 ]). In the present study the expression of CXCR5 and CXCR4 was 22.6 ± 0.7-fold higher and 3.5 ± 0.1-fold higher in RA tissue than in non-RA tissue, respectively. These results indicated that, of the chemokine receptors studied, CXCR5 was the most upregulated in RA (Table 3 ). The upregulation of CXCR2 could not be calculated for mathematical reasons because the signal intensity of CXCR2 in non-RA tissue after correction for the background was zero. CXCR2 was only visible on the non-RA autoradiogram upon prolonged exposure, at which point the housekeeping genes were saturated and were therefore unsuitable for quantification purposes. Out of the 33 chemokines present on the arrays, 29 of these ligands were visible on the RA membranes and 21 on the non-RA membranes (Fig. 1 and Table 3 ). These included CXCL13 , CXCL12 , CXCL8 , CXCL1-3 and CXCL5 , which are ligands for the chemokine receptors CXCR5, CXCR4 and CXCR2. Several chemokines were visible on RA microarrays but not on non-RA microarrays (namely CXCL13 , CCL21 and CCL24 ), suggesting that these genes might be induced in the inflamed synovium. In contrast, there were no chemokine signals that were present on non-RA membranes and were absent on RA membranes. Where chemokine signals were detectable on RA and non-RA microarrays, it was possible to quantify the degree of upregulation or downregulation using the criteria described earlier for chemokine receptors. Of these chemokines, the following showed upregulation: CCL18 (4.5 ± 0.4-fold increase), CXCL9 (3.6 ± 0.1-fold increase), CXCL5 (3.5 ± 0.3-fold increase), and CXCL8 (3.3 ± 0.2-fold increase). No chemokines displayed a downregulation with a RA/non-RA ratio less than 0.3. The upregulation of CXCL9 in RA synovia is in agreement with the only microarray study of RA synovia, in which this chemokine was also shown to be increased [ 31 ]. In our study only five chemokines ( CXCL7 , CCL13 , CCL20 , CCL17 and CCL25 ) could not be detected at all, whether in RA or non-RA samples. The rapid screening of several genes at once made array technology a very attractive method. Its use has revealed disadvantages, however, including the requirement for large amounts of RNA (which are not always available from human tissue biopsies), a susceptibility to experimental variability and a lack of standard optimum methods for statistical analysis [ 32 ]. Arrays also present the risk of cross-hybridization leading to false positive or negative results [ 31 ]. However, the array approach remains a valuable tool if the samples can be pooled and if it is used in conjunction with alternative methods such as RT-PCR. RT-PCR analysis of CXCR5 To confirm the array results and to examine individual patients, RT-PCR was performed on the total RNA from each patient sample (Fig. 2 ). PCR primers were run through the BLAST program (available through the UK MRC HGMP-RC website: ) to ensure the gene specificity of the RT-PCR results and to exclude the possibility of cross-hybridization with other genes. Overall, CXCR5 RNA was more abundant in RA patients than in non-RA patients, confirming the microarray data. CXCR5 expression was detected in the synovia of all eight RA patients and showed some degree of patient-to-patient variation. The difference in CXCR5 expression between RA patients and non-RA patients was unlikely to be due to differences in the relative amount of cDNA produced by different RT reactions since the PCR reactions were normalized using the ribosomal gene L27 . RT-PCR showed that the difference between RA patients and non-RA patients was less marked for CXCR2 and CXCR4 than for CXCR5 (data not shown). Immunohistochemistry To identify the cell types expressing CXCR5 , and since RNA expression and protein expression do not always correlate, the protein expression of this receptor and three specific cell markers (CD20, CD3 and CD68) was investigated by immunohistochemistry of paraffin-embedded sections. Seven out of eight RA patients presented substantial lymphoid follicles in their synovia. The specific cell markers CD20 and CD3 confirmed the presence of B cells and T cells, respectively, in these infiltrates. In every RA patient where lymphoid follicles occurred, CXCR5 + cells were always present in these structures; this indicates a correlation between the expression of CXCR5 and the occurrence of lymphoid follicles. Serial sections indicated that CXCR5 was expressed by CD20 + B cells (Fig. 3a,3c ). It was not possible to colocalize CXCR5 and CD3 in serial sections, so a double-label immunohistochemistry technique was developed. Sections were treated with anti-CD3 followed by alkaline phosphatase and Vector red substrate. Anti-CXCR5 was added to the same sections, and the colour developed using peroxidase and DAB-Nickel. CD3 expression alone gave a light red colour (Fig. 3e ) and CXCR5 expression alone produced a grey–black colour (Fig. 3f ). Where these two proteins colocalized a dark red colour was obtained (Fig. 3f ). Using this technique it was evident that in the RA synovium there was a population of CD3 + T cells that expressed CXCR5 (Fig. 3f ). These were localized exclusively in lymphoid follicles in the synovia of five out of the eight RA patients. The patient with diffuse infiltration was negative for CXCR5 + /CD3 + cells. Serial sections treated with anti-CXCR5 and the macrophage marker anti-CD68 suggested that CXCR5 + cells in the intima included macrophages (Fig. 4a,4b ). The endothelial cells of synovial postcapillary venules were positive for CXCR5 in the RA synovium (Fig. 4e ). In non-RA tissue, CXCR5 was localized in the intima and endothelial cells (Fig. 5 ). Intimal cells were widely positive for CXCR5 and serial sections indicated that these included CD68 + macrophage-like cells (Fig. 5a,5b ). No lymphocytic infiltrates were present in these synovial samples due to their non-inflamed nature. Sections treated with CD20 and CD3 antibodies were negative, showing that no B cells or T cells could be detected. In non-RA tissue and RA tissue, fibroblasts were negative for CXCR5, as were neutrophils in RA synovia, indicating selectivity in the cell types expressing this receptor. For all immunohistochemistry experiments in this study, the use of isotype-matched immunoglobulin controls or sera instead of primary antibodies resulted in negative staining of RA sections and non-RA sections (Figs 3b,3d,3g,3h , 4c,4d,4f and 5c,5d,5f ). RT-PCR on isolated RA monocytes/macrophages To further investigate whether macrophages themselves are producing CXCR5 and to confirm the results of immunohistochemistry, we performed RT-PCRs on monocytes/macrophages isolated from the PB and SF of four additional RA patients (Fig. 6 ). CXCR5 was strongly expressed in all four samples and there was little difference between PB and SF. Discussion The major finding of the present study is that CXCR5 is upregulated in the RA synovium. The cells expressing this chemokine receptor are B lymphocytes, T lymphocytes, macrophages and endothelial cells. The increased numbers of B lymphocytes, T lymphocytes and macrophages producing CXCR5 in the RA synovium are probably responsible for the increased expression of the receptor in this chronically inflamed tissue. The majority (seven out of eight) of the RA synovia included in this study contained substantial lymphoid aggregates but only one out of nine non-RA patients presented a very small infiltrate. These cell aggregates contained CD20 + B cells that expressed CXCR5. The expression of CXCR5 has been reported in mature B cells and secondary lymphoid organs but as far as the authors are aware this is the first report of a chemokine receptor expressed by B cells in the RA synovium and its ectopic lymphoid structures. Our findings are particularly interesting in view of the functional role of B cells in RA. This includes autoantibody production, antigen presentation, a role in lymphoid follicle and germinal centre formation, and the promising results of the anti-CD20 treatment in RA [ 33 , 34 ]. The microarrays showed that the mRNA for CXCL13, the only known ligand of CXCR5, was present in the RA synovium and not in the non-RA synovium. Furthermore, other reports have shown a CXCL13 message in RA synovia, together with its protein that localizes to follicular dendritic cells, endothelial cells and synovial fibroblasts, suggesting that these cells produce the chemokine [ 1 , 25 ]. Taken together with our data, this indicates that CXCR5 on B cells may be important in the recruitment of these cells into the RA synovium, in addition to their positioning and retention within the synovial infiltrates. In this regard, the role of CXCR5 on B cells in secondary lymphoid organs has been well documented [ 35 , 36 ]. CXCR5 guides B cells into the B-cell follicles and also directly promotes the recruitment of these cells into Peyer's patches via high endothelial venules [ 27 , 28 , 37 , 38 ]. In addition CXCR5-deficient mice exhibit impaired development of lymph nodes and Peyer's patches, and the tissue architecture of these organs is severely disturbed showing a lack of B-cell follicles [ 27 , 28 ]. Our double immunohistochemistry data indicate that there is a population of CXCR5 + CD3 + T cells present in the RA synovium. CXCR5 + T cells have been shown in secondary lymphoid tissue where some of these cells localize within germinal centres [ 20 , 39 ], and it is proposed that CXCR5 enables them to enter B-cell follicles guided by CXCL13 [ 36 ]. Within these follicles they may provide B-cell help and have therefore been named follicular B helper T cells, since purified human tonsillar CD4 + CXCR5 + T cells efficiently stimulate the production of immunoglobulins by B cells [ 39 , 40 ]. These follicular B helper T cells are CD57 + , whereas the majority of the CXCR5 + T cells that are present in interfollicular and T-cell areas of the lymphoid tissue are CD57 - and are poor B-cell helpers [ 41 ]. Since lymphoid neogenesis occurs in the RA synovium it is possible that the CXCR5 expression on T cells as shown in the present study is involved in the positioning of these cells within the synovium and in providing B-cell help, although further studies are required to characterize this synovial T-cell population. Whether the two populations of CXCR5 + CD57 + and CXCR5 + CD57 - T cells are present in the RA synovium and what their role could be is still unknown. However, CD57 + T cells are reported to be present in the RA synovium and SF, where levels of this marker are elevated compared with controls [ 42 , 43 ]. Furthermore, an involvement of CD57 + T cells has been shown in disease activity of RA [ 44 ]. Immunohistochemical experiments indicated that CD68 + cells in the synovial intima express CXCR5. Intimal cells comprise two cell types: macrophage-like cells and fibroblast-like cells. In RA the former macrophage-like cells are numerous, comprising up to 80% of this cell layer [ 45 ]. It has been reported that in the RA synovium anti-CD68 reacts strongly with intimal macrophages, but fibroblasts also show some reactivity with this antibody [ 45 ]. Therefore, since macrophages are abundant in the RA intima and because of their strong reactivity with anti-CD68, it is likely that intimal macrophages are positive for CXCR5. In the normal non-RA intima, macrophages are positive for CD68 and fibroblasts are negative, making it more certain that macrophages express CXCR5 in this cell layer [ 45 ]. Consequently, RT-PCR was performed to verify that RA macrophages/monocytes can express CXCR5 . The RT-PCR did indeed demonstrate CXCR5 mRNA in macrophages from RA SF, as well as PB monocytes from the same RA patients. Since the CXCR5 mRNA is expressed at similar levels in RA PB and RA SF it is suggested that the contribution of monocytes/macrophages to the upregulation of CXCR5 in the RA synovium is due to their increased number, rather than due to an increased abundance of CXCR5 transcripts per cell. CXCR5 mRNA has also been found in normal human PB monocytes by RT-PCR [ 21 ]. Studies by ourselves and other workers have shown that monocytes/macrophages express several other CXC chemokine receptors in RA, including CXCR1, CXCR2 and CXCR4 [ 15 , 16 , 46 ]. Furthermore, RA monocytes/macrophages express CC chemokine receptors such as CCR1, CCR2, CCR3 and CCR5 [ 14 ], illustrating their broad profile of chemokine receptor expression. Endothelial cells are another cell type expressing CXCR5 in the synovium. There have been several reports of endothelial cells in the RA synovium expressing chemokine receptors, including CXCR3 and CXCR4, in addition to the Duffy antigen that is a non-signalling chemokine receptor [ 18 , 47 - 49 ]. In the RA synovium there is increased turnover of blood vessels with enhanced formation of new blood vessels together with enhanced vascular regression [ 50 , 51 ]. These mechanisms are regulated by the balance of angiogenic and angiostatic factors, and these factors include chemokines. Some chemokines are angiogenic (e.g. CXCL8, CXCL12, CCL1 and CCL2) and other chemokines are angiostatic (e.g. CXCL9 and CXCL10), and activation of their respective chemokine receptors results in the stimulation of or inhibition of endothelial cell proliferation [ 47 , 52 - 57 ]. CXCL13 has been shown to have an angiostatic function, inhibiting the angiogenic effects of FGF-2 on human umbilical vein endothelial cells [ 58 ]. In addition, the presence of CXCR5 in a variety of cultured human endothelial cells – from umbilical and saphenous veins, for example – may mediate the angiostatic effects of CXCL13 [ 58 , 59 ]. Our data showing the presence of CXCR5 on endothelial cells in the synovium and the presence of its ligand in this tissue [ 1 , 25 ]suggest that CXCR5 may play an angiostatic role in RA pathophysiology, although the angiostatic effects of CXCL13 could potentially be acting through CXCR3, which is also expressed by the RA synovial endothelium [ 48 , 60 ]. In the present study mRNA for other chemokine receptors were detected in the RA synovium, such as CXCR1, CXCR2, CXCR4, Bob, CCR1, CCR2a, CCR7, CCR9 and CX3CR1 (CXCR3 was not on the microarray). All of these showed variable degrees of increased mRNA expression in RA, although the upregulation was less compared with that of CXCR5. Several previous reports have shown the expression of chemokine receptors by leukocytes from RA joints. These have included CCR4–CCR6, CXCR3, CXCR4 and CX3CR1 by T lymphocytes [ 9 , 12 , 13 , 19 ] and CCR1–CCR5 and CXCR1–CXCR4 by monocytes/macrophages [ 14 - 16 , 18 ]. Such reports mainly focused on selected cell types and certain chemokine receptors. The present study took a different approach. Ours was primarily a whole-tissue study examining the mRNA expression of a wide range of chemokine receptors in RA and control synovia. While our study is in general accord with previous reports, differences may in part be due to the RA ST used. This tissue was highly infiltrated and, in all but one sample, had extensive lymphoid follicles bearing resemblance to those of secondary lymphoid organs. This feature may be responsible for the particular upregulation of the constitutive chemokine receptor CXCR5. In addition, our RA patients had long-standing disease (Table 1 ) and the patient sample may also have influenced the types of chemokine receptors expressed. Conclusion Our study demonstrates the expression of CXCR5 on B cells, on T cells, on monocytes/macrophages and on endothelial cells in the RA synovium. The expression of a marker shared by cells that are known to play a central role in the process of chronic inflammation is of particular interest and suggests that targeting CXCR5 could provide a powerful new treatment for RA. Abbreviations DAB = 3,3'-diaminobenzidine; H&E = haematoxylin and eosin; PB = peripheral blood; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; RA = rheumatoid arthritis; RT = reverse transcription; SF = synovial fluid; ST = synovial tissue. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CS carried out the microarray work, the RT-PCR and the double immunohistochemisty, and drafted the manuscript. AH carried out the single colour immunohistochemistry. AB isolated the peripheral blood and synovial fluid monocytes, and isolated the RNA after adhesion. BA participated in the design of the study. CB and MS collaborated on the study or coordinated the collection of samples in Birmingham, and contributed to the writing of the manuscript. JM conceived the study, and participated in its design and in the writing the manuscript. All authors read and approved the final manuscript.

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1065317

Effect of adalimumab on neutrophil function in patients with rheumatoid arthritis

Neutrophils are known to be targets for the biological activity of tumour necrosis factor (TNF)-α in the pathogenensis of rheumatoid arthritis (RA). Therefore, these cells may be among the targets of anti-TNF-α therapy. In this study we evaluated the effect of therapy with adalimumab (a fully human anti-TNF-α mAb; dosage: 40 mg subcutaneously every other week) on certain phenotypic and functional aspects of neutrophils obtained from 10 selected patients with RA and 20 healthy control individuals. Peripheral blood neutrophils were obtained at baseline and during anti-TNF-α therapy (2, 6 and 12 weeks after the first administration of adalimumab). All patients had been receiving a stable regimen of hydroxychloroquine, methotrexate and prednisone for at least 3 months before and during the study. Baseline neutrophil chemotaxis was significantly decreased in RA patients when compared with control individuals ( P < 0.001). Two weeks after the first administration of adalimumab, chemotactic activity was completely restored, with no differences noted between patients and control individuals; these normal values were confirmed 6 and 12 weeks after the start of anti-TNF-α therapy. Phagocytic activity and CD11b membrane expression on neutrophils were similar between RA patients and control individuals; no modifications were observed during TNF-α neutralization. The production of reactive oxygen species, both in resting and PMA (phorbol 12-myristate 13-acetate)-stimulated cells, was significantly higher in RA patients at baseline ( P < 0.05) and was unmodified by anti-TNF-α mAb. Finally, we showed that the activation antigen CD69, which was absent on control neutrophils, was significantly expressed on neutrophils from RA patients at baseline ( P < 0.001, versus control individuals); however, the molecule was barely detectable on cells obtained from RA patients during adalimumab therapy. Because CD69 potentially plays a role in the pathogenesis of arthritis, our findings suggest that neutrophils are among the targets of anti-TNF-α activity in RA and may provide an insight into a new and interesting mechanism of action of anti-TNF-α mAbs in the control of inflammatory arthritis.

Introduction Tumour necrosis factor (TNF)-α has been found to play a central role in the pathogenesis of rheumatoid arthritis (RA), which has led to the rational development of novel drug therapies that neutralize the deleterious effects of this cytokine [ 1 , 2 ]. Several studies have shown dramatic therapeutic effects of anti-TNF-α antibodies, both in experimental collagen-induced arthritis and in the treatment of inflammatory diseases such as rheumatoid arthritis (RA) [ 3 - 5 ], psoriatic arthritis [ 6 ], juvenile rheumatoid arthritis [ 7 ] and Crohn's disease [ 8 ]. The role played by phagocytic cells in the pathogenesis of these inflammatory diseases [ 9 - 11 ] and the capacity of TNF-α to prime and/or activate phagocytic cells [ 12 ] suggest, at least in part, that downregulation of phagocyte activity may be involved in the mechanism of action of anti-TNF-α therapy [ 9 ]. There is increasing evidence that inhibition of TNF-α may be associated with the development of adverse consequences such as carcinogenesis, autoimmune disorders and, importantly, infectious diseases caused by Gram-positive and Gram-negative bacteria, mycobacteria and fungi (for review, see Olsen and Stein [ 2 ]). Again, the role played by TNF-α in the activation of phagocytic cells and the involvement of these cells in the host defence against infections suggest that impairment in phagocytic cell activity may heighten the risk for infection during TNF-α neutralization [ 13 ]. Few data have been reported on the effect of anti-TNF-α therapy on neutrophil function ex vivo . Decreased influx of neutrophils in inflamed joints was reported by Taylor and coworkers [ 14 ] in RA patients treated with infliximab (a chimeric anti-TNF-α mAb) and by Den Broeder and coworkers [ 15 ] in patients treated with adalimumab (a fully human anti-TNF-α mAb). However, no significant impairment in ex vivo neutrophil function was observed in RA patients treated with etanercept (a soluble human p75 TNF receptor) [ 16 ] or with adalimumab [ 15 ]. In this work we evaluated certain phenotypic and functional aspects of neutrophils obtained from RA patients during treatment with adalimumab. To this end, chemotaxis, phagocytosis and reactive oxygen species (ROS) production were assessed in peripheral blood neutrophils, together with membrane expression of CD11b and CD69 – two functionally different activation molecules [ 17 ]. Methods Reagents The anti-CD69 mAb (IgG 2a , clone HP-4B3) was obtained from Calbiochem (La Jolla, CA, USA). The anti-CD11b was OKM1 (mouse IgG 2 isotype; Ortho Diagnostics, Raritan, NJ, USA). FITC-conjugated goat anti-mouse IgG was from Immunotech SA (Marseille, France). Irrelevant class-matched mAbs were used as controls for nonspecific binding (Becton Dickinson, San Jose, CA, USA). Lymphoprep gradient (density 1.077 g/ml) was purchased from Nyegaard (Oslo, Norway). RPMI 1640 was obtained from HyClone Laboratories (Logan, UT, USA). Bovine serum albumin (BSA), N -formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol 12-myristate 13-acetate (PMA), lucigenin (bis- N -methylacridinum nitrate) and zymosan A were from Sigma Chemical Company (St. Louis, MO, USA). Patients Peripheral blood samples were collected from10 selected and consenting RA patients who satisfied the American College of Rheumatology 1987 criteria [ 18 ], who had active disease (defined as a disease activity score 28 > 3.2) [ 19 ], and who were enrolled in a European open-label, multicentre, multinational phase IIIb study (the Adalimumab Research in Active RA [ReAct] study [ 20 ]). The study was approved by the ethical committee of the Ospedale L Sacco (Milan, Italy). The mean age of the patients was 61.4 years (range 40–83 years); eight were rheumatoid factor positive and two were rheumatoid factor negative. Three months before and during the study, all patients received hydroxychloroquine (200 mg twice daily), methotrexate intramuscularly (7.5–15 mg/week), and no more than 10 mg/day prednisone. Adalimumab was administered subcutaneously every other week (40 mg). Peripheral blood samples were obtained before anti-TNF-α therapy and immediately before administration of adalimumab at weeks 2, 6 and 12. Controls were 20 healthy individual who were matched to the patients with respect to age and sex. Ex vivo neutrophil function Peripheral blood neutrophils were obtained by density gradient centrifugation (Lymphoprep) [ 21 ]. The purified cells consisted of a more than 95% pure population of viable neutrophils, as assessed by morphology and trypan blue exclusion test. Neutrophil chemotaxis was evaluated using a modified Boyden chamber assay, with blind well chambers and 3 μm micropore filters (Millipore, Bedford, MA, USA) [ 22 ]. Briefly, 200 μl of the cell suspension, containing 2.5 × 10 6 neutrophils/ml in RPMI1640 + 0.4% BSA were layered on top of the filter, and the lower compartment was filled with 200 μl of the chemotactic factor (see below). Following incubation at 37°C for 90 min in a humidified atmosphere with 5% carbon dioxide, the filters were fixed with ethanol and stained with haematoxylin–eosin. The chemotactic response was then determined by evaluating the number of cells × high power field that had migrated through the entire thickness of the filter; triplicate chambers were used in each experiment and five fields were examined in each filter. In all cases the person scoring the assay had no knowledge of the experimental grouping. The chemoattractants were zymosan-activated serum (1 mg/ml for 30 min at 37°C) at a 10% (vol/vol) final dilution in RPMI 1640, and the synthetic peptide FMLP at 10 -8 mol/l final concentration. Phagocytosis was evaluated using C3-coated zymosan (C3Zy) as particles for uptake [ 23 ]. C3Zy was prepared incubating zymosan in normal human serum (5 mg/ml) for 30 min at 37°C followed by extensive washing. The neutrophil suspension (200 μl) was incubated with C3Zy (cell to particle ratio, 1:5) for 30 min at 37°C in a shaking water bath. Cytocentrifuge slides of the mixtures were then immediately prepared and stained with May Grunwald–Giemsa. The number of particles ingested per cell (phagocytic index [PI]) were established by direct light microscopy (1000 × magnification) of at least 200 cells. In all cases the person scoring the assay had no knowledge of the experimental grouping. Lucigenin-amplified chemiluminescence was used to evaluate production of ROS by neutrophils [ 23 ]. For the measurement of chemiluminescence, 1 × 10 5 neutrophils were mixed in 3 ml polystyrene vials with 5 × 10 -5 mol/l lucigenin in a final volume of 700 μl. The vials were placed in the Luminometer 1251 (LKB Wallace, Turku, Finland) in the dark and allowed to equilibrate for 5 min at 37°C with intermittent shaking previously to record the background of the light output in mV. PMA (final concentration 5 ng/ml) was added with an appropriate dispenser (1291; LKB Wallace) and chemiluminescence was recorded continuously. Background counts were subtracted from the values obtained after neutrophil stimulation. Levels of neutrophil membrane expression of CD69 and CD11b were evaluated as previously reported [ 23 ]. Briefly, 2 × 10 5 neutrophils were washed in phosphate-buffered saline (PBS) and resuspended with 100 μl PBS containing 0.1% NaN 3 , 10% human AB serum (to prevent nonspecific binding of mAb to Fc receptors) and predetermined saturating concentrations of the anti-CD69 or anti-CD11b mAbs. After incubation for 60 min at 4°C the cells were washed twice with PBS/NaN 3 /0.1% BSA and the pellets were resuspended in 100 μl of the same buffer containing FITC-conjugated goat anti-mouse IgG in a saturating concentration and incubated for 30 min at 4°C. The cells were then washed twice in PBS and resuspended in 0.5 ml of ice-cold 2% paraformaldehyde in PBS (pH 7.2). The percentage of neutrophils positive for CD69 or CD11b was quantified within 24 hours on a FACSscan flow cytometer (Becton Dickinson). A relative measure of antigen expression was obtained using the mean fluorescence intensity (MFI), converted from log to linear scale, after subtraction of the cells' autofluorescence and the fluorescence of cells incubated with irrelevant isotype control mAbs. Statistical analysis Data are expressed as mean ± standard error of the mean. Statistical analysis was performed using the Student's t-test for unpaired or paired data as appropriate. P < 0.05 was considered statistically significant. Results The chemotactic activity of neutrophils obtained from RA patients at baseline was significantly impaired as compared with that in neutrophils from control individuals; the defect was evident both using zymosan-activated serum ( P < 0.001; Fig. 1 ) and FMLP ( P < 0.02; Fig. 2 ) as chemoattractant. Two weeks after the start of therapy with adalimumab, the neutrophil chemotactic responsiveness was significantly improved (Figs 1 and 2 ), with no differences between patients and control individuals. The improvement was evident and persistent during anti-TNF-α therapy at weeks 6 and 12 (Figs 1 and 2 ). The phagocytic capacity of neutrophils was similar between control individuals (PI 0.99 ± 0.03) and RA patients at baseline (PI 1.19 ± 0.32), and no changes were observed during anti-TNF-α therapy (week 2: 1.11 ± 0.03; week 6: 1.17 ± 0.09; week 12: 1.03 ± 0.04). The CD11b molecule was spontaneously expressed on more than 90% of neutrophils both in control individuals and in RA patients before and during anti-TNF-α therapy (data not shown). The level of both spontaneous and FMLP-induced CD11b membrane expression (MFI) was also similar between controls (MFI for spontaneous: 155.3 ± 3.7; MFI for FMLP-induced: 591.3 ± 13.9) and RA patients at baseline (MFI for spontaneous: 159.2 ± 8.5; MFI for FMLP-induced: 558.7 ± 27.1), as well as during adalimumab therapy (MFI for spontaneous, week 2: 166.3 ± 12.2; MFI for spontaneous, week 6: 161.0 ± 16.7; MFI for spontaneous, week 12: 154.4 ± 14.9; MFI for FMLP-induced, week 2: 503.6 ± 33.1; MFI for FMLP-induced, week 6: 547.8 ± 27.7; MFI for FMLP-induced, week 12: 610.2 ± 41.8). Both spontaneous and PMA-induced production of ROS by RA neutrophils was slightly increased at baseline as compared with controls ( P < 0.05) and the differences persisted at all time points examined during adalimumab therapy (Fig. 3 ). Although control neutrophils stained with anti-CD69 mAb yielded very low fluorescence, just above that of unstained cells (%CD69 + cells: 1.3 ± 0.5; MFI: 1.0 ± 0.3), CD69 was significantly expressed on neutrophils from RA patients at baseline (%CD69 + cells: 22.8 ± 5.4; MFI: 7.6 ± 1.4; P < 0.001 versus controls; Fig. 4 ). As shown in Fig. 4 , a significant inhibition of CD69 expression on RA neutrophils was induced by adalimumab therapy; the inhibition was already evident at week 2 after the start of therapy (%CD69 + cells: 5.5 ± 0.9; MFI: 2.6 ± 0.6; P < 0.01 versus RA baseline) but it was complete at weeks 6 and 12, when no differences were observed between RA patients and control individuals (Fig. 4 ). Discussion The first aim of the study was to determine whether anti-TNF-α therapy could downregulate neutrophil function, thus reducing the antimicrobial host defence in patients with RA. Our ex vivo functional assays do not support this possibility. In fact, we demonstrated that TNF-α neutralization in RA patients did not modify neutrophil activities such as phagocytosis, which were normal at baseline, or ROS production, which was slightly increased at baseline. In agreement with previous studies [ 24 , 25 ], we found impaired chemotaxis of neutrophils from RA patients toward two different chemoattractants. Unexpectedly, TNF-α neutralization induced complete reversal of the neutrophil chemotactic defect. Various mechanisms may account for the defective neutrophil migration in RA patients, such as saturation of membrane receptors with immune complexes [ 25 ], cytokine (TNF-α)-induced desensitization [ 26 - 28 ] and drug-induced cell toxicity [ 29 - 33 ]. Of particular relevance are the observations that TNF-α-primed neutrophils are less responsive to chemoattractants [ 26 - 28 ] and are more susceptible to the inhibitory effect of methotrexate on chemotaxis [ 31 ]. Because circulating TNF-α has been demonstrated in RA patients [ 34 ], it is possible that anti-TNF-α therapy improves neutrophil migration by removing the deleterious effect exerted by soluble and/or membrane bound TNF-α on these cells. The second aim of the study was to determine whether downregulation of phagocyte activities are involved in the anti-inflammatory activity of anti-TNF-α therapy. The lack of activity on phagocytosis, ROS production or CD11b membrane expression, and the improved migration of neutrophils did not implicate neutrophils as targets of the therapeutic effect of anti-TNF-α. The improved chemotactic responsiveness we observed in patients during adalimumab therapy does not explain the decreased influx of neutrophils into synovial joints previously observed in RA patients during anti-TNF-α therapy [ 14 , 15 ]. However, there is evidence that anti-TNF-α mAbs downregulate the expression of cytokine-inducible adhesion molecules on endothelial cells [ 35 , 36 ]. The decreased activation of endothelial cells in the synovial microvasculature, rather than a defective neutrophil migration, could be responsible for the decreased homing of neutrophils to the inflamed joints. We recently found that both synovial fluid and peripheral blood neutrophils from RA patients have increased membrane expression of CD69 [ 37 ], and this observation was confirmed in the present study. This activation molecule is not constitutively expressed on neutrophils but it may be induced on these cells in vitro by several cytokines, such as granulocyte–macrophage colony-stimulating factor, interferon-γ and interferon-α [ 23 , 38 ]. Although a specific ligand for this molecule has not been identified, a role for CD69 in the pathogenesis of RA was previously suggested by Laffon and coworkers [ 39 ], who found that CD69 + T lymphocytes were detectable at high levels in synovial fluid and synovial membrane from RA patients and correlated with disease activity. Furthermore, Murata and coworkers [ 40 ] recently reported that CD69-null mice were protected from collagen-induced arthritis, and that transfer of neutrophils from wild-type mice could restore arthritis in these animals. These data suggested a crucial role for CD69 + neutrophils in the pathogenesis of arthritis and implicate the molecule as a possible therapeutic target for human arthritis. In the present study we observed that CD69 was downregulated (or inhibited) on neutrophils from RA patients during adalimumab therapy. The mechanism underlying this inhibition is not clear because, in our experience, TNF-α per se is not an inducer of CD69 on neutrophils. However, it is possible that other and as yet undefined CD69 inducers are indirectly inhibited by TNF-α neutralization. In agreement with our data, Moore and coworkers [ 41 ] recently reported decreased CD69 expression on natural killer cells obtained from mice treated with anti-TNF-α. Conclusion In this study we found that administration of the anti-TNF-α mAb adalimumab to patients with RA does not interfere with the neutrophil activities that are required to maintain an adequate antimicrobial host defence capacity. On the other hand, the inhibitory activity of the mAb on CD69 membrane expression on neutrophils indicates that these cells are among the possible targets of anti-TNF-α activity in RA, and may provide an insight into a new and interesting mechanism of action of anti-TNF-α mAbs in the control of inflammatory arthritis. Abbreviations BSA = bovine serum albumin; C3Zy = C3-coated zymosan; FMLP = N -formyl-methionyl-leucyl-phenylalanine; mAb = monoclonal antibody; MFI = mean fluorescence intensity; PBS = phosphate buffered saline; PI = phagocytic index; PMA = phorbol 12-myristate 13-acetate; RA = rheumatoid arthritis; ROS = reactive oxygen species; TNF = tumour necrosis factor. Competing interests The author(s) declare that they have no competing interests. Authors' contributions FC conceived the study, participated in conducting neutrophil functional assays and drafted the manuscript. FM conducted the neutrophil functional assays. PB conducted the immunofluorescence assays. PS-P participated in study design and coordination, and helped to select patients. FA helped with monitoring patients before and during the study. MC participated in coordination of the study. All authors read and approved the final manuscript. AD helped to perform statistical analysis.

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1065318

Repression of anti-proliferative factor Tob1 in osteoarthritic cartilage

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal ( n = 9) and osteoarthritic ( n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1β and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage ( P < 0.01). One of the significantly repressed genes, Tob1 , encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo . Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression.

Introduction Osteoarthritis is the most common disabling condition of humans in the western world. Although osteoarthritis is mainly a disease and functional loss of the articular cartilage covering the joint surfaces, it is clearly the cells that are the active players during the disease process [ 1 ]. Whatever pleomorphisms the cellular reaction patterns display at first sight during the osteoarthritic disease process, they can be basically summarized in three categories (reviewed in [ 2 ]). First, the chondrocytes can degenerate or proliferate. Second, chondrocytes can activate or deactivate their synthetic anabolic or catabolic matrix-degrading activity by increasing or decreasing anabolic or catabolic gene expression. Last, chondrocytes can undergo phenotypic modulations implicating an overall severely altered gene expression profile of the cells in the diseased tissue. In fact, several distinct phenotypes of chondrocytes are known to occur in vitro , in vivo during fetal development and potentially also in the disease process itself, but new markers are required for the more accurate characterization of cellular behavior [ 3 ]. This will allow further analysis of the underlying pathology to develop therapeutic approaches that could delay, stop, or even reverse cartilage degeneration. In many laboratories single and multiple gene analyses have been performed on normal and osteoarthritic cartilage specimens; however, a global overview of disease-associated changes is not available. This highlights the need for establishing a broader gene expression profile of osteoarthritic chondrocytes by modern screening technologies so as to characterize more properly the cellular events and regulatory pathways directly involved in cartilage destruction. In the present study, we designed a custom-made oligonucleotide-based microarray to screen for differentially expressed genes in normal and osteoarthritic cartilage specimens. We found that Tob1 , a gene involved in cell cycle regulation and cell quiescence [ 4 , 5 ], was significantly repressed in osteoarthritic chondrocytes. This was confirmed by quantitative polymerase chain reaction (qPCR) and further analyzed in adult articular chondrocytes in vitro and in vivo . Materials and methods Donors for mRNA expression analysis For the study of mRNA expression levels within the tissue, cartilage from human femoral condyles of normal knee joints was used. Normal articular cartilage ( n qPCR = 10, age range 45–88 years, mean age 64.1 years; n array = 9, age range 37–83 years, mean age 59 years) was obtained from donors at autopsy, within 48 hours of death. Osteoarthritic cartilage samples from late-stage osteoarthritic joint disease were obtained from patients undergoing total knee replacement surgery ( n qPCR = 15, age range 63–85 years, mean age 74.5 years; n array = 10, age range 57 to 84 years, mean age 76 years). The cartilage was frozen in liquid nitrogen immediately after removal and stored at -80°C until required for RNA isolation. Cartilage was considered to be normal according to a macroscopic scoring system of the opened joint: this mainly included normal synovial membrane, normal synovial fluid, no significant overall softening or surface fibrillation (except on the tibial plateau, which is basically found in all specimens depending on age). The Mankin's grade of histological plugs taken was less than 3. Osteoarthritic cases fulfilled the criteria published by the American College of Rheumatology [ 6 ]. Cases of rheumatoid origin were excluded from the study. Isolation of primary human articular chondrocytes; stimulation with interleukin (IL)-1β and bone morphogenetic protein (BMP)-7 Normal human knee articular cartilage was obtained from six normal cases at autopsy within 48 hours of death. Cartilage pieces were finely chopped and chondrocytes were isolated enzymatically as described previously [ 7 ]. Chondrocytes were either plated in high-density monolayer cultures or cultured in alginate beads. Cultures were maintained for 48 hours in serum-free Dulbecco's modified Eagle's medium/F12 medium (Gibco BRL, Eggstein, Germany) supplemented with 1% penicillin/streptomycin solution (Gibco BRL) and 50 μg/ml ascorbate (Sigma, Taufkirchen, Germany) and 10% fetal calf serum (Biochrom, Berlin, Germany). After 48 hours, primary (non-passaged) chondrocytes were stimulated with 1 ng/ml IL-1β (R&D System, Minneapolis, MN, USA) in DMEM/F12 medium, 100 ng/ml recombinant human BMP-7 (Stryker Biotech, Hopkinton, MA, USA) or cultivated in medium alone for 24 hours with no medium change afterwards. The same experiments were performed in parallel in the presence and in the absence of 10% fetal calf serum. At the end of the culturing/stimulation period the cells were washed in sterile phosphate-buffered saline (PBS), lysed in 350 μl of lysate RLT buffer/10 6 cells and stored at -80°C. Culture of HCS-2/8 cells The human HCS-2/8 chondrosarcoma cell line (around passage 50–55) [ 8 , 9 ] was cultured in DMEM (PAA, Linz, Austria) supplemented with 20% fetal bovine serum (Gibco BRL) and with 50 μg/ml ascorbate (Sigma) in a humidified atmosphere of 5% CO 2 at 37°C as described [ 9 ]. Cells were seeded at 10 5 cells/cm 2 and grown for 3 days to obtain subconfluent stage cultures, at 2 × 10 5 /cm 2 and cultured for 7 days to obtain confluent stage cultures, and at 6 × 10 5 /cm 2 and grown for 10 days for over-confluent stage cultures. RNA isolation from articular cartilage and isolated articular chondrocytes Total RNA from both cartilage tissue and isolated chondrocytes was isolated as described previously [ 10 , 11 ]. The quality of total RNA samples was checked by agarose-gel electrophoresis and with the Bioanalyzer RNA 6000 Nano assay (Agilent, Waldbronn, Germany). Construction of the SensiChip cartilage microarray The SensiChip technology is a two-color microarray platform using the Planar Wave Guide technology for microarray detection [ 12 ], which increases signal-to-noise ratios and thereby the sensitivity of hybridization experiments. The arrays were spotted in duplicate with 70-mer oligonucleotides representing the 3' untranslated region (UTR) of about 340 human cartilage-relevant genes, whereas one single gene was represented by one 70-mer oligonucleotide. Expression profiling with the SensiChip two-color DNA-microarray platform Total RNA (250 ng) from osteoarthritic cartilage (10 samples) and pooled normal cartilage was amplified and labeled with Cy3-UTP and Cy5-UTP respectively (Amersham Pharmacia) using the MessageAmp aRNA kit (Ambion). After clean-up of the complementary RNA (cRNA) with the RNeasy kit (Qiagen), 5 μg of Cy3-labeled cRNA from osteoarthritic cartilage was mixed with 5 μg of Cy5-labeled cRNA from pooled normal cartilage. cRNA was fragmented by incubation with 40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate for 15 min at 95°C and desalted with a Microcon YM-10 concentrator (Millipore). Mixed Cy-dye labeled cRNA samples (600 ng) were hybridized for 16 hours on a SensiChip microarray (Qiagen) spotted in duplicate with 70-mer oligonucleotides representing the 3' UTR of selected genes. The gene-specific oligonucleotide sequences were designed by Operon by using GenBank accession numbers and proprietary algorithms. After washing steps performed in accordance with the manufacturer's standard protocol, arrays were scanned with the SensiChip Reader. The resulting array images were analyzed with SensiChip View 2.1 software (Qiagen) to quantify gene-specific signal intensities. For quality control of RNA labeling and hybridization efficiency, oligonucleotides representing human housekeeping genes, negative and external bacterial spiking controls were also included. These sequences were prelabeled with fluorescent Cy3 and Cy5 dyes, and mixed in different concentrations into the hybridization solutions containing the labeled cRNA samples from human cartilage. Expression data analysis All microarray scans were inspected visually and checked for quality on the basis of the performance of negative, housekeeping and externally added Cy3/Cy5-prelabeled spiking controls. Raw signal intensities from each scan were imported into the gene expression analysis software Resolver version 4.0 (Rosetta Biosoftware, Seattle, WA, USA). The software employs an error-modeling approach for the analysis of microarray data [ 13 ]. An error model specific for the SensiChip microarray platform was designed by Rosetta Biosoftware based on expression data from repeated hybridizations of the same RNA material to determine the variation of signal intensities. A complete description of the statistical methods used is available in the technology section of the Rosetta Biosoftware website . All scans were pre-processed and normalized with the SensiChip error model to calculate P values and error bars for every gene expression profile. The P value represented the probability that an observed gene regulation was due to a measurement error. Gene regulation was considered as statistically significant if the calculated P value was below a threshold of 0.05. For normalization of expression data, the average brightness of the Cy3 and Cy5 channels respectively was used that was calculated from spots within a range from 30% to 85% of the signal intensity distribution of all spots. Scans from multiple experiments (replicates) were combined by averaging expression data with an error-weighted algorithm (also described in the statistical methods document available on the Rosetta Biosoftware website). Real-time quantitative PCR using TaqMan technology Real-time PCR was used to detect human Tob1 , collagen type II , Ki-67 , matrix metalloproteinase (MMP)-13 and glyceraldehyde-3-phosphate dehydrogenase mRNA expression levels in human articular cartilage RNA samples. The primers (MWG Biotech, Ebersberg, Germany) and TaqMan probes (Eurogentec, Liège, Belgium) were designed using Primer Express™ software (Perkin Elmer). To be able to obtain quantifiable results for all genes, specific standard curves using sequence-specific control probes were performed in parallel to the analyses. Thus, for each gene a gene-specific cDNA fragment was amplified by the gene-specific primers (Table 1 ) and cloned into pGEM T Easy (Promega, Mannheim, Germany) or pCRII TOPO (Invitrogen, Karlsruhe, Germany). The cloned amplification product was sequenced to confirm correct cloning. Cloned standard probes were amplified with the plasmid amplification kit (Qiagen), linearized and used after careful estimation of the concentration (gel electrophoresis, photometry, and a fluorimetric assay for deoxyribonucleic acids (Picogreen; Molecular Probes, Eugene, OR, USA)). For the standard curves concentrations of 10, 100, 1000, 10,000, 100,000, and 1,000,000 molecules per assay were used (all in triplicate). For the analyses of the different genes, a separate master mixture was made up for each of the primer pairs and contained a final concentration of 200 μM NTPs, 600 nM Roxbuffer and 100 nM TaqMan probe. For all genes the final reaction mixture contained, besides cDNA and 1 U polymerase (Eurogentec), forward and reverse primers, the corresponding probes, and MgCl 2 at concentrations given in Table 1 . All experiments were performed in triplicate. Immunofluorescence Immunofluorescence studies were performed on paraformaldehyde-fixed paraffin-embedded specimens of normal ( n = 5) and osteoarthritic ( n = 5) articular cartilage. Sections were first incubated with the primary antibodies overnight, then with biotin-labeled goat anti-mouse antibodies (Dianova, Hamburg, Germany) and then with peroxidase-labeled streptavidin (Dianova). Subsequently, the tyramide amplification system (PerkinElmer, Boston, MA, USA) was used for signal amplification. Finally, the signals were detected with Cy5-labeled streptavidin (Dianova). Nuclear staining was again performed with 4,6-diamidino-2-phenylindole. The sections were evaluated by a (fluorescence) microscope (Olympus AX70) and photographed digitally. To obtain optimal staining results various enzymatic pretreatments were tested, including hyaluronidase (Boehringer, Mannheim, Germany; 2 mg/ml in PBS pH 5 for 60 min at 37°C), pronase (Sigma, Deisenhofen, Germany; 2 mg/ml in PBS pH 7.3 for 60 min at 37°C), and bacterial protease XXIV (Sigma; 0.02 mg/ml; PBS pH 7.3 for 60 min at 37°C). Finally, the mouse monoclonal antibodies against Tob1 (Assay Designs, Ann Arbor, MI, USA) were used at a dilution of 1:20 without pretreatment of the sections. Amplification and cloning of Tob1 cDNA RNA was isolated from differentiated ADTC5 cells (Ricken Library) in accordance with the extraction method with Trizol ® (Invitrogen) and reverse-transcribed into cDNA with SuperScript II™ reverse transcriptase (Invitrogen) by following the manufacturer's recommendation. PCR amplification of a 607 base pair Tob1 cDNA fragment (nucleotides 402–1008 of the sequence in GenBank accession no. NM_009427) was performed with gene-specific primers (forward, 5'-GGAGCCCCCAGGTGTTCATGC-3'; reverse, 5'-CTCGTTGAGGCCTCCGTAGG-3') by a standard method, and amplification products were cloned into pCR ® -BluntII-TOPO ® vector (Invitrogen). In situ hybridization In situ hybridization of sectioned appendicular skeleton from newborn mice was performed with digoxigenin-labeled antisense riboprobes transcribed from the Tob1 cDNA fragment. Hindlegs of newborn mice were fixed overnight in 4% paraformaldehyde resolved in PBS. After stepwise transfer through solutions with increasing ethanol concentration, the specimens were incubated in xylene and finally embedded in paraffin wax. For in situ hybridization, paraffin-embedded samples were cut into slices 7 μm thick and mounted on microscope slides. The sections were hybridized with digoxigenin-11-UTP-labeled antisense riboprobes, which were transcribed with T7 RNA polymerase from the Tob1 cDNA fragment cloned into pCR ® -BluntII-TOPO ® (Invitrogen), after linearization of the plasmid with Bam HI. In situ hybridization was performed as described by Dietz and colleagues [ 14 ]. After detection of hybridization products, the sections were mounted under coverslips in Kaiser's glycerol gelatin (Merck) and photographed under a Zeiss Axioplan 2 microscope. Results Construction of the SensiChip cartilage microarray A microarray covering 340 human cartilage relevant genes was constructed, where one single gene was represented by one 70-mer oligonucleotide (Fig. 1a ). Most genes were selected from the literature and have important roles in anabolic or catabolic pathways during osteoarthritis (for example, cartilage matrix proteins such as collagens, relevant degrading enzymes such as MMPs and aggrecanases, and genes from important catabolic [IL-1, tumor necrosis factor-α] and anabolic [BMP, transforming growth factor-β] signaling pathways). Gene expression analysis: differentially expressed genes Total RNAs from 10 late-stage osteoarthritic cartilage samples were hybridized separately against a pool of mixed total RNAs from nine normal cartilage donors on the customized SensiChip microarrays. Merging of expression profiles obtained from all 10 late-stage osteoarthritic cartilage samples used for hybridizations resulted in about 200 significantly regulated genes that were differentially expressed between normal and osteoarthritic cartilage, with P < 0.01 (Fig. 2 and Table 2 ; the whole data set is in Additional file 1 ). Tob1 is repressed in osteoarthritic chondrocytes One of the differentially expressed genes was the human transducer of ERBB2,1 ( Tob1 ; GenBank accession no. NM_005749). Tob1 was transcriptionally downregulated in all 10 human osteoarthritic cartilage samples to, on average, one-sixth (Fig. 1 ). Corresponding P values were less than 0.05 for all human OA samples. Confirmation of Tob1 expression and regulation by (quantitative) PCR and immunostaining in normal and osteoarthritic articular cartilage Conventional PCR confirmed the expression of Tob1 , both in normal ( n = 3) and osteoarthritic ( n = 3) chondrocytes, with a weaker signal detected in the osteoarthritic samples (Fig. 3a ). To validate and quantify differential regulation of Tob1 , qPCR was performed on a set of normal ( n = 10) and osteoarthritic ( n = 15) samples. These experiments confirmed both its expression in normal articular cartilage and a highly significant decrease in Tob1 transcript levels in osteoarthritic samples (7.8-fold; P < 0.001; Fig. 3b ). Immunolocalization with monoclonal antibodies against Tob1 showed the presence of Tob1 protein in normal ( n = 5) and osteoarthritic ( n = 8) articular chondrocytes (Fig. 3c,d ). A somewhat weaker staining was observed in the osteoarthritic specimens than in the normal specimens, but this was not quantifiable because of the immunostaining technology used. Correlation of Tob1 expression to markers for chondrocyte anabolism, catabolism, and proliferation Next we examined whether Tob1 gene expression levels were correlated with the expression of marker genes of cell proliferation ( Ki-67 ) and anabolic ( collagen type II ) and catabolic ( MMP-13 ) activation of articular chondrocytes. This analysis showed highly significant correlations between these genes in osteoarthritic compared with normal chondrocytes (Fig. 4 ). Expression of Tob1 in articular chondrocytes in vitro Tob1 was expressed in isolated human adult articular chondrocytes in vitro . The mRNA expression levels of Tob1 in vitro were comparable to those of osteoarthritic chondrocytes in situ and were therefore significantly lower than those of normal chondrocytes in situ (oligo-array, Fig. 1d ; qPCR, Fig. 3b ). It is noteworthy that Tob1 was more strongly expressed in cells cultured without serum than with it. No significant regulation of Tob1 was found by two major anabolic (BMP-7) and catabolic (IL-1β) mediators in adult articular cartilage in cultured chondrocytes in vitro (data not shown). Expression of Tob1 in the fetal growth plate and during chondrocyte differentiation in vitro In situ hybridization on mouse fetal growth plate cartilage was performed to assess differential expression in the different cartilage zones. This showed that the expression of Tob1 was concentrated in the hypertrophic zone (zone of terminal differentiation and cessation of proliferation). Cells of the resting and proliferating zones (that is, areas of proliferation and matrix synthesis) showed no or very much weaker staining (Fig. 3e ). In addition, osteoblasts were positive (not shown). Expression profiling in HCS-2/8 cells, which are known to show a more differentiated phenotype in high-density cultures than when cultured in subconfluent or confluent status [ 15 ] showed an inverse relationship between Tob1 expression and the proliferation marker Ki-67 (Fig. 5 ). Discussion Differential gene expression analysis, as performed by us on normal and osteoarthritic chondrocytes, reveals long lists of differentially expressed genes of potential interest for furthering the understanding, diagnosis and/or modulation of osteoarthritis. The genes identified might be interesting with regard to any of these three aspects, but careful validation is needed to confirm the relevance of the findings obtained. In this regard, three levels of validation have to be achieved: (1) technical validation of screening results, (2) functional validation of the gene in situ or in vitro , and finally (3) establishment of relevance of the gene for the (physiology and/or) pathophysiology of the tissue. In our oligonucleotide-based array screen we detected many known differentially expressed genes. Thus, many marker genes behaved as expected from previous investigations: stromelysin I ( MMP-3 ) [ 7 ] and the cartilage transcription factor SOX9 [ 16 ] were significantly downregulated, whereas many constituents of the extracellular matrix were significantly upregulated ( collagen types II [ 17 ], III [ 18 ], VI [ 19 ], COMP [ 20 ], and fibronectin [ 21 ]). Further, MMP-13, the major collagenase of osteoarthritic cartilage [ 22 , 23 ], was induced [ 7 ]). Taken together, these findings validated this gene array technology as a reliable tool for identifying differentially expressed genes. In addition, many genes previously unknown to be differentially regulated in osteoarthritic cartilage were detected. Among the new differentially expressed genes we identified Tob1 as being significantly downregulated in osteoarthritic compared with normal articular chondrocytes. For technical validation (validation level I), this was confirmed by conventional and quantitative PCR at a very high significance level. Immunostaining provided additional evidence of the presence of Tob1 in normal and osteoarthritic chondrocytes. Tob1 , originally identified as binding partner of Erb ('transducer of Erb' [ 24 ]), is a member of a larger family of proteins, which share common protein domains and are known to exert anti-proliferative and phenotype-stabilizing effects on various cell types including osteoblasts ([ 24 , 25 ]; reviewed in [ 4 ] and [ 5 ]). Thus, to obtain insights into the functional activity of Tob1 in articular cartilage (validation level II), we correlated Tob1 expression with the expression of the Ki-67 antigen, a well-established gene expressed only by cells in the proliferation phase [ 26 ]. We found a highly significant inverse correlation between Tob1 expression and proliferative activity of chondrocytes. It is noteworthy that after isolation from the articular matrix Tob1 was also repressed in normal articular chondrocytes in vitro . This might well reflect the fact that adult articular chondrocytes show an increased proliferative activity and also enhanced anabolic [ 27 ] and catabolic activity [ 7 ] after removal from the tissue. The fact that cells cultured with serum in vitro showed even lower Tob1 expression levels than cultures without serum further supports this notion, because serum is known to increase proliferation of chondrocytes in vitro [ 28 ]. In addition, the chondrocytic cell line HCS-2/8 showed an inverse relationship between proliferative activity and cell differentiation on the one hand and Tob1 expression on the other. Interestingly, fetal chondrocytes in situ selectively express Tob1 in the hypertrophic zone, which is in contrast to other zones where no proliferative activity is seen [ 25 ]. This indicates that Tob1 expression in chondrocytes is inversely related to proliferation in a similar way to that seen in T cells [ 29 ]. Another basic effect of Tob1 is also observed in chondrocytes: a repression of Tob1 is needed before activation of otherwise quiescent T cells [ 29 , 30 ]; similarly, there is a clearcut inverse correlation between (anabolic and catabolic) chondrocyte activity and Tob1 expression. In many respects the downregulation of Tob1 fits well into the scenarios taking place during osteoarthritis (validation level III), which suggests that Tob1 is a potential key molecule of cell phenotype regulation in osteoarthritic chondrocytes. Thus, in osteoarthritic cartilage an increase in proliferation [ 31 - 35 ] is found, whereas hardly any proliferative activity exists in normal articular adult cartilage [ 31 , 32 ]. These cells seem to be G0-arrested, quiescent and phenotypically stable, in other words exactly the cell type that would be expected to express high levels of Tob1 [ 4 , 29 ]. It is noteworthy that both phenotypic instability [ 36 ] and anabolic activation [ 17 ] are key features of osteoarthritic chondrocytes, fitting well to the downregulation of Tob1 . Tob1 seems in many circumstances and, in particular, in skeletal cells to interact with the BMP pathway [ 37 ]. Tob1-knockout mice develop osteopetrosis due to a lack of inhibition of BMP-stimulated bone growth [ 37 ]. In addition, overexpression of Tob1 reduces BMP2 signaling [ 38 ]. Although in Tob1 -knockout mice no specific 'hyperplastic' cartilage phenotype was obvious, BMP-2 and BMP-7 are reported to have important functions in cartilage homeostasis [ 39 , 40 ]. The presence of Tob1 could therefore explain why, despite the presence of BMPs within articular cartilage [ 39 ], normal chondrocytes show only very low anabolic activity. By the same argument, osteoarthritic chondrocytes BMPs might have much more anabolic potential, a feature recently suggested in studies in vitro [ 27 ]. In sum, our study provides for the first time compelling evidence of the expression and presence of Tob1 as a new intracellular mediator in adult articular chondrocytes and its downregulation in the osteoarthritic disease process. Tob1 fits well functionally with the cellular biological changes found in this condition such as proliferation, activation and the loss of a differentiated phenotype. Our data, together with the knowledge from other cellular systems in the literature, suggest that Tob1 is a key molecule in the scenario of cellular alterations of osteoarthritis. Conclusions Oligonucleotide-based microarray analysis was used to screen for differences in gene expression levels in between normal and osteoarthritic chondrocytes. Among other genes, Tob1 was identified as being significantly downregulated in osteoarthritic chondrocytes. Correlative gene expression studies on cellular features such as cell proliferation, cell activation and the loss of a differentiated phenotype suggest that downregulation of Tob1 expression might be an important aspect of cellular processes in osteoarthritic cartilage degeneration. Abbreviations BMP = bone morphogenetic protein; cDNA = complementary DNA; cRNA = complementary RNA; IL = interleukin; MMP = matrix metalloproteinase; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; qPCR = quantitative polymerase chain reaction; UTR = untranslated region. Competing interests The author(s) declare that they have no competing interests. MG, JS, UD, and EB are all employed by Sanofi-Aventis as research scientists. The publication is a result of a scientific collaboration between industry and the other academic authors. The protein Tob1 is not pursued as a project within the osteoarthritis portfolio of Sanofi-Aventis; therefore the industry-affiliated authors have stated that they and the company have no competing interests. Authors' contributions MG performed the gene expression analysis. JS cultured the HCS-2/8 cells and contributed to the bioinformatic analysis of obtained data sets. JH performed the collection and processing of human material (including RNA isolation). UD performed the in situ hybridization analysis. MT contributed the HCS-2/8 cell line. EB participated in the design of the study and coordinated the gene expression experiments including the bioinformatic analysis. TA wrote most of the manuscript and participated in the design of the study. His group contributed the TaqMan, conventional PCR and immunohistochemical analyses (together with JH). All authors contributed to writing and correcting the manuscript and have approved the final version. Supplementary Material Additional File 1 An Excel file that contains details of 200 significantly regulated genes that were differentially expressed between normal and osteoarthritic cartilage with P values <0.01. Click here for file

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1065319

Tapasin gene polymorphism in systemic onset juvenile rheumatoid arthritis: a family-based case–control study

Juvenile rheumatoid arthritis (JRA) comprises a group of chronic systemic inflammatory disorders that primarily affect joints and can cause long-term disability. JRA is likely to be a complex genetic trait, or a series of such traits, with both genetic and environmental factors contributing to the risk for developing the disease and to its progression. The HLA region on the short arm of chromosome 6 has been intensively evaluated for genetic contributors to JRA, and multiple associations, and more recently linkage, has been detected. Other genes involved in innate and acquired immunity also map to near the HLA cluster on 6p, and it is possible that variation within these genes also confers risk for developing JRA. We examined the TPSN gene, which encodes tapasin, an endoplasmic reticulum chaperone that is involved in antigen processing, to elucidate its involvement, if any, in JRA. We employed both a case–control approach and the transmission disequilibrium test, and found linkage and association between the TPSN allele (Arg260) and the systemic onset subtype of JRA. Two independent JRA cohorts were used, one recruited from the Rheumatology Clinic at Cincinnati Children's Hospital Medical Center (82 simplex families) and one collected by the British Paediatric Rheumatology Group in London, England (74 simplex families). The transmission disequilibrium test for these cohorts combined was statistically significant (χ 2 = 4.2, one degree of freedom; P = 0.04). Linkage disequilibrium testing between the HLA alleles that are known to be associated with systemic onset JRA did not reveal linkage disequilibrium with the Arg260 allele, either in the Cincinnati systemic onset JRA cohort or in 113 Caucasian healthy individuals. These results suggest that there is a weak association between systemic onset JRA and the TPSN polymorphism, possibly due to linkage disequilibrium with an as yet unknown susceptibility allele in the centromeric part of chromosome 6.

Introduction Juvenile rheumatoid arthritis (JRA) is the most common chronic arthritic condition of childhood, encompassing pauciarticular, polyarticular, and systemic-onset disease subtypes. JRA is typically considered autoimmune in etiology, with characteristic T-cell abnormalities and chronic synovitis. The extent of synovitis may range from minimal to severe, and vary in terms of number of joints involved, with systemic onset disease typically associated with the greatest morbidity. JRA is probably a collection of diseases with complex overlapping etiologies, with each subtype influenced by multiple genetic susceptibility loci and mediated by environmental effects [ 1 ]. The MHC on the short arm of chromosome 6 has been intensively analyzed, and associations with both HLA and non-HLA genes have been reported. Genetic associations with MHC alleles have been documented primarily within the HLA class II region, but also with certain class I alleles. These associations are largely JRA subtype and age specific [ 2 ], and are strongest for pauciarticular and polyarticular disease [ 1 ]. For systemic onset JRA (SoJRA), associations with HLA-B8 , HLA-Bw35 [ 3 , 4 ] and HLA-DR4 [ 3 , 5 ] have been observed, whereas HLA-DPB1*0401 was reported to have a protective effect in one Caucasian population [ 6 ]. Associations with HLA-DRB1*0401 and HLA-DRB1*0405 have been reported in a Japanese population [ 7 ]. Most of these associations have not been replicated. In the present study we targeted the tapasin gene ( TPSN ), which is in the class II region of the MHC, 180 kilobases centromeric of HLA-DP. The tapasin protein is necessary for the proper assembly and peptide-presenting function of HLA class I molecules [ 8 ]. The TPSN gene has a polymorphism in exon 4 that results in a nonconservative amino acid substitution of Arg/Thr at amino acid 260 (ref SNP ID: rs2071888) [ 9 , 10 ]. Three intronic polymorphisms of TPSN have also been described, none of which appear to be in linkage disequilibrium (LD) with HLA class I alleles or the extended HLA-A1 , HLA-B8 , HLA-DR3 haplotype, in at least one healthy Caucasian population [ 11 ]. Furthermore, using a large UK Caucasian sample, Ahmad and coworkers [ 12 ] recently reported that TPSN polymorphisms are not in LD with more telomeric MHC haplotypes. In the present study we report an association between the exon 4 TPSN polymorphism and susceptibility to SoJRA, involving the TPSN allele Arg260 (01 allele). Methods The study cohort included 88 SoJRA affected families recruited in Cincinnati (US cohort) and 74 simplex (with one affected offspring) SoJRA families identified by the British Paediatric Rheumatology Study Group (UK cohort). Unaffected siblings were available for analysis in the US but not in the UK cohort. An additional 113 healthy unrelated control individuals, primarily Caucasians from the Midwest and New England, resembled the SoJRA-affected families in terms ethnicity and served as the control population. Ethics approvals were obtained from the participating institutions, and informed consent was obtained from parents and/or children. All affected children met American College of Rheumatology criteria for a diagnosis of JRA; they were subgrouped as pauciarticular, polyarticular, or SoJRA. Genomic DNA was purified from peripheral blood cells by standard techniques and analyzed for TPSN alleles (Arg260/01 and Thr260/02) by polymerase chain reaction and restriction site enzyme digestion. Briefly, a 298-base-pair fragment of exon 4 of the TPSN gene containing the polymorphism was amplified and then digested with Bfa I, which recognizes the 01 allele, and Sfc I, which recognizes the 02 allele. The primers used were Tsn 479 forward (5'-CCC ACC CTC TAC CCC TGG A-3') and Tsn 641 reverse (5'-CAG CAC CTG GGT AAG GGA CA-3'). HLA types were determined for a subgroup of the participants using DNA-based low-resolution methodology (Geno-Vision Inc., Exton, PA, USA), and serologically using standard typing sera and microcytotoxicity assays. Preliminary association analysis was conducted by χ 2 testing on contingency tables comparing the three genotypic frequencies between cases and control individuals to yield a χ 2 with two degrees of freedom. Family-based association analysis was performed using the transmission disequilibrium test (TDT). The TDT [ 13 , 14 ] is a family-based association test that compares within a cohort the number of times a particular parental allele is transmitted to an affected offspring versus the number of times it is not transmitted. To allow inclusion of families with missing data for a single parent, the Transmit program [ 15 ], which uses population allele frequencies to weight the possible parental genotypes, was used for the TDT analysis. In the Transmit program, genotypes of unaffected siblings (or siblings whose disease status is unknown) are used to infer parental genotypes, thus increasing the power to detect association. We applied this test first to the combined US and the UK data and then to each set separately. The significance level of the combined results was also calculated using Fisher's method of combining P values for two independent analyses that test the same hypothesis. LD between TPSN and the HLA region (limited to the Cincinnati cohort) was evaluated using the EH program [ 16 , 17 ]. The Geno-Pdt test in the PdT 5.1 program was also used as a test for association and linkage in the US SoJRA-affected families [ 18 - 20 ]. Results The distribution of tapasin genotypes among SoJRA-affected children was compared with that in their healthy siblings, as well as with that in unrelated healthy control individuals using a two degrees of freedom χ 2 test in the US cohort (Table 1 ). The differences did not reach statistical significance. The allelic frequencies of tapasin in the independent cohorts from the USA (Table 1 ) were in Hardy–Weinberg equilibrium (healthy individuals from the USA: χ 2 = 0.3049, P = 0.58; SoJRA-affected individuals from the USA: χ 2 = 2.004, P = 0.156). In the UK data only SoJRA-affected individuals were available for Hardy–Weinberg equilibrium testing, and the result was borderline (χ 2 = 5.26, P = 0.02). We then tested for evidence of linkage of TPSN to JRA by applying the family-based TDT only to the cohorts of affected individuals for whom parental and sibling information was available. These families included 82 US SoJRA familes (389 individuals, including family members) and 74 UK SoJRA families. The TDT test, as implemented in the Transmit program, detected the preferential transmission of the TPSN 01 allele in the UK and US SoJRA families ( n = 156; χ 2 = 4.2, 1 degree of freedom [df]; P = 0.04; Table 2 ). When the US SoJRA cohort was analyzed alone, this preferential transmission for the TPSN allele 01 was even more significant (χ 2 = 6.0, 1 df; P = 0.01; Table 2 ). However, SoJRA families from the UK alone as a subgroup failed to show significant preference for TPSN allele 01 transmission (χ 2 = 0.075, 1 df; P = 0.78; Table 2 ). When the SoJRA UK cohort was analyzed, it was recognized that information for one of the parents was missing in 22 families (29.7% of the total). There was no information from unaffected siblings of the probands for these UK families, which is necessary for the Transmit program to narrow down the range of the possible parental genotypes. When one parent is missing, the Transmit program assigns and weights possible haplotypes to the missing parent using the information from the known parent and the siblings of the proband, and averages the probability of all transmissions to the proband. Because there were no data regarding the genotypes of unaffected siblings for the UK families, the Transmit program was unable to infer parental genotypes and thus had less power to detect the preferential transmission of the 260Arg allele. In the US SoJRA-affected cohort parental information was missing for 32 of the 82 families (39%), but for 12 of these (14.6%) there was information regarding unaffected siblings. Although 24.4% of the cohort was unavailable for TDT calculation, 62 families (75.6%) were available. We combined the two P values using Fisher's method [ 21 ] to obtain a χ 2 with four degrees of freedom and found the combination of the two analyses to be significant (χ 2 = 9.7, 4 df; P = 0.05). Overall, when the two cohorts were analyzed together the tapasin 260Arg allele was transmitted more often than the 260Thr allele, suggesting association and linkage between the TPSN polymorphism and SoJRA. In order to include information from the unaffected siblings for association testing, we also applied an alternative association test to the US SoJRA population – the pedigree disequilibrium test (PDT) [ 18 , 19 ]. A new version of PDT, the genotype-based association test for pedigrees (Genotype-PDT), was applied to the data. Genotype-PDT [ 20 ] tests for linkage and underlying patterns of association at the genotypic level. It is more conservative and has lesser type 1 error when compared with the TDT test implemented in the Transmit program. Genotype-PDT also uses information from affected individuals, unaffected siblings, and their nuclear families. Therefore, we were only able to apply this test to the US SoJRA cohort. The genotype-PDT test revealed association and linkage of the tapasin 260Arg allele with SoJRA at the genotypic level (χ 2 = 6.727, 1 df; P = 0.034) in the US SoJRA cohort. Furthermore, we wished to control for possible transmission distortion of tapasin 260Arg allele in SoJRA-affected families. This allele could also be preferentially transmitted to the unaffected siblings of the SoJRA-affected individuals from their parents, and our statistical significance could be falsely inflated because of allele-specific segregation bias (altered transmission of an allele independent of its role in disease). We therefore applied the TDT test to the unaffected siblings from the US SoJRA cohort. In contrast to the affected siblings, there was no significant preference toward 260Arg allele transmission to healthy siblings, suggesting no segregation bias (χ 2 = 1.043, 1 df; P = 0.3; Table 2 ). These data provide evidence of a genotypic association and linkage between the TPSN 260Arg allele and susceptibility to SoJRA. Discussion Although JRA is the most common rheumatologic disease in childhood, the SoJRA subtype comprises less then 20% of cases and is a rare disease. In the past, because of the small sample sizes, studies conducted by single centers failed to establish strong genetic associations. The present study was therefore done with collaboration between two different centers. These two centers recruited mainly Caucasian families with one SoJRA-affected offspring. An association of the TPSN allele 260Arg with SoJRA was detected when both cohorts were analyzed and in the US cohort by itself. In the UK cohort the statistical analysis did not reveal a significant association. This discrepancy may be due to the limited sample size for the UK data, the different ethnic backgrounds of the two cohorts, and/or gene–environment interactions. In general, it is suggested that studies using independent controls are more powerful than those using related (family-based) controls, but they may be biased if cases and controls have different ethnic backgrounds because of population stratification. Family-based control studies are less powerful because of overmatching, but they are robust to population stratification. In the present study we used family-based control association tests, which allowed us to analyze SoJRA family cohorts recruited by two different centers (US and UK). Statistical programs designed to test genetic linkage based on TDTs (i.e. linkage in the presence of association) calculate the transmission of alleles from heterozygous parents to affected individuals. In the absence of one parent, the family becomes uninformative regarding single nucleotide polymorphisms and cannot be included in the analysis. This decreases the sample size, thus reducing the power to detect association or genetic linkage in rare diseases. Recently, programs such as Transmit and PDT have become available that are designed to calculate the possible genotypes of the missing parent from unaffected children or other family members such as grandparents. However, in cohorts consisting of simplex families (mother, father and the affected child), which do not have unaffected siblings or grandparents, and when there are families with missing parents, these programs are unable to achieve as much power to detect genetic association. The TDT test in the Transmit program was unable to infer the missing parental information (22 families, 29% of the data) from the UK cohort, which decreased the sample size to 52 families (71% of the cohort). In contrast, in the US SoJRA cohort the presence of unaffected siblings made 62 families (75.6% of the cohort) available for testing and increased the power to detect linkage in the presence of association. In order to detect whether the group of children with earlier age at onset of SoJRA (<6 years at onset) is in association with TPSN allele 260Arg, both cohorts were dichotomized by age at disease onset and analyzed using the Transmit program. TPSN allele 260Arg was still preferentially transmitted in both of the age onset groups but there was no statistical significance at the 5% level. When the age at onset groups <6 years and ≥6 years were pooled together from US and UK cohorts and analyzed, there was still no statistically significant association with TPSN allele 260Arg. The other possible reason for the lack of significant linkage in the UK cohort when analyzed alone might be ethnic difference, with a different polymorphism associated with SoJRA and a different disease frequency. Although both cohorts consisted of Caucasians, there might still have been ethnic differences between them. Therefore, it could be that the association of TPSN allele 260Arg with SoJRA in the US population is due to LD, with different SoJRA susceptibility alleles located on chromosome 6 being due to differing recombination processes between the US and UK Caucasian populations. Because weak associations between SoJRA and HLA-DR alleles [ 5 - 7 ] have previously been noted, we compared the available class II allele frequencies, including HLA-DR , HLA-DP and HLA-DQ , in SoJRA patients from the US cohort ( n = 69) with healthy control individuals ( n = 66). No statistically significant differences were found (data not shown). However, it is worth noting that we detected a small trend toward a lower HLA-DPB*0401 frequency in SoJRA patients (28%) as compared with healthy control individuals (35%), which is consistent with a previous report [ 6 ] that suggested a possible protective role for this particular HLA allele in SoJRA. Furthermore, we applied a test for LD (using the EH program) to assess LD between TPSN alleles and the HLA alleles in SoJRA patients. We included 34 SoJRA-affected children from the US cohort and 38 healthy individuals for whom HLA typing data were available. There was no evidence for LD between the TPSN and any of the HLA alleles with the SoJRA-affected individuals or the healthy individuals. Because HLA typing was not available for all patients in the US and UK SoJRA patient groups, these calculations were done using very small sample sizes, and so the possibility of LD between the TPSN and HLA loci in these groups cannot be completely eliminated. The landmark cytokines that contribute directly to the clinical features or autoimmune process of SoJRA, namely IL-6, tumor necrosis factor (TNF)-α, and IL-1, are also known to be important regulators of apoptosis. SoJRA's characteristic clinical and laboratory features, such as fever, skin rash, hypergammaglobulinemia, hypoalbuminemia, elevated erythrocyte sedimentation rate, and fibrinogen levels, may all be explained by cytokine-activated inflammatory and/or immune responses. Elevated blood level of IL-6 in SoJRA is known to correlate with fever episodes [ 22 , 23 ]. Some of these cytokines were evaluated for their associations with SoJRA. The non-MHC cytokine gene polymorphisms that have been associated with SoJRA are the IL-6 5' flanking polymorphism [ 24 ], the TNF-α 5' flanking polymorphism [ 25 ], and the macrophage migration inhibition factor polymorphism [ 26 ]. Recent cytotoxicity studies also implicate natural killer cell dysfunction in this process [ 27 ]. Functional differences between the tapasin proteins encoded by the two alleles (Arg versus Thr at 260) have not, to our knowledge, been described. Given what is known about the function of tapasin, it is conceivable that polymorphisms might affect the quality or quantity of peptides presented by class I molecules, thereby influencing the immune response. It is also worth noting that the TPSN gene is separated from Daxx , an effector of Fas ligand and transforming growth factor-β mediated apoptosis [ 28 , 29 ] by only a single gene ( BING2 ). Apoptosis plays a key role in regulating the immune response in part by balancing excess cellular proliferation, and several of the key cytokines that have been implicated in the pathogenesis of SoJRA, such as TNF-β, IL-6, and IL-1, are known to influence apoptotic pathways. Thus, it is perhaps more tempting to speculate that the TPSN 01 polymorphism (TPSN 260Arg) associated with SoJRA might be in LD with another susceptibility allele in a gene such as Daxx (or other genes in the region that play roles in apoptosis). Furthermore, the TPSN 260Arg allele might be part of a haplotype in the HLA region that contributes to susceptibility to SoJRA. It will be important to examine additional SoJRA populations to determine whether TPSN is associated with disease. If so then further genetic studies of this region, including LD testing and exploration of candidate gene alleles in the region, may be of considerable interest. Conclusion In conclusion, our studies support the existence of a weak association, possibly due to a linked gene in the region, between the TPSN 01 allele and susceptibility to SoJRA. Abbreviations df = degrees of freedom; IL = interleukin; JRA = juvenile rheumatoid arthritis; LD = linkage disequilibrium; MHC = major histocompatibility complex; PDT = pedigree disequilibrium test; SoJRA = systemic onset juvenile rheumatoid arthritis; TDT = transmission disequilibrium test; TNF = tumor necrosis factor. Competing interests The author(s) declare that they have no competing interests. Authors' contributions HB carried out the molecular genetic study in US, genotyped the US cohort, did the statistical analysis of both US and UK cohorts, and drafted the manuscript. MF carried out the molecular study in UK and participated in drafting the manuscript. MT confirmed the genotypes of US SoJRA patients. SDT participated in the coordination of the study and drafting the manuscript. NAT genotyped the UK SoJRA cohort. PW coordinated the UK study and participated in drafting the manuscript for the UK cohort.

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1065320

CXCR3/CXCL10 expression in the synovium of children with juvenile idiopathic arthritis

The accumulation of T cells in the synovial membrane is the crucial step in the pathophysiology of the inflammatory processes characterizing juvenile idiopathic arthritis (JIA). In this study, we evaluated the expression and the pathogenetic role in oligoarticular JIA of a CXC chemokine involved in the directional migration of activated T cells, i.e. IFNγ-inducible protein 10 (CXCL10) and its receptor, CXCR3. Immunochemistry with an antihuman CXCL10 showed that synovial macrophages, epithelial cells, and endothelial cells bear the chemokine. By flow cytometry and immunochemistry, it has been shown that CXCR3 is expressed at high density by virtually all T lymphocytes isolated from synovial fluid (SF) and infiltrating the synovial membrane. Particularly strongly stained CXCR3 + T cells can be observed close to the luminal space and in the perivascular area. Furthermore, densitometric analysis has revealed that the mRNA levels for CXCR3 are significantly higher in JIA patients than in controls. T cells purified from SF exhibit a definite migratory capability in response to CXCL10. Furthermore, SF exerts significant chemotactic activity on the CXCR3 + T-cell line, and this activity is inhibited by the addition of an anti-CXCL10 neutralizing antibody. Taken together, these data suggest that CXCR3/CXCL10 interactions are involved in the pathophysiology of JIA-associated inflammatory processes, regulating both the activation of T cells and their recruitment into the inflamed synovium.

Introduction The trafficking and accumulation of immunocompetent cells are essential components in the pathophysiology of the inflammatory processes. A number of recent data suggest that most of these events are regulated by chemokines, a superfamily of 8–10 kDa molecules that has been divided into four branches (C, CC, CXC, and CXXXC) according to variations in a shared cysteine [ 1 , 2 ]. The current roster approaches more than 50 related proteins. Structural variations of chemokines have been associated with differences in their ability to regulate the trafficking of immune cells during inflammatory disorders. The biological activity of chemokines is mediated by seven-transmembrane-domain, G-protein-coupled receptors classified as C, CC, CXC, or CXXXC chemokine receptors according to the type of chemokine bound. Chemokine receptors are constitutively expressed on some cells, whereas they are inducible on others [ 3 ]. Three CXC chemokines (IP-10/CXCL10, Mig/CXCL9, and I-TAC/CXCL11) that are produced in response to IFNγ allow for the accumulation of activated lymphocytes by interacting with a specific receptor (CXCR3) [ 2 ]. Although the interactions of chemokine receptors are often characterized by considerable promiscuity, CXCR3 is selective in the recruitment of Th1 cells, B cells, and NK (natural killer) cells but not of nonlymphoid cells. Juvenile idiopathic arthritis (JIA) is characterized by chronic inflammation of the synovium in multiple joints. Early studies of the synovial membrane in JIA have shown the presence of a dense infiltrate of activated T cells clustered around activated dendritic cells, suggesting that lymphocyte recruitment is crucial in the pathogenesis of the disease [ 4 , 5 ]. There is also strong evidence of an up-regulation of IFNγ expression in synovial tissue relative to that in peripheral blood of patients with JIA [ 6 , 7 ], indicating a Th1 type polarization of local inflammatory response. Taken together, these data suggest that lymphocyte-specific CXC chemokines could be involved in the mechanisms promoting the development of inflammatory events in JIA patients. In this study, using immunohistochemical and molecular studies of tissue sections and flow cytometry evaluation of cells recovered from synovial fluid, we evaluated the role of CXCR3/CXCL10 interactions in the regulation of T-cell migration into the joints of patients with JIA. We have demonstrated the presence of IP-10/CXCL10 in the synovial tissue and its release into the synovial fluid, where it exerts chemotactic activity toward activated CXCR3 + T cells. Taken together, our data suggest that the local production of CXCL10 is involved in the pathophysiology of JIA-associated inflammatory processes. Materials and methods Study populations We analyzed synovial tissue from nine patients with oligoarticular JIA who were undergoing arthroscopic synovectomy. All the patients fulfilled the revised criteria for JIA according to the International League of Associations for Rheumatology (ILAR) classification [ 8 ] and were managed at the Pediatric Rheumatology Unit of Padua University. The procedure was performed in the case of persistently inflamed joints that did not respond either to systemic anti-inflammatory therapy or to intra-articular steroid injections. In all these patients, gadolinium-enhanced MRI showed marked thickening of the synovial membrane throughout the joint. The patients' mean age at onset of the disease was 70.6 months (range 34–156); the average disease duration at synovectomy was 29.5 months (range 2–60). As controls, three synovial tissue specimens obtained from children with noninflammatory arthropathy were analyzed by immunochemistry. These subjects had presented with either hexadactylism, bone dysplasia, or bone fracture. Paired samples of peripheral blood (PB) and synovial fluid (SF) from 20 consecutive patients undergoing intra-articular steroid injection were examined. These patients' mean age at onset of the disease was 77 months (range 13–264) and the mean disease duration was 17 months (range 2–108). Patients who were having systemic anti-inflammatory treatment at the time were excluded from the study. Since the local ethics committee was not established yet at the beginning of the study, institutional review board approval was not requested, but informed consent was obtained from the parents of all the children included in this study. Phenotypic evaluation of lymphocytes from peripheral blood and synovial fluid The commercially available conjugated or unconjugated monoclonal antibodies used were from the Becton Dickinson (Sunnyvale, CA, USA) and PharMingen (San Diego, CA, USA) series and included CD3, CD4, CD8, CD45R0, CD45RA, and isotype-matched controls. Fluorescein-isothiocyanate-labelled mouse antihuman CXCR3 (R&D Systems Inc, Minneapolis, MN, USA) was also used, and the frequency of PB and SF cells positive for this reagent was determined by flow cytometry as previously reported [ 9 ]. Specifically, cells were scored using a FACSCalibur analyzer (Becton Dickinson) and data were processed using the Macintosh CELLQuest software program (Becton Dickinson). For immunofluorescence analysis, control mouse IgG 1 and IgG 2a were obtained from Becton Dickinson. Chemotactic activity of synovial fluid The CXCR3-positive cell line 300-19 (kindly provided by Dr B Moser, Theodor-Kocher Institute, University of Bern, Switzerland) was used to evaluate the chemotactic activity of SF. The cells were grown in RPMI 1640 medium supplemented with 1% glutamine, 5% human serum, 1% kanamycin, and 100 U/ml human recombinant IL-2. Cells were periodically expanded by restimulation with phytohemagglutinin (1 μg/ml) in the presence of irradiated blood mononuclear cells (10:1 ratio of feeder cells : 300-19 cells) and were used for experiments after a culture period of 10 to 14 days. Cell migration was measured in a 48-well modified Boyden chamber (AC48, Neuro Probe Inc, Gaithersburg, MD, USA). The chamber contains two sections. Chemotactic stimuli were loaded in the bottom section, and cells were put into the top compartment. Polyvinylpyrrolidone-free polycarbonate membranes with 3- to 5-μm pores and coated with fibronectin were placed between the two chamber parts. Only the bottom face of filters was pretreated with fibronectin; this treatment maximizes attachment of migrating cells to filters, increasing their adherence. SF samples or control medium (30 μl) was added to the bottom wells, and 50 μl of 300-19 cells resuspended in RPMI 1640 medium was added to the top wells. The chamber was incubated at 37°C with 5% CO 2 for 2 hours. The membranes were then removed, washed with PBS on the upper side, fixed, and stained with DiffQuik (Dade AG, Düdingen, Switzerland). Cells were counted in three fields per well at magnification ×800. All assays were performed in triplicate. In blocking experiments, cell suspensions were preincubated before chemotaxis assay for 30 min at 4°C with antihuman IP-10 antibodies at 20 μg/ml. In a few experiments, T cells purified from SF were evaluated for their migratory capability in response to CXCL10 (20 ng/ml and 200 ng/ml, R&D Systems). Data are expressed as a migration index, which is the ratio between the number of migrating cells in the presence of the stimulus and that in medium alone. Immunohistochemical analysis Expression of CXCR3 and CXCL10 was detected by immunohistochemistry with anti-CXCR3 and anti-IP-10 antibodies, respectively (R&D Systems). Paraffin-embedded sections (4 μm thick) from patients and controls were used for immunostaining with the standard avidin–biotin complex method (Vectastain ABC kit; Vector Laboratories, Burlingame, CA, USA), as previously described [ 10 ]. Briefly, for the microwave antigen-retrieval procedure, slides were placed in a 2-L glass beaker containing 0.01 mol/L citrate buffer, pH 5.9, and microwaved at full power (800 W for 5 min, three times) before cooling and equilibration in PBS. To neutralize endogenous peroxidase activity, we pretreated slides with 3% hydrogen peroxide for 5 min. Primary antibodies were applied at a concentrations of 1:100 for both antibodies (anti-hCXCR3 monoclonal antibody and anti-hIP-10/CXCL10 polyclonal antibody) for 1 hour in a humidified chamber at 37°C. Immunoreactivity was detected using biotinylated secondary antibodies (1:50 rabbit antigoat and 1:1000 goat antirabbit antibodies diluted in PBS–bovine serum albumin buffer) incubated for 45 min, followed by a 30-min incubation with avidin–peroxidase (1:200) and visualized by a 7-min incubation with the use of 0.1% 3,3'-diaminobenzidene tetrahydrochloride as the chromogen. Thereafter the slides were rinsed and washed with PBS for 5 min, and the sections were counterstained with Mayer's hematoxylin. The last steps were performed at room temperature. Control slides were incubated with Tris-buffered saline containing isotype-matched antibodies instead of the primary antibody; they were invariably negative (data not shown). The intensity of antibody staining was classified as strong, moderate, weak, and negative. Parallel control slides were prepared either lacking primary antibody or lacking primary and secondary antibodies, or were stained with normal sera to control for background reactivity. Immunohistochemistry for the characterization of inflammatory infiltrate, endothelial cells, and synovial cells was carried out using the following monoclonal antibodies CD45 (1:20), CD45RO (1:100), CD20 (1:100), CD68 (1:50), CD4 (1:100), CD8 (1:100), CD31 (1:30) (all from Dako Glostrup, Denmark), and cytokeratin–CAM 5.2 (1:1 Becton Dickinson). The immunoreaction products were developed using the avidin–biotin–peroxidase complex method as described above. Confocal microscopy In order to evaluate the expression of CXCL10 by synovial macrophages, confocal microscopy experiments were performed in three patients with JIA. Paraffined sections were prepared for immunofluorescent labelling. Briefly, primary antibodies against CD68 and IP-10 (diluted 1:50 and 1:1 00, respectively, in PBS with 5 g/L bovine serum albumin and 1 g/L gelatin, respectively) and secondary antibodies (goat antimouse IgG and donkey antigoat IgG) conjugated with Texas red or Alexa 488 (Sigma, Milan, Italy) were used. Double labelling using both antibodies on the same section was performed. Primary antibodies and secondary antibodies were incubated for 1 hour at room temperature. Nuclear staining was carried out with DAPI (4' 6-diamidino-2-phenyindole; Sigma) in PBS. Slides were stored at 4°C and analyzed within 24 hours. As a control, the primary antibody was omitted. Immunofluorescence was observed with a Leica TCS SL spectral confocal and multiphoton system (Leica, Heidelberg, Germany). We used an argon laser at 488 nm in combination with a helium neon laser at 543 nm to excite the green (CD68) and red (IP-10) fluorochromes simultaneously. Emitted fluorescence was detected with a 505–530-nm band-pass filter for the green signal and a 560-nm long-pass filter for the red signal. RT-PCR RNA was extracted from the tissues using TRIzol reagent (Invitrogen, San Giuliano Milanese, Milan, Italy). The concentration of RNA was estimated by spectrophotometer. The RNA was treated with DNase I (Invitrogen) to remove any genomic DNA that might contaminate the RNA preparations. Complementary DNA (cDNA) was prepared using a synthesis kit (SuperScript II DNA Preamplification System; Invitrogen). A cDNA reaction mixture from 0.1 μg of RNA was used for DNA amplification by PCR. A typical amplification reaction included 2 units of Taq polymerase (Takara, Shiga, Japan), 20 pmol of sense and antisense oligonucleotide primers, and 200 μM each of dATP, dCTP, dGTP, and dTTP. Amplification was carried out for 30 cycles: 1 min at 92°C, 1 min at 55°C, and 1 min at 72°C. The amplified DNA was electrophoresed on a 2% agarose gel (Invitrogen), stained with ethidium bromide, visualized under ultraviolet light, and photographed. The primer sequences used were as follows: for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-TCC-ATG-ACA-ACT-TTG-GTA-TCG-3' (sense) and 5'-GTC-GCT-GTT-GAA-GTC-AGA-GGA-3' (antisense); for CXCR3, 5'-TTG-ACC-GCT-ACC-TGA-ACA-TA-3' (sense) and 5'-ACG-TCT-ACC-CTG-CTT-TCT-CG-3'. The expected sizes for the cDNA amplicons were as follows: 376 bp for GAPDH, 377 bp for IP-10, and 456 bp for CXCR3. All assays were performed in triplicate. The number of cycles (30) was chosen to ensure that the amount of products synthesized was proportional to the amount of specific mRNA in the original preparation. After PCR amplification, PCR products (15 μl) were subjected to electrophoresis on 2% agarose gels containing 0.03 μg/ml ethidium bromide. The quantification of transcript level was carried out by scanning photographs of gels and analyzing the area under peaks, using Quantity one Biorad software. Levels of mRNA expression were normalized by calculating them as a percentage of 3GAPDH mRNA expression levels [ 11 ]. The band intensity for 3GAPDH did not differ significantly between experiments. Statistical analysis Data were analyzed with the assistance of the Statistical Analysis System. Data are expressed as means ± standard deviation. Mean values were compared using the ANOVA test. Results Immunohistochemical analysis of the expression and cellular distribution of CXCL10 in the synovial membrane during JIA Immunohistochemical analysis was used to investigate the pattern of expression of this chemokine in synovial membranes from nine children with JIA and three age-matched controls. All the JIA synovial tissues showed moderate or strong staining for CXCL10 (Table 1 ). As shown in Fig. 1a and, at higher magnification, in Fig. 1b , CXCL10 was demonstrated on the surface of three types of cells, specifically macrophages, epithelial cells, and endothelial cells, as determined by cell morphology. Most of the IP-10-expressing cells were macrophages. Matched controls revealed no CXCL10 staining (Fig. 1c,d ). In order to verify whether macrophages express CXCL10 morphology, data were confirmed by the use of confocal microscopy. As shown in Fig. 2 , double staining with CD68 and CXCL10 clearly demonstrated that CD68 + macrophages showed an intense coexpression of the chemokine. CXCL10 is present in synovial fluid from patients with JIA and mediates chemotactic activity To evaluate if CXCL10 is released into the SF and is capable of inducing T-cell migration, the chemotactic activity of supernatants from the SF of four patients with JIA was tested on a T-cell clone expressing high levels of CXCR3 (300-19). As shown in Fig. 3 , SF of all the patients we studied exerted significant chemotactic activity on the CXCR3 + T-cell line. The addition of an anti-CXCL10 neutralizing antibody (α CXCL10) but not of a control antibody inhibited chemotactic activities, suggesting the presence of IP-10/CXCL10 in SF and its responsibility in the chemotaxis of CXCR3 + cells. In a second set of experiments, T cells purified from SF exhibited a definite migratory capability per se, which was significantly enhanced in response to CXCL10. Two representative experiments are represented in Fig. 4 . Immunohistochemical and flow cytometry analysis of the expression of CXCR3 by PB, SF, and synovial-tissue T lymphocytes in JIA The possibility that CXCL10 in synovial fluid and membrane might account for the recruitment of CXCR3 + T lymphocytes from the bloodstream to the synovium was investigated by immunohistochemical analysis of the expression of this chemokine receptor. All the JIA patients showed CXCR3-expressing lymphocytes infiltrating the synovium, with strong or moderate staining intensities (see Table 1 ). Particularly strongly stained cells were observed close to the perivascular area (as in Fig. 5a,b , showing two different magnifications of the same slide). In a few cases, a follicular pattern of strongly marked lymphocytes was visible close to the luminal space (Fig. 6 ). The control synovial tissues revealed no CXCR3 staining (Fig. 5c,d ). Densitometric analysis showed that CXCR3 mRNA levels were significantly higher in patients with JIA than in controls (CXCR3:GADPH ratio 2.25 ± 1.8 vs 0.6 ± 0.49, P < 0.05) (Fig. 7 ). Flow cytometry analysis confirmed the selective recruitment of CXCR3 + lymphocytes into the synovium. We analyzed paired samples of PB and SF from 20 children with JIA, and in 18 of these patients, T lymphocytes isolated from the SF showed greater expression of CXCR3 with than did those from PB, both in terms of percentage of positive cells and of the MFI ( P = 0.01) (Table 2 ). Flow cytometry profiles for one representative patient are shown in Fig. 8 . Taken together, these results strongly suggest a role for the CXCL10 released into the synovial compartment in the accumulation of its selective CXCR3-receptor expressing T cells. Discussion JIA is characterized by a persistent accumulation in the synovial membrane of T lymphocytes most of which express surface markers indicative of activation, such as CD45RO, and a type-1 cytokine profile [ 4 , 5 ]. The cellular infiltrate is defined largely by the composition of locally produced chemokines as well as by the diversity of circulating leukocytes expressing the relevant receptors. Our principal findings are that in JIA, CXCL10/IP-10 is strongly expressed in synovial membranes and is released into synovial fluid (SF), where it exerts a definite chemotactic activity on CXCR3 + T-cell clones and on T cells purified from SF; and that there is an accumulation of CXCR3 expressing T lymphocytes from the bloodstream to the synovial fluid and membrane. These findings suggest a role for CXCL10 in the mechanism of T-cell activation and recruitment into the inflamed synovium. The high expression of CXCR3 by T cells retrieved from the synovia of patients with JIA might be considered a by-product of the in vivo cell hyperactivity of the tissue T-cell compartment in this disease. In fact, recent data clearly indicate that CXCR3 and its ligands become functional on recently activated T cells [ 12 ]. After antigenic challenge or in response to stimulation through the T-cell receptor (TCR), T cells express CXCR3, respond with chemotaxis to CXCR3 ligands, and produce IFNγ. Furthermore, in the presence of persistent antigenic stimulations, CXCR3 expression is maintained and poised for rapid up-regulation with reactivation. We and other authors have previously shown that CXCR3/CXCL10 interaction is involved in the pathogenesis of other Th1-mediated processes, such as Crohn's disease and sarcoidosis [ 13 , 14 ]. A similar sequence of events could take place in the synovia of children with JIA. In fact, as previously reported [ 15 ], the evaluation of the molecular organization of the TCR revealed that T cells proliferating in children with JIA show a preferential usage of definite TCR gene regions, indicating an ordered immune response in which a specific TCR has been triggered and CXCR3 expression is induced [ 16 ]. CXCL10 was expressed by macrophages in synovial membrane of patients with JIA but not of controls. This finding suggests that CXCL10 is part of the matrix of cytokines that regulates the accessory activity of macrophages at sites of inflammatory lesions in the synovial microenvironment. Since large amounts of type 1 inflammatory cytokines, such as IFNγ, tumor necrosis factor α, IL-15, and IL-18, have been detected in JIA synovium [ 7 ], it is likely that these cytokines act in concert, sustaining the local proinflammatory responses and up-regulating CXCL10 expression. In turn, since CXCL10 is known to be capable of up-regulating cytokine synthesis in human Th1 cells, it is likely that macrophage-derived chemokines as IL-18 and IL-15 could participate in the maintenance of the default Th1/Tc1 polarization seen during JIA inflammation. It should be noted that, as shown in Fig. 2 , anti-IP-10 almost completely inhibited the migration of the CXCR3 + 300-19 T cells in response to synovial fluid. Given the ability of I-ITAC/CXCL11 and Mig/CXCL10 to favor T-cell recruitment [ 17 ], we are currently investigating whether this non-ERL chemokine may influence entry of T cells into the JIA synovia. It remains to be established whether synovial endothelial cells express CXCL10 (Fig. 1a ). In a previous report it has been shown that human umbilical-vein-derived endothelial cell monolayers stimulated with IFNγ and tumor necrosis factor α produce IP-10/CXCL10, retaining it on their surface, and that this leads to a rapid adhesion of T lymphocytes. This effect was drastically reduced by anti-CXCR3 monoclonal antibody [ 18 ]. Furthermore, it is known that unstimulated human umbilical-vein-derived endothelial cells are able to retain IP-10 added exogenously, through binding to cell-surface proteoglycans [ 19 ]. Finally, recent data have definitively demonstrated that human endothelial cells may express a previously unrecognized receptor for CXC chemokines named CXCR3B and derived from an alternative splicing of the CXCR3 gene [ 20 ]. This receptor shows higher affinity for CXCL10 than classic CXCR3, mediates the inhibition of endothelial-cell growth, and accounts for the known angiostatic capability of CXCL10. Thus, it is possible that nonspecific binding of IP-10 may be responsible for the CXCL10 positivity we observed on endothelial cells. Further studies are in progress to determine whether synovial endothelial cells express CXCR3B in vivo and, if this be the case, to determine the putative role of CXCR3B/IP-10 interactions on the balance of angiogenic/angiostatic events in the JIA synovia. Previous studies on chemokines and their receptors in modulating the recruitment of leukocytes at the sites of inflammation suggested that targeting these molecules with engineered agents might have therapeutic utility in down-modulating inflammatory responses. Results of CXCR3 or IP-10/CXCL10 blockade have already been reported in animal models. Recently, some authors have shown a rapid and marked improvement of adjuvant-induced arthritis in rats treated with IP-10 DNA vaccine [ 21 ]. Moreover, anti-mCXCR3 neutralizing antibodies were found to inhibit Th1 lymphocyte recruitment to peripheral inflammatory sites in a mouse model [ 22 ]. Further studies are needed in animal models to explore the therapeutic potential of CXCR3- or CXCL10-antagonists, with the ultimate goal of offering new clues for immune intervention in Th1-mediated diseases such as JIA and rheumatoid arthritis. Conclusion Our results provide the first evidence of the functional role of CXCR3/CXCL10 interactions in mediating recruitment of T cells at sites of synovial inflammation in JIA. An in-depth molecular study of mechanisms regulating overexpression of CXCR3/CXCL10 might help in defining the role of these molecules in synovial inflammatory responses, offering new insights into elements controlling the immune response within joints. Abbreviations cDNA = complementary DNA; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; IFNγ = interferon γ ; IL = interleukin; JIA = juvenile idiopathic arthritis; PB = peripheral blood; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; RT-PCR = reverse transcriptase PCR; SF = synovial fluid; TCR = T-cell receptor; Th1 = T helper cell type 1. Competing interests The author(s) declare that they have no competing interests. Authors' contributions GM conceived and coordinated the study and drafted the manuscript. FZ participated in the design of the study. FC performed the immunohistochemistry and helped to draft the manuscript. MB and MF carried out the chemotaxis. AC performed the flow cytometry experiments. MV participated in the immunohistochemistry. FZ participated in the design of the study. CA conceived the study and helped in the draft of the manuscript. All authors read and approved the final manuscript

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1065321

Arthritis imaging using a near-infrared fluorescence folate-targeted probe

A recently developed near-infrared fluorescence-labeled folate probe (NIR2-folate) was tested for in vivo imaging of arthritis using a lipopolysaccharide intra-articular injection model and a KRN transgenic mice serum induction mouse model. In the lipopolysaccharide injection model, the fluorescence signal intensity of NIR2-folate ( n = 12) and of free NIR2 ( n = 5) was compared between lipopolysaccharide-treated and control joints. The fluorescence signal intensity of the NIR2-folate probe at the inflammatory joints was found to be significantly higher than the control normal joints (up to 2.3-fold, P < 0.001). The NIR2-free dye injection group showed a persistent lower enhancement ratio than the NIR2-folate probe injection group. Excessive folic acid was also given to demonstrate a competitive effect with the NIR2-folate. In the KRN serum transfer model ( n = 4), NIR2-folate was applied at different time points after serum transfer, and the inflamed joints could be detected as early as 30 hours after arthritogenic antibody transfer (1.8-fold increase in signal intensity). Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results. We conclude that in vivo arthritis detection was feasible using a folate-targeted near-infrared fluorescence probe. This receptor-targeted imaging method may facilitate improved arthritis diagnosis and early assessment of the disease progress by providing an in vivo characterization of active macrophage status in inflammatory joint diseases.

Introduction Rheumatoid arthritis (RA) is a common chronic inflammatory and destructive arthropathy that consumes substantial personal, social, and economic costs. The synovial membrane in patients with RA is characterized by hyperplasia, by increased vascularity, and by an infiltration of inflammatory cells, including activated macrophages [ 1 ]. Activated macrophages presenting in large numbers of arthritic joints play an active role in RA [ 2 ] and other inflammatory diseases [ 3 ] by producing cytokines that drive subsequent inflammatory reaction. Folate receptor (FR) is a 38-kDa glycosyl phosphatidylinositol-anchored protein that binds the vitamin folic acid with high affinity (< 1 nM) [ 4 , 5 ]. With the exception of the kidney and the placenta, normal tissues express low or undetectable levels of FR [ 4 ]. Previously it has been reported that FR has three isoforms: FR-α, FR-β, and FR-γ. Among them, FR-β, a nonepithelial isoform of FR, is expressed on activated synovial macrophages but not on resting synovial macrophages [ 6 ]. Folate derivatization might therefore be exploited to target activated macrophages involved in inflammatory joint disease. Turk and colleagues [ 7 , 8 ] have recently used folate- 99m Tc for assaying the participation of activated macrophages in an adjuvant-induced arthritis model, and have shown that folate- 99m Tc selectively targets activated macrophages. This suggests that folate-linked imaging agents warrant further scrutiny as possible tools for evaluating arthritis. A newly synthesized folic acid and near-infrared fluorochrome conjugate (NIR2-folate) was recently used as a FR-targeting imaging probe in vivo [ 9 , 10 ]. Fluorescence in the near-infrared spectrum (700–900 nm) was used for in vivo imaging because it allows efficient photon migration through the tissues and has minimal autofluorescence [ 11 ]. The use of near-infrared fluorescent (NIRF) in vivo imaging probes has been shown to significantly enhance tumor detection [ 12 - 15 ], to facilitate identification of small preneoplastic lesions [ 16 ], and to allow objective assessment of new therapeutic paradigms [ 17 ] in animal studies. The NIRF imaging technology has recently been extended to arthritic studies. In vivo NIRF imaging of arthritis in experimental animals was demonstrated using a protease-sensitive probe and NIRF-labeled antibody [ 18 - 21 ]. The goal of the present study is to determine whether a fairly abundant FR on activated macrophages in the arthritic inflammatory process could serve as a target for NIRF-enhanced optical imaging. Materials and methods Imaging probe The folate-targeting optical probe NIR2-folate, consisting of a near-infrared fluorochrome (NIR2) and folic acid, was synthesized and characterized as previously described [ 9 , 10 ]. Briefly, folic acid was first reacted with 2,2'-(ethylenedioxy) bis(ethylamine) using di-isopropylcarbodimide as the coupling agent in dimethyl sulfoxide. The N -hydroxysuccinimide-activated ester of NIR2 [ 22 ] was then coupled with the amino-derivatized folic acid. The final conjugate was purified by C-18 reverse-phase HPLC and confirmed by mass spectroscopic analysis. The NIR2-folate has an excitation wavelength maximum at 662 nm and an emission wavelength maximum at 686 nm. Animal preparation and arthritis models All animal studies were approved by the Institutional Animal Care Committee. Carbon dioxide inhalation was used for euthanasia. C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME, USA) weighing 19–21 g, 12 weeks old, were handled in accordance with government guidelines. Lipopolysaccharide (LPS) intra-articular injection and KRN transgenic mice serum transfer served as two mice arthritis models in this study. The LPS induction arthritis model was achieved according to published protocols [ 23 , 24 ]. Mice were anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) intraperitoneally, and then LPS (Sigma, St Louis, MO, USA), 10 μg in 20 μl saline, was injected intra-articularly into the right ankle joint through the Achilles tendon using a 30-gauge needle. As a control, the same volume of normal saline was injected in the opposite ankle joint of the same animal. The KRN transgenic mice were a gift from Dr D Mathis and Dr C Benoist (Joslin Diabetes Center, Boston, MA, USA). Blood was obtained from arthritic adult KRN mice, and the sera containing arthritogenic autoantibodies were pooled [ 18 , 25 , 26 ]. One hundred micoliters of KRN mice serum were intravenously injected into healthy C57BL/6 mice, and the NIR2-folate probe was then given at different time points after serum transfer to detect early inflammatory changes. Experimental groups In the LPS induction model, the three experimental groups of animals were injected intravenously with NIR2-folate probe (2 nmol per animal, n = 12), with free NIR2 (2 nmol per animal, n = 5), or with 600-fold of folic acid (1200 nmol per animal) 5 min prior to NIR2-folate probe injection (2 nmol per animal, n = 5) to demonstrate the competition effect of free folic acid against the probe. In the KRN serum transfer model, four animals were intravenously injected with 100 μl KRN serum and the NIR2-folate probe was given 24 hours ( n = 1) or 96 hours ( n = 3) after serum transfer. In vivo NIRF reflectance imaging and lesion assessment All animals were imaged in a prone position using a home-built NIRF reflectance imaging system, which has been described elsewhere [ 27 ]. For fluorescence acquisition, a 615–645 nm excitation filter and a 680–720 nm emission filter (Omega Optical, Brattleboro, VT, USA) were used. Images were analyzed using commercially available software (Digital Science 1D software; Kodak, Rochester, NY, USA). Following data acquisition, postprocessing and visualization were performed using the in-house program CMIR Image. The enhancement ratio of the inflamed joint was used to demonstrate the effectiveness of the probe, which was defined by the fluorescence signal intensity (SI) at the affected ankle joint divided by the fluorescence SI at the opposite ankle joint. NIRF images were acquired preinjection and postinjection at different time points. Histology, immunohistochemistry, and immunofluorescent microscopy assessment Ankles were excised and fixed in phosphate-buffered formalin for 24 hours, and were subsequently decalcified in 10% EDTA for 48 hours, paraffin embedded, cut into 8-μm sections, and stained with H&E. Immunohistochemistry was performed using an anti-activated macrophage antibody [ 28 ] (Mac-3, 1:500 dilution, rat anti-mouse monoclonal antibody; BD Biosciences, San Diego, CA, USA) and a goat anti-human folate receptor polyclonal antibody (sc-16387, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), revealed with biotinylated rabbit anti-rat and donkey anti-goat secondary antibodies (1:250 dilution; Santa Cruz Biotechnology). The staining procedure was performed with a modified avidin–biotin–peroxidase complex technique. The slides were visualized with a chromogen of diaminobenzidine (Vectastain; Vector Laboratories, Burlingame, CA, USA). Sections were counterstained with hematoxylin (Vector Laboratories). Positive immunoreactions appeared as dark brown staining on a blue background. Control sections were processed identically but with incubation of the nonspecific isotype immunoglobulin (Vector Laboratories). Immunofluorescence staining was performed using Mac-3 rat anti-mouse monoclonal antibody (1:500 dilution) and FITC-conjugated anti-rat secondary antibody (1:250 dilution; Vector Laboratories). The inflamed ankles were cut into 10-μm thick slices using a Leica CM 1900 cryotome (Leica, Bannockburn, IL, USA). Slices were analyzed using an inverted epifluorescence microscope (Axiovert; Zeiss, Thorn-Wood, NY, USA). FITC and Cy5.5 channels were used for Mac-3 and NIR2-folate fluorescence signal detection. A cooled CCD camera (Sensys; Photometrics, Tucson, AZ, USA) adapted with a bandpass filter was used for image capture, and IPLab software (Scanlytics, Fairfax, VA, USA) was used for image analysis. Statistical analysis Data are presented as the mean and standard error of the mean. Statistical analysis of the fluorescence SI and the enhancement ratio between different groups was conducted using a two-tailed paired Student t test. The paired Student t test was used for analyzing the SI difference between bilateral ankles in the same mouse. P < 0.05 was considered to indicate a statistically significant difference. All statistics were analyzed using Stata 7.0 (Stata, College Station, TX, USA) for Windows (Microsoft, Redmond, WA, USA). Results Establishment of a LPS-induced arthritis model Progressive discoloration and swelling of the ankle joints was noted 24 hours after LPS intra-articular injection. Abundant polymorphonuclear cell infiltration was noted in the synovial lining layer and the subsynovial adipose tissue in histologic sections 48 hours after LPS injection. Immunohistochemistry revealed Mac-3-positive and FR-positive cells scattered among polymorphonuclear cells and subsynovial tissues in adjacent tissue sections (Fig. 1 ). These findings indicate that arthritis can be induced by LPS, and that the presence of active macrophages within inflammatory tissues can be used as a target for the NIR2-folate probe. NIRF imaging of a LPS-induced mice arthritis model The NIR2-folate probe was injected 48 hours after LPS induction ( n = 12). The fluorescence SI of the inflamed joints was significantly higher than the opposite ankle joint at 2 min, and 12, 24, 48, and 72 hours after probe injection (468 ± 51 arbitrary units [AU] versus 303 ± 33 AU, 400 ± 31 AU versus 181 ± 18 AU, 310 ± 18 AU versus 137 ± 8 AU, 209 ± 14 AU versus 111 ± 7 AU, and 144 ± 14 AU versus 80 ± 4 AU; P < 0.001 in all sets) (Fig. 2 ). There was no significant difference in the preinjection fluorescence SI in bilateral ankle joints (85 ± 6 AU versus 82 ± 7 AU, P > 0.05). The average enhancement ratio of the inflamed joint was up to 2.3-fold in the first 12 and 24 hours after probe injection, and remained at 1.8-fold 72 hours after probe injection (Fig. 3 ). In comparison, the NIR2-free dye group ( n = 5) showed a persistent lower enhancement ratio than the probe injection group at all time points (Fig. 3 ). The average enhancement ratios of the inflamed ankles in the NIR2-free dye group and the NIR2-folate group at 24-hour, 48-hour, and 72-hour time points were 1.6 ± 0.1 versus 2.3 ± 0.1, 1.3 ± 0.1 versus 1.9 ± 0.1, and 1.3 ± 0.03 versus 1.8 ± 0.1 ( P < 0.05), respectively. To understand the possible mechanism, folic acid was used to compete with the probe. In the folic acid competition study ( n = 5), 600-fold folic acid (1.2 μm per animal) was given intravenously 5 min before the NIR2-folate injection. The enhancement ratio of the arthritic joint in the folic acid competition group was significantly lower than that of the NIR2-folate injection group (1.1 ± 0.1 versus 1.6 ± 0.1, P < 0.05). Colocalization of NIRF signal with Mac-3 immunofluorescence Immunofluorescence of the LPS-treated arthritic joint showed scattered Mac-3-positive cells in the inflammatory tissues in the FITC channel (Fig. 4a ), whereas NIR2-folate uptake cells were seen in the near-infrared channel using an inverted epifluorescence microscope (Fig. 4b ). In the superimposed image (Fig. 4c ), the Mac-3-positive cells colocalized well with NIR2-folate uptake cells. Establishment of a KRN serum transfer mice arthritis model There was no visible swelling or discoloration at peripheral joints in the first 2 days after KRN serum transfer. Progressive discoloration and swelling of the peripheral joints was noted 3 days after serum transfer in sick KRN mice (Fig. 5a ). In histological sections, Mac-3-positive cells intermingled among polymorphonuclear cells, and pannus formation was noted in the affected joints (Fig. 5b,c ). NIRF imaging of a KRN serum transfer mice arthritic model NIR2-folate was first given intravenously 4 days after KRN mice serum transfer. At this time point, discoloration and swelling of the affected peripheral joints was clearly observed (Fig. 5a ). An intense fluorescence signal was found in peripheral joints (Fig. 5d ). The NIR signal of the affected joints was 1.5-fold to 3.5-fold (average, 2.4-fold) higher than that of the unaffected joints. To evaluate its ability for early detection of the inflammatory process, NIR2-folate was then given intravenously at a much earlier time point – 24 hours after serum transfer. No gross swelling or discoloration at peripheral joints could be observed (Fig. 6a ). Six hours after the NIR2-folate probe injection (30 hours after serum transfer), however, the NIRF reflectance imaging showed a 1.8-fold increase in the fluorescence signal at the right wrist joint as compared with the opposite site (Fig. 6b ). The correlated histology showed an increased amount of inflammatory cells at the affected joint compared with the opposite wrist (Fig. 6c,d ). Abundant Mac-3-positive cell infiltration at the right wrist joint region was also revealed by immunohistochemistry (Fig. 6e ). Discussion Activated macrophages are thought to be intimately involved in the pathogenesis of RA by directly destroying articular tissue, secreting metalloproteinases, and attracting or activating other immune cells via the release of cytokines [ 2 , 29 ]. The quantitation of activated macrophages in joint tissues might consequently be of diagnostic value because activated macrophage content correlates well with articular destruction and poor disease prognosis in humans [ 2 , 30 ]. Because FR expression may coincide with macrophage activation [ 6 ], we hypothesized that arthritic joints could be imaged using folate-derivatized fluorescent imaging agents. The present studies demonstrated that the folate-targeted NIRF probe can indeed selectively target activated macrophages in vivo , and that folate-linked imaging agents can facilitate the noninvasive analysis of inflammatory activity in situ . Two different animal arthritis models were used in this study. The LPS induction model was established by intra-articular injection of LPS, which induces transient synoviocyte hyperplasia and polymorphonuclear cell infiltration [ 23 , 24 , 31 , 32 ]. The advantage of the LPS induction model is that the opposite ankle joint could be used as an internal control, thus demonstrating the effectiveness of the probe in statistical analysis. The entity of this model, however, is a bacterial toxin-induced arthritis that resembles pyogenic arthritis instead of RA. The second model was established by transferring serum of sick KRN mice into healthy B6 mice, which induces synovial polymorphonuclear cells and macrophage infiltration by arthritogenic immunoglobulins [ 18 , 26 , 33 ]. The KRN serum transferred model resembles human RA because both are chronic symmetric joint diseases with pannus formation and destructive bone and cartilage erosion, predominantly of the distal joints. The enhancement ratio of inflamed joints in the LPS model was slightly increased in the NIR2-free dye injection group during the first 24 hours after NIR2 injection. This might be due to nonspecific phagocytosis by activated macrophages, or due to NIR2-free dyes pooled at the interstitial space because of increased vascular permeability at the inflammation tissues. However, the enhancement ratio of the inflammatory joints in the NIR2-folate injection group was significantly higher than that of NIR2 injection group, which was more prominent 48 hours after injection (Fig. 3 ). Most of the NIR2-free dye began to be washed out from the inflamed joints, but NIR2-folate remained at the inflamed joints 72 hours after injection. The data indicate that the NIR2-folate probe has significant advantages over nonspecific fluorochromes for in vivo imaging, the latter often being used for nontargeted image enhancement [ 34 , 35 ]. Histological colocalization of the infiltrated Mac-3-positive and FR-positive cells was found to correlate well in the inflammatory tissues (Fig. 1 ). The NIR2-folate uptake cells colocalized with Mac-3-positive cells using fluorescence microscopy (Fig. 4 ), which indicates that uptake of folate conjugates at inflammatory joints is mediated by activated macrophages. In addition, the in vivo competition study confirmed that free folate was able to compete with the NIR2-folate probe for FR binding. The average enhancement ratio of arthritic joints in the folic acid competition group was significantly lower than in the NIR2-folate group postadministration. The results support the fluorescent probe uptake being receptor dependent. Another important finding of this study is the potential of applying this technique in early assessment of RA. Our results indicate that the folate-linked NIR fluorescence probe could detect mild inflammatory changes as early as 30 hours after arthritogenic antibody transfer, before any morphological changes can be observed. A sensitive imaging modality for assessment of early events in RA could provide valuable information for diagnosis and treatment [ 36 ]. 99m Tc-folate has recently been used to assay the participation of activated macrophages in adjuvant-induced arthritis mice models using gamma scintigraphy as the imaging modality [ 7 ]. In contrast, optical imaging is a noninvasive method and does not depend on radiolabeled contrast agents such as those in nuclear medicine; there is thus no exposure of the patient to ionizing radiation. The present hindrance of optical imaging is that tissue penetration of light in living tissue may attenuate the SI. The near-infrared fluorescence probe allows the most efficient photon migration through the tissues [ 11 ]. In addition, there is less soft tissue around peripheral joints, which gives the near-infrared optical imaging a competitive role in the diagnosis of peripheral joint disease, especially in detection of early arthritis or assessment of treatment effects. Conclusions The results indicate that it is feasible to image the activated macrophage status in inflamed joints in vivo at an early stage. The FR-targeting probe not only offers better assessment at early stages in inflammatory disease, but also improves the evaluation of future anti-inflammatory treatments. This technique may therefore represent a step toward the level of molecular diagnosis of arthritis. Abbreviations AU = arbitrary units; FITC = fluorescein isothiocyanate; FR = folate receptor; H&E = hematoxylin and eosin; HPLC = high-performance liquid chromatography; LPS = lipopolysaccharide; NIRF = near-infrared fluorescent; RA = rheumatoid arthritis; SI = signal intensity. Competing interests The author(s) declare that there are no competing interests. Authors' contributions WC and CT participated in all experimental design, data collection and analysis, and drafted the manuscript. UM participated in the KRN experiments and drafted the manuscript. RW participated in the design and helped to draft the manuscript. All authors read and approved the final manuscript.

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1065322

The role of regulatory T cells in antigen-induced arthritis: aggravation of arthritis after depletion and amelioration after transfer of CD4+CD25+ T cells

It is now generally accepted that CD4 + CD25 + T reg cells play a major role in the prevention of autoimmunity and pathological immune responses. Their involvement in the pathogenesis of chronic arthritis is controversial, however, and so we examined their role in experimental antigen-induced arthritis in mice. Depletion of CD25-expressing cells in immunized animals before arthritis induction led to increased cellular and humoral immune responses to the inducing antigen (methylated bovine serum albumin; mBSA) and autoantigens, and to an exacerbation of arthritis, as indicated by clinical (knee joint swelling) and histological scores. Transfer of CD4 + CD25 + cells into immunized mice at the time of induction of antigen-induced arthritis decreased the severity of disease but was not able to cure established arthritis. No significant changes in mBSA-specific immune responses were detected. In vivo migration studies showed a preferential accumulation of CD4 + CD25 + cells in the inflamed joint as compared with CD4 + CD25 - cells. These data imply a significant role for CD4 + CD25 + T reg cells in the control of chronic arthritis. However, transferred T reg cells appear to be unable to counteract established acute or chronic inflammation. This is of considerable importance for the timing of T reg cell transfer in potential therapeutic applications.

Introduction Rheumatoid arthritis (RA) is the most common autoimmune disease in humans, affecting 1% of the population in western countries. Histologically, RA is characterized by hyperplasia and infiltration of the synovial membrane with mononuclear cells, development of an aggressive tissue called pannus and secretion of proteases, which are responsible for the destruction of articular cartilage and adjacent bone. It is well established that macrophages and synovial fibroblasts are effector cells of joint destruction, and it is presumed that autoreactive CD4 + T cells are involved in their activation [ 1 ]. There is now a large body of evidence that, in rodents, regulatory T cells (T reg ) actively control the activation of autoreactive T cells and thus maintain immunological self-tolerance. Apart from adaptive T reg cells, which can be induced by antigen-specific stimulation of conventional peripheral T cells under tolerogenic conditions (for review [ 2 ]), there is no doubt that naturally occurring T reg cells exist in healthy mice as well as in humans and rats, and these are characterized by constitutive expression of CD25 [ 3 - 5 ]. Absence of these cells in vivo results in a multi-organ autoimmune syndrome [ 3 , 6 ]. These CD4 + CD25 + T reg cells leave the thymus as committed 'professional' suppressor T cells [ 7 - 9 ], proliferate in the periphery, and acquire an effector/memory-like phenotype [ 10 ]. In unmanipulated mice, T reg cells can also be found in the CD25 - compartment, based on the expression of the integrin α E β 7 [ 10 , 11 ], possibly reflecting differences in developmental stages of these cells. The exact role played by naturally occurring CD4 + CD25 + T reg cells in the pathogenesis of arthritis remains controversial. Arthritis is part of the autoimmune syndrome induced by transfer of CD25-depleted splenocytes into lymphopenic hosts [ 3 ], and CD4 + CD25 + cells are protective in collagen-induced arthritis [ 12 ]. However, Bardos and coworkers [ 13 ] ruled out a role for naturally occurring CD4 + CD25 + T reg cells in proteoglycan-induced arthritis. To clarify this issue, we used the antigen-induced arthritis (AIA) model. AIA is a Tcell-dependent experimental arthritis that is induced by intra-articular injection of antigen (methylated bovine serum albumin [mBSA]) into knee joints of preimmunized mice [ 14 , 15 ]. This results in an acute inflammatory reaction, which is characterized by exudation of neutrophils and fibrin, which later proceeds to a chronic arthritis with synovial hyperplasia, infiltration of mononuclear cells, and cartilage and bone destruction – histopathological changes similar to those that occur in RA. Autoimmune responses against cartilage constituents such as collagen types I and II and proteoglycans are involved in rendering the disease chronic [ 16 , 17 ]. Beyond the 100% incidence of arthritis, another major advantage of the AIA model is that the time point of induction of arthritis is known, allowing manipulation of CD4 + CD25 + T reg cell number in vivo at defined stages in the disease. Using depletion of CD25-expressing cells or transfer of CD4 + CD25 + cells, in the present study we demonstrated that T reg cells modulate the onset of AIA but are ineffective at later stages, calling into question their value as a new therapeutic approach to established chronic arthritis. Methods Animals, arthritis induction and assessment For all animal experiments, female C57Bl/6 mice (Charles River, Sulzfeld, Germany; age range 6–10 weeks) were used. Animals were kept under standard conditions, fed a standard diet and given free access to water. All animal studies were approved by the government commission for animal protection. At 21 and 14 days before arthritis induction, mice were subcutaneously injected with 100 μg mBSA (Sigma, Deisenhofen, Germany), emulsified in complete Freund's adjuvant (Sigma) supplemented to 2 mg/ml heat-killed Mycobacterium tuberculosis (strain H37RA; Becton Dickinson [BD], Heidelberg, Germany). Simultaneously, mice received 5 × 10 8 heat-inactivated Bordetella pertussis (Chiron-Behring, Marburg, Germany) intraperitoneally. Arthritis was induced by intra-articular injection of 100 μg mBSA in 25 μl phosphate-buffered saline (PBS) into the right knee joint cavity. Arthritis severity was monitored by measurement of lateral joint diameter using a vernier caliper (Oditest, Kroeplin Längenmesstechnik, Schlüchtern, Germany). Histological severity of arthritis was scored in a blinded manner by two investigators (PKP and MG) in frontal knee joint sections, stained with haematoxylin and eosin and prepared as described previously [ 14 ]. Briefly, at least four sections per knee joint were semiquantitatively examined on a 0–3 point scale for each of the following: extent of synovial hyperplasia, mononuclear infiltration, cartilage destruction and pannus formation. Antibodies and reagents The following antibodies were grown and purified from the culture supernatants in our laboratory: anti-CD25 (PC61), anti-CD3 (145 2C11), anti-CD4-FITC and FITC-labelled anti-CD4-F(ab) (GK1.5), anti-CD8 (TIB105), anti-CD28 (37.51) and anti-Mac-1 (M1/70). The following antibodies and secondary reagents were purchased from BD Pharmingen (Heidelberg, Germany): PE-Cy5-labelled anti-CD4 (H129.9), biotinylated anti-α E β 7 (M290), biotinylated anti-CD25 (7D4), allophycocyanine or FITC-conjugated anti-CD25 (PC61), streptavidin-allophycocyanine and streptavidin-PE, and matched antibody pairs for ELISPOT analysis of IFN-γ (R4-6A2 and biotinylated XMG1.2) and IL-4 (BVD4 1D11 and biotinylated BVD6-24G2) production. In vivo depletion Mice were injected with 0.5 mg purified anti-CD25 antibody (PC61) 4 and 2 days before intra-articular antigen injection. Polyclonal rat IgG, purified from normal rat serum, was used as control. The degree of depletion was determined by fluorescence-activated cell sorting, using a non-cross-reactive biotin-labelled anti-CD25, FITC-labelled anti-CD4 and streptavidin-conjugated allophycocyanine. Measurement was performed using FACSCalibur ® (BD) and data were analyzed using WinMDI . Preparation, pre-activation and transfer of regulatory T cells Pooled spleen and lymph node cells from naive C57Bl/6 donors or, if indicated, from immunized mice were incubated with anti-CD4-FITC (clone GK1.5) and anti-CD25-biotin (clone 7D4; BD). CD4 + T cells were isolated using an anti-FITC-Multisort-Kit (Miltenyi Biotech, Bergisch-Gladbach, Germany) in accordance with the manufacturer's instructions. CD4 + T cells were sorted into CD25 - and CD25 + cells using anti-biotin MicroBeads (Miltenyi Biotech). Purity was greater than 92% for CD4 + CD25 - and greater than 80% for CD4 + CD25 + cells. CD25-expressing and α E β 7 -expressing subsets were sorted by FACS. Briefly, pooled spleen and lymph node cells from naive mice were stained with anti-CD25-FITC, anti-α E β 7 -biotin and streptavidin-PE. The stained cells were enriched with anti-FITC and anti-PE MicroBeads, using the AutoMACS separation unit (Miltenyi Biotech). Thereafter, the cells were sorted into subsets according to their expression of CD25 or α E β 7 using a FACSDiVa cell sorter (BD). The purity was 90–95%, as determined by FACS. For activation, cells were cultured for 24–72 hours in the presence of plate-bound anti-CD3 (3 μg/ml), anti-CD28 (10 μg/ml) and rhIL-2 (100 U/ml; Chiron, Ratingen, Germany) in RPMI 1640 containing 10% fetal calf serum (FCS; Gibco, Karlsruhe, Germany). Thereafter, cells were washed with PBS and transferred intravenously via lateral tail vein into mice at the time point of AIA induction or at later time points when indicated. Delayed-type hypersensitivity reaction Seven days after arthritis induction, mice were challenged by intradermal injection into their ears of 5 μg mBSA in 10 μl PBS. Ear thickness was measured before injection and 24 and 48 hours later using a vernier caliper (Kroeplin). Proliferation assay and ELISPOT analysis Single cell suspension from spleens and lymph nodes (inguinal, popliteal, axillary) were cultured at a density of 1 × 10 6 /ml in RPMI 1640, containing 10% FCS, 2 mmol/l L-glutamine, 10 mmol/l Hepes, 1 mmol/l sodium pyruvate, 0.5 μmol/l 2-mercaptoethanol and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin; all from Gibco) in the presence of medium alone or 25 μg/ml mBSA for 72 hours in 96-well tissue culture plates (Greiner Bio One, Nürtingen, Germany). Cells were pulsed with 0.5 μCi [ 3 H]thymidine (Amersham-Buchler, Braunschweig, Germany) for the last 18 hours of culture. Thereafter, cells were harvested onto 96-well glass fibre filters (Packard Bioscience, Groningen, The Netherlands), and [ 3 H]thymidine incorporation was measured with a scintillation counter (Top-Count; Packard Bioscience). For ELISPOT analysis, PVDF-membrane 96-well microplates (Millipore, Eschborn, Germany) were coated overnight at 4°C with the primary antibody diluted in sterile PBS. After washing, plates were blocked for 2 hours with RPMI 1640 containing 10% FCS. Thereafter 2 × 10 5 (IL-2 and IFN-γ) or 1 × 10 6 (IL-4) cells were cultured in duplicate wells for 24 (IL-2 and IFN-γ) or 48 hours (IL-4). After washing again plates were incubated overnight at 4°C with the secondary antibody diluted in PBS/1% BSA. Extravidin–alkaline phosphatase conjugate (1:30,000 in PBS/1% BSA) and BCIP/NBT solution (bromochloroindolyle phophate/nitroblue tetrazolium; both from Sigma) were used for spot development. The number of spots was quantified using a KS-ELISPOT-Reader (Carl Zeiss, Oberkochen, Germany). Determination of serum IgG by ELISA Microplates (96-well; Greiner Bio One) were coated with antigen (0.125 μg/ml mBSA), collagen type I (from rat tail tendon) and type II (10 μg/ml), and proteoglycans (10 μg/ml both from bovine cartilage) and left overnight, as described previously [ 14 ]. After washing, plates were incubated with serially diluted serum samples and the amount of bound IgG was determined using anti-mouse IgG-peroxidase conjugate (ICN, Eschwege, Germany) and ortho-phenylendiamine (Sigma) as substrate. Extinction was measured at 492 nm against 620 nm with an ELISA reader (Tecan, Crailsheim, Germany). Cell transfer for in vivo homing assay For in vivo homing assay, cells were sorted with a modified protocol and labelled with 111 indium, as described elsewhere [ 10 ]. Briefly, CD4 + cells were enriched by negative selection. Enriched CD4 + T cells were stained with FITC-conjugated anti-CD4-F(ab) and anti-CD25-allophycocyanine and sorted into CD4 + CD25 + or CD4 + CD25 - cells by FACS (BD). Cells were labelled with 111 In (Indiumoxin; Amersham-Buchler) for 20 min at room temperature; 1 × 10 6 labeled cells were injected intravenously, and 24 hours later mice were killed and the distribution of radioactivity in various organs and the rest of the body was measured in a γ-counter (Wallac Counter, Turku, Finnland). Alternatively, a proportion of these cells was labelled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) by incubation with 5 μmol/l CFSE (Molecular Probes, Leiden, The Netherlands) in RPMI 1640 for 5 min at room temperature. After washing, 1 × 10 6 cells were injected intravenously. Twenty-four hours later single cell suspensions were prepared from the draining and nondraining peripheral and mesenteric lymph nodes, the spleen and the peripheral blood, and stained with anti-CD4 and analyzed by FACS. Dead cells were excluded using propidiumiodide. Statistical analysis Data are expressed as mean ± standard error of mean, unless otherwise indicated. Experimental groups were tested for statistically significant differences with the Mann–Whitney U-test using SPSS 10.0 (SPSS Inc, Chicago, IL, USA). Results Depletion of CD25-expressing cells exacerbates antigen-induced arthritis Mice were injected intraperitoneally with 0.5 mg anti-CD25 (PC61) 4 and 2 days before induction of arthritis (i.e. 19 and 17 days after first immunization). Depletion of CD25-expressing cells was confirmed using FACS at the time of AIA induction (day 0) using an antibody that recognizes a different epitope on the CD25 molecule. In the PC61-treated group there was a 70.9 ± 11.4% ( n = 3) reduction in CD4 + CD25 + cells in the spleens as compared with control mice injected with rat IgG (Fig. 1 ). Of note, the anti-CD25 treatment almost completely depleted cells with high expression of CD25, which are considered T reg cells, in contrast to CD4 + T cells with low or intermediate levels of CD25 expression. After intra-articular antigen injection, knee joint swelling of the CD25-depleted mice was significantly greater from day 3 onward than in the control group injected with rat IgG (Fig. 2a ). Histological examination of knee joint sections 14 days after AIA induction revealed increased hyperplasia and infiltration of the synovial membrane, as well as increased articular damage in those animals (Fig. 2b–d ). In summary, this indicates a marked exacerbation of AIA by depletion of CD25-expressing cells. Increased cellular and humoral immune responsiveness in CD25-depleted mice To assess how in vivo cellular immune responses against mBSA are influenced by depletion of CD25-expressing cells, delayed-type hypersensitivity (DTH) reaction against the same antigen was tested by intradermal injection of mBSA into the ears of mice at day 7 after induction of AIA. Anti-CD25 treated mice mounted a significantly stronger DTH response than did rat IgG-treated controls (Fig. 3a ). For analysis of the cellular immune responses ex vivo , draining lymph node cells of arthritic animals were harvested 14 days after AIA induction, restimulated with mBSA, and analyzed for proliferative response and cytokine production. As expected from the increased DTH reaction, the proliferative response to mBSA was significantly increased in cells from CD25-depleted mice as compared with that in rat IgG-treated controls (Fig. 3b ). Importantly, even without antigenic stimulation the lymph node cells from CD25-depleted mice proliferated fourfold as much as cells from mice treated with control IgG. These data imply that a substantial proportion of the T-cell compartment is still activated 14 days after intra-articular antigen challenge in the absence of T reg cells. Compatible with these findings is that the production of cytokines in response to mBSA was greater in CD25-depleted mice. Importantly, both T-helper-1 (IFN-γ) and T-helper-2 (IL-4) responses were aggravated by depletion of T reg cells, indicating that both types of response are subject to suppression by T reg cells (Fig. 3c ). Again, cytokine secretion from T reg -depleted animals was increased even without antigenic stimulus. In accordance with this, serum levels of IgG directed against mBSA as well as levels of the cartilage-specific autoantigens collagen type I, collagen type II and proteoglycans, were found to be increased in CD25-depleted mice (Fig. 3d ). Taken together, these data clearly demonstrate that CD4 + CD25 + T reg cells regulate the severity of arthritis by limiting the cellular and humoral immune responses against the inducing antigen mBSA as well as some arthritis-related autoantigens. Transfer of CD4 + CD25 + cells To further characterize the suppressive potential of CD4 + CD25 + T reg cells, we performed cell transfer studies. In a first set of experiments we transferred T reg cells freshly isolated from naive (Fig. 4a ) or mBSA/CFA immunized (Fig. 4b ) mice into mBSA-immunized recipients at the time of intra-articular antigen challenge (day 0). With this protocol, a slight decrease in the severity of clinical arthritis (knee joint swelling) could be induced. Accordingly, the histological severity of AIA was also found to be reduced, albeit not statistically significantly (Fig. 4a, b ). It is known that T reg cells must be activated via their T-cell receptor to exert their suppressive function. Because we were unable to use antigen-specific (i.e. T-cell receptor transgenic) T reg cells, we opted to pre-activate the CD4 + CD25 + cells by in vitro culture in the presence of anti-CD3, anti-CD28 and IL-2 in order to increase their suppressive potential. Transfer of 1 × 10 6 pre-activated cells significantly suppressed both knee joint swelling and histological arthritis score (Fig. 4c ). This effective suppression of AIA development was a consistent finding in different experiments, even with the use of lower cell numbers (for instance 2 × 10 5 cells; data not shown). In the next step, we attempted to cure established arthritis by transfer of T reg cells. Surprisingly, 1 × 10 6 pre-activated CD4 + CD25 + cells had no influence on either knee joint swelling or histological arthritis score when transferred at day 1 (Fig. 5a ) or day 7 (Fig. 5b ) after induction of arthritis. Also, the transfer of 1 × 10 6 pre-activated α E β 7 -expressing T reg cells, which are highly effective in preventing AIA [ 10 ], had no effect on disease at this time point (Fig. 5c ). Taken together, our data demonstrate that T reg cells can inhibit arthritis development when transferred at the time of arthritis induction. However, we were unable to demonstrate any therapeutic effect of T reg cell transfer (in numbers that are effective in prevention) when performed after disease onset. Transferred CD4 + CD25 + T reg cells do not suppress humoral or cellular immune responses Because CD25-depletion caused a substantial increase in both cellular and humoral immunoreactivity against mBSA, we examined whether transfer of CD4 + CD25 + T reg cells can suppress these responses. Neither DTH reactivity against mBSA (analyzed 7 days after AIA induction; Fig. 6a ) nor mBSA-induced proliferation (Fig. 6b ) and cytokine production by draining lymph node cells (Fig. 6c ) at day 14 after induction of AIA were found to be suppressed in the recipients of 1 × 10 6 pre-activated CD4 + CD25 + cells. Thus, transfer of T reg cells into immunized animals does not eliminate or induce functional modification to the previously primed mBSA-specific immune response. In contrast, transfer of CD4 + CD25 - cells did significantly enhance the proliferation as well as the cytokine production in the recipients. Accordingly, serum levels of IgG directed against mBSA and the cartilage-specific autoantigens collagen type I and type II, and proteoglycans were also not significantly diminished in T reg cell recipients compared with the saline-treated control group. Recipients of CD4 + CD25 - cells had higher levels of IgGs (Fig. 6d ). Homing properties of CD4 + CD25 + T reg cells Because the mechanism of suppression of T reg cells in vitro is cell contact dependent, localization of cells might be important for their regulatory activity. Therefore, we investigated the migration behaviour of CD4 + CD25 + and CD4 + CD25 - cells in vivo . For these experiments CD4 + cells were enriched by negative selection and sorted by FACS into CD4 + CD25 + and CD4 + CD25 - populations with preferential use of F(ab)-fragments or antibodies, which do not interfere with migration in vivo . Cells were labelled with 111 In and injected intravenously into AIA mice 7 days after induction of arthritis. After 24 hours radioactivity was measured in different organs. Compared with CD4 + CD25 - cells, CD4 + CD25 + T reg cells were less abundant in secondary lymphoid organs such as lymph nodes and spleen. Thus, CD4 + CD25 + cells recirculate through these organs less than do CD4 + CD25 - cells. In the liver, more radioactivity was recovered in recipients of CD4 + CD25 + cells as compared with CD4 + CD25 - cells. Importantly, CD4 + CD25 + cells also had a significantly better capacity to enter the inflamed joint than did CD4 + CD25 - cells (Fig. 7a ). The level of radioactivity detected in the arthritic joints was low but similar to levels found in transfer experiments with effector T cells [ 18 ]. As a control, some mice were injected with CFSE-labelled cells. FACS analysis of the secondary lymphoid organs revealed the presence of viable cells 24 hours after transfer and excluded the possibility that the difference in migration pattern is due to leakage of radioactivity (Fig. 7b ). The migration behaviour of CD4 + CD25 + T reg cells does reflect their more activated phenotype, and their ability to enter inflamed joints makes it possible that they act directly at the site of inflammation. Discussion Our findings provide clear evidence that CD4 + CD25 + T reg cells are critical for regulating the severity of AIA in mice. We showed this by manipulating the T reg cell numbers using two different approaches: depletion of CD25-expressing cells and transfer of purified CD4 + CD25 + T reg cells. It is important to stress that we depleted CD25-expressing cells in the interval between immunization and AIA induction, because CD25-depletion before immunization profoundly increases the resulting humoral and cellular immune responses [ 3 , 12 ]. These data are consistent with studies conducted in collagen-induced arthritis; however, in these experiments CD25-expressing cells were depleted before immunization with collagen type II, and the resulting more severe arthritis could be interpreted as the result of stronger immunization state [ 12 ]. With our experimental design, we were able to examine the effect of T reg cells in ongoing joint inflammation directly. Because CD25 is also expressed on activated conventional T cells, it could be assumed that injection of an anti-CD25 antibody would deplete not only T reg cells but also effector T cells, but the exacerbated AIA in CD25-depleted mice argues against such a depletion of effector T cells. Accordingly, in control experiments lymph node cells from CD25-depleted mice isolated at the time of induction of AIA were able to mount a similar anti-mBSA response in vitro as compared with control mice (data not shown). Furthermore, CD4 + CD25 + cells isolated from immunized donors can suppress development of AIA (Fig. 4b ). Taken together, these data imply that the CD25 + compartment in immunized mice largely consists of T reg cells. AIA induction in CD25-depleted mice resulted in a much more severe arthritis in the acute and chronic stages of disease. We recently showed, with the use of a depleting anti-CD4 antibody, that this acute stage of AIA is already under the control of T cells [ 15 ]. Nevertheless, early AIA is dominated by cells of the innate immune system [ 19 ], and the exacerbation of arthritis in CD25-depleted mice could be due to a lack of suppression of these cells by T reg cells. In accordance with this view, CD4 + CD25 + T reg cells are able to suppress innate immune cells in a model of bacteria-induced colitis [ 20 ]. In later stages exacerbated arthritis in CD25-depleted mice is accompanied by increased mBSA-specific proliferation and IgG production. This enhanced responsiveness emerged during arthritis development and is due to sustained T cell activation. Such prolonged T cell activation in the absence of CD4 + CD25 + cells has also been described in other disease models [ 21 ] and is probably the cause of the increased AIA severity. Moreover, the PC61 antibody used in our study has a half-life of approximately 3 weeks in vivo (Sutmuller R, personal communication), which makes it possible that T reg cell function is not only impaired by depletion but also by blockade of IL-2 binding to CD25 by the PC61 antibody. IL-2 or IL-2 signalling via CD25 has been shown to be critical to the regulatory action of T reg cells [ 22 , 23 ]. Also, activation-induced cell death of pathogenic T cells, which is regulated by IL-2, could be impaired by withdrawal of IL-2 signalling and therefore contribute to the observed high levels of cellular immune responses in our study [ 24 ]. The fact that depletion of CD4 + CD25 + T reg cells enhances the immune response against the foreign antigen mBSA clearly demonstrates that their suppressive effect is not strictly limited to autoreactive T cells. Taking into consideration that T reg cells are also critically involved in the control of immune responses against pathogens [ 25 , 26 ], their physiological function is not just to prevent autoimmunity but also to control the extent of inflammatory reactions in order to prevent tissue damage to the host. Further support for the influence of CD4 + CD25 + T reg cells on arthritis development came from the transfer experiments. When transferred at the time of induction of AIA, CD4 + CD25 + cells were able to ameliorate ongoing disease. Analysis of the recipients did not reveal a remarkable long-lasting suppression of systemic mBSA-specific immune reactions. Thus, prevention of AIA appears to be possible without inducing anergy or abrogating previously induced T-cell effector functions [ 27 ]. In contrast to this, transferred CD4 + CD25 - cells significantly enhance cell-mediated and humoral immune responses. Furthermore, the homing data presented here demonstrate that CD4 + CD25 + cells can migrate into the arthritic knee joint. Functional T reg cells have repeatedly been found within such effector sites and/or draining lymph nodes, for instance in tolerated allografts [ 28 ], in Langerhans islets and pancreatic lymph nodes in inflammation-induced diabetes [ 29 ], in chronically inflamed skin in a Leishmania infection model [ 30 ], and in the mucosa and mesenteric lymph nodes in inflammatory colitis in severe combined immunodeficient (SCID) mice [ 31 ]. Interestingly, two recent papers [ 32 , 33 ] reported an accumulation of functional T reg cells in the inflamed joints of patients with RA, juvenile arthritis and other rheumatic diseases. It is most likely that the transferred CD4 + CD25 + T reg cells act in the draining lymph node as well as in the inflamed tissue. Within such a scenario, it could be possible that T reg cells inhibit the activation of effector T cells and their subsequent migration to the joints. Such a mechanism was recently speculated in modulation of virally induced immunopathology by T cells [ 26 ]. Huehn and colleagues [ 11 ] recently demonstrated that CD4 + CD25 + T reg cells can be divided into subsets based on the expression of the integrin α E β 7 . Moreover, this marker identifies CD25 - T reg cells [ 34 ]. Both α E β 7 -expressing subsets had better capacity to reach the inflamed joint and to prevent arthritis in the AIA model, as compared with α E β 7 - T reg cells [ 10 ]. Thus, suppression at the site of inflammation is also an important part of the activity of T reg cells. How this effect is mediated is unclear but an involvement of IL-10 or transforming growth factor-β is possible [ 20 , 35 , 36 ]. If these hypotheses are correct, then they could explain why the transfer of T reg cells after arthritis induction is not effective. On the one hand, transfer of T reg cells 24 hours after intra-articular antigen challenge might be too late to inhibit activation of effector T cells and their migration to the joint. Indeed, T-cell activation is an early event in AIA because CD4 + T cell depletion ameliorates the acute stage of the model [ 15 ]. On the other hand, it could be possible that the suppressive function of regulatory T cells is switched off under the inflammatory conditions present in the inflamed tissue by factors such as IL-6 or glucocorticoid-induced tumor necrosis factor family-related gene (GITR) and GITR-ligand interactions, abrogating the suppressive effect of T reg cells [ 37 ]. With this in mind, it could be interesting to investigate whether the accumulated T reg cells in patients with arthritis function properly in vivo and whether these patients could really benefit from a therapeutic enhancement of T reg function, as suggested by some enthusiastic investigators in this field. In this regard, data on the curative effects of T reg cells in experimental disease models are conflicting. To best of our knowledge, a curative effect of CD4 + CD25 + T reg cells has only been demonstrated in the colitis model induced by transfer of CD45RB high T cells into SCID mice [ 31 , 38 ]. In contrast, other authors were unable to demonstrate such an inhibitory effect of T reg cells on SCID colitis when they were transferred 1 week after administration of pathogenic CD45RB high T cells [ 39 ]. Because arthritis in the AIA model has a hyperacute onset, it could be assumed that the time window for an ameliorative effect of T reg cell transfer ends very shortly after intra-articular injection of antigen. However, further studies on the role of T reg cells in other arthritis models are clearly needed to clarify whether enhancement in T reg cell function might be beneficial in experimental arthritis and perhaps in human disease. Conclusion Our data show that T reg cells are critically involved in the control of immune responses that are responsible for the pathogenesis of chronic arthritis. Transfer of such cells can modulate the severity of ongoing inflammatory arthritis but they cannot suppress established disease. Thus, timing of T reg cell transfer for therapeutic purposes is of considerable importance. Abbreviations AIA = antigen-induced arthritis; CFSE = 5,6-carboxyfluorescein diacetate succinimidyl ester; DTH = delayed-type hypersensitivity; ELISA = enzyme-linked immunosorbent assay; FACS = fluorescence-activated cell sorting; FCS = fetal calf serum; IFN = interferon; IL = interleukin; mBSA = methylated bovine serum albumin; PBS = phosphate-buffered saline; RA = rheumatoid arthritis; SCID = severe combined immunodeficient; T reg = regulatory T cell. Competing interests The author(s) declare that they have no competing interests. Authors' contributions OF purified the anti-CD25 hybridoma and purified the monoclonal antibodies from the supernatant; planned and conducted all animal experiments, including ELISA and ELISPOT analysis; and drafted the manuscript. PKP and MG scored the histological changes in arthritic joints. KS, JH and AH conducted the migration experiments, as well as the α E β 7 transfer experiments. RB supervised the project and participated together with AS and AR in the design of the study and its coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.

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1065323

Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis

The aim of the study was to characterise CCR7 + and CCR7 - memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-γ by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4 + , CCR7 - memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4 + CCR7 - memory T cells were enriched for CCR5 + and interferon-γ + cells, whereas CD4 + CCR7 + memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4 + memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7 + T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7 + cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5 + cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.

Introduction Migration and accumulation of memory T cells in the synovium is a critical step in the pathogenesis of chronic arthritides [ 1 - 3 ]. Chemokines are a large family of small secreted proteins (8–15 kDa) that control lymphocyte trafficking in physiological and pathological processes. The evaluation of type and distribution of chemokines and their receptors in the synovium is therefore crucial to an understanding of the mechanisms of synovial T cell recruitment. From a functional point of view, chemokines can be broadly classified into two groups: inflammatory and homeostatic [ 4 ]. The inflammatory chemokines are induced by proinflammatory stimuli and control the migration of leukocytes to the site of inflammation. CCR5 and CXCR3 are classical examples of receptors for inflammatory chemokines [ 5 ]. The homeostatic chemokines regulate the basal traffic of lymphocytes and other leukocytes through peripheral lymphoid tissues. CCR7 is an example of a receptor for homeostatic chemokines. CCR7 and its ligands (CCL19 and CCL21) have also been shown to have a pivotal role in the development and maintenance of secondary lymphoid organ microarchitecture [ 4 , 5 ]. Recently, the CCR7 chemokine receptor has been identified as an important marker of memory T cell differentiation. It has been proposed that CCR7 + memory T cells represent a pool of 'central' memory T cells homing to lymph nodes, where they undergo further differentiation into CCR7 - memory T cells, which migrate to the peripheral tissues to perform their effector functions [ 6 ]. However, this model has been disputed by other investigators [ 7 , 8 ] and CCR7 + naive and memory T lymphocytes have been detected in both normal and inflamed human tissues [ 9 ]. Previous studies have shown that Th1-polarised [ 10 , 11 ], CCR5 + and CXCR3 + lymphocytes are enriched in synovial inflammatory infiltrates and in synovial fluid (SF) lymphocytes from patients with adult rheumatoid arthritis (RA) [ 12 , 13 ] and juvenile idiopathic arthritis (JIA) [ 14 - 16 ]. CCR5 and CXCR3 ligands, namely RANTES (or CCL5) and macrophage inhibitory protein-1α (MIP-1α, or CCL3), and interferon-inducible protein-10 (IP-10, or CXCL10) and ITA-C (CXCL11), respectively, have also been detected in rheumatoid synovium [ 17 ]. Limited information is available on CCR7 expression in synovial lymphocytes from patients with chronic arthritis. Naive CD45RA + T cells with a CCR7 phenotype have been found to infiltrate the synovial tissue in patients with RA [ 16 ]. The CCR7 ligands CCL19 and CCL21 have been detected in endothelial cells and in the perivascular infiltrate in RA synovium, suggesting their potential involvement in lymphoid neogenesis that occurs in inflamed synovial tissue [ 18 - 20 ]. No information is so far available on the expression of CCR7 in memory T cells homing to the synovial microenvironment in relation to expression of the inflammatory chemokine receptors CCR5 and CXCR3. In this study we therefore investigated the expression of CCR7, CCR5 and CXCR3 on SF and peripheral blood (PB) memory CD4 + T cells from patients with JIA, chemotaxis of the latter cells to the ligands of these receptors, and the distribution of cells positive for CCR7, CCR5 and CXCR3 in the inflamed synovium. Methods Patients Immunophenotypic and functional characterisation of freshly isolated PB and/or SF lymphocytes was performed in a total of 25 patients with JIA (14 female, 9 male) undergoing therapeutic arthrocentesis. According to ILAR Durban classification criteria [ 21 ], 15 patients had persistent oligoarticular JIA (pOJIA), 6 had extended oligoarticular JIA (eOJIA) (which means a total of five or more joints involved after the first 6 months of disease and therefore a polyarticular course) and 4 had rheumatoid factor (RF)-negative polyarticular JIA. Several clinical (number of active joints, number of joints with limited range of motion, and physician global assessment of overall disease activity) and laboratory parameters (erythrocyte sedimentation rate, C-reactive protein, white blood cell and platelet counts, and hemoglobin serum concentration) of disease activity were recorded, together with the ongoing treatment, at the time of the study. Paired serum and SF samples from 28 additional patients with JIA (16 with pOJIA, 6 with eOJIA, 4 with RF negative polyarticular JIA, and 2 with systemic JIA) were tested for CCL21 concentrations. The clinical characteristics of patients with JIA and the ongoing treatment at the time of the study are reported in Tables 1 and 2 . For each patient, SF was collected at the time of intra-articular steroid injection. Paired serum sample was obtained, with permission, on the occasion of concomitant routine venipuncture. Both SF and sera were stored at -80°C immediately after centrifugation. A previous steroid injection into the same joint in the previous 6 months was considered to be an exclusion criterion. Peripheral blood and/or sera from 15 age-matched healthy subjects attending our clinic for routinary pre-operative examinations for minor surgical procedures were used as controls. Synovial tissue from six patients (two with pOJIA, one with eOJIA and three with RF-negative polyarticular JIA) was obtained, with permission, at the time of synoviectomy. Samples were taken from patients and healthy controls, and stored after parental permission in accordance with the informed consent approved by the ethical committee of the 'G. Gaslini' Institute. Cell preparation and flow cytometry PB and SF mononuclear cells (MNC) were isolated from heparinised blood and SF samples by Ficoll–Hypaque (Sigma, St Louis, MO, USA) density gradient centrifugation. Cells were washed, resuspended in complete medium (RPMI 1640 with L-glutamine, penicillin/streptomycin, nonessential amino acids and 10% fetal bovine serum; Sigma) and depleted of adherent cells by adherence to plastic for 1 h at 37°C in 5% CO 2 . To analyse the expression of CCR7 on CD4 + memory T cells in SF and PB MNC, cells were triple-stained with CD45RO-TC (Caltag, Burlingame, CA, USA), CD4–FITC (BD Biosciences, San Jose, CA, USA) and anti-CCR7–PE (BD Pharmingen, San Diego, CA, USA) monoclonal antibodies (mAbs) and analysed by flow cytometry (CellQuest software and FACScan; BD Biosciences). CCR7 expression was evaluated by gating on the CD45RO + CD4 + lymphocyte population. CD45RO + cells were purified from PB and SF MNC by negative selection with a CD45RA mAb (Caltag) and goat anti-mouse IgG-coated magnetic beads (Immunotech, Marseille, France), in accordance with the manufacturer's instructions. Recovered cells were 95% enriched for CD45RO + cells. CCR5 or CXCR3 expression was investigated by three-colour staining of freshly isolated SF and PB CD45RO + cells with fluorescein isothiocyanate (FITC)-conjugated CD4 (BD Biosciences), anti-CCR7–phycoerythrin (PE) and anti-CCR5–CyChrome mAbs (BD Pharmingen) or CD4-TC (where TC stands for Tri-color), anti-CCR7–PE and anti-CXCR3–FITC (R&D System, Minneapolis, MN, USA), respectively, gating on the CD4 + CCR7 + and CD4 + CCR7 - lymphocyte populations. For interferon (IFN)-γ intracellular staining, freshly purified SF CD45RO + cells (10 6 ) were incubated for 5 hours in the presence of phorbol 12-myristate 13-acetate (20 ng/ml; Sigma), the calcium ionophore A-23187 (250 ng/ml; Sigma) and brefeldin-A (5 μg/ml; Sigma). Cells were washed in phosphate-buffered saline (PBS) with 1% fetal calf serum (staining buffer) and surface stained with CD4–TC (Caltag) and anti-CCR7–PE (BD Pharmingen) mAbs for 30 min at 4°C in the dark. Cells were washed in staining buffer and fixed in 4% paraformaldehyde for 20 min at 4°C in the dark. Afterwards, the cells were washed twice with permeabilisation buffer (PBS containing 1% fetal calf serum and 0.1% saponin [Sigma]) and stained with FITC-conjugated mAbs against human IFN-γ (Caltag) for 30 min at 4°C in the dark. Cells were then washed in staining buffer and analysed by flow cytometry, gating on the CD4 + CCR7 + and CD4 + CCR7 - lymphocyte subsets. Although stimulation with phorbol 12-myristate 13-acetate and calcium ionophore downregulates the intensity of CD4 and CCR7 expression, the proportion of cells positive for each marker was similar before and after stimulation. Isotype matched, PE-, FITC-, TC- and CyChrome-conjugated mAbs of irrelevant specificity were tested as negative controls in all of the above experiments. The results of flow cytometry experiments were expressed as percentage positive cells or as mean fluorescence intensity; that is, the staining intensity of a test mAb minus that of an isotype-matched, irrelevant control mAb. The threshold for calculating the percentage positive cells was based on the maximum staining obtained with irrelevant isotype-matched mAb, used at the same concentration as the test mAb. Negative cells were defined such that less than 1% of cells stained positive with control mAbs. Cells labelled with test antibody that were brighter than those stained with isotypic control antibody were defined as positive. Mean fluorescence intensities of the isotype control and of test mAbs were used to evaluate whether the differences between the peaks of cells were statistically significant with respect to the control. The Kolmogorov–Smirnov test for the analysis of histograms was used, in accordance with the CellQuest software user's guide. Differences between paired PB and SF MNC of patients with JIA on the one hand, and PB MNC of healthy controls on the other, were evaluated by the Kruskal–Wallis analysis of variance (ANOVA) test and the Wilcoxon rank test. Chemotactic assays Migration assays were performed in 24 transwell plates (pore size 5 μm, polycarbonate membrane; Costar, Cambridge, MA, USA). Freshly purified SF CD45RO + cells (5 × 10 5 ) were dispensed in the upper chamber in 100 μl, and 600 μl of different chemokines at 100 ng/ml (R&D System) or medium alone was added to the lower chamber. Migration was performed in migration medium (RPMI 1640, 0.1% bovine serum albumin; Sigma). Plates were incubated for 2 hours at 37°C. After removal of the transwell inserts, cells from the lower compartments were collected. Furthermore, 0.5 ml of 5 mM EDTA was added to the lower chamber for 15 min at 37°C to detach adherent cells from the bottom of the wells. Detached cells were pooled with the previously collected cell suspensions and counted by staining with trypan blue. To evaluate the percentage of migrated CD4 + lymphocytes, cell suspensions were double-stained with CD4–PE and CD3–FITC mAbs (BD Biosciences) before and after migration and analysed by flow cytometry. The percentage input was calculated as follows: 100 × (cells migrated to chemokine/total cell number). Differences between cells that migrated to a given chemokine and the same cells that migrated in medium alone were calculated with non-parametric Wilcoxon rank test. CCL21 serum and SF concentrations Forty-three sera (15 from controls) and 28 SFs were tested for CCL21 by an enzyme-linked immunosorbent assay kit from R&D System (Minneapolis, USA), in accordance with the instructions of the manufacturer. Serum levels of CCL21 were compared in three groups of patients (12 patients with JIA with a polyarticular course, 16 patients with JIA with an oligoarticular course and 15 healthy controls) with the use of the non-parametric Kruskal–Wallis ANOVA test. Correlations between all the variables considered were evaluated with the non-parametric Spearman rank test. Differences between paired serum and SF chemokine concentrations were evaluated by the Wilcoxon rank test. Immunohistochemical studies Tissue specimens with sizes between 5 and 12 mm were treated for single and double immunohistochemical stainings with a standard technique as reported previously [ 22 ]. In brief, all specimens were fixed in 4% formalin for 24 hours, then dehydrated and embedded in paraffin. Sections 4 μm thick were layered on polylysine-coated slides. Slides were deparaffinised in xylene, and rehydrated in a descending sequence of ethanol concentrations (100–70%). Three different immunohistochemical techniques, namely alkaline phosphatase–anti-alkaline phosphatase (APAAP) for CCR7, avidin–biotin complex for CD21, and indirect immunoperoxidase (CD3, CD4, CD45RO, CD20, CCR5, CXCR3, CCL19 and CCL21), were performed after 30 min of warming in an oven in citrate buffer, pH 6, with subsequent inhibition of endogenous peroxidase. For single staining, tissue sections were incubated overnight at 4°C with the anti-CCR7 murine mAb, clone 2H4 (Pharmingen). Incubation of tissue sections with anti-CCL21 goat antiserum (R&D), anti-CCR5, clone 2D7 (Pharmingen), anti-CXCR3, clone 1C6 (Pharmingen), anti-CD3 (Dako, Glostrup, Denmark), anti-CD4, clone 4B12 (Neomarkers, Fremont, CA, USA), anti-CD20, clone L26 (Dako) and anti-CD45RO, clone UCHL1 (Menarini, Firenze, Italy) and anti-CD31 clone JC70A (Dako) was performed overnight at 4°C. Sections were subsequently reacted for 30 min at room temperature (20–25°C) with (1) anti-mouse Ig antibody conjugated to peroxidase-labelled dextran polymer (EnVision; Dako) for CD3, CD45RO, CCR5 and CCR7 stainings, (2) anti-goat secondary biotinylated antibody, followed by high-sensitivity streptavidin–horseradish peroxidase conjugate for CCL21 determination (Cell and Tissue Staining kit; R&D), and (3) APAAP-conjugated rabbit anti-mouse Ig (1:25 dilution; Dako) antibody for CCR7 determination. The chromogenic diaminobenzidine substrate (Dako) was applied for 10 min. All washings were performed by incubating the sections in PBS. For CCR7 determination the alkaline phosphatase reaction was performed with a medium containing Tris-HCl buffer pH 8.2, naphthol AS-TR salt (Sigma) and levamisole (Sigma), for 20 min at 98°C. Slides were counterstained with Mayer's haematoxylin. For double CCR7/CCL21 staining, the sections were subjected to peroxidase reaction with goat CCL21 and were washed three times in Tris-buffered saline. Subsequently, the APAAP technique (see above) was applied with the mouse CCR7 Ab (at room temperature, for 3 hours). The secondary reagents were applied for 30 min each. For CCR7 and CCL21, a reactive lymph node from a 10-year-old boy was considered as positive control. Reactions in the absence of primary antibody and with irrelevant antibodies of the same isotypes (anti-cytomegalovirus, clones DDG9 and CCH2; Dako) were performed as negative controls. Slides were evaluated on two different occasions by two blinded observers (MG and AG) and an expert pathologist (CG). Each specimen was evaluated for the pattern of lymphocyte infiltration in three different categories: (1) aggregates of T cells (CD4) and B cells (CD20) with germinal centre (GC)-like reaction (presence of CD21-positive cells), (2) aggregates of T and B cells without GC-like reaction, and (3) diffuse lymphocytic infiltrate without lymphoid organisation [ 20 ]. For each sample a semiquantitative score for the overall degree of T lymphocyte infiltration (CD3) was used (range 0–3). For the assessment of chemokine receptor expression each sample was subjected to microscopical analysis of: (1) the lining layer and sublining zone of the superficial subintima [ 23 ]; (2) perivascular infiltrates of the sublining layer without lymphoid organisation; (3) aggregates of T and B cells. Because CCR7, CXCR3 and CCR5 can be expressed by several cell types (lymphocytes, dendritic cells, B cells and plasma cells) [ 18 , 24 ], only areas characterised by a clear lymphocyte infiltration (as defined by anti-CD3 and anti-CD4 positivity) were taken into consideration. The following semiquantitative global score was based on a visual inspection of four different high-power fields (40×) at each level: absent (-, no positive cells per high-power field), weakly positive (+, 1–10 positive cells per high-power field), moderately positive (++, 10–20 positive cells per high-power field), and strongly positive (+++, more than 20 positive cells per high-power field). The assignment of each sample to one of the above categories was based on the predominant pattern observed. Minor differences between the observers were resolved by mutual agreement. Intra observer and interobserver variability was less than 5%. Results Phenotypic and functional characterisation of CCR7 + and CCR7 - CD4 + memory T cells isolated from SF Expression of CCR7 on CD4 + memory T cells from the PB and SF of 10 patients with JIA was investigated by three-colour immunofluorescence analysis and compared with that detected on the same PB cell subset from eight age-matched healthy controls. The heterogeneity test between the three subgroups was highly significant (Kruskal–Wallis ANOVA test, P = 0.0001). At post hoc analysis, in the PB from patients with JIA, the percentage of CCR7 + cells in the CD4 + CD45RO + subpopulation (median 65.5%, range 50–90%) was significantly lower than in PB from controls (median 76%, range 73–89%, P = 0.03, Mann–Whitney U -test). A further decrease in CCR7 + cells was observed in memory CD4 + cells isolated from SF (median 41.2%, range 12–59%) in comparison with paired PB. Thus, even a variable proportion of SF memory CD4 + T cells are positive for CCR7; this subpopulation is clearly enriched in CCR7 - cells in comparison with paired PB. Next we investigated the expression of CCR5, CXCR3 and IFN-γ in SF CD4 + CD45RO + CCR7 + and CCR7 - cells from 10 consecutive patients and compared it with that detected in paired PB from 5 of these patients and in the PB of 5 healthy controls. To this end, purified CD45RO + cells were stained with anti-CD4, anti-CCR7 and respectively, anti-CCR5, CXCR3 or IFN-γ mAbs in three-colour immunofluorescence. In SF, CCR5 + cells were found to be enriched in the CD4 + CD45RO + CCR7 - lymphocyte subset (median 85%, range 74–99%) as compared to the CD4 + CD45RO + CCR7 + cell fraction (median 65%, range 46–84%, P = 0.005; Wilcoxon test; not shown). These data were in line with our previous observation of a higher expression of CCR7 in 'early' CD27 + memory T cells and a prevalent CCR7 - CCR5 + phenotype in 'effector' CD27 - T cells in SF from patients with JIA [ 25 ]. The median percentage of CD4 + , CD45RO + , IFN-γ positive cells was 27% (range 21–46%) for CCR7 + cells and 40% (range 24–69%) for CCR7 - cells ( P = 0.005) (Fig. 1a,c ), as assessed by intracellular staining. Accordingly, the mean fluorescence intensity for IFN-γ was lower for CCR7 + cells (median 136, range 84–184) than for paired CCR7 - CD4 + memory T cells (median 190, range 127–307, P = 0.005). CXCR3 was highly expressed on both CCR7 + and CCR7 - subsets of SF memory CD4 + T cells. However, in all patients with JIA, SF CCR7 + memory CD4 + T cells showed a higher expression of CXCR3 (median 86%, range 74–93%) than the CCR7 - counterpart (median 76%, range 62–85, P = 0.005) (Fig. 1b,d ). In comparison with SF, PB of patients with JIA showed a lower expression of CXCR3 in both CCR7 + (median 38.5%, range 24–55%) and CCR7 - (median 27%, range 17–40%) CD4 + memory T cells. A similar expression was also found in circulating CCR7 + (median 40%, range 32–55%) and CCR7 - (median 17%, range 13–45%) CD4 + memory T cells from age-matched healthy controls. Taken together, these results show that the CD4 + CD45RO + CCR7 - subpopulation is enriched in 'effector' CCR5 and IFN-γ expressing cells, whereas the CD4 + CD45RO + CCR7 + subpopulation shows a lower expression of CCR5 and IFN-γ and a higher degree of coexpression with CXCR3. Different localisation of CCR7, CXCR3 and CCR5 positive cells in synovial tissue We next addressed the following questions: (1) is CCR7 expressed in synovial tissue, (2) how does its expression correlate with the pattern of lymphocytic infiltration, and (3) how is CCR7 expression related to that of CXCR3 and CCR5, two Th1-associated chemokine receptors? To this end, synovial tissues obtained at synoviectomy from six patients with JIA were analysed for the expression of CCR7, CXCR3 and CCR5 in areas characterised by a clear lymphocyte infiltration (Table 3 ). Different patterns in the synovial inflammatory infiltrate were observed in the individual patients (Table 3 ). One patient (no. 6) showed T and B cell aggregates with the presence of a GC reaction, as demonstrated by the presence of CD21 + follicular dendritic cells [ 20 ]. In three patients (nos 3, 4 and 5) clusters of T and B cell aggregates in the absence of follicular dendritic cells were observed [ 20 ]. Two patients (nos 1 and 2) displayed diffuse lymphocytic infiltrates as perivascular aggregates in the sublining layer or scattered throughout the synovium up to the lining layer [ 20 ]. CCR7-positive cells were detected both in cases showing a diffuse lymphocytic infiltrate (Fig. 2a,b ) and in those displaying a more organised lymphoid structure (Fig. 2c–e ). In the former, CCR7 expression was detected mostly in the perivascular lymphocytic infiltrates of the sublining layer (Fig. 2b,o ) and, only occasionally, in scattered cells in the sublining zone of the superficial subintima (see also Fig. 4c below). In the latter, CCR7-positive cells were localised inside and around lymphoid aggregates (Fig. 2e ). Because naive CD45RA + T cells with a CCR7 + phenotype had also previously been found to infiltrate the synovial tissue from patients with RA [ 19 ], serial sections were stained with CCR7 and CD45RO antibodies. A clear positivity for CCR7 was detected in lymphocytic infiltrates staining heavily for CD45RO (not shown). CXCR3 was abundantly expressed in all lymphocyte-infiltrated areas examined (Table 3 ). In fact, CXCR3-positive cells were detected in lymphoid aggregates (Fig. 2f ) and in perivascular infiltrates of sublining layer (Table 3 ). In many areas, CXCR3 and CCR7 displayed a similar pattern of tissue distribution, especially at the level of lymphocyte aggregates (Fig. 2e,f ). Conversely, CCR5-positive cells were detected mainly in the lining layer and in the sublining zone of the superficial subintima and, to a smaller extent, in the perivascular infiltrates of the sublining layer (Fig. 2p ) and in the T and B cell aggregates (Fig. 2n ) (Table 3 ). Altogether, even if a certain degree of co-localisation of the two chemokine receptors was found (Table 3 ), CCR5 positive cells showed a substantially different tissue distribution from that of CCR7, either in T and B cell aggregates (Fig. 2e,g,l,n ) or in diffuse lymphocytic infiltrates (Fig. 2o,p ). Conversely, a variable degree of co-localisation was found for CCR5 and CXCR3 at the level of the sublining and lining layer (Table 3 ). Chemotaxis of SF CD4 + memory T cells to inflammatory and homeostatic chemokines In further experiments, chemotaxis of freshly isolated SF memory T cells in response to CCR7, CCR5 and CXCR3 ligands was investigated, and migrated CD4 + CD45RO + T cells were detected by flow cytometry. Chemotactic assays were performed with SF CD45RO + cells isolated from eight patients with JIA (five with pOJIA, three with eOJIA) and tested in the presence or absence of two inflammatory chemokines that bind to CCR5 (CCL3) and CXCR3 (CXCL11), respectively, and of homeostatic chemokines binding to CCR7 (CCL21 and CCL19). CD4 + CD45RO + T cells migrated significantly to both CCL3 and CXCL11 ( P = 0.02 for both chemokines). Similar responses were observed when CCL19 was tested ( P = 0.02). Chemotaxis of CD4 + memory T cells to CCL21 approached but did not reach statistical significance ( P = 0.1) (Fig. 3 ). The latter finding might be related to the limited number of the samples tested. In the patients studied, the variability of chemotaxis of SF CD4 + memory T cells did not show any significant correlation with disease form, degree of disease activity and treatment at the moment of sampling. Expression of CCL21 in SF and synovial tissue To gain further insight into the relevance of the interactions between CCR7 and its ligand CCL21 in vivo , sera and SF CCL21 concentrations were tested in 28 consecutive patients with JIA and in 15 healthy controls. The heterogeneity test between the three subgroups was highly significant (Kruskal–Wallis ANOVA test, P = 0.0045). Concentrations of CCL21 were significantly higher in SF (median 1769.5 pg/ml, range 110–25,556 pg/l) than in paired sera from patients with JIA (median 268 pg/ml, range 57.6–5146.9 pg/ml, P < 0.0001; Wilcoxon test; Fig. 4a ). A strong correlation was found between paired serum and SF CCL21 concentrations ( r = 0.91, P = 0.001; Spearman's test). No significant difference was observed in CCL21 serum concentrations between patients with JIA with oligoarticular course (median 229.2 pg/ml, range 67–3948 pg/ml), patients with JIA with polyarticular course (median 378 pg/ml, range 65–5146 pg/ml) and age-matched healthy controls (median 282.2 pg/ml, range 76–2349 pg/ml, P = 0.3; Kruskal–Wallis ANOVA test). Similarly, no significant difference was found in SF CCL21 concentrations between patients with JIA with an oligoarticular course and patients with a polyarticular course ( P = 0.52; Mann–Whitney U -test). Finally, no significant correlation was found between CCL21 serum concentrations and several clinical and laboratory parameters of disease activity in patients with JIA (see the Methods section; not shown). The expression of CCL21 was also analysed in synovial tissues by immunohistochemistry. CCL21 was detected in all specimens. In the samples characterised by lymphoid organisation, CCL21 staining was observed in the perivascular lymphocytic aggregates and in the vascular endothelium within follicular structures, a pattern reminiscent of that observed on staining for CCR7 [ 19 ]. A similar pattern was detected in tissues showing a diffuse lymphocytic infiltration (Fig. 4b ). Moreover, a clear-cut expression of CCL21 was also observed in flat wall vessels of the superficial subintima of the sublining layer (Fig. 4c,d ) [ 23 ]. Discussion In this study we have investigated the role of CCR7 in the recruitment of CD4 + memory T cells into the inflamed joints of patients with JIA, and attempted the functional and anatomical dissection of these cells according to their expression of CCR7, CXCR3, CCR5 and IFN-γ. We detected two populations of SF CD4 + memory T cells: the CD4 + CD45RO + CCR7 - subset, which was enriched in 'effector' CCR5 and IFN-γ positive cells, and the CD4 + CD45RO + CCR7 + subset, which was less well represented and showed higher CXCR3 coexpression. SF CD4 + memory T cells displayed chemotactic activity to both inflammatory and homeostatic chemokines representing the physiological ligands of these receptors. Of the three chemokine receptors studied, CXCR3 proved to be the most widely expressed in synovial tissue, with a clear distribution both in lymphoid aggregates and in perivascular infiltrates of sublining layer and in the lining layer. Conversely, CCR7-positive and CCR5-positive cells in the synovial tissue displayed a different distribution, showing an even higher differentiation in their expression in respect to SF. In fact, CCR7 + cells were detected in synovial tissues irrespective of the pattern of lymphoid organisation and were localised mainly in lymphoid aggregates and in perivascular infiltrates of the sublining layer. Notably, CCL21, the CCR7 ligand, was found in the SF as well as in perivascular lymphocytic aggregates and in the vascular endothelium of follicular structures. In contrast, CCR5 + cells were detected mainly in the lining layer and in the sublining zone of the superficial subintima and, to a smaller extent, in the perivascular infiltrates of sublining layer and in the T and B cell aggregates. These findings in synovial tissue are in line with the results of the phenotypic characterisation of SF CCR7 + and CCR7 - memory CD4 + T cells performed in the present study and with previous observations showing a variable degree of coexpression of CXCR3 and CCR5 on T cells isolated from inflamed tissues [ 14 , 26 , 27 ]. To our knowledge, this is the first demonstration of a different anatomical localisation of cells positive for CCR7, CCR5 and CXCR3 infiltrating the inflamed synovium; this finding may have functional implications for the intra-tissue migration of T cells. During the past decade several studies have focused on the capacity of memory T cells to differentiate in the context of inflamed tissues. Many of these studies used a member of the tumour necrosis factor receptor family, CD27, to distinguish recently activated CD27 + from 'effector' CD27 - memory CD4 + T cells [ 28 ]. Notably, a clear enrichment of the latter subpopulation has been found in SF of patients with RA and JIA [ 29 , 30 ]. In a recent study we showed that CD27 + memory T cells in SF of patients with JIA expressed CCR7 more highly than CCR5, whereas CD27 - T cells displayed a prevalent CCR7 - CCR5 + phenotype [ 25 ]. Notably, the immunohistochemical characterisation of rheumatoid synovial tissue in adult RA has shown a prevalent localisation of CD4 + CD27 + T cells in the perivascular lymphocytic aggregates, with a relative increase in CD27 - T cells in diffuse lymphocytic infiltrates [ 31 ]. Thus, it is conceivable that the functional and phenotypic characterisation of CCR7 + and CCR7 - memory CD4 + T cells and the different tissue distribution between CCR7 and CCR5 found in the present study might reflect the same behaviour already observed for CD27 + and CD27 - memory T cells, yielding more insight into the migratory properties of memory T cells into and within the synovial tissue. The partial overlap of CCL21 and CCR7 expression in the inflamed synovium might suggest that the CCR7/CCL21 system, probably in synergy with CXCR3 and its ligands, is involved in the recruitment of memory T cells, as already shown for naive T cells [ 19 ]. However, the possibility cannot be ruled out that CCR7 expression in CD4 + memory T cells isolated from SF was upregulated after the reactivation of these cells at the site of inflammation [ 32 ]. CCL21, together with other homeostatic chemokines such as CXCL13, has been shown to have a fundamental function in the development of secondary lymphoid organs by interacting with CCR7 [ 4 , 33 ]. Mice whose CCR7 or CCL21 genes have been knocked out exhibit marked deficiencies in the structural and cellular composition of lymph nodes [ 34 ]. A sequence of events similar to that taking place in lymph node organogenesis is supposed to be involved in the development of organised lymphoid structures in inflamed tissues, such as the rheumatoid synovium [ 18 - 20 ]. Indeed, up to 20% of synovial tissue biopsies from patients with RA show the typical features of the GC reaction. Other patients show aggregates of T and B cells in the absence of an evident follicular organisation [ 20 ], whereas in more than 50% of synovial tissue samples from RA [ 20 ] and a considerable proportion of patients with JIA (M Gattorno, unpublished data), diffuse T and B lymphocytic infiltrates in the absence of aggregates or follicular structures are observed. Interestingly, in the individual patients with RA, the pattern of lymphocytic infiltration was found to persist unaltered over time, and showed similar features in all biopsies taken from different joints at the same type [ 20 ]. In our study, both CCR7 and its ligand CCL21 were found to be abundantly expressed in synovial biopsies, irrespective of the pattern of lymphoid infiltration. These observations support the hypothesis that CCR7 and its ligands have a direct function in the recruitment of memory T cells to the inflamed synovium, one that is independent of their ability to organise in lymphoid structures. In this respect, the recent demonstration of different regulation of CCL21 in lymphoid and non-lymphoid tissues is noteworthy. Lymphotoxin-α directs the formation of lymph nodes and Peyer's patches through the induction of adhesion molecules and the production of chemokines, including CCL21, by the mesenchymal organiser cells during the early developmental steps [ 4 , 35 ]. Lymphotoxin-α-deficient mice show a marked impairment of lymphoid organisation in secondary lymphoid organs, but normal recruitment of naive and memory T cells to peripheral inflamed tissue through the CCR7/CCL21 system [ 36 ]. CCL21 and CCR7 might therefore either regulate lymphoid neogenesis by a lymphotoxin-dependent mechanism or recruit T cells to the inflamed tissues by a lymphotoxin-independent mechanism. Taken together, our findings suggest that CCR7 + memory T cells can be directly recruited, with the possible contribution of other chemokines such as CXCR3 ligands, to the synovium, where they undergo further differentiation leading to the downregulation of CCR7 from the cell surface and the concomitant upregulation of CCR5. This differentiation might be driven either by antigen-dependent or antigen-independent mechanisms. In fact, cytokines produced in the synovial microenvironment (namely interleukin-7 and interleukin-15) might allow the proliferation, expansion and differentiation of CCR7 + memory T cells into effector cells, marked by the downregulation of CCR7, the upregulation of CCR5 and the production of IFN-γ [ 37 ]. In this model, CCR5 could represent the major chemokine receptor used for CD4 + memory T cell locomotion within the inflamed tissue, according to a step-by-step navigation model through different chemoattractant gradients [ 38 ]. In contrast, the enrichment of CCR7 - CCR5 + cells infiltrating the lining and sublining layer could be also related to the presence of other relevant effector cells, such as the granzyme B + cytotoxic cells [ 39 ]. Conclusion The present study delineates a coordinated pattern of expression of homeostatic and inflammatory chemokines in the inflamed synovium, with potential implications for the mechanisms regulating the intra-tissue migration and local differentiation of inflammatory cells. Abbreviations ANOVA = analysis of variance; APAAP = alkaline phosphatase–anti-alkaline phosphatase; eOJIA = extended oligoarticular JIA; FITC = fluorescein isothiocyanate; GC = germinal centre; IFN = interferon; JIA = juvenile idiopathic arthritis; mAb = monoclonal antibody; MNC = mononuclear cells; PB = peripheral blood; PBS = phosphate-buffered saline; pOJIA = persistent oligoarticular JIA; RA = rheumatoid arthritis; RF = rheumatoid factor; SF = synovial fluid; TC = Tri color. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MG conceived and coordinated the study, performed patients' selection and wrote the manuscript. IP, AM and VP participated in the study design and helped to draft the manuscript. IP, FM, SC and FF performed the cytofluorimetric analysis and chemotaxis studies. AU performed the enzyme-linked immunosorbent assay for CCL21 determination in sera and SF, and helped to draft the manuscript. AG, AF and CG performed the immunohistochemical analysis of synovial tissue. All authors read and approved the final manuscript.

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1065324

Multilevel examination of minor salivary gland biopsy for Sjögren's syndrome significantly improves diagnostic performance of AECG classification criteria

The recently observed low reproducibility of focus score (FS) assessment at different section depths in a series of single minor salivary gland biopsies highlighted the need for a standardized protocol of extensive histopathological examination of such biopsies in Sjögren's syndrome. For this purpose, a cumulative focus score (cFS) was evaluated on three slides cut at 200-μm intervals from each of a series of 120 salivary biopsies. The cFS was substituted for the baseline FS in the American–European Consensus Group (AECG) criteria set for Sjögren's syndrome classification, and then test specificity and sensitivity were assessed against clinical patient re-evaluation. Test performances of the AECG classification with the original FS and the score obtained after multilevel examination were statistically compared using receiver operating characteristic (ROC) curve analysis. The diagnostic performance of AECG classification significantly improved when the cFS was entered in the AECG classification; the improvement was mostly due to increased specificity in biopsies with a baseline FS ≥ 1 but <2. The assessment of a cFS obtained at three different section levels on minor salivary gland biopsies can be useful especially in biopsies with baseline FSs between 1 and 2.

Introduction Sjögren's syndrome (SS) is characterized by diffuse chronic inflammation of exocrine glands, which leads to symptoms and complaints referred to as 'sicca syndrome' [ 1 ]. No single instrumental or laboratory parameter is available for the diagnosis of SS, which relies instead on the evaluation of multiple clinical, serological, functional, and morphological parameters [ 2 ], such as those proposed and validated by a group of investigators sponsored by the European Community (now the European Union) [ 3 , 4 ] and recently revised by the American-European Consensus Group (AECG) [ 5 ]. The presence of chronic inflammatory infiltrates in lip salivary glands, as assessed with minor salivary gland biopsy (MSGB), is one of the parameters included in most criteria sets proposed for SS classification [ 3 , 5 - 9 ]. Salivary gland inflammation is assessed by scoring the degree of infiltration according to the method of Greenspan and Daniels, who defined the focus score (FS) as the number of inflammatory infiltrates of at least 50 cells present in 4 mm 2 of gland surface unit [ 10 , 11 ]. Different criteria sets consider as positive a FS ≥ 1 or FS ≥ 2 [ 3 , 9 ]. Although the methodology of sampling, processing, and examining MSGBs has been standardized [ 10 , 11 ], the reproducibility of the routine histopathological evaluation in the diagnosis of SS at different section levels within the same biopsy specimen has been recently challenged [ 12 , 13 ]. To avoid any bias that might therefore arise, the examination of multiple levels of tissue has been recommended, to maximize the number of foci, the glandular area, and the technical quality of the material, although the number of sections required has not yet been standardized [ 12 ]. In this study, we tried to standardize a protocol for histopathological MSGB evaluation in which the FS is assessed by examining a larger area of the biopsy tissue, and we investigated how the FS obtained affects the number of patients classified as having SS, as compared with the routine method, using the classification criteria recently proposed by the AECG [ 5 ]. The diagnostic accuracy of the test was validated against the clinical re-evaluation of the patients performed by two experienced rheumatologists after at least 1 year of follow-up. Materials and methods Selection criteria We retrospectively studied a consecutive series of patients thoroughly investigated at our hospital between 1998 and 2002 for suspected primary SS, including a follow-up of at least 1 year after the diagnostic evaluation. Patients with secondary SS or who had been diagnosed by biopsy as having nonspecific inflammation, fibrosis, and atrophy of the gland were excluded [ 10 - 12 ]. Less-than-optimal tissue area (biopsy section area less than 4 mm 2 ) was not considered a criterion for exclusion, provided that at least one normotrophic glandular lobule had been sampled. Baseline clinical and histopathological evaluation All patients had undergone thorough clinical and instrumental evaluation [ 3 , 4 ], including MSGB performed as suggested by Daniels [ 11 ]. The diagnosis of SS was established for all patients according to the classification criteria proposed by the AECG [ 5 ]. MSBG samples were fixed in formalin, processed, and embedded in paraffin according to standardized laboratory methods. Baseline histopathological slides containing 4-μm-thick sections stained with hematoxylin and eosin were reviewed by a pathologist, blinded to clinical and laboratory data, who recorded for each patient the number of glands, the sample surface area, the presence of alterations suggestive of nonspecific sialoadenitis, and the baseline FS [ 10 , 11 ]. The lymphocytic focus and the focus score were defined according to Greenspan and Daniels [ 10 , 11 ]. In individual biopsies, lobules with acinar atrophy and diffuse fibrosis were excluded from diagnostic evaluation. The histological parameter was considered as negative in the absence of any inflammatory infiltrate (FS = 0) and in the presence of less than 1 focus per 4 mm 2 (0 < FS < 1) [ 5 ]; the presence of one or more foci per 4 mm 2 was considered positive when the adjacent glandular parenchyma was histologically normal. We further classified patients with a positive FS into two groups, those with fewer than two foci per 4 mm 2 (1 ≤ FS < 2) and those with two or more (FS ≥ 2). The area of the biopsy sections was assessed with video-assisted morphometric software capable of measuring the area of delineated surfaces (ImageDB System, Casti Imaging, Cazzago di Pianiga, Italy). The comparison of automated and manual area measurements of a smaller series of MSGB sections did not show a significant difference (data not shown). This prompted us to choose the automated system to simplify the examination of the large number of samples involved in the study. Serial histopathological re-evaluation Sample blocks were recut at two additional levels, about 200 and 400 μm deeper than the original section. Sections 4 μm thick corresponding to these levels were collected on separate slides and stained with hematoxylin and eosin. Considering that an infiltrate of 50 lymphocytes in our section had a mean diameter of 50 μm, we assumed that the interposition of 200 μm between the evaluated sections was enough to ensure that the FS recorded at each level was independent of the other two and that if the same focus was present in two section levels, the focus itself was large enough to justify repeated scoring. The two new sections were blindly examined by the same pathologist, who again recorded the area and the focus score for each level. For each patient, the total number of foci at all three levels and the total surface area measured at all levels were used to calculate a cumulative FS (cFS) for the three sections. Reclassification of patients The cFS obtained after re-evaluation was entered in the AECG criteria set [ 5 ], to obtain a re-classification of each patient. To compare the diagnostic performance of the original classification and the reclassification, a 'gold standard' was needed independent of the AECG criteria set. We adopted as reference standard the opinion of experienced clinicians, analogously to what had been done by the European Community Study Group on Diagnostic Criteria for Sjögren's Syndrome when SS and control patients were selected to validate the proposed criteria [ 3 - 5 ]. Briefly, three experienced rheumatologists, blinded to the results of the histopathological re-evaluation, performed a clinical evaluation of each patient and reviewed the patient's charts including the original clinical, laboratory, and instrumental evaluation, and the subsequent documentation covering at least 1 year of follow-up and treatment response. On this basis they were requested to judge whether individual patients had SS. Statistical analysis Quantitative data are shown as means ± standard deviation (SD). Specificity and sensitivity were assessed with their 95% confidence intervals (CI). Differences in frequencies were evaluated by means of chi-square statistics or the Fisher exact test, as appropriate. Given the known limitations of diagnostic accuracy as a parameter for measuring the diagnostic performance of a test, specificity and sensitivity were compared using receiver operating characteristic (ROC) curves [ 14 ]. A P value of less than 0.05 was considered to indicate statistical significance. All tests were two-sided. Analyses were performed with Statistica for Windows (StatSoft Inc, 2002, Tulsa, OK, USA) and MedCalc software. Results Baseline examination The study series comprised 138 patients, 65 of whom had a baseline FS = 0, 14 with 0 < FS < 1, 18 with 1 ≤ FS < 2, and 41 with FS ≥ 2. Eighteen patients had incomplete clinical data that hampered either the AECG classification or the clinical re-evaluation. These patients (8 with FS = 0, 3 with 0 < FS < 1, 3 with 1 ≤ FS < 2, and 4 with FS ≥ 2) were excluded from further analysis. The final series included 120 patients, for whom demographic, biopsy, and clinical data and the result of the clinical re-evaluation are presented in Table 1 . Histological re-evaluation In 96 (80%) of the 120 biopsies, the FS group did not change after serial sectioning and calculation of the cFS. In 14 of these biopsies, the FS group changed but this did not affect that patient's negative or positive status. In the biopsies for the other 10 patients, 1 (1.7%) of the 57 with a baseline FS = 0 and 1 (9%) of the 11 with a baseline score of 0 < FS < 1 switched to a FS consistent with SS according to AECG criteria (FS ≥ 1). At clinical re-evaluation, these two patients were considered not to have SS. Seven (46%) of the 15 patients with a baseline score of 1 ≤ FS < 2 and one (3%) of 37 with a baseline FS ≥ 2 switched to a grade inconsistent with SS (FS < 1). On clinical re-evaluation, 7 of these 8 patients were assessed as not having SS. Patient reclassification according to AECG criteria When the cFSs were entered in the AECG criteria set [ 5 ], the baseline classifications of the 63 non-SS patients were not changed, while the classifications of 7 of the 57 patients originally classified as having SS were changed to non-SS (Table 2 ). The classification was changed in 6% of the 120 patients. Six of these seven patients had a baseline score of 1 ≤ FS < 2 and one had a baseline FS ≥ 2. On clinical re-evaluation, all these seven patients were judged not to have SS. The clinical re-evaluation also refuted 7 of the 113 (6.2%) classifications that had not been changed at biopsy revision. Considering the clinical re-evaluation as the reference gold standard, the number of false-negative AECG classifications did not change (3 of 63 AECG non-SS cases), while the number of false positives was reduced from 11 to 4 (63.6% reduction). Comparison of sensitivity and specificity between baseline and multilevel FS evaluation In the present series of 120 patients fully evaluated for SS, the sensitivity and specificity of the baseline AECG criteria set were 93.9% and 84.5%, respectively. Reclassification with cFS did not affect sensitivity, whereas specificity changed to 94.4% ( P = 0.056), increasing the accuracy from 88.3% (95% CI 81.2–93.5) to 94.2% (95% CI 88.3–97.6). Pairwise comparison of the ROC curves showed a statistically significant difference between patient classification before and after multilevel FS evaluation (difference between areas: 0.049 [SE 0.021]; 95% CI 0.009–0.089; P = 0.016) (Fig. 1 ). Sensitivity and specificity did not change for biopsies with FS = 0 or FS < 1 (inconsistent with SS), while specificity increased substantially in biopsies consistent with SS (FS ≥ 1) (Table 2 ). Pairwise comparison of the ROC curves showed a statistically significant difference ( P = 0.013) only in biopsies with 1 ≤ FS < 2 (difference between areas: 0.43 [SE: 0.17]; 95% CI 0.09–0.76; P = 0.013; Fig. 1 ). The diagnostic accuracy of the MSGB histological analysis considered independently of other criteria changed from 85.8% (95% CI 78.3–91.5) to 90.8% (95% CI 84.2–95.3), but the comparison of the ROC curves did not show a statistically significant difference ( P = 0.15). Discussion In the present study, we show that the histopathological evaluation of salivary gland biopsies with multilevel sectioning and assessment of a cumulative focus score (cFS) changes the baseline classification in 6% of patients evaluated for SS and increases the diagnostic performance of the criteria recently proposed by the AECG for SS classification [ 5 ]. In particular, multilevel evaluation improved the diagnostic accuracy of biopsies with a baseline FS between 1 and 2, which is the most critical cutoff in SS histopathological evaluation. The present study was prompted by a recent paper documenting that MSGB grading of inflammation was scarcely reproducible at different section depths, and that the difference between grades recorded at baseline and at deeper levels was sufficient to change the biopsy from positive to negative or vice versa in 10% of grade I (FS = 0), 44.4% of grade II (0 < FS < 1), 88.8% of grade III (1 ≤ FS < 2), and 40% of grade IV (FS ≥ 2) biopsies [ 13 ]. The authors of that paper recommended that multiple sections of MSGB should be examined to improve the reliability of the histopathological grading. However, they did not suggest how many sections should be examined or how to deal for diagnostic purposes with the different scores obtained at different levels, nor did they give a clinical interpretation of their results by entering them in a criteria set for SS patient classification. On this basis, we aimed at assessing if the histopathological evaluation of a larger area of MSGB tissue, as obtained by cutting the biopsy sample at additional section levels, could increase the diagnostic performance of the histopathological study and of the AECG criteria set proposed for the classification of SS. We chose a minimum requirement of three different section levels, by analogy with the procedure standardized for the histopathological study of endomyocardial biopsies [ 15 ], assuming that a 200-μm distance should ensure the detection of independent foci on each section while reducing the chance of missing the smaller ones, thus allowing estimation of the overall density of inflammatory foci with sufficient precision. With reference to the diagnostic gold standard, when patients were classified according to the AECG criteria set including the cFS, specificity increased by 9.8%, and the pairwise comparison of the ROC curves showed a statistically significant improvement of the diagnostic performance, mostly due to the increased test specificity in biopsies with 1 ≤ FS < 2, whereas the increase was minimal in FS ≥ 2 and null in biopsies inconsistent with SS (0 < FS < 1). One advantage of the proposed method of MSGB evaluation is that specificity is increased without affecting sensitivity; on the other hand, it was shown that improving sensitivity by means of increasing the cutoff value of positive FS resulted in a substantial reduction of specificity [ 16 ]. To explain the increased specificity observed with examination of multilevel salivary gland biopsies, it should be considered that, because of the uneven distribution of inflammatory infiltrates in the gland [ 14 ], the examination of a single tissue section might easily either overestimate or underestimate the FS, while the observation of a larger area of biopsy sample would allow a more precise quantification of the focus distribution, provided that the sections are distant enough to avoid recutting and rescoring of the same focus. In accordance with this hypothesis, and confirming previous results [ 13 ], after multilevel examination the higher numbers of FS changes proven to be relevant for classification and clinical diagnosis were seen in patients with mild to moderate MSGB inflammatory infiltrates (1 ≤ FS < 2), while very few relevant changes were recorded in patients with negative or highly positive biopsies (FS < 1 or FS ≥ 2). We suggest that in mild inflammation, lymphocytic foci are unevenly distributed through the gland, so that positive baseline sections can occasionally be followed by sections with less or no inflammation, whereas negative or highly positive biopsies (FS < 1 and ≥ 2) are likely to be more homogeneous. Our observations also confirmed the common knowledge that no single test can be reliably applied to the diagnosis of SS [ 2 - 9 ]. In fact, the performance of the test was significantly improved when the cFS was entered in the criteria set, but not when the histopathological test was considered alone. One potential limit of the present study is represented by the need to introduce a gold standard reference to assess the diagnostic accuracy of the test, independent of the widely accepted AECG criteria set for SS classification. In fact, after clinical re-evaluation, which we adopted as a gold standard, some patients appeared to have been misclassified according to AECG criteria. This only partial correspondence between the judgement of experienced clinicians and classification criteria is a well-known problem in the diagnosis of rheumatological disorders and justifies the requirement of a wide criteria set for patient classification. In the absence of single, straightforward diagnostic parameters, a thorough patient's chart and follow-up revision by experienced rheumatologists was chosen as reference gold standard, by analogy with what has been done in many rheumatological studies, including that of the European Community Study Group on Diagnostic Criteria for SS [ 3 - 5 ]. Accordingly, a multicenter study would be useful to better standardize the procedure of evaluating FSs by oral pathologists, backed by a larger panel of experienced clinicians, because the clinical performance of SS classification criteria could be improved. Conclusion The assessment of a cumulative focus score (cFS) obtained at three different section levels on minor salivary gland biopsies, cut at least 200 μm apart, can improve the diagnostic accuracy of the criteria set used for SS classification, especially in biopsies with a baseline FS between 1 and 2. Since the value of the MSGB biopsy has been confirmed by the recent AECG revision of the SS classification criteria [ 5 ], the increase of the diagnostic performance of the histological study will further help to correctly identify SS patients. Abbreviations AECG = American-European Consensus Group; cFS = cumulative FS; CI = confidence interval; FS = focus score; MSGB = minor salivary gland biopsy; ROC = receiver operating characteristic; SE = standard error; SS = Sjögren's syndrome. Competing interests The author(s) declare that they have no competing interests. Authors' contributions PM participated in the design of the study, performed the histopathological analysis, coordinated the study, and drafted the manuscript. AM and RC reviewed and discussed patients' charts for clinical re-evaluation. OE performed all salivary gland biopsies. CV participated in case collection and data analysis. CT participated in the design of the study and performed the statistical analysis. ES and CM conceived the study and participated in its design. CM also participated in the clinical re-evaluation of patients. All authors read and approved the final manuscript.

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1065325

Inhibition of antithrombin by hyaluronic acid may be involved in the pathogenesis of rheumatoid arthritis

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, proinflammatory processes, and proliferation of fibroblast-like cells. Abnormalities in these processes are primary features of rheumatoid arthritis (RA) in synovial tissues. Tissue destruction in joints causes the accumulation of large quantities of free hyaluronic acid (HA) in RA synovial fluid. The present study was conducted to investigate the effects of HA and several other glycosaminoglycans on antithrombin, a plasma inhibitor of thrombin. Various glycosaminoglycans, including HA, chondroitin sulfate, keratan sulfate, heparin, and heparan, were incubated with human antithrombin III in vitro . The residual activity of antithrombin was determined using a thrombin-specific chromogenic assay. HA concentrations ranging from 250 to 1000 μg/ml significantly blocked the ability of antithrombin to inhibit thrombin in the presence of Ca 2+ or Fe 3+ , and chondroitin A, B and C also reduced this ability under the same conditions but to a lesser extent. Our study suggests that the high concentration of free HA in RA synovium may block antithrombin locally, thereby deregulating thrombin activity to drive the pathogenic process of RA under physiological conditions. The study also helps to explain why RA occurs and develops in joint tissue, because the inflamed RA synovium is uniquely rich in free HA along with extracellular matrix degeneration. Our findings are consistent with those of others regarding increased coagulation activity in RA synovium.

Introduction Thrombin is a multifunctional protease that can activate hemostasis and coagulation through the cleavage of fibrinogen to form fibrin clots. Increasing fibrin deposition is a predominant feature of rheumatoid arthritis (RA) in synovial tissue, which contributes to chronic inflammation and progressive tissue abnormalities [ 1 ]. Thrombin also acts as a mitogen to stimulate the abnormal proliferation of synovial cells during RA pathogenesis. In this regard, thrombin can elevate the expression of nuclear factor-κB, interleukin-6, and granulocyte colony-stimulating factor in fibroblast-like cells of the RA synovium [ 2 , 3 ]. By a similar mechanism, thrombin can upregulate the transcription of vascular endothelial growth factor receptor and thereby induce the permeability, proliferation, and migration of capillary endothelial cells or their progenitors during angiogenesis [ 4 - 6 ]. Thrombin also plays an important role in the proinflammatory process by stimulating neutrophil adhesion to vessel walls and releasing prostacyclin [ 7 ]. Thus, thrombin is essential for enhancing synovial thickness and inflammation during the pathogenesis of RA. The principal plasma inhibitor of thrombin is antithrombin, a single-chain 51 kDa glycoprotein that is synthesized in liver. The inhibitory activity of antithrombin on thrombin is significantly enhanced by heparin, a type of glycosaminoglycan (GAG) [ 8 ]. The GAG family comprises large anionic polysaccharides with similar disaccharide repeats of uronic acid and hexosamine. Physiologically important GAGs include hyaluronic acid (HA), chondroitin sulfates, keratan sulfate (KS), heparin, and heparan, which are the major components of joint cartilage, synovial fluid, and other soft connective tissues [ 9 , 10 ]. Along with the destruction of RA joint tissue, a remarkable quantity of various GAG molecules, especially HA, are released from the extracellular matrix of the synovium [ 9 , 10 ], which is a key feature of RA progression. Because GAGs and heparin share a similar molecular structure, we investigated how HA and other GAGs affect antithrombin activity. Methods Highly purified HA, chondroitin sulfate A (CSA), chondroitin sulfate B (CSB), chondroitin sulfate C (CSC), KS, heparin, or heparan (Seikagaku, Tokyo, Japan) were incubated for 24 hours with human antithrombin III at 150 μg/ml (Sigma, St. Louis, MO, USA) at 37°C in working buffer (100 mmol/l Tris-HCl, pH 7.5) containing 5 mmol/l CaCl 2 or FeCl 3 . The concentration of antithrombin was determined according to its physiologic level in synovial fluid [ 11 , 12 ]. The reaction was stopped with EDTA. Residual activity of antithrombin was analyzed using the chromogenic Actichrome AT III (American Diagnostica, Greenwich, CT, USA) kit, which quantifies antithrombin III activity as follows. After exposure to GAGs, antithrombin was incubated with the thrombin reagent provided with the kit and residual thrombin activity was determined by incubation with the thrombin-specific chromogenic substrate in the kit. Absorbance was measured at a wavelength of 405 nm. Hence, the inhibitory ability of antithrombin on thrombin was inversely proportional to the residual thrombin activity. This assay method is usually used in the clinical setting. We prepared a series of control tests in which HA, CSA, CSB, CSC, and KS were digested in 0.1 mol/l phosphate buffer (prepare 100 ml of the buffer with 94 ml of 0.1 M KH 2 PO 4 and 6 ml of 0.1 M K 2 HPO 4 , pH 6.2) at 37°C for 2 hours with 0.1 units/ml hyaluronidase (Seikagaku, Japan) before incubation with antithrombin. Hyaluronidase preferentially digests HA rather than other GAGs. To determine whether HA can prevent heparin from stimulating antithrombin, we simultaneously incubated heparin (10 μg/ml) and various concentrations of HA with antithrombin (150 μg/ml) at 37°C for 24 hours in the presence of 5 mmol/l CaCl 2 . To investigate the effect of HA on antithrombin in the presence of other metal ions, we incubated HA (1 mg/ml) and human antithrombin III (150 μg/ml) at 37°C for 24 hours in the presence of CaCl 2 , FeCl 3 , KCl, MgCl 2 , and NaCl at various concentrations. Residual antithrombin activity was measured as described above. Results In the absence of heparin, antithrombin partly inhibited thrombin activity. Low concentrations of HA did not significantly affect antithrombin activity, regardless of the presence or absence of Ca 2+ or Fe 3+ . However, HA concentrations above 250 μg/ml considerably suppressed the inhibitory ability of antithrombin against thrombin in the presence of Ca 2+ or Fe 3+ , and 1 mg/ml HA completely blocked antithrombin activity under the same conditions. Consequently, thrombin activity was gradually elevated by increasing HA concentrations between 250 and 1000 μg/ml. However, HA at concentrations above 1000 μg/ml progressively lost the ability to prevent inhibition of thrombin activity by antithrombin. Furthermore, HA after digestion with hyaluronidase inhibited antithrombin activity at relatively low concentrations (100 μg/ml) in the presence of Ca 2+ . This observation indicated that the inhibitory effect of HA on antithrombin was not caused by impurities in the reagent. The control without antithrombin indicated that HA does not directly affect thrombin (Fig. 1 ). CSA, CSB, and CSC also inhibited the antithrombin effect in the presence of Ca 2+ but to a lesser extent than did HA (Fig. 2 ). KS did not significantly affect antithrombin activity. Exposing CSs and KS to hyaluronidase did not clearly change this effect, indicating that CSs themselves inhibit antithrombin (data not shown). In contrast to HA, heparin and heparan clearly stimulated thrombin inhibition by antithrombin (Fig. 2 ). However, the stimulatory effect of heparin was considerably decreased in the presence of HA and Ca 2+ . Moreover, the ability of HA to prevent heparin activity was progressively strengthened with increased concentrations of HA within the range 250–1000 μg/ml (Fig. 3 ). Other metal ions, including K + , Mg 2+ , and Na + , did alter the effect of HA on antithrombin (Fig. 4 ). Discussion The destruction of joint tissue is a primary feature of RA. In the inflamed RA synovium, proliferating macrophages and colonizing lymphocytes, together with persistent angiogenesis, produce large amounts of matrix metalloproteinases that destroy the surrounding cartilage and extracellular matrix of connective tissue [ 13 ]. Because GAGs are the basic structural components of joint cartilage, synovial fluid, and soft tissues [ 9 , 10 ], the RA synovium produces an abundance of free GAGs during tissue destruction. Among these, HA is a predominant component of the articular surface and synovial fluid, in which the HA concentration is between 1500 and 2500 μg/ml [ 14 , 15 ]. Pitsillides and coworkers [ 14 ] found that the ratio of free HA to bound HA was significantly increased in the RA (4.53 ± 0.40) as compared with the healthy (1.87 ± 0.42) synovium, although the total concentration of hyaluronan was not increased in the rheumatoid synovium. Their histochemical staining also showed that hyaluronan was concentrated in the lining layer of noninflamed synovial membrane but was more uniformly distributed throughout rheumatoid samples. On the other hand, the HA level is very low among various other tissues. For example, the concentration of serum HA from healthy individuals averages 16 ng/ml, which is 1 × 10 5 fold lower than that in synovial fluid [ 16 , 17 ]. The present study found that HA at concentrations between 250 and 1000 μg/ml significantly blocked the ability of antithrombin to inhibit thrombin. This finding helps to explain why RA occurs and develops in joint tissue, because the inflamed RA synovium is uniquely rich in free HA and other GAGs, along with extracellular matrix degeneration. Although the HA levels are higher in RA than in healthy sera [ 18 ], we demonstrated that the relatively low levels of HA do not prevent antithrombin activity and thus cannot cause blood clots in the circulation. Hence, only the conditions in the RA synovium can drive the pathogenesis of thrombin-related RA, which includes abnormal angiogenesis, extreme proliferation of fibroblast-like cells, excessive fibrin deposition, and proinflammatory processes. Thus, thrombin-related RA worsens because of the snowball effect of HA release in inflamed joints. Our notion is supported by many other studies. Jones and coworkers [ 11 ] found that antithrombin activity is selectively depressed in RA synovial fluid as compared with that in osteoarthritis, although the concentration of the antithrombin–thrombin complex was significantly increased. Ohba and coworkers [ 12 ] also found high levels of thrombin activity in RA synovial fluid. These findings support the notion that inhibiting antithrombin activity plays an essential role in RA pathogenesis. Wang and coworkers [ 10 ] recently constructed a model of arthritis by injecting various GAGs into mice. We postulate that the injected GAGs significantly disrupted the inhibition of thrombin by antithrombin, which therefore caused connective tissue disease through abnormally activated angiogenesis, proinflammatory processes, and fibrin deposition. On the other hand, heparan, which has an almost identical structure to that of heparin but contains fewer sulfates, stimulated antithrombin activity in a similar manner to heparin. These observations indicate that the diverse effects of GAGs on antithrombin are due to differences in their molecular configurations. Heparin pentasaccharide can form complexes with antithrombin and expose a reactive proteinase binding loop on the protein surface [ 19 , 20 ]. Because the molecular structure of HA is analogous to that of heparin, HA might exert its effect by binding to the heparin-binding region of antithrombin. However, such binding did not stimulate the activity of antithrombin as did heparin and heparan; in fact, it blocked the ability of antithrombin to inhibit thrombin. In the present study, the stimulatory effect of heparin on antithrombin was considerably decreased in the presence of HA, supporting the notion that HA could compete with heparin for the heparin-binding region of antithrombin. Remarkably, HA affected the inhibition by antithrombin only within the range 250–1000 μg/ml. At concentrations above 2000 μg/ml, HA either lost its inhibitory effect or elevated the ability of antithrombin to inhibit thrombin. The physiologic level of free HA in the RA synovium is just within the range 500–1000 μg/ml [ 14 ]. Some clinical studies have shown that injecting HA into articular rheumatoid joints can ameliorate inflammation [ 21 , 22 ]. Although further investigation is required to elucidate the exact mechanism by which HA inhibits antithrombin, the results of the present study do not refute the notion that optimal proteoglycan uptake can improve overall articular function in patients with arthritis. Why HA inhibited antithrombin more after than before hyaluronidase digestion remains obscure. Perhaps the small HA molecule can easily bind and thus exert a more inhibitory role on antithrombin. Nagaya and coworkers [ 23 ] found high hyaluronidase activity in the synovial fluid and serum of RA patients, implying an abundance of small HA molecules in the RA synovium. Maneirio and coworkers [ 24 ] reported that HA at various molecular weights had different effects on the interleukin-1 induced synthesis of both nitric oxide and prostaglandin E 2 in chondrocytes. How Ca 2+ and Fe 3+ are involved in inhibiting antithrombin by HA is also poorly understood. Some investigators found that Ca 2+ dramatically promotes the ability of heparin to drive antithrombin activity [ 8 , 25 , 26 ]. Thus, both Ca 2+ and Fe 3+ ions might play similar roles in HA-induced changes in the configuration of antithrombin. Synovial fluid from RA patients contains a far greater abundance of free iron than that from patients with osteoarthritis [ 27 , 28 ]. It was reported that Fe 3+ stored in the RA synovium perpetuates inflammation by supporting the production of oxygen radicals and by promoting hyaluronic acid degradation, as well as the release of lysosomal enzymes [ 29 ]. Telfer and coworkers [ 30 ] recently found that proinflammatory cytokines produced in the RA synovium increased the accumulation of iron in synovial fluid. On other hand, Davies and coworkers [ 31 ] reported that neutrophils from synovial fluid and the circulation of RA patients could increase the release of free Ca 2+ at inflammatory sites. Caruthers and coworkers [ 32 ] also showed that calcium signaling is altered in T lymphocytes from RA patients. Genome-wide single nucleotide polymorphism analysis has shown that peptidylarginine deiminase (PADI4), an enzyme that post-translationally catalyzes peptidyl arginine to citrulline, is closely associated with RA [ 33 ]. We recently found that recombinant human PADI4 protein inactivated human antithrombin III via citrullination in vitro . We also detected an increased level of citrullinated antithrombin in the plasma of RA patients [ 34 ]. PADI4 is extensively expressed in RA synovial tissue [ 35 , 36 ]. Thus, we suggested that the citrullination of antithrombin is one potential pathway through which PADI4 contributes to the pathogenesis of RA [ 34 ]. This notion does not contradict the current findings. We postulate that the genetic, single nucleotide polymorphism-associated disorder of PADI4 and its excessive citrullination of antithrombin play important roles in initiating the RA pathogenic process, whereas inhibition of antithrombin by HA contributes to the development of RA rather than its initiation, because free HA in the synovium achieves high concentrations along with RA progression. Because of abundant Fe 3+ and altered Ca 2+ metabolism together with significant hyaluronidase activity in the RA synovium, thrombin-related RA specifically worsens in joint tissue as a result of antithrombin inactivation by local PADI4 and free HA (Fig. 5 ). HA is an important component of the extracellular matrix. Thrombin and antithrombin play key roles in hemostasis and are involved in the pathogenic processes of many diseases [ 6 , 37 , 38 ]. The findings presented here should also be useful in investigating the nature of other diseases. Conclusion At concentrations of 250–1000 μg/ml in vitro , HA blocked the thrombin-inhibitory ability of antithrombin in the presence of Ca 2+ and Fe 3+ . This finding suggested that the high concentration of free HA in diseased RA synovium locally blocks antithrombin under physiologic conditions and thereby deregulates the activity of thrombin. These processes in turn drive the thrombin-related pathogenesis of RA, which includes extensive fibrin deposition, extreme angiogenesis, and abnormal fibroblast-like cell proliferation. Our findings are consistent with those of previous reports regarding increased coagulation activity in the RA synovium. Abbreviations CS = chondroitin sulfate; GAG = glycosaminoglycan; HA = hyaluronic acid; KS = keratan sulfate; PADI = peptidylarginine deiminase; RA = rheumatoid arthritis. Competing interests The author(s) declare that they have no competing interests. Authors' contributions XC designed and executed the study and prepared the manuscript. RY and KY supervised the project, evaluated data, and assisted in preparing the manuscript.

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1065326

Prescription channeling of COX-2 inhibitors and traditional nonselective nonsteroidal anti-inflammatory drugs: a population-based case–control study

This pharmacoepidemiologic study was conducted to determine whether risk factors for upper gastrointestinal bleeding influenced the prescription of cyclo-oxygenase (COX)-2 inhibitors and traditional nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) at the time when COX-2 inhibitors were first included in the formulary of reimbursed medications. A population-based case–control study was conducted in which the prevalence of risk factors and the medical histories of patients prescribed COX-2 inhibitors and traditional nonselective NSAIDs were compared. The study population consisted of a random sample of members of the Quebec drug plan (age 18 years or older) who received at least one dispensation of celecoxib ( n = 42,422; cases), rofecoxib ( n = 25,674; cases), or traditional nonselective NSAIDs ( n = 12,418; controls) during the year 2000. All study data were obtained from the Quebec health care databases. Adjusting for income level, Chronic Disease Score, prior use of low-dose acetylsalicylic acid, acetaminophen, antidepressants, benzodiazepines, prescriber specialty, and time period, the following factors were significantly associated with the prescription of COX-2 inhibitors: age 75 years or older (odds ratio [OR] 4.22, 95% confidence interval [CI] 3.95–4.51), age 55–74 years (OR 3.23, 95% CI 3.06–3.40), female sex (OR 1.52, 95% CI 1.45–1.58), prior diagnosis of gastropathy (OR 1.21, 95% CI 1.08–1.36) and prior dispensation of gastroprotective agents (OR 1.57, 95% CI 1.47–1.67). Patients who received a traditional nonselective NSAID recently were more likely to switch to a coxib, especially first-time users (OR 2.17, 95% CI 1.93–2.43). Associations were significantly greater for celecoxib than rofecoxib for age, chronic NSAID use, and last NSAID use between 1 and 3 months before the index date. At the time of introduction of COX-2 inhibitors into the formulary, prescription channeling could confound risk comparisons across products.

Introduction Although randomized clinical trials have confirmed the advantage of cyclo-oxygenase (COX)-2 inhibitors over traditional nonselective nonsteroidal anti-inflammatory drugs (NSAIDs) with respect to gastrotoxicity [ 1 - 8 ], a large number of spontaneous reports have incriminated COX-2 inhibitors [ 9 ]. Numerous editorials and letters have been published that question the safety of these products [ 10 - 17 ]. The randomized clinical trial is the design best suited to determine drug efficacy, but it is inadequate for the evaluation of effectiveness, which applies to heterogeneous patient populations and patterns of drug use observed in a real life setting. In addition to pharmacological differences across products, the dosages used for the various indications [ 18 ] and past experience with the drug (through the 'depletion of susceptibles' effect) [ 19 ] account for differences in the risk of adverse effects. In an observational setting, such as postmarketing surveillance, the decision to prescribe one product over another is influenced by the characteristics of the patient, the prescriber and the health care system [ 20 ]. In the absence of randomization, it is consequently of utmost importance, when comparing the risks associated with individual drug classes, to determine whether the patient populations are indeed comparable. The present study was conducted to compare the prevalence of selected risk factors for upper gastrointestinal bleeding among patients prescribed COX-2 inhibitors with those among patients prescribed traditional nonselective NSAIDs, and to compare the characteristics of patients prescribed celecoxib and rofecoxib, which are the two COX-2 inhibitors marketed in Canada at the time of the study. Methods Design A case–control analysis was conducted in which the prevalence of selected gastrointestinal risk factors and medical histories of patients prescribed COX-2 inhibitors (the cases) were compared with those of users of traditional nonselective NSAIDs (the controls). Setting The study involved prescriptions acquired through community pharmacies by members of the Quebec public drug program. Identification of eligible patients and acquisition of study variables were conducted via linkage with four administrative health care databases containing information on beneficiaries, health professionals, pharmaceutical services and medical services. Study population The study targeted all ambulatory adult residents (aged 18 years or older) of the province of Quebec who were members of the public drug coverage program. In Quebec, coverage of prescribed medications was universal for all elderly residents (those aged 65 years or older) regardless of income as well as for all welfare recipients. The program was broadened in 1997 to include patients who do not have access to a private insurance program regardless of age. For everyone, the program now includes a deductible payment and a co-payment, with a monthly premium that depends on the beneficiary's income. In practice, the program includes the following segments of the population: the great majority of community-dwelling elderly persons (>94%), welfare recipients and patients younger than 65 years who do not have access to private insurance (e.g. the self-employed). A sample of 100,000 drug plan members who received at least one celecoxib or rofecoxib prescription between 1 January and 31 December 2000 was randomly selected. A sample of 60,000 nonselective NSAID users was also randomly selected during the same time period, and patients who used low-dose aspirin (acetylsalicylic acid [ASA] ≤325 mg/day) only were excluded from the comparison group. The study population included both new (incident) users and longer time (prevalent) users. The status of patients with respect to being a user of COX-2 inhibitor or nonselective NSAID was determined at the end of the study year. Patients who had received both a COX-2 inhibitor and a nonselective NSAID were considered to be COX-2 inhibitor users. The index date was defined as the date of first dispensation of a COX-2 inhibitor or, for the traditional nonselective NSAID group, the date of the first dispensation of a nonselective NSAID. The following inclusion criteria were applied: participants were required to have been a resident of Quebec for at least 2 years before the index date; and they were required to have had continuous coverage of medical and pharmaceutical services for at least 2 years before the index date. These criteria were verified through the beneficiary database. Study variables The dependent variable was the prescription of COX-2 inhibitors (celecoxib or rofecoxib) or traditional nonselective NSAIDs. The independent variables were selected risk factors for upper gastrointestinal bleeding: patient demographic characteristics (age, sex); prescribed dosage; concomitant use of corticosteroids or anticoagulants; history of gastropathy (using four indicators: prior diagnosis of gastropathy, history of upper gastrointestinal procedures, prior dispensation of gastroprotective agents, and prior referral to a gastroenterologist); and prior history of NSAID use. Comparisons were controlled for prescriber specialty, patient overall health status (using the Chronic Disease Score [CDS]) [ 21 ], income level, past use use of low-dose ASA, acetaminophen, antidepressants and benzodiazepines, and time period. Risk factors for gastrointestinal events Patient demographic characteristics included age, sex and income level, which were sought from the beneficiary database. For reasons of confidentiality, only age on 1 July 2000 was available. Income level was indirectly derived from the type of coverage (amount of deductible payment and co-payment), which was assigned to the patient based on their income. History of gastropathy was assessed during the year before the index date through the presence of a diagnosis consistent with upper gastrointestinal bleeding in the medical services database. When present, this diagnosis was found to be reliable [ 22 ]. However, because it is not mandatory for the physician to be reimbursed, it is often missing. Consequently, three other markers were used: presence of an upper gastrointestinal procedure (e.g. gastroscopy, radiological examination) in the medical database; prior referral to a gastroenterologist, using physician specialty in the medical database; and prior dispensation of gastroprotective agents in the prescription database. Prescribed daily dosage of the COX-2 inhibitors and the traditional nonselective NSAIDs was derived from the dose per unit, quantity dispensed and prescribed duration. Daily dosages were subsequently categorized into low, standard and high, (for each product the dosage thresholds are listed in Table 1 ). Standard dosages were the recommended anti-inflammatory dosages. The threshold for low-dose corresponded to the maximum approved over-the-counter dosage, or, for products available on perscription only, dosages below the recommended prescribed anti-inflammatory dosage. High dosages were those above the maximum recommended anti-inflammatory dosage. Details regarding the dispensation of acetaminophen, low-dose ASA, corticosteroids (excluding asthma-related drugs) and anticoagulants during the year before the index date were obtained from the prescription database. Past use of NSAIDs was assessed through records of the dispensation of these agents during the year before the index date. Patterns of use were defined using three categories of recency (last dispensation ≤1 month, >1 to 3 months, and >3 to 12 months before the index date). For recent users, two categories of duration of use were obtained: chronic (defined as at least one dispensation in each quarter of the previous year) and nonchronic (defined as less than one dispensation in each quarter). Covariables Other variables may influence the prescription of NSAIDs and could act as confounders if they are also associated with risk factors for gastrointestinal events. Patient overall health status was assessed through records on medications dispensed during the year before the index date using the CDS [ 21 ]. Scores are weighted according to the number of different chronic diseases under treatment and the severity of the diseases. The CDS has been found to predict subsequent mortality and hospitalization rates. Because health status at the index date was the variable most likely to influence the physician's prescription, dispensing data for the year before were used for the calculation. Based on the distribution of scores, four categories were defined: 0, 1–4, 5–9 and ≥10. In addition, prescriptions of antidepressants and benzodiazepines were also considered to confirm the findings of a previous unpublished study that demonstrated an association between antidepressant and benzodiazepine use and prescription of COX-2 inhibitors. Prescriber specialty at the index date was determined from the prescription database. Index dates were categorized into three time periods during the study year in order to account for differences in the date of entry of COX-2 inhibitors into the formulary of reimbursed medications (July 1999 and April 2000 for celecoxib and rofecoxib, respectively). The time periods considered were January–June, July–September and October–December 2000. Statistical analysis The strength of the association between each patient characteristic and prescribed drug class was measured using odds ratios. The concomitant effect of patient characteristics was examined using multivariate logistic regression. Three models were used: COX-2 inhibitors as a class versus traditional nonselective NSAIDs, celecoxib versus traditional nonselective NSAIDs, and rofecoxib versus traditional nonselective NSAIDs. All data were analyzed using the SAS statistical package (SAS versions 6.12 and 8.0 for Windows; SAS Institute Inc., Cary, NC, USA). The level of statistical significance was set at 0.05 and the statistical uncertainty of the estimates was assessed using 95% confidence intervals. Ethical considerations No patient or physician identifiers were provided to the researchers; only scrambled identifiers were used throughout the study. The study was approved by the Université de Montréal Health Sciences Ethics Committee. Results After applying the selection criteria, 42,422 celecoxib, 25,674 rofecoxib and 12,418 traditional non-selective NSAID users were identified for the study. The characteristics of the study population are presented in Table 2 . Because of the very large sample size, all differences were statistically significant and therefore P values are not reported. Patients treated with celecoxib were on average slightly older than those treated with rofecoxib or traditional nonselective NSAIDs, and a larger proportion of women were treated with COX-2 inhibitors as opposed to traditional nonselective NSAIDs. For each of the four indicators of prior history of gastropathy, there was a larger proportion of COX-2 inhibitor users with a positive history as compared to nonselective NSAID users. For all indicators used, the proportion was also greater for celecoxib than for rofecoxib. Very few patients had used anticoagulants during the year before the index date, but again the prevalence of use was greater for COX-2 inhibitors than for traditional nonselective NSAIDs. Using the data presented in Table 2 , we were able to determine that, overall, very few patients had used a nonselective NSAID for the first time during the month before the index date. The proportion of patients who had received their last NSAID prescription in the distant past (between 3 and 12 months before index date) was greater for celecoxib than for rofecoxib. Of the patients treated with rofecoxib, 72.8% had not received any NSAIDs during the prior year, which means that it was often used as a first treatment obtained under prescription. This proportion was lower for celecoxib (63.7%) and traditional nonselective NSAIDs (55.2%). Only 6.3% of rofecoxib users had received their last NSAID prescription between 1 and 3 months before the index date, as compared with 7.8% among celecoxib users and 15.7% among nonselective NSAID users. The great majority of NSAIDs were prescribed by general practitioners (85.9% of traditional nonselective NSAIDs, 85.3% of celebrex and 88.3% of rofecoxib prescriptions). Dosage levels were highly heterogeneous across products. A large proportion of traditional nonselective NSAIDs were prescribed at dosages lower than those recommended for anti-inflammatory indications (22.1%) in comparison with celecoxib (3.4%) and rofecoxib (18.2%). Conversely, the majority of COX-2 inhibitors were prescribed at standard anti-inflammatory dosages (65.3% of celecoxib and 73.0% of rofecoxib prescriptions). A relatively high proportion of COX-2 inhibitors, especially celecoxib, were prescribed at dosages in excess of standard recommendations (31.2% of celecoxib and 8.8% of rofecoxib prescriptions). There was a strong correlation between dosage and age. For all products, the proportion of low dosages increased with age, and conversely the proportion of high dosages decreased with age (data not shown). This relationship was also found for overall health status; the higher the CDS, the higher was the proportion of prescriptions for low dosages (30.4% of all prescriptions were of low dosages for patients with a CDS 10+ versus 14.5% for those with a CDS of 0). Results of the multivariate logistic regression are presented in Table 3 . Increasing age and female sex were both associated with greater likelihood of receiving a COX-2 inhibitor. Compared with patients aged 18–54 years, older patients were more likely to receive a COX-2 inhibitor, but this association was greatly confounded by dosage category. Income level marginally influenced the choice of product; patients with lower income favoured the less costly traditional nonselective NSAIDs. According to crude odds ratio estimates, there was a positive association between each indicator of history of gastropathy and the probability of receiving a COX-2 inhibitor. However, when all the indicators were fitted simultaneously in the multivariate model, a history of gastrointestinal procedures was no longer significant; this finding is probably attributable to correlation between the various indicators. The analyses revealed an association between the CDS scores and the probability of receiving a COX-2 inhibitor, although no trend was observed. Use of acetaminophen, corticosteroids, anticoagulants, antidepressants and benzodiazepines during the year before the index date were all associated with the prescription of COX-2 inhibitors. On the other hand, patients who had received low-dose ASA during the previous year were more likely to receive a traditional nonselective NSAIDs than a COX-2 inhibitor. Specialists were less likely to prescribe a COX-2 inhibitor than were general practitioners. Results from the multivariate logistic regression models specific for celecoxib and rofecoxib are presented in Table 4 . As shown, the strength of the association with gastrointestinal risk factors was significantly greater for celecoxib than for rofecoxib for age, past use of NSAIDs between 1 and 3 months before the index date, and recent chronic NSAID use. Point estimates of odds ratio for sex, other patterns of NSAID use, prior dispensation of gastroprotective agents, prior referral to a gastroenterologist, prior gastrointestinal procedures, prior use of antidepressants and benzodiazepines, and anticoagulants were greater for celecoxib than for rofecoxib, but the difference was not significant. Because rofecoxib was only included in the list of reimbursed medications in April 2000, it was not available for half of the first time period, which explains its lower likelihood of being prescribed than nonselective NSAIDs (odds ratio 0.24, 95% confidence interval 0.22–0.26). However, for the second period (July–Sept) there was no significant difference between rofecoxib and celecoxib. Discussion This study provides empirical evidence that channeling exists in the prescription of COX-2 inhibitors. Patients with risk factors for gastropathy were more likely to receive a COX-2 inhibitor than a traditional nonselective NSAID. Age, sex and history of gastropathy are well known independent risk factors for gastrointestinal bleeding, and it is therefore not surprising that they influenced prescribing practices. The effect of sex may be explained by greater use of over-the-counter NSAIDs in the past, not recorded in the databases, for the treatment of dysmenorrhoea. The effect of corticosteroids and anticoagulants is also not surprising, given that these drugs represent contraindications to the prescription of traditional nonselective NSAIDs. These findings are consistent with those obtained in a recent study conducted in a UK primary care setting [ 23 ] but they contradict those reported in a elderly Medicare population in the USA [ 24 ]. In the latter study it appeared that there was over-treatment with COX-2 inhibitors in patients without risk factors, and under-treatment in patients who had at least one risk factor. The effect of past NSAID use is more difficult to interpret because of the lack of data regarding reasons for discontinuation of NSAIDs. Although past NSAID use has been found to be associated with decreased incidence of gastrointestinal bleeding, the impact that such a 'depletion of susceptibles' effect may have on prescribing practices remains to be clarified. Regardless of the underlying mechanism, it can be concluded from these results that past NSAID use is likely to confound risk comparisons across drug classes because it is an independent risk factor for gastrointestinal problems as well as influencing prescribing practices. Patients who had received acetaminophen in the past were more likely to switch to a COX-2 inhibitor than to a traditional nonselective NSAIDs. Patients who had received antidepressants and benzodiazepines were also more likely to receive a COX-2 inhibitor than a traditional nonselective NSAID. This empirical finding is difficult to interpret. It may be hypothesized that physicians may be more likely in general to prescribe newer agents to patients who are anxious. More studies are needed to explore further the interaction between patients and physicians in order to elucidate this issue. Although there was an association between physician specialty and prescription of COX-2 inhibitors or traditional nonselective NSAIDs, the results did not confound the associations between patient characteristics and prescription practices. Results for patient overall health status and prescribing practices were highly confounded by dosage. This suggests that, for the sickest patients, prescribing practices are largely determined by dosage rather than by drug class. Patients with a high level of comorbidity still receive traditional nonselective NSAIDs but at lower dosages. Such findings are likely to be time-sensitive because COX-2 inhibitors were just introduced into the Canadian market during the study period, and there might have been reluctance in the medical community to prescribe newer agents to sicker patients. Comparisons between the two COX-2 inhibitors indicated that for several risk factors under investigation the channeling process is stronger for celecoxib than rofecoxib. However, these findings should be interpreted with caution because for several of the risk factors investigated the differences between products were not statistically significant. On the other hand, celecoxib was not always at a disadvantage; past chronic NSAID use, which, according to the depletion of susceptibles effect, places patients at a lower risk for upper gastrointestinal events [ 19 ], was associated with a greater probability of being prescribed celecoxib than rofecoxib. Many risk factors for gastrointestinal bleeding could not be ascertained in this study, such as smoking status and alcohol use, which are known risk factors for gastrointestinal events and have also been found to influence prescribing practices [ 23 ]. Also, there were no data on indications but we controlled for dosage, which, according to Griffin and coworkers [ 18 ], is more likely to influence the risk of gastrointestinal bleeding than indication per se . Dosage had a very large impact on the results, and its exclusion would have produced spurious differences across products. There were no data on over-the-counter use of NSAIDs such as aspirin and ibuprofen. Therefore, it was not possible to explore the concomitant use of nonprescribed NSAIDs. Finally, data are generalizable only to recent use (in the previous year). We were not able to explore the impact of more distant history. Nevertheless, the use of retrospective data obtained from administrative databases allowed us to examine the various associations in a truly observational setting without influencing prescribing practices in any way. In addition, the large sample size allowed us to conduct comparisons across individual products. Conclusion Our results provide empirical evidence that the introduction of a new class of medications into the market results in the channeling of patients at high risk for adverse effects. However, as shown by the present study, differences across individual products cannot be predicted from their order of entry into the formulary. Other factors, such as marketing strategies, play a major role as well. Neverthless, one may conclude that selective prescribing results in a positive association between risk factors and drug use, which could confound risk comparisons across products. Abbreviations ASA = acetylsalicylic acid; CDS = chronic disease score; COX = cyclo-oxygenase; NSAIDs = nonsteroidal anti-inflammatory drugs. Competing interests This study was funded through an unrestricted grant from Pharmacia Corporation. YM was a paid consultant for this study. JFB, TD, NM and SP declare that they have no competing interests. SZ was an employee of Pharmacia Corporation at the time the study was conducted. Authors' contributions YM, as principal investigator of the study, designed and coordinated the study, interpreted study results, and wrote the manuscript. TD conducted the statistical analyses. JFB participated in the design of the strategy for the sampling of the study population and helped to draft the manuscript. NM assisted in the conduct of the statistical analyses and contributed to the interpretation of the study results. SP assisted in the review of the literature and determined the relevance of the study. SZ conceived the study and participated in its design. All authors read and approved the final manuscript.

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1065327

Endothelin-1 in osteoarthritic chondrocytes triggers nitric oxide production and upregulates collagenase production

The mechanism of endothelin-1 (ET-1)-induced nitric oxide (NO) production, MMP-1 production and MMP-13 production was investigated in human osteoarthritis chondrocytes. The cells were isolated from human articular cartilage obtained at surgery and were cultured in the absence or presence of ET-1 with or without inhibitors of protein kinase or LY83583 (an inhibitor of soluble guanylate cyclase and of cGMP). MMP-1, MMP-13 and NO levels were then measured by ELISA and Griess reaction, respectively. Additionally, inducible nitric oxide synthase (iNOS) and phosphorylated forms of p38 mitogen-activated protein kinase, p44/42, stress-activated protein kinase/Jun-N-terminal kinase and serine-threonine Akt kinase were determined by western blot. Results show that ET-1 greatly increased MMP-1 and MMP-13 production, iNOS expression and NO release. LY83583 decreased the production of both metalloproteases below basal levels, whereas the inhibitor of p38 kinase, SB202190, suppressed ET-1-stimulated production only. Similarly, the ET-1-induced NO production was partially suppressed by the p38 kinase inhibitor and was completely suppressed by the protein kinase A kinase inhibitor KT5720 and by LY83583, suggesting the involvement of these enzymes in relevant ET-1 signalling pathways. In human osteoarthritis chondrocytes, ET-1 controls the production of MMP-1 and MMP-13. ET-1 also induces NO release via iNOS induction. ET-1 and NO should thus become important target molecules for future therapies aimed at stopping cartilage destruction.

Introduction Cartilage degradation in osteoarthritis (OA) and rheumatoid arthritis constitutes a major structural change in the joint, which may severely impair its function and cause pain and disability. This degradation is accompanied by the release in the synovial fluid of degraded matrix constituents that primarily result from an increased matrix catabolism [ 1 ]. Various factors are directly involved in this process. Endothelin-1 (ET-1), a potent vasoconstrictor and promitogen peptide for many cell types, including chondrocytes, was recently identified as one such factor [ 2 , 3 ]. ET-1 binds to the specific endothelin A or endothelin B receptors expressed on chondrocytes [ 4 ] and triggers a cascade of intracellular events, including phospholipase C activation [ 5 ], an increase in intracellular calcium [ 6 , 7 ], prostaglandin production [ 8 ] and nitric oxide (NO) release [ 9 ]. The effect of ET-1 on DNA and protein synthesis in chondrocytes is biphasic. The potent initial stimulatory effect of ET-1 decreases progressively with time and is followed by an inhibition [ 3 , 8 ]. The inhibitory effect seems to be mediated by NO and cGMP, both produced in response to ET-1 stimulation [ 8 , 9 ]. Additionally, we have recently demonstrated that ET-1 is significantly increased locally in OA cartilage and synovial membrane when compared with normal tissues. In OA cartilage, ET-1 is involved in cartilage catabolism through metalloprotease (MMP) regulation and the induction of type II collagen breakdown [ 2 ]. MMPs are a family of structurally related zinc-dependent neutral endopeptidases classified into subgroups of collagenases, gelatinases, stromelysins, membrane-type MMPs and other MMPs [ 10 ]. When activated, MMPs degrade a broad spectrum of substrates, including collagens and other matrix macromolecules. As a whole, MMPs play an important role in the extracellular matrix remodelling that occurs under physiological and pathological conditions. Among all the MMPs, we have recently demonstrated an induction in the synthesis, secretion and activation of two collagenases (MMP-1 and MMP-13) by ET-1 [ 2 ]. These MMPs play an active role in the progression of OA pathology as they are the most effective at initiating collagen destruction during the inflammatory process and the remodelling phase of the disease [ 11 , 12 ]. Another deleterious agent in joint cartilage is the NO radical [ 13 , 14 ], which downregulates DNA [ 8 ] and matrix synthesis [ 14 ] and upregulates matrix degradation via increased MMP synthesis [ 15 ]. Indeed, inhibition of NO production was shown to slow down the progression of OA. It has been demonstrated that, in vitro , NO could also upregulate MMP synthesis and activity in joint chondrocytes and cartilage [ 15 ]. In vivo in an OA animal model, selective inhibition of the inducible nitric oxide synthase (iNOS) provides a protective effect on OA joint tissues more specifically in regard to the degradation of the extracellular matrix and the downregulation of MMP [ 16 ]. The aim of the present study was to further investigate the role of ET-1 in human OA chondrocytes, focusing on NO, MMP-1 and MMP-13 production as well as the relevant signalling pathways activated by ET-1 in human OA chondrocytes in regard to these factors. Materials and methods Specimens Human cartilage was obtained with the consent of 12 OA patients (mean ± standard error of the mean age, 58 ± 6 years) undergoing total knee replacement. The Institutional Ethics Committee Board of Notre Dame Hospital in Montreal, Canada approved the study protocol. Tissue specimens were embedded in paraffin, were sectioned and stained with Safranin O and fast green, and were evaluated using the Mankin histological/histochemical scale [ 17 ]. Only tissues corresponding to a moderate degree of OA severity (Mankin 3–7) were included in this study. Cartilage was sectioned from the tibial plateaus, rinsed and finely chopped, and the cells released by enzymatic digestion performed as previously described [ 2 , 11 ]. The cells were seeded in culture flasks at the density of 10 4 cells/cm 2 and were grown to confluence in DMEM (Gibco BRL, Burlington, ON, Canada) containing 10% heat-inactivated FCS (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Gibco BRL). Only first-passage-cultured cells were used. MMP-1 and MMP-13 quantification MMP-1 and MMP-13 protein levels were determined in the culture media using specific ELISA assays. The ELISA assay (Amersham Biosciences Corp., Baie d'Urfé, QC, Canada) for MMP-1 specifically detected the total human MMP-1 (i.e. active MMP-1, the latent enzyme and the enzyme complexed with inhibitors such as tissue inhibitor of matrix metalloproteinases 1). The sensitivity of this assay is 1.7 ng/ml, and there is no significant cross-reactivity or interference with MMP-3, MMP-2 and MMP-9. The MMP-13 ELISA assay (R&D Systems Inc., Minneapolis, MN, USA) is a monoclonal polyclonal-based assay specific for both the active and latent MMP-13. Its sensitivity is 0.032 ng/ml, and there is no cross-reactivity with MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9 and MT1-MMP. Results are expressed as nanograms per 5 × 10 5 cells. The effect of ET-1, protein kinase inhibitors and a guanylate cyclase inhibitor (LY83583) on MMP-1, MMP-13 and NO production MMP-1 production, MMP-13 production and NO production were studied in the absence of and in the presence of ET-1, using various inhibitors: 1 μM SB 202190 (inhibitor of p38 mitogen-activated protein [MAP] kinase), 10 μM PD 98059 (a selective mitogen-activated protein kinase kinase 1/2 [MEK1/2] inhibitor), 100 nM Wortmannin (a phosphatidyl inositol 3 kinase inhibitor), 4 μM KT5720 (a protein kinase A [PKA] inhibitor), or 2 μM LY83583 (an inhibitor of NO-dependent soluble guanylate cyclase inhibitor). All inhibitors were purchased from Calbiochem EDM Biosciences Inc. (San Diego, CA, USA), and the active concentrations chosen are based on the literature or were assayed in preliminary experiments [ 18 , 19 ]. ET-1 was purchased from (Sigma-Aldrich, Oakville, ON, Canada). Confluent OA chondrocytes were preincubated for 30 min with these inhibitors and then 10 nM ET-1 was added for 24 hours. Following incubation, the MMP-13 and MMP-1 protein levels and NO levels were determined in the media of six independent cultures as described in the following. NO determination Nitrite (NO 2 - ), a stable end product of NO, was measured in the media of cultured cells using a spectrophotometric method based on the Griess reaction [ 20 ]. To examine the effects of ET-1 on NO production, a dose–response curve was performed by incubating OA chondrocytes for 24 hours with increased concentrations (0–100 nM) of ET-1, or by pretreating with protein kinase inhibitors or a guanylate cyclase inhibitor and ET-1 as already described. NO production was also evaluated in the presence of the iNOS inhibitor L-NIL (L-N 6 (1-iminoethyl)lysine) (Calbiochem EDM Biosciences Inc.). Chondrocytes were preincubated for 30 min with 0–50 μM L-NIL and were then incubated for 24 hours with 10 nM ET-1. The media were collected and the released NO levels were determined. Results are expressed as nanomoles per 5 × 10 5 cells ± standard error of the mean or as a percentage of the control cultures. Western blot Confluent OA chondrocytes were incubated in the presence of or in the absence (control) of 10 nM ET-1, and the cells were lysed in 0.2 ml lysis buffer (25 mM HEPES, 5 mM MgCl 2 , 1 mM EDTA, 1 mM PMSF, 10 μg/ml pepstatin, 10 μg/ml leupeptin, pH 7.5). The protein concentration of the lysate was determined with the Bradford dye assay (Bio-Rad Laboratories, Hercules, CA, USA). For western blot, 10 μg lysate protein was separated by electrophoresis on a 10% SDS discontinuous gradient polyacrylamide gel. Separated proteins were then transferred electrophoretically onto a nitrocellulose membrane (Hybond C extra; Amersham, Pharmacia Biotech, Chalfont St Giles, UK). The membranes were immersed overnight in the Super Block Blocking buffer (Pierce, Rockford, IL, USA), rinsed and incubated for 24 hours at 4°C with one of the mouse monoclonal primary antibodies (New England Biolabs, Mississauga, ON, Canada) specifically recognizing phosphorylated p38 or total p38 (dilution, 1/1000), phosphorylated p44/42 (dilution, 1/5000), phosphorylated Akt (dilution, 1/2000), phosphorylated stress-activated protein kinase/Jun-N-terminal kinase (SAP/JNK) (dilution, 1/1000), or actin C-terminal fragment (dilution, 1/5000). iNOS was detected with a rabbit polyclonal antibody (dilution, 1/1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Following incubation with primary antibody, membranes were carefully washed and reincubated for 1 hour at 4°C with a second antibody (anti-rabbit IgG). Anti-mouse horseradish peroxidase-conjugated IgG (dilution, 1/25,000) was used for the detection of the monoclonal antibody, and sheep anti-rabbit horseradish peroxidase-conjugated IgG (dilution, 1/40,000) was used for the polyclonal antibody. Detection was performed using the Super Signal Ultra Western blot chemiluminescence system (Pierce) [ 11 ]. Apoptosis Apoptosis was investigated in OA chondrocytes cultured on Lab-Tec chamber slides (Nalge Nunc International, Naperville, IL, USA). At confluence, the cells were rinsed and incubated at 37°C for 72 hours in DMEM containing 2.5% heat-inactivated FCS in the absence of or in the presence of 10 nM human recombinant ET-1. Apoptotic cells were detected by in situ staining using the TUNEL method (Trevigen Inc., Gaithersburg, MD, USA). Both pro-apoptotic Bad and anti-apoptotic Bcl2 proteins were determined by immunocytochemical detection using specific anti-Bad and anti-Bcl2 antibodies (Upstate Biotechnology, Lake Placid, NY, USA). The results are expressed as the mean percentage of positively stained cells according to a previously published method [ 21 , 22 ]. Statistical analysis Data are expressed as the mean ± standard error of the mean of five or six independent cultures. Statistical significance was assessed by the Mann–Whitney test, and P < 0.05 was considered significant. Results ET-1 induces MMP-1 and MMP-13 production The effects of ET-1 and those of various inhibitors on MMP-1 production and MMP-13 production are shown in Fig. 1 . At 10 nM ET-1 the production of both enzymes was significantly increased ( P < 0.005). SB202190, a p38 inhibitor, completely suppressed the ET-1-stimulated production of both enzymes, whereas the phosphatidyl inositol 3 kinase inhibitor Wortmannin and the PKA inhibitor KT5720 partially but significantly ( P < 0.01) decreased the level of MMP-13 only. Interestingly, the most potent inhibitor of MMP-1 and MMP-13 production was LY83583, an inhibitor of NO-dependent soluble guanylate cyclase and of cGMP. This agent not only suppressed the ET-1-induced stimulation, but also decreased the level of both enzymes below the basal level: a significant difference was found for both MMP-13 and MMP-1 when compared with the ET-1 stimulation ( P < 0.005) and for MMP-13 when compared with the control ( P < 0.05). Although a decrease in MMP-13 was noted with the MEK1/2 kinase inhibitor PD98059 at the concentration tested, it did not reach statistical significance. With this inhibitor, no effect was found on MMP-1 production. ET-1 induces NO production The effects of ET-1 on NO release and on iNOS expression are shown in Fig. 2 . Figure 2a shows that ET-1 greatly stimulated NO production and was released in a concentration-dependent manner. Incubation with increasing concentrations of ET-1, from 0.1 to 100 nM, augmented almost 12-fold the linear accumulation of NO. To determine the mechanism involved in the ET-1-induced NO production, the effects of the major intracellular signalling pathways were investigated. Figure 2b shows that the ET-1-induced NO release was significantly inhibited by p38 inhibition and prevented by KT5720, a PKA inhibitor. No significant effect was noted for MEK1/2 inhibition by PD98059 and by Wortmannin. Moreover, the guanylate cyclase inhibitor LY83583 reduced the NO production as significant differences were found when compared with either the ET-1 stimulation ( P < 0.05) or with the control ( P < 0.05), and this inhibitor also decreased both the endogenous and ET-1-induced iNOS level (Fig. 2d ). The ET-1-induced NO release occurs via iNOS as shown in Figure 2c : complete inhibition of iNOS by 50 μM allosteric iNOS inhibitor L-NIL, as expected, almost completely inhibited NO release. Figure 2d shows the effects of various inhibitors on iNOS expression, as determined by western blot analysis of cell extracts. The 24-hour incubation of cells with ET-1 results in an increase of iNOS protein (Fig. 2d , lane 2). The ET-1-induced iNOS protein expression was completely suppressed by SB202190 and LY83583, and was partially suppressed by Wortmannin and KT5720. PD98059 had no effect. Intracellular protein kinase phosphorylation in the presence of ET-1 Figure 3a–d show the effects of ET-1 on the phosphorylation of p38, Akt, p44/42 and SAP/JNK kinases as detected by western blot of cell extracts. ET-1 at 10 nM induced p38, Akt, p44/42, and SAP/JNK phosphorylation in a time-ordered manner. For p38, the maximal effect following cell exposure to ET-1 was obtained at 10 min. For Akt, the maximal effect was observed at 2 min of cell exposure and this effect persisted during 30 min, followed by a decline at 45 min. At this time (45 min), both p38 kinase and Akt phosphorylated forms were diminished. The maximal effect was obtained at 15 min for p44/42 kinase and at 45 min for SAP/JNK. The SAP/JNK phosphorylated forms were not detected at 60 min, whereas that of p44/42 decreased but was still present even at 60 min. ET-1 did not affect apoptosis As ET-1 induces NO release and because the accumulation of NO causes apoptosis, we explored this potential effect. OA chondrocytes incubated in the absence of (control) or in the presence of ET-1 (10 nM) for 72 hours showed that ET-1 did not affect apoptosis (TUNEL reaction; data not shown) or the production of either anti-apoptotic Bcl2 or pro-apoptotic Bad proteins. A similar percentage of positively stained cells was found for Bcl2 (42.8 ± 5.1% and 43.2 ± 4.3% for the control and for ET-1, respectively) and for Bad (10.1 ± 3.8% and 9.5 ± 2.1%, respectively). Discussion This study shows an overproduction of NO, MMP-1 and MMP-13 in human OA chondrocytes stimulated by ET-1. This result goes beyond previous results [ 2 ], which showed that human OA synovial tissue and joint cartilage express the ET-1 gene and overproduce ET-1, resulting in an excessive synthesis of MMP-1 and MMP-13 in the same tissues. In addition, the result goes beyond these findings and enlightens on the mechanism by which ET-1 accomplishes this action. Strong evidence was obtained for the key role played by NO, whose production and release were also upregulated by ET-1. NO induces smooth muscle cell relaxation by activating soluble guanylate cyclase and by increasing the intracellular concentration of cGMP. LY83583 suppresses the effect of NO by inhibiting this NO-dependent production of cGMP [ 23 ]. In the present study, LY83583 was also shown to strongly inhibit MMP-1 and MMP-13 production by unstimulated and ET-1-stimulated OA chondrocytes, showing the key role of cGMP for the synthesis of these enzymes. This finding confirms a previous observation that cGMP is necessary for protein synthesis [ 9 ], and brings further evidence that an excess of NO is harmful to cells. It is generally accepted that progressive tissue destruction in rheumatoid arthritis and in OA results from an excessive breakdown mediated by various proteolytic enzymes and other catabolic agents such as free radicals and NO [ 1 , 13 , 24 , 25 ]. Our results suggest that ET-1 should also be added to the list of potential deleterious agents that may account for articular cartilage destruction in rheumatic diseases. The action of ET-1 seems to be dual via an increase in MMP and NO production. ET-1-induced stimulation of MMP-1 and MMP-13, as well as the induction of iNOS gene expression with subsequent NO overproduction by OA chondrocytes, may interfere with the proinflammatory cytokine pathways. Indeed, we and other workers have shown that IL-1β upregulates the synthesis of ET-1 [ 3 ], which in turn can induce IL-1β gene transcription and consequently the production of the protein [ 26 ]. We previously demonstrated [ 2 ] that MMP-13 expression was induced similarly by ET-1 and IL-1β; however, although they both enhanced MMP-1 expression, the effect of IL-1β was more potent on this enzyme. Interestingly, using a specific immunoassay measuring the C telopeptide of type II collagen fragments on OA cartilage explants, we also found that the level of the cleaved collagen fragments were significantly increased in the presence of both IL-1β and ET-1 with a more potent effect observed for ET-1. This could be explained by a putative synergy between ET-1 and IL-1β as ET-1 induces IL-1β and as IL-1β has a positive feedback on ET-1 synthesis [ 19 , 27 ]. NO is an important signalling molecule at physiological concentrations [ 28 ], but when overproduced via iNOS gene activation it is toxic to cells [ 29 ]. NO triggers the transcription of several proinflammatory genes [ 28 , 30 ], interacts with the cysteine residues of many proteins (S nitrosylation) and may alter their structure and function. In the presence of the superoxide anion, NO generates peroxynitrite and hydroxyl radicals that are cytotoxic, inducing peroxidation of lipids and damaging other molecules, such as DNA, and matrix macromolecules. This finally results in the inhibition of many cellular processes that impair the capacity of the cells to synthesize matrix macromolecules and to repair damaged tissue [ 8 , 31 ]. In addition to the findings already discussed, the present study sheds more light on the major signalling pathways involved in the ET-1-induced MMP-1 and MMP-13 production and in NO production. In OA chondrocytes, ET-1 seems to stimulate the production of these enzymes through activation of, at least, two kinases, p38 MAP kinase and PKA. As shown by western blot analysis of the cell extracts, incubation of cells for a short period of time with ET-1 results in the phosphorylation of p38 MAP, p44/42, SAP/JNK and Akt kinases. This effect occurs within minutes following a challenge with ET-1, and disappears after 45 and 60 min for the p-38 and SAP/JNK kinases, respectively. The activation of these kinases is probably necessary for the induction by ET-1 of MMP-1 production and MMP-13 production. The inhibition of p38 kinase is associated with a suppression of the ET-1-induced stimulation of both enzymes, whereas the inhibitions of adenyl cyclase-dependent PKA kinase is associated with a partial suppression of the ET-1-induced stimulation of MMP-13 production only. This suggests that these inhibitors are specific for the ET-1-activated pathways since they do not affect the basal levels of MMP-1 and MMP-13. Another point also deserves consideration. Tardif and colleagues [ 32 ] have described two OA chondrocyte populations distinctive by their MMP-13 content and their response to IL-1β. One population contains small amounts of MMP-13 protein and is highly sensitive to IL-1β stimulation; the other population is enriched in MMP-13 protein but poorly responds to the cytokine. The cell heterogeneity of OA cartilage may explain some variability of the results observed in our study, particularly in the case of using low doses of the MEK1/2 inhibition followed by ET-1 stimulation. In fact, when MAP kinase pathways (extracellular signal-regulated kinase, JNK and p38) are activated in chondrocytes, their inhibition is dependent of the inhibitor concentration used, particularly for SB 203580 and PD 98059 [ 18 ]. PD 98059, which had no effect in the present study at the concentration of 10 μM on ET-1-induced iNOS expression and NO production, was demonstrated in other studies to suppress NO induction in human chondrocytes, as shown by Gemba and colleagues [ 18 ]. The phosphorylation of p38 MAP kinase by ET-1 was also described in osteoblast-like cells [ 33 ] and in cardiac myocytes [ 34 ], while in chondrocytes overproducing MMP-1 and MMP-13 this MAP kinase was shown to be phosphorylated principally by IL-1β [ 35 ]. Activation of PKA was shown to be required for the upregulation of iNOS, and for the subsequent production and release of NO by several cell types such as vascular smooth muscle cells [ 36 ], cardiac myocytes [ 37 ] and human macrophages [ 38 ]. It is also associated with the cytokine-induced NO production in human OA articular chondrocytes [ 39 ]. Our results suggest that the activation of PKA is also required for the ET-1-induced upregulation of iNOS and for subsequent production of NO by human OA chondrocytes. However, PKA activation seems to be less required for the ET-1-induced upregulation of MMP-13 and not at all necessary for the upregulation of MMP-1 since the inhibition of PKA with KT5720 does not affect the ET-1-induced overproduction of this enzyme. In the present study, subtle differences are shown in the pattern of inhibition of the ET-1-induced overproduction of MMP-1 and MMP-13. The effect of ET-1 on MMP-13 production was more sensitive to the inhibitors of protein kinases than on MMP-1 production. As suggested earlier, these variable responses point to possible different cell populations producing these two enzymes or to relevant signalling pathways eliciting the ET-1-induced stimulations [ 35 ]. We also tested the hypothesis that ET-1 may act in OA through induction of apoptosis. This was based on the findings that cells of the superficial layer disappear during cartilage degeneration [ 40 ], that ET-1 is preferentially produced in this layer [ 2 ], and that NO may induce apoptosis and cell death at high concentrations [ 29 ]. Indeed, chondrocyte death may represent one of the contributing factors in cartilage destruction. However, as shown in the present study, ET-1 does not appear to induce chondrocyte apoptosis or cell death. Using the TUNEL technique (which was recently shown to detect both apoptosis and cell death [ 29 ]), and using Bcl2 and Bad protein determination, no differences were found between ET-1-treated cultures and control cultures. Conclusion The present study shows that ET-1 causes an overproduction of NO, MMP-1 and MMP-13 in human OA chondrocytes. The signalling pathway used by ET-1 in these cells was also demonstrated. The fact that ET-1 possesses the biological properties described acknowledges this peptide as an important catabolic factor contributing to the cartilage destruction via induction of the deleterious molecules such as MMPs and NO. NO seems to be a key molecule that is produced in parallel with the ET-1-induced overproduction of the MMPs. Blocking the effects of ET-1 may thus become a useful therapeutic approach aimed at stopping cartilage destruction in rheumatic conditions such as rheumatoid arthritis and OA. Abbreviations DMEM = Dulbecco's modified Eagle's medium; ELISA = enzyme-linked immunosorbent assay; ET-1 = endothelin-1; FCS = foetal calf serum; IL = interleukin; iNOS = inducible nitric oxide synthase; L-NIL = L-N 6 (1-iminoethyl)lysine; MAP = mitogen-activated protein; MEK1/2 = mitogen-activated protein kinase kinase 1/2; MMP = metalloprotease; NO = nitric oxide; OA = osteoarthritis; PKA = protein kinase A; SAP/JNK = stress-activated protein kinase/Jun-N-terminal kinase; TUNEL = terminal deoxynucleotidyl transferase-medulated dUTP nick end labelling. Competing interests The author(s) declare there are no competing interests. Authors' contributions CAM executed the study, contributed to the preparation of the manuscript and performed statistical analysis. MR-B and FSS assisted in the experiments and in the isolation of chondrocytes from human cartilage. JCF, JM-P and J-PP assisted with the design of experiments and obtained human tissues. DRM evaluated and interpreted data and assisted with the preparation of the manuscript. FM designed the study, supervised the project, evaluated and interpreted data, and prepared the manuscript.

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1065328

Autoantibodies specific for apoptotic U1-70K are superior serological markers for mixed connective tissue disease

Modifications occurring on autoantigens during cell death have been proposed to have a role in the initiation of autoimmune diseases. Patients suffering from mixed connective tissue disease (MCTD) produce autoantibodies directed to U1 small nuclear ribonucleoprotein (snRNP), and antibodies against a 70 kDa protein component, the U1-70K (70K) protein, are the most prominent. During apoptosis, 70K is cleaved by caspase-3 to a 40 kDa product, which remains associated with the complex. Autoantibodies preferentially recognizing the apoptotic form of 70K have been described previously, and an apoptosis-specific epitope on 70K has been identified. This study shows that 29 of 53 (54%) MCTD sera preferentially recognize the apoptotic form of 70K over intact 70K. Moreover, we show that antibodies directed to an apoptosis-specific epitope on 70K are more specifically associated with MCTD than other anti-70K antibodies, suggesting that apoptotic 70K is a better antigen for the detection of these antibodies in MCTD patients. Longitudinal analysis of 12 MCTD patients showed in several patients that early sera are relatively enriched with antibodies recognizing an apoptosis-specific epitope, and that the levels of these apoptosis-specific antibodies decrease in time. These findings indicate that the early detection of apoptotic 70K is of considerable interest for anti-U1 snRNP-positive patients.

Introduction Patients suffering from autoimmune diseases are characterized by the presence of autoantibodies directed to a wide range of autoantigens. Mixed connective tissue disease (MCTD) is a relatively rare systemic autoimmune disease and includes a group of patients with overlapping clinical symptoms of systemic lupus erythematosus (SLE), systemic sclerosis (SSc), rheumatoid arthritis and polymyositis/dermatomyositis. Sharp and colleagues were the first to describe MCTD as a distinct rheumatic disease [ 1 ], but whether MCTD can be regarded as a distinct disorder has been a subject of discussion [ 2 ]. A characteristic serological feature that distinguishes MCTD patients from patients with other connective tissue diseases is high levels of autoantibodies directed against the U1 small nuclear ribonucleoprotein (snRNP) particle [ 1 , 3 ]. The U1 snRNP is a highly conserved RNA–protein complex, located in the nucleus, where it is involved in the processing of pre-mRNA [ 4 , 5 ]. It consists of the U1 snRNA molecule and several proteins: the U1A, U1C and U1-70K (70K) proteins are components specific for the U1 snRNP, whereas the seven Sm proteins (B/B', D1, D2, D3, E, F and G) are shared with other U snRNPs [ 6 ]. Most U1 snRNP components are autoantigenic in MCTD and SLE. Autoantibodies directed against U1A, U1C, 70K and the U1 snRNA molecule are mainly found in MCTD patients, whereas autoantibodies targeting Sm-D, Sm-B/B' and the E.F.G complex are more specifically associated with SLE [ 7 , 8 ]. The mechanisms through which such autoantigens, generally highly conserved and ubiquitously expressed molecules, escape tolerance and are recognized by the immune system as non-self remain unclear, but it is proposed that cell death is important in the initiation of autoimmune responses [ 9 , 10 ]. Recently, secondary necrosis has also been put forward as a source of proteolytically modified autoantigens [ 11 ], but the modifications that occur on autoantigens during apoptosis were studied most extensively. Apoptotic modifications on autoantigens include specific cleavage by caspases or granzyme B, (hyper)phosphorylation, dephosphorylation, citrullination, methylation and transglutaminase cross-linking [ 10 , 12 , 13 ], and it is thought that these modifications might be seen by the immune system as novel 'cryptic' epitopes. It is believed that these novel epitopes induce the primary immune response, and that secondary immune responses and epitope spreading result in autoantibodies that are directed against unmodified regions of the autoantigens and antigens that are associated with the initially modified autoantigen [ 9 ]. One of the apoptotic modifications occurring on the U1 snRNP is the cleavage of 70K at residue 341 by caspase-3 [ 14 , 15 ]. Antibodies against 70K are in general the first autoantibodies to appear in anti-U1 snRNP (often referred to as anti-RNP) positive patients, indicating that 70K is important as an initial autoantigen [ 16 ]. The molecular and immunological characteristics of the major apoptotic isoform of 70K, a 40 kDa cleavage product that remains associated with the U1 snRNP complex [ 17 ], and its role in the triggering of the primary and possibly secondary autoimmune response, are therefore intriguing. Recently it was shown that sera of some anti-U1 snRNP positive patients contain antibodies that specifically bind to the apoptotic form of 70K, which displays an epitope that is not present on the intact form [ 18 , 19 ]. This epitope is dependent on the region between amino acids 180 and 205, partly overlapping with the RNA-binding domain and overlapping with the most common T cell epitope [ 20 ]. In this study we analyzed a cohort of MCTD and control patients for the presence of autoantibodies against intact and apoptotic 70K. Moreover, we longitudinally analyzed sera from another group of MCTD patients. Our results show that, early in disease, autoantibodies directed against the apoptotic form of 70K (70K apop ) are more strongly represented than autoantibodies against the intact form. Longitudinal studies also show that autoantibodies against 70K apop are not significantly correlated with disease flares. Methods Patient sera All patients were seen at the Department of Rheumatology of the University Medical Centre Nijmegen or the St Maartenskliniek Nijmegen (The Netherlands), and were classified in accordance with standard criteria for each disease. All MCTD patients ( n = 53) tested positive for anti-U1 snRNP autoantibodies by counter-immunoelectrophoresis, and for antibodies against one or more components of the U1 snRNP complex by immunoblotting. Most of the sera (91%) were also RNP positive as shown by U1 snRNA co-immunoprecipitation. Longitudinal serum collections were obtained from 12 MCTD patients and have been described previously [ 21 ]. From each patient, over a period of 4–15 years (average 10 years), 8 to 33 serum samples (average 18 samples) were available and were analyzed. During the follow-up study, the patients were regularly monitored for clinical and serological parameters. At each visit the disease activity was measured in accordance with a validated SLE disease activity index described by Ter Borg and colleagues [ 22 ]. Medication was given as indicated by the clinical status. Additionally, patient sera were collected from SLE ( n = 48), polymyositis/dermatomyositis ( n = 26), primary Sjögren's syndrome ( n = 18), SSc ( n = 10), rheumatoid arthritis ( n = 3), Raynaud's phenomenon ( n = 3) and undefined connective tissue disease ( n = 1). Informed consent was obtained from all participants in accordance with the medical ethical regulations of the local ethics committee. Sera were stored at -70°C until use. Cell lines, induction of cell death and preparation of cell extracts Jurkat (human T cell leukemia) suspension cells were grown in RPMI 1640 medium (Gibco-BRL), supplemented with 1 mM sodium pyruvate, 1 mM penicillin, 1 mM streptomycin and 10% heat-inactivated fetal calf serum (Gibco-BRL), in a humidified 37°C incubator containing 5% CO 2 . Cells were maintained at a concentration of 10 6 cells/ml and were induced to undergo apoptosis by the addition of 10 μg/ml anisomycin. Eight hours after induction, apoptotic cells were harvested by centrifugation at 800 g for 10 min and washed with PBS. Apoptotic and non-apoptotic Jurkat cells were resuspended in Nonidet P40 (NP40)-containing lysis buffer (25 mM Tris-HCl, pH 7.6, 100 mM KCl, 10 mM MgCl 2 , 0.25 mM dithioerythritol, 1% NP40, Complete™ protease inhibitor cocktail [Roche]) at a concentration of 10 8 cells/ml. Cells were lysed on ice for 30 min and subsequently centrifuged for 30 min at 12,000 g and 4°C. Supernatants were used immediately or stored at -70°C. SDS–polyacrylamide gel electrophoresis and western blotting Cell extracts of 1.3 × 10 7 non-apoptotic Jurkat cells and 1.3 × 10 7 apoptotic Jurkat cells, either separately or mixed, were separated by SDS–polyacrylamide gel electrophoresis. Directly after gel electrophoresis, proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell) by semi-dry electroblotting. Staining of the membrane with Ponceau S (Sigma) was used to verify protein transfer. Probing western blots with patient sera All incubation steps were performed at approximately 20°C on a shaking table. Western blots containing non-apoptotic and apoptotic Jurkat cell extracts were pre-blocked with 5% non-fat dried milk in PBS containing 0.1% NP40 (MPBS/NP40) for 2 hours. Subsequently, membranes were incubated with patient serum, diluted 1000–5000-fold in MPBS/NP40, for 1 hour. After extensive washing with PBS containing 0.1% NP40 (PBS/NP40), membranes were incubated with horseradish peroxidase-labeled rabbit anti-human IgA/IgG/IgM antibody (Dako Immunoglobulins), diluted 1000-fold in MPBS/NP40, for 1 hour. After several washes with PBS/NP40 and PBS, bound antibodies were detected by enhanced chemiluminescence. Antibody reactivities against 70K and 70K apop were scored ranging from 0 to 5 by three researchers independently. In each experiment several control antibodies were used. Results In this study, patient sera were analyzed for the presence of autoantibodies against 70K and its apoptotic product (70K apop ), on western blots containing extracts of non-apoptotic and apoptotic Jurkat cells. Two positive controls for the detection of 70K and 70K apop were included in each experiment: anti-70K mouse monoclonal antibody 2.73 [ 23 ], which displays higher reactivities with 70K than with 70K apop , and serum from MCTD patient B16, which reacts with both 70K and 70K apop . The position of 70K apop on western blots was confirmed by a recombinant monoclonal antibody recognizing both 70K and 70K apop (Fig. 1a ) [ 24 ]. The results show that in these apoptotic cells 70K is converted almost completely into 70K apop . Besides positive controls for 70K and 70K apop , mouse monoclonal antibody ANA125 directed against Sm-B/B' (Fig 1a ), and anti-U1-A/U2-B" mouse monoclonal antibody 9A9 (not shown) were also used. To be able to detect autoantibody reactivities to the intact 70K and its apoptotic 40K fragment simultaneously and to facilitate a direct comparison of these reactivities, a mixture of apoptotic and non-apoptotic cell extracts was used to prepare western blots. An additional advantage of this approach was that differences between blots could be excluded, thereby allowing a more accurate comparison of reactivities with 70K and 70K apop in a single patient serum. Serum antibody reactivities against 70K and 70K apop were scored ranging from 0 to 5. Figure 1b shows a western blot containing such a mixture of non-apoptotic and apoptotic Jurkat cell extracts, probed with a serial dilution of serum from MCTD patient B16. It can be seen that the signals for 70K and 70K apop increase when the serum is applied at a lower dilution, indicating that the western blot assay can be used for semi-quantitative interpretation. Autoantibodies against 70K are more easily detected with 70K apop The presence of high levels of autoantibodies directed against components of the U1 snRNP, such as 70K, is one of the criteria for the diagnosis of MCTD [ 2 ]. However, anti-70K antibodies are also found in some SLE and SSc patients [ 3 ]. To compare the disease specificity of anti-70K apop and anti-70K autoantibodies, sera from a group of MCTD patients and from a group of patients suffering from a variety of autoimmune disorders were analyzed. As shown in Table 1 , most MCTD patients (54%) displayed antibody reactivities that preferentially recognized 70K apop over the intact 70K protein. Seven patients (13%) reacted with 70K and 70K apop with similar efficiencies, and only 6% of the MCTD patients reacted preferentially with the intact 70K protein. Fourteen sera (27%) did not react detectably with either 70K polypeptide, although the sera were anti-RNP positive by several techniques. These results indicate that 70K apop is a better antigen than the intact 70K protein for the detection of anti-70K autoantibodies. Antibody reactivity with 70K apop was found in only 2% of sera from control groups, whereas antibody reactivity with 70K was found in 5% of patient sera from control groups. Autoantibodies against 70K apop are not correlated with disease activity It has been described that, in some patients with MCTD, antibody titers against the U1 snRNA molecule are correlated with disease activity, and could even possess prognostic value [ 21 ]. In contrast, most studies did not find a correlation between disease activity and antibody responses to 70K, either by serum analysis using recombinant protein as antigen in ELISA [ 21 , 25 ] or by analysis on western blots using native protein from cell extracts [ 26 ]. Only one study, using ELISA with recombinant 70K as technique, has reported decreasing disease activity concomitant with decreasing anti-70K antibody levels [ 27 ]. Because apoptotic modifications on autoantigens, such as the cleavage of 70K, are believed to be involved in the primary autoimmune response, we proposed that immune complexes containing anti-70K apop antibodies might also be important for triggering disease flares. Serum samples were collected longitudinally from 12 MCTD patients by a follow-up during variable time intervals (4–15 years; average 10 years). All samples were analyzed for the presence of autoantibodies against 70K and 70K apop on western blots containing non-apoptotic and apoptotic Jurkat cell extracts, and the presence of these autoantibodies was compared with the disease activity of each patient. The overall conclusion of this longitudinal study was that no significant correlations between antibody titres against either 70K apop or 70K and disease exacerbations could be observed. Autoantibodies against 70K apop are more prevalent early in disease As mentioned above, it has been proposed that apoptotic modifications trigger the primary immune response towards self proteins and that, through secondary immune responses and epitope spreading, autoantibodies directed against unmodified regions on the autoantigen appear at later stages of the disease. To investigate this possibility for the 70K autoantigen, the longitudinal serum collection [ 21 ] of 12 MCTD patients was re-examined, now for antibodies against 70K and for antibodies against 70K apop . Three patients produced antibodies reacting strongly with 70K apop , whereas no or only weak reactivity against 70K was observed. In one of these three patients, autoantibodies against 70K apop were more prevalent in early serum samples, and the level decreased in time. Eight patients were found to have high titres of antibodies with reactivities to both 70K and 70K apop . Interestingly, in three of these patients early serum samples showed a higher reactivity with 70K apop than with 70K, whereas later samples showed comparable reactivities with both antigens, or higher reactivities with 70K. An example of this type of reactivity profile is shown in Fig. 2 . One of the 12 patient sera did not detectably contain antibodies directed against 70K or 70K apop . These results thus support the idea that antibodies against 70K apop appear earlier in the disease than antibodies against the complete 70K protein. Discussion Greidinger and colleagues recently showed that antibodies against the 70K protein in RNP-positive patients are often accompanied by antibodies directed against the apoptotic cleavage product of this autoantigen and that the B cell epitopes recognized on the apoptotic product are antigenically different from those contained in the intact form of the 70K protein [ 18 , 19 ]. This study is the first to confirm and extend these findings and strongly suggests that the reactivity of a patient serum with anti-70K antibodies depends on the presence of antibodies against epitopes shared by 70K and 70K apop , and on the presence of antibodies against epitopes exclusively present on 70K apop . The major apoptosis-specific epitope on 70K has been shown to be located in the region containing the RNA-binding domain, and its formation depends on amino acids 180–205, overlapping with the most common T cell epitope [ 20 ]. Other apoptosis-specific epitopes on 70K have not yet been described. In our recent studies, monoclonal recombinant human antibodies against 70K were isolated from phage display libraries derived from SLE patients, and several of these monoclonal antibodies preferentially recognized 70K apop on a western blot and in immunoprecipitation experiments ([ 24 ], and D Hof, unpublished results). It is believed that the apoptotic cleavage of 70K leads to the exposure of a neo-epitope that, if presented to the immune system, triggers the autoimmune response. Greidinger and colleagues showed that a mutant consisting of the amino-terminal 205 amino acids, was indeed able to induce an anti-70K apop antibody response in mice, with subsequent epitope spreading. Interestingly, some of the immunized mice developed pulmonary lesions comparable to lesions found in lungs of MCTD patients. This finding supports the hypothesis that apoptosis-specific epitopes, and antibodies directed against them, might have a pathological role in the triggering and maintenance of the human autoimmune response to 70K [ 19 ]. In our study, a minority of MCTD sera (4%) contained autoantibodies exclusively reacting with intact 70K. We suggest that these sera derive from patients in a relatively late disease phase and primarily contain antibodies resulting from expanded epitope spreading. Most epitopes recognized by these sera might therefore be dependent on the carboxy-terminal part of the protein, which is cleaved off during apoptosis and is not present on 70K apop . Patients that tested negative in our western blot experiments might either have low levels of anti-70K antibodies or might not produce such antibodies at all. Instead, other components of the U1 snRNP, such as the U1 RNA molecule, U1A or U1C, might be targeted by these sera and might explain their anti-U1 snRNP reactivity. We show here that most U1 snRNP-positive patient sera preferentially recognize 70K apop , which is most probably explained by the presence of antibodies targeting an apoptotic 70K epitope. These results are in line with reports by Greidinger and colleagues [ 18 , 19 ], who found that about 50% of their RNP-positive sera contained 70K apop autoantibodies. How disease flares are induced is not completely understood. Correlations between serum levels of certain autoantibodies and disease activity have been reported for MCTD and SLE [ 21 , 22 ], but it can be disputed whether these antibodies contribute to the disease flares or are merely epiphenomena. Our data show that antibodies against 70K apop are not significantly correlated with disease activity, suggesting that there is no important role for 70K apop in the initiation of disease flares. However, it is possible that the variations in antibody levels against the apoptosis-specific epitope are masked by the presence of antibodies against other epitopes on 70K and U1-70K apop . Furthermore, a polyspecific secondary antibody was used to detect bound serum antibodies, and as a consequence variations in isotype-specific antibody levels might have remained undetected. It has been reported that the first autoantibodies to appear in anti-RNP-positive patients are generally antibodies against 70K [ 16 , 27 ]. Our results suggest that 70K apop drives the primary autoimmune response to 70K, because in several patients antibodies against an epitope associated with 70K apop preceded the appearance of reactivity with intact 70K. The fact that the first serum samples from relatively few patients exclusively contain anti-70K apop antibodies might be due to the stage of disease development at which the patient enters the rheumatological clinic. It is likely that the first symptoms, later followed by the diagnosis of the disease, had been established years before the start of the longitudinal study. Moreover, it is possible that autoantibodies, especially those generated by the primary immune response, were already present before the manifestation of clinical symptoms and that subsequent epitope spreading might have occurred before the patient entered the rheumatological clinic. For example, anti-cyclic citrullinated peptide autoantibody is a very specific marker for rheumatoid arthritis, and such antibodies can be detected in patients up to 10 years before the occurrence of the first clinical symptoms [ 28 , 29 ]. In our opinion this might explain why a relative enrichment of anti-70K apop antibodies could not be detected in the early sera of all patients. During apoptosis, the U1 snRNP complex is modified in several ways. In addition to cleavage of 70K, U1 snRNA and the Sm-F protein are cleaved, and phosphorylated serine–arginine proteins associate with the complex [ 30 ]. Apoptotic modifications of the U1A and U1C proteins have not yet been described. 70K can be cleaved by caspase-3 and granzyme B, and it can be oxidatively fragmented in the presence of metals, resulting in products of 40, 60 and 55 kDa, respectively. Correlations between the recognition of specific 70K fragments and disease manifestations are interesting. For example, patients suffering from Raynaud's phenomenon preferentially recognize the oxidatively modified 55 kDa fragment of 70K [ 31 ]. The findings that early MCTD sera are enriched for antibodies against the 40 kDa apoptotic fragment (70K apop ) and that most sera show a higher reactivity with this fragment suggest that caspase-3 cleaved 70K has a role in breaking tolerance in these patients. Although granzyme B is postulated to have a role in breaking tolerance [ 32 ] to 70K, it is unknown whether specific patient groups preferentially recognize the 60 kDa cleavage product generated by granzyme B, which would be interesting to study in more detail. Conclusions Analysis of a group of MCTD patient sera by western blotting showed that the majority of patient sera recognized 70K apop more efficiently than the intact form of the 70K protein. The fact that the presence of these antibodies in most patients precedes the occurrence of other anti-70K antibodies suggests that 70K apop is particularly important for the early detection of this disease in patients. Abbreviations 70K = U1-70K; 70K apop = apoptotic cleavage product of U1-70K protein of about 40 kDa; MCTD = mixed connective tissue disease; NP40 = Nonidet P40; RNP = ribonucleoprotein; SLE = systemic lupus erythematosus; snRNP = small nuclear ribonucleoprotein; SSc = systemic sclerosis; U1-70K = 70 kDa protein component of the U1 snRNP complex. Competing interests JMHR works and holds shares in ModiQuest BV, a company producing antibodies for research purposes, but will not gain or lose financially from publication of this paper. Authors' contributions DH conceived of the study, participated in the design of the study and was involved in the analysis of the immunoassay results. KC performed and analyzed the immunoassays. DR collected the patient sera. FH provided patient data. GP participated in the design of the study and in the analysis of the immunoassay results. WV conceived of the study and participated in the design of the study. JR conceived of the study and was involved in its design and coordination. All authors read and approved the final manuscript.

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1065329

High-resolution optical coherence tomographic imaging of osteoarthritic cartilage during open knee surgery

This study demonstrates the first real-time imaging in vivo of human cartilage in normal and osteoarthritic knee joints at a resolution of micrometers, using optical coherence tomography (OCT). This recently developed high-resolution imaging technology is analogous to B-mode ultrasound except that it uses infrared light rather than sound. Real-time imaging with 11-μm resolution at four frames per second was performed on six patients using a portable OCT system with a handheld imaging probe during open knee surgery. Tissue registration was achieved by marking sites before imaging, and then histologic processing was performed. Structural changes including cartilage thinning, fissures, and fibrillations were observed at a resolution substantially higher than is achieved with any current clinical imaging technology. The structural features detected with OCT were evident in the corresponding histology. In addition to changes in architectural morphology, changes in the birefringent or the polarization properties of the articular cartilage were observed with OCT, suggesting collagen disorganization, an early indicator of osteoarthritis. Furthermore, this study supports the hypothesis that polarization-sensitive OCT may allow osteoarthritis to be diagnosed before cartilage thinning. This study illustrates that OCT, which can eventually be developed for use in offices or through an arthroscope, has considerable potential for assessing early osteoarthritic cartilage and monitoring therapeutic effects for cartilage repair with resolution in real time on a scale of micrometers.

Introduction Osteoarthritis (OA) is the leading cause of chronic disability in developed countries, symptomatically affecting about 14% of the adult population in the United States alone. Among the signs of early OA are collagen disorganization, an increase in water content, a decrease in superficial proteoglycan, and alterations in glycosaminoglycans [ 1 ]. The later changes include cartilage loss (thinning effect), fibrillation, and surface erosion. Current imaging technologies are limited in their ability to monitor changes in articular cartilage [ 2 ]. Furthermore, symptoms are an unreliable indicator of disease progression [ 3 ]. Since the cartilage response to intervention cannot be monitored in a noninvasive or minimally invasive manner, assessing the effectiveness of these drugs and following the progression of the disease remain a challenge. This deficiency is the basis of the current US National Institutes of Health OA initiative to find solutions to this major healthcare dilemma [ 3 ]. A diagnostic technique capable of high-resolution imaging of articular cartilage in vivo could be invaluable to detect the onset of disease, follow its progression, and monitor therapeutic effectiveness. Other imaging technologies play an important role in managing OA, but they have limitations. While conventional x-rays have an obvious role in managing arthritis, this technology lacks the resolution to monitor changes within the cartilage [ 2 , 4 ]. Magnetic resonance imaging is invaluable for globally evaluating the joint noninvasively, with a typical clinical resolution of 250–300 μm at 10T [ 5 ]. However, the resolution of this technique is problematic, since cartilage is typically less than 2–3 mm thick and the evaluation would rely heavily on the interpretation of a few pixels [ 6 , 7 ]. In addition, its high cost, relatively long imaging time, large size of equipment, and limited availability could limit its widespread clinical use. Arthroscopy is also widely used in the diagnosis of joint disorders [ 8 ]. While it provides magnified views of the articular surface, it is unable to assess subsurface. Optical coherence tomography (OCT) is a recently developed imaging technique that can generate cross-sectional images of tissue microstructure [ 9 , 10 ]. OCT is analogous to ultrasound, but measures the intensity of infrared light rather than sound. It is an attractive imaging alternative for OA because it permits imaging in near-real time with unprecedented high resolution (4–15 μm), 10 to 100 times as fine as that of current clinical imaging modalities. Since OCT is based on fiber-optic systems, the apparatus is compact, roughly the size of an ultrasound unit. Imaging catheters can be constructed with diameters less than 0.006 inches (Lightlab Imaging Inc, Westford, MA, USA; ). Recently, OCT has been adapted for high-resolution imaging in nontransparent tissue. In addition, a variety of spectroscopic techniques can be incorporated, such as absorption, dispersion, and polarization spectroscopy [ 11 - 13 ]. Preliminary work demonstrated the feasibility of OCT in assessing joint cartilage pathologies in vitro [ 11 , 14 ]. Microstructures such as fibrillations, cartilage thinning, and new bone growth can be identified on OCT images [ 14 ]. Comparison with histology reveals strong correlation between OCT images and corresponding histological sections. In addition, OCT has demonstrated superior qualitative and quantitative performance against both 30- and 40-MHz ultrasound, the current clinical technology with the highest resolution [ 15 , 16 ]. Polarization-sensitivity OCT imaging of articular cartilage has also been performed [ 11 , 14 ]. With this technique, the OCT image changes with change in the polarization state of the incident light. In the previous in vitro study, polarization-sensitive changes on OCT images of cartilage were directly correlated with collagen organization [ 11 ], as assessed by picrosirius staining. Loss of both polarization sensitivity and collagen organization were noted to take place before cartilage thinning and fibrillation, making it a potential additional marker of early OA in addition to structural imaging. These results have been recently confirmed also in tendons and ligaments, and also in studies with theoretical modeling [ 17 , 18 ]. Through this work, quantitative methods have now been developed and are being studied, including the use of the fast Fourier transform or rate of peak change with rotation of the source optical axis. This study extends our previous in vitro work [ 11 , 14 ]. In this study, observations on the ability of OCT to perform in vitro imaging of the human knee were confirmed in vivo using a novel handheld probe. Materials and methods The principle behind OCT has been described in detail previously [ 9 , 10 ]. A schematic drawing of the OCT system used in this study is shown in Fig. 1a . In this study, a novel, compact, handheld OCT imaging probe capable of performing lateral scanning of the articular cartilage subsurface during open knee surgeries was used. The probe had dimensions of ~1.5 cm in diameter and ~15 cm in length (see Fig. 1b ) and was developed and used to deliver, focus, scan, and detect the returning beam. It consisted of a four-lens relay and a scanning mirror. The measured resolution was approximately 11 μm (axial) and 30 μm (transverse) with a working distance (as defined by the distance between the distal end of the probe and the beam focus) of about 2.5 cm, which provided enough space to perform noncontact imaging. A 532-nm visible beam (green) with a very low power (<0.2 mW) was coupled into the handheld probe for aiming purposes. OCT images were stored in digital format and also recorded on a super VHS tape for future analysis. The protocol for OCT imaging during open knee surgery was approved by the investigational review board of the Massachusetts Institute of Technology and West Roxbury Veterans Association Hospital. Six patients 65 to 75 years of age who had been diagnosed with severe OA and were scheduled for treatment through partial or total knee replacement surgery were contacted about 4 weeks before surgery and their informed consent was obtained. Patients underwent routine surgical preparation procedures, and OCT imaging did not commence until the articular surface of the femur/tibia was fully exposed. OCT imaging was performed under sterile conditions. Both visually normal and visually abnormal regions were imaged. Imaging planes were marked with sterile dye (methylene blue) for tissue registration. During imaging, the probe did not contact the cartilage surface and the air distance between the probe and the cartilage surface was maintained at ~2.5 cm to insure that the imaged sites remain in focus. Images of 512 × 256 pixels (transverse × axial) were generated at four frames per second. Each OCT image corresponded to a two-dimensional tissue cross section 5 mm wide by 2.6 mm deep. Multiple sites on the articular surface were imaged within the allotted 10-min imaging period. After OCT imaging, surgery resumed as usual. Upon completion of the surgical procedures, the methylene blue dots were re-marked with India ink to improve visualization during post processing. The cartilage was then immediately fixed in 10% buffered formalin and then decalcified with EDTA followed by routine histological processing and stained with Masson trichrome blue. Results A representative OCT image and the corresponding histology of normal knee articular cartilage are shown in Fig. 2 . The OCT image (Fig. 2a ) reveals that the cartilage was thick and uniform with a rather smooth surface. The same characteristics can also be seen in the histology as shown in Fig. 2b . A banding pattern is seen in the OCT image (Fig. 2a , red arrows). Previous work showed that this pattern represents alternating maximum and minimum intensities of back scattering, which results from rotation of the polarization state of back-reflected light as it passes through the organized collagen. During the imaging process, it was noted that the position of the bands moved as the polarization state of the incident light was changed (induced by moving the fiber in the sampling arm). Fig. 3 illustrates a representative OCT image (Fig. 3a ) and the corresponding histology (Fig. 3b ) of moderately diseased cartilage. Regions of diminished back scattering are noted in the OCT image, which correlate with areas of hypocellularity and diminished matrix in histological preparations. On the OCT image, the banding pattern is disrupted and correlates with histologically abnormal staining and cellularity. Fig. 4 shows an OCT image (Fig. 4a ) and the corresponding histology (Fig. 4b ) of severely diseased cartilage. Distinctive thinning of the cartilage was observed only on the left portion of both OCT image and histology. In addition, an irregular cartilage surface is seen in the OCT image, with multiple fibrillations evident in the corresponding histology. The OCT image is highly heterogeneous and the cartilage and bone interface are poorly identified. No banding appearance or polarization sensitivity was observed on this image. On the right portion of the OCT image and the histology section, cartilage is absent and the bone is exposed to the surface. An OCT image of thick cartilage with no evidence of surface erosion and early degenerative changes is shown in Fig. 5 . The OCT structural image is relatively homogeneous but the banding pattern is lost. The abnormal region seen on histology consists of an area of hypocellularity over a region of hypercellularity. Fig. 6 shows normal and diseased cartilage in close approximation in two sections of cartilage. The region on the left of both images is presumed normal cartilage, while on the right, the polarization sensitivity and back-scattering intensity abruptly changes. In addition, since these two samples come from the femur (Fig. 6a ) and patella (Fig. 6b ), respectively, the figure confirms that the polarization phenomenon exists in areas other than the tibia. Discussion The current study demonstrates that osteoarthritic structural changes in cartilage can be visualized with OCT in vivo using a handheld probe. Structural changes including cartilage thinning and fibrillations were observed at a resolution substantially higher than that of any current clinical imaging technology. While normal cartilage demonstrates a banding pattern with a relatively homogeneous intensity (as seen in Fig. 2 ), areas of hypocellularity appear to lose this banding pattern (as seen in Fig. 3 ). These changes are dramatic enough to distinguish between adjacent areas of healthy and diseased tissue (as in Fig. 6 ). These results indicate that OCT may be able to be used by surgeons to aid in the evaluation of the microstructural integrity of articular cartilage during surgical procedures. It can ultimately be envisioned that OCT imaging will be performed with a surgical arthroscope or a needle arthroscope for assessing the articular cartilage in a minimally invasive fashion. Future efforts will be on the development of a small OCT arthroscope capable of being either used in combination with or integrated into a standard arthroscope. Endoscopic imaging using an OCT probe introduced through the accessory port of an endoscope has been demonstrated in the human gastrointestinal tract [ 19 , 20 ]. The collagen matrix in healthy cartilage is a highly organized structure [ 21 , 22 ]. The banding pattern seen on the OCT images (e.g. Figs 2 , 3 , and 6 ) are due to tissue birefringence and are related to collagen organization [ 11 , 14 ]. Changes in collagen organization, although not necessarily in collagen content, are among the earliest changes in OA [ 1 ]. It has been shown in animals that a decrease in birefringence, evident on histological evaluation, precedes fibrillations and can even be noted after chronic long-distance running [ 23 , 24 ]. The diminishing and absent banding pattern on the OCT images (e.g. Figs 3 , 4 , 5 , 6 ), an observation supported by in vitro work, represents a reduction and loss of the birefringence of the cartilage, which is caused by the reduction or loss of collagen structural organization [ 14 ]. This has recently been confirmed in experimental models of OA in the rat [ 25 , 26 ]. That study indicated that changes in the birefringent properties of cartilage (as with OA) are reflected in the polarization sensitivity of OCT images. In the current study, polarization changes were not quantitatively measured. However, as the fiber of the sample arm moved, it would induce a polarization state shift, allowing quick assessment of the polarization sensitivity of the area being imaged. Protocols are now available using fast Fourier transforms to quantitate single-detector OCT. Conclusion A true clinical need exists for monitoring therapeutic intervention with regard to osteoarthritic cartilage. This study demonstrates real-time, high-resolution OCT imaging of articular tissues in vivo during joint replacement surgery at resolutions on a scale of micrometers. Abnormalities such as cartilage thinning and fibrillations were detected and qualitatively correlated with the corresponding histology. In addition, birefringence changes between osteoarthritic and normal cartilage were noted in this study, indicative of a loss of collagen organization. OCT represents a promising new technology for the evaluation of articular cartilage in vivo . Abbreviations OA = osteoarthritis; OCT = optical coherence tomography. Competing interests The author(s) declare that they have no competing interests. Authors' contributions XL designed and constructed the OCT system. SM performed studies in patients, which included gaining their consent and postoperative observation. CP assisted in the construction of the OCT system. RG assisted with the construction of the handheld probe. DS advised on histological preparations. MH processed the tissues. JF consulted on the design of the OCT system. MB was involved with the engineering design, OCT protocol, evaluation of data, and writing of manuscript. All authors read and approved the final manuscript.

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1065330

TLR2 modulates inflammation in zymosan-induced arthritis in mice

The interplay between the innate and acquired immune systems in chronic inflammation is not well documented. We have investigated the mechanisms of inflammation in murine zymosan-induced arthritis (ZIA) in the light of recent data on the roles of Toll-like receptor 2 (TLR2) and Dectin-1 in the activation of monocyte/macrophages by zymosan. The severity of inflammation, joint histology, lymphocyte proliferation and antibody production in response to zymosan were analyzed in mice deficient in TLR2 and complement C3, and the effects of Dectin-1 inhibition by laminarin were studied. In comparison with wild-type animals, TLR2-deficient mice showed a significant decrease in the early (day 1) and late phases (day 24) of joint inflammation. C3-deficient mice showed no differences in technetium uptake or histological scoring. TLR2-deficient mice also showed a significant decrease in lymph node cell proliferation in response to zymosan and a lower IgG antibody response to zymosan at day 25 in comparison with wild-type controls, indicating that TLR2 signalling has a role in the development of acquired immune responses to zymosan. Although laminarin, a soluble β-glucan, was able to significantly inhibit zymosan uptake by macrophages in vitro , it had no effect on ZIA in vivo . These results show that ZIA is more prolonged than was originally described and involves both the innate and acquired immune pathways. C3 does not seem to have a major role in this model of joint inflammation.

Introduction Zymosan, a polysaccharide from the cell wall of Saccharomyces cerevisiae , is composed primarily of glucan and mannan residues [ 1 ]. In vitro , it has served as a model for the study of innate immune responses, because it is capable of stimulating inflammatory cytokine production [ 2 ] and can activate complement in the absence of immunoglobulins [ 3 ]. Zymosan is recognized and phagocytosed principally by monocytes and macrophages and leads to cellular activation [ 4 ]. Zymosan-induced arthritis (ZIA) in mice was first described by Keystone in 1977 [ 5 ]. Arthritis was induced by intra-articular injection of zymosan and was thought to be mediated by activation of the alternative pathway of complement and the release of lysosomal hydrolases from activated macrophages [ 6 ]. The recent discovery of pattern recognition receptors and their role in innate immunity has led to a re-evaluation of our concepts of zymosan-induced inflammation. Toll-like receptors (TLRs) are a family of type 1 transmembrane proteins that consists of an extracellular leucine-rich repeat domain and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin 1 (IL-1) receptor [ 7 ]. The ligands of TLR2 include lipopeptides and peptidoglycan [ 8 , 9 ], and TLR2 is a receptor for zymosan, acting in collaboration with CD14 and TLR6 [ 2 , 10 ]. Ligand binding to TLRs induces the activation of NF-κB and the production of the inflammatory cytokines IL-1, IL-6, IL-8, and IL-18 as well as the expression of the co-stimulatory molecule B7.1 [ 7 ]. Additionally, zymosan is able to induce maturation of dendritic cells in vitro and to stimulate their production of IL-2 [ 11 , 12 ], providing evidence for a link between the innate and the adaptive immune responses. The inflammatory response triggered by zymosan is linked to its phagocytosis, a process that is mediated by a set of different receptors from the TLRs. The non-opsonic recognition of zymosan by macrophages is mediated by Dectin-1. Dectin-1 is a type 2 membrane receptor with an extracellular C-type lectin-like domain fold and a cytoplasmic immunoreceptor tyrosine-based activation motif [ 13 ] and is expressed on macrophages, dendritic cells and neutrophils [ 14 - 16 ]. Dectin-1 mediates the binding of Saccharomyces cerevisiae and Candida albicans in a β-glucan-dependent manner and may also have a pro-inflammatory function [ 17 ]. In the light of the above findings, we have re-investigated ZIA to elucidate the roles of the innate and adaptive immune responses in this model and to compare the effects of TLR2 deficiency and complement C3 deficiency. The role of Dectin-1 in zymosan-induced inflammation was also investigated. Our results indicate that TLR2 is the major pathway of pro-inflammatory signalling in ZIA and is necessary for the development of specific immune responses to zymosan. Materials and methods Animals C3-deficient mice (C3 -/- ) on a C57bl/6 background were generated by Professor M Botto [ 18 ]. TLR2-deficient mice (TLR2 -/- ) on a C57bl/6 background were provided by Dr Kiyoshi Takeda (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University) [ 19 ]. Wild-type (WT) C57bl/6 mice were purchased from Charles River (L'Arbresle, France). All mice were bred in our animal house facility. Double knockout and double WT mice were generated by mating TLR2 -/- and C3 -/- mice. The genotypes of all mice used were confirmed by polymerase chain reaction analysis of genomic DNA extracted from mice tails. The primer sequences used were as follows: TLR2 sense, 5' -GTTCTCCCAGCATTTAAAATCATT-3' ; TLR2 antisense, 5' -GTCTCCAGTTTGGGAAAAGAACC-3' ; TLR2 NEO antisense, 5' -CGACACAGCTGCGCAAGCAAC-3' ; C3 sense, 5' -CTTCATAGACTGCTGCAACCA-3' ; C3 antisense, 5' -AACCAGCTCTGTGGGAAGTG-3' ; C3 NEO antisense, 5' -AAGGGACTGGCTGCTATTGG-3'. Induction of ZIA Zymosan A from Saccharomyces cerevisiae (Sigma, St Louis, MO, USA) (300 mg) was resuspended in 10 ml of endotoxin-free saline, boiled and homogenized by sonic emulsification. The suspension was autoclaved and stored in aliquots at -20°C. Arthritis was induced by intra-articular injection of 180 μg (6 μl) of zymosan through the suprapatellar ligament into the joint cavity. In specified experiments, the contralateral knee was injected with an equal amount of sterile saline (6 μl) as control. Laminarin was co-injected at a dose of either 500 μg or 100 μg together with 180 μg of zymosan into the knee joint. Approval was obtained from the local animal health committee for these experiments. Isotopic quantification of joint inflammation in vivo Joint inflammation was measured by 99m Tc uptake in the knee joint as described [ 20 ]. Mice were sedated by the intra-peritoneal administration of sodium pentobarbital (50 mg/kg) and then injected subcutaneously in the neck region with 10 μCi of 99m Tc. The accumulation of the isotope in the knee was determined by external gamma-counting after 15 min. The ratio of 99m Tc uptake in the inflamed arthritic knee to 99m Tc uptake in the contralateral control knee was calculated. A ratio higher than 1.1:1 indicated joint inflammation. Histological grading of arthritis Mice were killed at day 8 and at day 25. Knees were dissected and fixed for 2 weeks in 10% buffered formalin. Fixed tissues were decalcified for 2 weeks in 15% EDTA, dehydrated and embedded in paraffin. Sagittal sections (5 μm) of the whole knee joint were stained with safranin-O and counterstained with fast green/iron hematoxylin. Histological sections were graded by two observers unaware of animal genotype or treatment. Synovial cell infiltrate and exudate were scored from 0 (no cells) to 6 (maximum number of inflammatory cells). Cartilage proteoglycan depletion (damage), reflected by a loss of safranin-O staining intensity, was scored on a scale from 0 (fully stained cartilage) to 6 (totally unstained cartilage) in proportion to severity. For each histopathological measure the score (mean ± SEM) of all slides was calculated. T cell proliferation assay Mice were killed in accordance with the experimental protocol. Inguinal lymph nodes were removed and single-cell suspensions were incubated in RPMI supplemented with 2-mercaptoethanol, penicillin, streptomycin and 1% autologous serum. Lymph node cells (LNC; 4 × 10 5 per 200 μl per well) were plated in 96-well flat-bottomed plates and stimulated with zymosan at specified concentrations. Concanavalin A at 4 μg/ml was used as non-specific mitogen. The cells were incubated for 48 hours at 37°C in 5% CO 2 , then [ 3 H]thymidine (1 μCi per well) was added to the cultures for 18 hours. The cells were harvested, and [ 3 H]thymidine uptake was measured with a beta scintillation counter. Determination of interferon-γ production in vitro Culture supernatants from LNC cultured with or without 4 μg/ml zymosan were harvested after 72 hours for determination of interferon (IFN)-γ levels. Quantification of cytokine production was performed with an enzyme-linked immunosorbent assay (ELISA) kit specific for murine IFN-γ (Amersham Pharmacia, Dubendorf, Switzerland). TLR2 immunohistochemistry Immunohistochemistry was performed with affinity purified anti-mouse TLR2 antibody (clone 6C2; eBioscience, San Diego, CA, USA). Specificity of the antibody was tested on bone marrow cells derived from c57bl/6 TLR2 +/+ and TLR2 -/- mice. Dissected knees were embedded in Tissue-Tek OCT, then immediately frozen in precooled hexane and stored at -70°C until use. Sections 7 μm thick were cut on a motor-driven Leica cryostat with a retraction microtome and a tungsten carbide knife at a cabinet temperature of -25°C and mounted on Menzel Super Frost Color glass slides. Phagocytosis assay RAW 264.7 cells (5 × 10 5 to 10 6 per chamber) were plated on a Lab-Tek II Chamber Slide system (Nalge Nunc International). After adherence, cells were either preincubated with 100 or 500 μg/ml laminarin [ 21 ] for 20 min followed by the addition of 25 zymosan particles per cell, or laminarin was co-administrated with zymosan. After incubation for 3 hours at 37°C in 5% CO 2 , cells were washed twice with PBS and fixed for 10 min in acetone. Cell-bound and phagocytosed particles were stained by periodic acid Schiff, a stain specific for insoluble glucose polymers, and quantified by light microscopy. Quantification of IgG levels Serum levels of total IgG were quantified with ELISA. In brief, rabbit anti-mouse IgG (Dako, Carpinteria, CA, USA) was coated on 96-well plates (Nunc, Roskilde, Denmark). Murine sera from naive and ZIA mice (dilution 1:100,000) were added and incubated for 2 hours. Secondary alkaline-phosphatase-linked anti-mouse IgG (Sigma, Buchs, Switzerland) was added and p -nitrophenyl phosphate (Sigma, Buchs, Switzerland) completed the reaction. Serum levels of specific anti-zymosan IgG were also quantified by ELISA. Zymosan particles at 1 mg/ml were coated on 96-well plates and murine sera (dilution 1:100) were added and incubated for 2 hours. The reaction was developed as previously described. Statistical analysis The Wilcoxon rank sum test for unpaired variables (two-tailed) was used to compare differences between groups. The unpaired Student t -test was used to compare the groups with normally distributed values. A level of P < 0.05 was considered statistically significant. Results Zymosan-mediated inflammation in the knee joint is biphasic In experiments on WT C57bl/6 mice, we observed a biphasic course of inflammation, with an initial peak of 99m Tc uptake at day 1 (1.71 ± 0.08), followed by a decrease to a trough value at day 7 (1.29 ± 0.05) and a secondary increase in uptake at day 14. Inflammation measured by 99m Tc uptake persisted up to day 25 (1.40 ± 0.06) (Fig. 1a ). Histological assessment of the mice at day 8 showed a low score for cellular infiltration (1.00 ± 0.32) and for cartilage destruction (0.7 ± 0.2) (Fig. 1b ), whereas scoring at day 25 was characterized by an increase in cellular infiltration (2.5 ± 0.37) while cartilage destruction remained low (0.71 ± 0.24) (Fig. 1c ). Histology and immune responses at day 25 of ZIA To determine whether zymosan particles persisted in the joint at day 25, periodic acid Schiff staining was performed on joint tissues obtained at day 25 and showed persistence of zymosan particles in the synovial membrane of mice injected with zymosan (Fig. 2a ). To verify that joint inflammation was associated with the development of specific immune responses to zymosan, we assessed both humoral and cellular responses in WT mice. Proliferation of LNC in response to zymosan was significantly increased in day 25 WT ZIA mice compared with LNC of naive mice (3.5 stimulation index in ZIA WT mice versus 1.5 in naive mice; P < 0.001), whereas mitogenic response to the non-specific mitogen concanavalin A at 4 μg/ml showed no difference between groups (Fig. 2b ). No difference in proliferation in response to zymosan was observed between ZIA and naive mice at day 8 (data not shown). The humoral response to zymosan was measured by ELISA. In arthritic mice, the serum levels of anti-zymosan IgG antibodies were significantly increased at day 25 in comparison with those in untreated naive mice (antibody ratio for WT = 0.944 versus naive = 0.677; P < 0.02) (Fig. 2c ). In addition, in vitro stimulation of WT ZIA LNC with zymosan at 4 μg/ml induced the secretion of IFN-γ at 1200 pg/ml, whereas unstimulated LNC produced undetectable levels of IFN-γ (Fig. 2d ). Synovial expression of TLR2 and its role in ZIA We wished next to evaluate whether TLR2 might have a role in the recognition of zymosan in vivo and in mediating inflammation in ZIA. Specific antibody for TLR2 was used to stain synovium from WT mice that had developed ZIA at day 25. Figure 3a shows a representative example of the distribution of TLR2 expression in the synovial cell lining. Control antibody staining was negative (Fig. 3b ). Antibody specificity was confirmed by a lack of staining in TLR2 -/- mice (data not shown). To explore whether the deficiency of TLR2 had an effect on the course of ZIA, we measured knee joint inflammation in TLR2 +/+ and TLR2 -/- mice by 99m Tc uptake at different time points up to day 24 (Fig. 3c ). In two independent experiments we observed an attenuation of inflammation in TLR2 -/- mice at days 1, 3, 14, 17 and 24, although only the decrease observed at days 1 and 24 reached statistical significance ( P < 0.05). TLR2 deficiency ameliorates histological features of ZIA We compared the histological features of arthritic knee joints from TLR2 +/+ and TLR2 -/- mice (Fig. 3d ). In both groups, arthritis was histologically present in all knees that had been injected with zymosan. In TLR2 +/+ mice, on day 25 of ZIA, the synovial membrane was thickened, mainly as a result of invasion by inflammatory cells (see Fig. 1c ). In TLR2 -/- mice, synovial infiltrate was significantly decreased in comparison with TLR2 +/+ mice (4.9 ± 0.33 in TLR2 +/+ mice [ n = 15] versus 3.1 ± 0.67 in TLR2 -/- mice [ n = 12] on day 25 after arthritis onset; P < 0.045). TLR2 -/- mice showed no difference from WT mice in terms of cartilage destruction, as assessed by the loss of safranin-O staining at day 25 (Fig. 3d ). Effect of TLR2 deficiency on cellular responses The role of TLR2 on the cellular response to zymosan was examined by isolating LNC from ZIA mice. The proliferation of LNC induced by zymosan was significantly lower in cells isolated from TLR2 -/- mice than in TLR2 +/+ mice. A significant difference was found at both concentrations of zymosan studied (4 and 8 μg/ml; both P < 0.05) (Fig. 3e ). No differences were observed in proliferation stimulated by the non-specific mitogen concanavalin A (data not shown). The serum levels of anti-zymosan IgG antibodies, measured by ELISA, were decreased by 50% in TLR2 -/- mice at day 25 in comparison with the serum levels in controls (antibody ratio for WT = 1.00 versus TLR2 -/- = 0.51, P = 0.047) (Fig. 3f ). Lack of effect of C3 on inflammation in ZIA The availability of C3-deficient mice in a C57bl/6 background allowed us to reassess the role of C3 in ZIA. No effect, either in 99m Tc uptake or in histological scoring, was observed in C3-deficient ( n = 25) mice in comparison with WT mice ( n = 25). In addition, humoral and cellular responses were similar in C3 -/- and C3 +/+ mice (data not shown). Generation of TLR2/C3 double-deficient mice gave similar responses as TLR2 -/- mice, excluding a synergistic effect of double deficiency and confirming no role for the alternative pathway component of the complement cascade (Fig. 4a ). Histological scoring showed the presence of arthritis in both groups of animals. In TLR2/C-3 double-deficient mice, synovial infiltrate was significantly decreased in comparison with control (4.0 ± 0.65 in control mice [ n = 5] versus 1.9 ± 0.62 in TLR2/C-3 double-deficient mice [ n = 5] on day 25 after arthritis onset; P < 0.05) (Fig. 4b ). TLR2/C-3 double-deficient mice also showed a significantly decreased cartilage destruction in comparison with WT mice at day 25 (1.7 ± 0.12 in control mice [ n = 5] versus 0.9 ± 0.29 in TLR2/C-3 double-deficient mice [ n = 5]; P < 0.05) (Fig. 4b ). Stimulation of LNC with zymosan in vitro showed a significant decrease of stimulation in double-deficient mice compared with WT littermates, similar to that observed in TLR2 -/- mice (data not shown). In addition, a decreased production of zymosan-specific IgGs was observed in the double-deficient mice (ratio for WT = 0.944 versus double knockout = 0.616; P < 0.05) (Fig. 4c ). Dectin-1 has a minor role in inflammation in ZIA The identification of the β-glucan receptor Dectin-1 and its ability to bind zymosan particles in vitro stimulated us to study the role of Dectin-1 in vivo in ZIA. In vitro blockade of the Dectin-1 receptor by laminarin led to a 50% decrease in a phagocytosis assay with RAW 264.7 cells. This decrease was not dependent on the time of administration of laminarin, because it was not modified by preincubation or co-incubation with zymosan particles (Fig. 5a ). Co-administration of laminarin and zymosan in the knee joint of C57bl/6 mice showed a trend to a decrease of 99m Tc uptake in the early phase of inflammation in a laminarin-treated knee, compared with an untreated knee, at 4 hours and 1 day after administration, but did not reach statistical significance (Fig. 5b ). Discussion For more than 50 years zymosan has been a tool in the study of microbial recognition by the innate immune system. The mechanisms mediating the recognition and phagocytosis of zymosan in vivo are complex. Phagocytes, including monocytes, macrophages and dendritic cells, express receptors such as the TLRs, complement receptor 3, scavenger receptors (such as acetylated LDL receptors) and Dectin-1 [ 22 - 24 ], which have all been implicated in the cellular response to zymosan [ 25 ]. In addition, zymosan is capable of activating the alternative pathway of complement through C3 [ 3 ], which may serve to amplify the inflammatory response. To elucidate how zymosan induces inflammation in vivo , we re-investigated the ZIA model that was first studied in the 1970s. This model has been often used as a tool to dissect non-immune mechanisms of joint inflammation [ 26 - 28 ]. In our experiments we observed that ZIA was not as short lived as originally described. Arthritis persisted beyond day 14 and in fact beyond day 25. After an initial peak of inflammation at about day 3, inflammation subsided by day 7. Subsequently, inflammation returned to levels that could be as high (as measured by 99m Tc uptake) as the initial peak, suggesting that ZIA has early and late phases. Histologically, the joint inflammation was characterized by mononuclear cell infiltration in the sublining layer and hypertrophy of the lining layer as well as cartilage damage. Histological changes were milder at day 8 than at day 25. Zymosan particles were present in the synovium at day 25. We then investigated the role of TLR2 in ZIA because the macrophage inflammatory response to zymosan depends largely on its recognition by a heterodimer of TLR2 and TLR6 [ 2 , 10 ]. In TLR2 -/- mice there was a significant attenuation of the early and late inflammatory phases of ZIA, indicating that a ligand that activates the innate immune response through TLR2 can lead to a chronic local inflammatory reaction. In the absence of TLR2, joint inflammation was not totally blocked. This would suggest that, in vivo , the inflammatory response to zymosan is not dependent on TLR2 signalling alone and that receptors other than TLR2 might have a role. This is supported by the observation that inhibition of TLR2 and MyD88 by dominant-negative mutants blocked pro-inflammatory signalling but not zymosan uptake in vitro . Recent data have shown that Dectin-1 and SIGNR1 [ 29 ] on macrophages and pentraxin-3, an opsonin for the recognition of zymosan by Dectin-1, are involved in zymosan recognition and internalization [ 30 ]. We therefore investigated the role of Dectin-1 by using the β-glucan laminarin as a competitive inhibitor of zymosan [ 16 ]. We confirmed that laminarin inhibited zymosan uptake by RAW 264.7 cells and did not observe any difference in the blocking capacity of laminarin, whether administered before or at the same time as zymosan. In both cases and at two different concentrations, we observed a 50% decrease in cell-bound zymosan particles. On the basis of these results, we proceeded to assess the effect of laminarin on ZIA. Although there was a trend towards reduced 99m Tc uptake in the treated animals, this was not statistically significant. It is possible that 50% inhibition of zymosan phagocytosis is insufficient to modulate inflammatory signalling through TLR2. Furthermore, a redundancy in the multiple mechanisms that mediate zymosan phagocytosis could also explain the lack of effect of laminarin inhibition in vivo [ 31 ]. The biphasic course of ZIA and its modulation by TLR2 led us to study the acquired immune response to zymosan and the effects of TLR2 deficiency on it. We compared the cellular proliferative and antibody responses to zymosan in WT and TLR2 -/- mice at day 25. First, we were able to detect zymosan-induced lymphocyte proliferation and enhanced IFN-γ production in the draining LNC of mice with ZIA, and second, this was accompanied by the formation of a zymosan-specific IgG. In TLR2 -/- animals, the proliferative response was blunted and only reached 50% of that observed in WT ZIA animals. There was also a significant decrease in the zymosan-specific IgG response, which was about 50% lower than in WT mice. At day 8 we did not observe any difference between ZIA and naive WT mice in their proliferative response to zymosan (data not shown). These findings suggest that inflammation in the later phase of ZIA is paralleled by the development of acquired immune response to zymosan. The finding that zymosan particles persisted in the joint even at day 25 suggests that they could become a target for specific immune responses. The decrease in acquired immune responses in TLR2 -/- mice might be a result of the decreased antigen presentation efficiency of dendritic cells in the absence of TLR2 [ 32 ] or the lack of co-stimulatory signals through TLR2 expressed on activated T cells [ 33 ]. A significant role for the alternative pathway of complement in this model of inflammation was excluded by the phenotype observed in C3 -/- mice. Both phases of ZIA were comparable to that observed in WT controls. Mice with combined deletions of the C3 and the TLR2 genes did not show a significant decrease in 99m Tc uptake in comparison with TLR2 -/- mice. Histologically, we observed a significant decrease in cartilage damage in mice with the combined deficiency of TLR2 and C3, which did not occur in C3 -/- mice. We interpret this effect to be due in principle to the lack of TLR2, because TLR2 -/- mice also showed a diminished cartilage score (although it did not reach statistical significance). Combined with recent data showing that phagocytosis of zymosan is not mediated by complement receptor 3 [ 16 ], complement activation does not seem to contribute to zymosan-induced joint inflammation in vivo . The expression of TLR2 in arthritic synovium from WT mice in the ZIA model and the modulation of joint inflammation in TLR2 -/- animals show that TLR2 may have a general role in amplifying local inflammation. TLR2 has been shown to be expressed on neutrophils and lymphocytes as well as macrophages, and they all are participants in the inflammatory process in this model. The data in human arthritis would also support such a role for TLR2. Increased expression of TLR2 in synovial lining layer and by CD16 + peripheral blood mononuclear cells in RA indicate that its expression is upregulated during chronic inflammation [ 34 ]. TLR2 is also expressed on RA synovial fibroblasts, and incubation of cultured RA synovial fibroblasts with pro-inflammatory cytokines increases levels of TLR2 mRNA [ 35 ]. Although the precise role of TLR signalling in RA is unclear at present, increased TLR2 expression might modulate synovial inflammation if endogenous or exogenous TLR2 ligands gain access to the joint, thus amplifying specific and innate immune pathways of synovial inflammation. Furthermore, our results provide a model by which stimulation of the innate immune response can lead to chronic inflammation in the joint. These pathways may be of relevance to the development of reactive arthritis in man. Conclusion The results of the present study indicate that the biphasic joint inflammation in ZIA is mediated primarily by activation of the innate immune system. In the early phase of arthritis TLR2 plays a vital role, and in the later phase the development of a secondary immune response to zymosan may contribute to joint inflammation. Innate immune responses may be important amplificatory pathways of joint inflammation in man. Abbreviations ELISA = enzyme-linked immunosorbent assay; IFN = interferon; IL = interleukin; LNC = lymph node cells; TLR = Toll-like receptor; WT = wild-type; ZIA = zymosan-induced arthritis. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MF contributed to breeding and genotyping, performed technetium uptake measurements and immunoassays, and participated in coordination of the study. DT participated in technetium uptake measurements. VC performed histological stainings and scoring. NB performed statistical analysis and participated in the design of the study. AS conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.

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1065333

Balance between survivin, a key member of the apoptosis inhibitor family, and its specific antibodies determines erosivity in rheumatoid arthritis

Rheumatoid arthritis (RA) is a highly heterogeneous disease with respect to its joint destructivity. The reasons underlying this heterogeneity are unknown. Deficient apoptosis in rheumatoid synovial tissue has been recently demonstrated. We have therefore decided to study the synovial expression of survivin, a key member of the apoptosis inhibitor family. The levels of survivin and antibodies against survivin were assessed by an ELISA in matched blood and synovial fluid samples collected from 131 RA patients. Results were related to joint erosivity at the time of sampling. Monocytes were transfected with survivin anti-sense oligonucleotides and were assessed for their ability to produce inflammatory cytokines. Survivin levels were significantly higher in patients with destructive disease as compared with in RA patients displaying a non-erosive disease. High survivin levels were an independent prognostic parameter for erosive RA. In contrast, high levels of antibodies against survivin were found in patients with non-erosive RA, and were negatively related to erosivity. Survivin levels in RA patients were influenced by treatment, being significantly lower among patients treated with disease-modifying anti-rheumatic drugs. Specific suppression of survivin mRNA resulted in downregulation of IL-6 production. We conclude that survivin determines the erosive course of RA, whereas survivin antibodies lead to a less aggressive course of the disease. These findings together with decreased survivin levels upon disease-modifying anti-rheumatic drug treatment, and the downregulation of inflammatory response using survivin anti-sense oligonucleotides, suggest that extracellular survivin expression mediates the erosive course of joint disease whereas autoimmune responses to the same molecule, manifested as survivin targeting antibodies, mediate protection.

Introduction Rheumatoid arthritis (RA) is an inflammatory joint disease characterized by hyperplasia of synovial tissue and pannus formation growing invasively into the cartilage, followed by cartilage and bone destruction. Analyses of hyperplastic synovial tissues of patients with RA reveal features of transformed long-living cells such as the presence of somatic mutations, expression of oncogenes, and resistance to apoptosis [ 1 - 3 ]. Resistance to apoptosis further contributes to synovial hyperplasia and is closely linked to the invasive phenotype of synovial fibroblasts [ 4 , 5 ]. Apoptosis is a tightly regulated process of elimination of aged cells without disrupting cellular integrity (reviewed in [ 6 , 7 ]). Apoptosis may be initiated by extracellular stimuli through activation of death receptors on the cell surface, and intracellularly by the release of mitochondrial cytochrome c into the cytoplasm. Both pathways induce expression of apoptosis genes and activation of the caspase cascade, resulting in DNA fragmentation. The apoptosis signals are abrogated by the family of apoptosis-inhibiting proteins (IAPs). A number of disturbances in the apoptosis machinery have been pointed out in RA patients. Fibroblasts from RA synovia are relatively resistant to apoptosis induced by extracellular Fas stimulation. Moreover, co-culture of synovial fibroblasts from RA joints with T cells and B cells induces anergy of lymphocytes. Increased levels of soluble Fas in RA synovial fluid have been suggested as one possible explanation for this fact [ 8 ]. Indeed, administration of antagonistic anti-Fas antibodies or of Fas ligand has been shown effective in abrogation of arthritis in animal models [ 9 , 10 ]. Resistance to Fas-induced apoptosis in RA synovium correlates with a markedly increased expression of sentrin-1 [ 11 ]. Sentrin-1/SUMO is a molecule whose binding to a protein results in the prevention of ubiquitin-related processing and degradation of that protein. Sentrin-mediated protection has been shown for such proteins as p53 and IkBa. Upregulation of anti-apoptotic molecules belonging to the Bcl family and of the caspase-8 inhibitor FLIP has been repeatedly reported in RA [ 12 ]. Inhibited apoptosis has been shown to contribute to the pathogenesis of experimental arthritis [ 13 , 14 ]. Survivin is a 142-amino-acid protein that belongs to the IAP family, and it inhibits the activity of caspase 3, caspase 7, and caspase 9, but not of the upstream initiator protease caspase 8. Survivin can thereby downregulate, directly or indirectly, both death-receptor-mediated and mitochondria-mediated pathways of apoptosis [ 15 ]. Survivin has been also suggested to regulate cell division during mitosis. Indeed, survivin is the only one of IAPs that is tightly connected to the cell cycle being upregulated in the G 2 /M phase. Inside the dividing cell, survivin is found incorporated in centrosomes and mitotic spindles, and relocates to midbodies in the late telophase. Disruption of survivin function by negative mutation or by introduction of anti-sense oligonucleotides results in a cell-division defect [ 16 , 17 ]. Survivin is abundantly expressed in all the most common human cancers and in transformed cell lines [ 15 ], while most normal differentiated adult tissues do not express this molecule. A few adult tissues reported to express survivin include the spleen, the testes, the thymi, the placentas, and the colonic crypts. In the present study we demonstrate high levels of the anti-apoptotic protein survivin extracellularly in plasma and synovial fluid of patients with RA. In all the cases but one, high levels of survivin were associated with the erosive type of joint disease. Moreover, it is demonstrated that autoantibody responses to survivin led to a more benign (non-erosive) course of RA. The latter finding may have potential therapeutic consequences. Methods Participants Plasma and synovial fluid samples were collected from 131 RA patients who attended the rheumatology clinics at Sahlgrenska University Hospital, Göteborg for acute joint effusion. RA was diagnosed according to the American College of Rheumatology criteria [ 18 ]. At the time of synovial fluid and blood sampling all the patients received non-steroidal anti-inflammatory drugs. Disease-modifying anti-rheumatic drugs (DMARDs) were used by 96 patients, 67 of which used methotrexate (MTX). Forty-two of these 67 patients combined medication of MTX with the inhibitors of tumour necrosis factor alpha (TNF-α), two other patients combined MTX with sulfasalazine, one patient combined it with cyklosporine A, and the remaining 22 patients were treated with MTX alone. DMARDs other then MTX were used by 14 patients, six patients were treated with sulfasalazine, five patients were treated with cyklosporine A (one patient in combination with azathioprine, one patient with leflunomide, two with sulfasalazine, and the remaining patient with infliximab), four patients used parenteral or oral gold salt compounds, one patient used leflunomide, and one patient used azathioprine. The inhibitors of TNF-α were used in 47 patients (42 patients in combination with MTX, three patients in combination with azathioprine, one patient in combination with cyklosporine, and the remaining patient in combination with cyclophosphamide). The remaining 35 of 131 patients had no DMARD treatment at the time of blood and synovial fluid sampling. Recent radiographs of the hand and foot skeletons for all patients were studied. The presence of bone erosions, defined as the loss of cortical definition at the joint, was recorded in proximal interphalangeal joints, metacarpophalangeal joints, carpus joints, wrist joints, and metatarsophalangeal joints. The presence of one erosion was sufficient to fulfil the requirement of an erosive disease. We considered the presence of rheumatoid factor (RF) of any of the immunoglobulin isotypes as positive. Informed consent was obtained from the patients and the controls. The study was approved by the Ethics Committee of Sahlgrenska University Hospital. Analyses of survivin and antibodies to survivin Synovial fluid samples were obtained by arthrocentesis of knee joints. Synovial fluid was aspirated aseptically and transferred into tubes containing sodium citrate (0.129 mol/l; pH 7.4). We obtained blood samples simultaneously from the cubital vein and directly transferred them into sodium citrate medium. Blood samples from healthy individuals ( n = 34; age range, 18–62 years; mean age, 42 ± 7 years) were used as controls. Collected blood and synovial fluid samples were centrifuged at 800 × g for 15 min, aliquoted, and stored frozen at -20°C until use. Survivin levels were determined by a sandwich ELISA using a pair of matched antibodies (rabbit anti-human survivin; R&D Systems, Stockholm, Sweden). Briefly, 96-well polystyrene dishes (Nunc, Roskilde, Denmark) were coated with capture antibodies and were left overnight at room temperature. Following washing, plates were blocked with PBS–BSA containing 5% sucrose. Matched samples of plasma and synovial fluid were introduced into the parallel strips, at a dilution of one in 10 in PBS–BSA. Horseradish peroxidase-labelled detection antibodies and the corresponding substrate were used for colour development. Double-wave reading at 450 and 570 nm was used and the difference of absorbances was calculated. The obtained absorbance values were compared with the serial dilution of recombinant survivin and are presented as picograms per millitre. Antibodies of IgG and IgM class specific for survivin were measured in blood and synovial fluid samples by an ELISA. Briefly, 96-well polystyrene dishes (Nunc) were coated with human recombinant survivin (R&D Systems). Reconstituted survivin (0.5 μμg/ml) was introduced in each well and left overnight at room temperature. Following washing with PBS containing 0.1% Tween-20, plates were blocked with 1% ovalbumin (Sigma, St Louis, MO, USA) in PBS for 2 hours at room temperature. Matched samples of plasma and synovial fluid were introduced into the parallel strips, in a dilution of one in 100 using PBS–1% ovalbumin. This dilution was established as being on a linear scale in preliminary titration experiments. Horseradish peroxidase-labelled detection antibodies (rabbit F(ab')2-anti-human IgG and IgM; Dako, Glostrup, Denmark), ExtrAvidin peroxidase conjugate (Sigma), and the corresponding substrate were used for colour development. The absorbance at 405 nm was registered. Absorbances of the patient samples were compared with the mean values obtained in the control group of healthy individuals. Interaction with survivin transcription Peripheral blood mononuclear cells (PBMC) were prepared from heparinized blood of healthy individuals by separation on a Lymphoprep density gradient. We washed the cells, and resuspended in complete medium (Iscoves medium containing 1% l-glutamine, 5 × 10 -5 M β-mercaptoethanol, 50 μg/ml gentamycin sulphate, and 10% heat-inactivated FCS). We cultured PBMC in 24-well plates in a humidified atmosphere of 5% CO 2 at 37°C. In addition, we cultured the human monocytic cell line THP-1 (American Type Culture Collection, Manassas, VA, USA) in 10-ml culture flasks (Nunc) in RPMI 1649 medium supplemented with 10% FCS, 1% sodium pyruvate, gentamycin, and 2.5% Hepes in a humidified atmosphere of 5% CO 2 at 37°C. For the experiments, 4-day-old THP-1 cells were harvested, washed, and adjusted to 1 × 10 6 cells/ml. For the transfection experiments, phosphorothioated oligonucleotides containing the anti-sense-targeting human survivin gene [ 19 ] were synthesized by MWG Oligo (Ebersberg, Germany). The following anti-sense sequences were used: aSur 1, 5'-CCCAGCCTTCCAGCTCCTTG-3' ; and aSur 2, 5'-GCACCTAGTCTCCCTGCACC-3'. Irrelevant non-sense sequences were used as controls: non-sense 1, 5'-GTCCTCCACTGGCCTCACTC-3' ; and non-sense 2, 5'-CCCCGATTCACCTCGTCCGT-3'. Oligonucleotides were delivered to THP-1 cells using oligofectamine reagent (Invitrogen, Carlsbad, CA, USA). Before the transfection procedure we seeded THP-1 cells in 96-well tissue culture plates and cultured them overnight in RPMI medium free of antibiotics and FCS. Transfection was performed in RPMI medium supplemented with 2.5% Hepes and 100 mg/ml CaCl 2 . We mixed 0.6 μl oligofectamine with diluted oligonucletides and added it to the washed THP-1 cells. Following 4 hours of incubation at 37°C in a CO 2 incubator, the transfection procedure was discontinued by adding RPMI medium containing a threefold excess of FCS. At this time point, we also stimulated the cells with phytohaemagglutinine (PHA) (1.5 μg/ml) if required. Following 48 hours of stimulation, THP-1 cultures were aseptically collected, centrifuged at 1000 × g for 5 min, and kept frozen at -20°C until analysis. We prepared cell lysates by incubating the cell pellet for 1 hour in 1 mM EDTA buffer containing 6 M urea and proteinase inhibitors (Complete MiniTab; Boehringers, Ingelheim, Germany). These preparations were assessed for proliferation, survivin expression, and IL-6 levels. Cell survival and apoptosis in the transfected cultures were assessed by surface expression of annexin V and propidium iodide intake. Following transfection and stimulation for 48 hours, THP-1 cells were washed and stained with FITC-marked anti-annexin V antibodies and were subjected to flow cytometry (FACSort; Becton Dickinson, San Jose, CA, USA). The results were analysed using the CELLQuest software (Becton Dickinson). Proliferation of THP-1 cells was assessed by incubating the cell suspension with the test substance for 48 hours. The cells were then pulsed for 12 hours with 1 μCi [ 3 H]thymidine (specific activity, 42 Ci/mmol; Amersham, Bucks, UK). Cells were collected onto glass fibre filters. Thymidine incorporation was measured in a beta-counter. We compared the counts obtained in cells transfected with survivin anti-sense oligonucleotides and those incubated with oligofectamine alone. The results were expressed as a percentage. The level of IL-6 in supernatants was assessed by a bioassay. The effect of test samples on proliferation of the IL-6-dependent cell line B13.29 [ 20 ] was assessed following 72 hours of culturing. The results were analysed by incorporation of [ 3 H]thymidine (Amersham) during the last 4 hours of incubation at 37°C. Cells were collected onto a glass fibre filter. Proliferation in the presence of test samples was compared with that induced by standard dilutions of recombinant IL-6 (Genzyme, Cambridge, MA, USA). The results were further recalculated as in the proliferation assay. Statistical analysis We expressed the level of survivin and antibodies against survivin in the blood, in synovial fluid samples, as well as in cell lysates as the mean ± standard error of the mean. The survivin levels in the matched blood and synovial fluid samples were analysed by the paired Student t test. We further performed a comparison of survivin levels between the patient blood samples and the healthy controls using the Mann–Whitney U test. We stratified the patient material according to radiological findings (erosive RA versus non-erosive RA) and calculated the difference in survivin levels between the groups employing the Mann–Whitney U test. An arbitrary level of survivin corresponding to three standard deviations of the control group (300 pg/ml) was chosen as a cut-off. The RA patients were further stratified as having 'high' (>300 pg/ml) or 'low' (<300 pg/ml) levels of survivin. We performed the evaluation of survivin as a prognostic factor for the development of joint destruction, comparing the group having 'high' and 'low' survivin levels in a multivariate analysis. In order to control for the role of other prognostic factors (RF, disease duration, age, presence of antibodies against survivin), a multivariate logistic regression was performed. Odds ratios (with 95% confidence interval) are given for descriptive purposes. All tests were two-tailed and conducted at the 5% significance level. We evaluated a possible influence of the ongoing treatment on the survivin levels, and we stratified patient material according to DMARD treatment (treated versus untreated). For the simultaneous comparison of the survivin levels in more than two groups the equality of variance F test was employed. The inter-relation between the survivin levels and duration of the joint disease, age, white blood cell (WBC) count, and C-reactive protein was calculated employing the Spearman correlation coefficient. For all the statistical evaluation of the results, P < 0.05 was considered significant. All statistical evaluations were performed using StatView PowerPC software. Results Clinical and demographic data of the patient population and the control group are presented in Table 1 . The patient group showed no difference regarding gender compared with controls, while individuals from the control group were younger ( P < 0.05). After stratification of the RA patients with respect to radiological changes, the group with erosive joint disease (ERA) was, as expected, more often positive for RF compared with the group for non-erosive joint disease (NRA) (91% versus 23%, P < 0.0001), and had longer duration of RA ( P = 0.0002) as compared with NRA patients. With respect to treatment, 68% of ERA patients were treated with MTX, and 48% in combination with TNF-α inhibitors. Among NRA patients, only 28% were treated with MTX ( P < 0.025), and 12% with TNF-α inhibitors. NRA patients were significantly more often without DMARDs at the time of blood sampling compared with ERA patients (63% versus 20%, P < 0.0001). Extracellular survivin determines the erosive course of RA Plasma of the RA patients contained significantly higher levels of survivin as compared with the controls (330 ± 123 pg/ml versus 121 ± 2 pg/ml, P = 0.002). Survivin levels in plasma correlated strongly to their levels in synovial fluid ( r = 0.89). Evaluation of the survivin level was performed in RA patients with respect to the erosivity of joint disease (Fig. 1 ). Patients with ERA had a significantly higher level of survivin compared with NRA patients in plasma (430 ± 108 pg/ml versus 127 ± 5 pg/ml, P = 0.0022) and in the synovial fluid (434 ± 181 pg/ml versus 124 ± 2 pg/ml, P = 0.0029). The levels of survivin did not differ significantly between the patients positive for RF ( n = 90) and those who were RF-negative ( n = 41) (418 ± 107 pg/ml versus 151 ± 20 pg/ml, not significant). Survivin levels showed no significant correlation to the serum levels of C-reactive protein and WBC count, and neither to the synovial fluid leukocyte count and IL-6 levels. The RA patients were further stratified as having 'high' (>300 pg/ml) or 'low' (<300 pg/ml) levels of survivin, departing from the level of survivin that corresponded to a mean + three standard deviations of the control group as a cut-off. The difference in the mean survivin level between the 'high' and the 'low' groups was about 10-fold (1180 ± 309 pg/ml versus 97 ± 9 pg/ml). High levels of survivin were detected in 28 of 131 patients (21%). All but one (96%) of the patients with a high survivin level displayed erosive RA. A dominance of a high survivin level among the ERA patients was consequently found both in plasma and in synovial fluid samples. Comparison between the ERA patients having high and low levels of survivin (Table 2 ) revealed, beside erosivity, an association between high levels of survivin and increased circulating C-reactive protein as well as elevated WBC counts. In contrast, age, gender, RF-positivity, and duration of the disease were similar in the ERA patients with high levels of survivin as compared with those with low levels. The level of survivin was also studied in RA synovial fluid samples separated with respect to the cell pellet and the supernatant by centrifugation ( n = 9). Survivin levels found in supernatants and in the lysates of synovial fluid cells obtained from the same sample revealed a strong correlation ( r = 0.87, P < 0.0001). These data indicate that survivin is produced and secreted locally in the joints of RA patients. To evaluate the predictive value of high survivin levels for the development of destructive joint disease, a logistic regression model was constructed, taking erosive changes at radiological examination of the hand and foot skeletons as a dependent variable. We found that high levels of survivin were significantly associated with erosive changes (odds ratio, 18.76; 95% confidence interval, 2.45–143.65; P = 0.0048). To determine whether survivin was independently associated with erosive RA, we developed a multivariate logistic regression model with radiological changes as the dependent variable and with RF, duration of RA, gender, and the survivin level as independent variables. After adjusting for the presence of RF, gender, and the duration of RA, a high level of survivin was significantly associated with erosive RA (odds ratio, 16.02; 95% confidence interval, 2.02–127.19; P = 0.013). Our data thus demonstrate that RA patients having high levels of survivin are 16 times more likely to develop erosive joint disease compared with those with low levels of survivin. Taking into account the fact that the increased survivin levels were observed predominantly among the ERA patients, we assessed the effect of DMARD treatment on survivin levels in this patient group. To analyse the putative influence of anti-rheumatic treatment on the level of survivin, ERA patients were stratified with respect to their treatment modality at the time of sampling into three groups. Group 1 included patients receiving MTX ( n = 18), group 2 included patients treated with combination of MTX and TNF-α inhibitors ( n = 42), group 3 included patients treated with DMARDs other than MTX ( n = 10), and group 4 included patients having no treatment with DMARD at the time of sampling ( n = 18) (Fig. 2 ). The highest level of survivin, both in blood and in synovial fluid, was found in the group of patients having no DMARD at the time of sampling (blood, 666 ± 473 pg/ml and synovial fluid, 830 ± 610 pg/ml, respectively). This was significantly higher than in the patients treated with MTX (322 ± 174 pg/ml, P = 0.02) and in the patients treated with other DMARDs (280 ± 82 pg/ml, P < 0.001). These three groups of patients were similar with respect to the duration of the disease, age, WBC counts in blood and synovial fluid, and levels of C-reactive protein. Patients treated with combination of MTX and TNF-α inhibitors exhibited no significant difference in survivin plasma levels compared with the patients treated with MTX alone. This was despite the fact that patients obtaining TNF-α inhibitors were younger ( P < 0.05) and had lower levels of WBC and C-reactive protein ( P < 0.05). Autoantibodies specific for survivin relate to the non-erosive course of RA An ELISA was used for the evaluation of antibodies against survivin of IgG and IgM isotypes in plasma and in synovial fluid of 129 patients with RA and of 34 healthy controls. The absorbance values revealed a significantly higher antibody reactivity with human recombinant survivin in the case of RA patients compared with the controls (Fig. 3 ). This was true both for IgG (0.19 ± 0.02 versus 0.11 ± 0.012, P = 0.022) and for IgM (0.60 ± 0.03 versus 0.28 ± 0.03, P < 0.0001) isotypes of antibodies. There was a weak, although significant, correlation between the antibodies of IgG and IgM isotypes in blood ( r = 0.389, P < 0.001), but not in synovial fluid ( r = 0.146, not significant). No significant difference in the IgG antibody levels was found between blood and synovial fluid (0.19 ± 0.02 versus 0.20 ± 0.03, not significant), while the level of IgM antibodies was significantly higher in blood samples than in synovial fluid samples (0.60 ± 0.03 versus 0.43 ± 0.03, P = 0.031). Stratification of the patient material with respect to radiological changes revealed that the level of antibodies against survivin was higher in NRA patients compared with ERA patients (Fig. 4 ). The difference was most pronounced in synovial fluid samples (IgG, 0.18 ± 0.02 versus 0.22 ± 0.02, P = 0.038; IgM, 0.31 ± 0.03 versus 0.59 ± 0.03, P = 0.0007). Among the ERA patients, a distinct group of patients with high extracellular levels of survivin was outlined. These patients had significantly higher levels of antibodies against survivin both in blood (IgG, 0.25 ± 0.02 versus 0.15 ± 0.02, P < 0.0001; IgM, 0.64 ± 0.03 versus 0.55 ± 0.03, not significant) and in synovial fluid (IgG, 0.21 ± 0.02 versus 0.16 ± 0.02, not significant; IgM, 0.40 ± 0.03 versus 0.27 ± 0.03, P = 0.023) as compared with those ERA patients with low survivin levels. However, no significant correlation between the level of extracellular survivin and the level of antibodies against survivin was observed ( r = 0.05). Influence of survivin expression on inflammatory responses PBMC from healthy individuals and from RA patients were stimulated with various B-cell and T-cell mitogens, superantigen, and TNF-α (10–100 ng/ml lipopolysaccharide, 0.5–5 μg/ml Concanavalin A, 10–100 ng/ml TNF-α, 10–100 ng/ml TSST-1, 0.5–5 μg/ml PHA) for 6–48 hours. Supernatants and cell lysates were evaluated for survivin expression by an ELISA. Detectable levels of survivin were not found in supernatants. In the cell lysates, levels of survivin varied in response to the aforementioned stimuli (Fig. 4 ). In the tested panel, the T-cell mitogen PHA was found to be a potent inducer of survivin expression both by PBMC originating from RA patients ( n = 3) and by PBMC from healthy controls ( n = 6) Stimulation of THP-1 with PHA was therefore used in the subsequent transfection experiments. To assess the role of survivin in the inflammatory process, the human mononuclear cell line THP-1 was transfected with oligonucleotides targeting different regions of survivin mRNA. Oligonucleotides were delivered in complex with oligofectamine as described in Materials and methods. Successful transfection with the inhibitory sequence was confirmed by a downregulation of survivin expression in THP-1 lysates as assessed by ELISA. THP-1 cells displayed, as expected, high spontaneous intracellular expression of survivin, which correlated well with their proliferative activity. Following the transfection procedure, cells were stimulated with PHA (1.5 μg/ml) for 48 hours and the cultures were assessed for proliferation and secretion of IL-6. Two different anti-sense sequences were tested, and both anti-sense oligonucleotides downregulated survivin expression (from 100% to 30–44%, P < 0.05). In contrast, non-sense oligonucleotides showed no significant suppression of survivin expression as compared with the THP-1 cultures incubated with oligofectamine alone (Fig. 5a ). In the THP-1 cultures displaying suppressed survivin expression, a significant downregulation ( P < 0.01) of IL-6 production was observed, decreasing from 100% to 21–30% (Fig. 5c ). To assess whether low survivin expression was related to apoptosis and cell death in the transfected cell cultures, cell proliferation and the expression of annexin V were measured using FACS analysis. THP-1 cells transfected with anti-sense oligonucleotides showed no significant difference regarding annexin V expression (24–37% versus 20–27%, not significant) or proliferation rate (57–68% versus 64–80%, not significant) (Fig. 5b ) compared with the cells transfected with non-sense oligonucleotides. These data indicate that the production of inflammatory cytokine IL-6 participating in the regulation of inflammatory responses is directly related to survivin expression by monocytes. Discussion Suppression of apoptosis has been suggested as a key mechanism supporting selection and accumulation of distinct lymphocyte subsets in chronically inflamed joint tissues [ 21 ]. Indeed, synovial T cells in RA are highly differentiated and would not normally be expected to survive for a prolonged time within inflamed joints unless their death was actively inhibited [ 22 ]. In the present study we demonstrate that high expression of survivin, a member of the IAP family, is a new and potentially important mechanism of apoptosis suppression in patients with RA. Survivin is known as a multipotent inhibitor of apoptosis, neutralizing several caspases at the final steps of the apoptosis cascade, thus abrogating signals from both the death-receptor-dependent and mitochondrial pathways of apoptosis. Together with previous findings of upregulation of other caspase inhibitors (Bcl and FLIP) [ 12 , 13 ], high levels of survivin give new insights in numerous alterations of the apoptosis machinery during the course of RA. We observed that survivin levels were clearly increased in synovial fluid and plasma of RA patients compared with the healthy controls. Survivin expression was originally considered a reflection of cell proliferation. Indeed, survivin is continuously overexpressed in cancer cells [ 23 ]. Survivin gene transcription is repressed by wild-type p53 [ 24 - 26 ]. Multiple mutations and functional dysregulation of p53 have been demonstrated in the synovial tissue of RA patients [ 3 , 27 ] and constitute one of the possible reasons for increased survivin production in this non-malignant condition. Notably, high survivin levels (over three standard deviations of the mean of healthy blood donors) were registered exclusively in patients with erosive joint disease and were associated with markers of inflammation such as WBC count and C-reactive protein levels, as well as with the absence of immunosuppressive treatment. This category of RA patients typically displays chronic joint inflammation, progressive joint destruction, and early mortality [ 28 , 29 ]. Altogether these findings place survivin at the centre of attention as a potential prognostic factor for the destructive course of disease in RA. Indeed, using logistic regression analysis, we demonstrated that RA patients having high levels of survivin had a 16 times higher risk to develop destructive joint disease as compared with the patients with low levels of survivin. Moreover, in a multivariant model we showed that the role of survivin is independent of the presence of RF, the duration of the rheumatic disease, and gender. Interestingly, survivin expression has been shown to be an important prognostic factor in acute leukaemia [ 30 , 31 ], and a predictor of recurrence in soft-tissue sarcomas [ 32 ] and urinary bladder cancer [ 33 , 34 ]. In the latter case, extracellular urinary survivin levels were used for the evaluation of treatment and recurrence of cell carcinoma. Survivin expression determined locally in the inflamed joints and also systemically in circulation of patients with RA was measured extracellularly. Whether survivin found extracellularly originates from dead cells or is a subject of active secretion is presently unknown. The number of in vitro leukocyte-activating stimuli (e.g. lipopolysaccharide, PHA, TSST-1, Concanavalin A) will not induce secretion of survivin. This observation suggests, but does not prove, that extracellular survivin found in synovial fluid originates from dead cells. Alternatively, some other cells (e.g. fibroblasts) or endogenous stimuli give rise to secretion of this molecule. Little is known about extracellular functions of survivin. Survivin has been suggested to function as a self-antigen in patients with haematologic malignancies and solid tumours. In our patient material we demonstrate the presence of antibodies to survivin in the plasma and synovial fluid of patients with RA. Interestingly, reactivity against survivin was significantly higher in the patient group with non-erosive RA. Notably, patients with non-erosive RA have extracellular survivin levels undistinguishable from these of the healthy controls. The association of a high level of antibodies against survivin with non-erosive joint disease may be a reflection of a protective autoimmune mechanism existing in these patients. To assess the role of survivin in the inflammatory process, we first studied its inducibility in differentiated mature human PBMC. Most of the pro-inflammatory stimuli including lipopolysaccharide, Concanavalin A, TSST-1, and TNF-α leading to a significant release of inflammatory cytokines and chemokines, failed to induce survivin expression by PBMC. In contrast, downregulation of survivin expression using specific anti-sense oligonucleotides resulted in the decrease of IL-6 production by human monocytes. These two observations suggest that the regulatory role of survivin in inflammation is mediated by an increase of cytokine production. The connection between survivin expression and production of IL-6 deserves special attention in the view of recent success of the neutralization of IL-6 for alleviation of RA [ 35 ]. These observations support the regulatory role of survivin in the pathogenesis of arthritis. Studying the variability of survivin levels in patients with RA, we observed that in most cases survivin levels were inclined to decrease in survivin-positive patients and almost never converted from absent to high in survivin-negative cases (data not shown). We also showed that the decrease of survivin levels could be mediated by treatment with DMARDs. This suggests survivin to be a transient phenomenon in the course of RA and may explain a relatively low frequency of patients having high survivin levels (21%) in the cohort tested. However, the results of our study may be affected by the fact that most of the patients were treated with DMARDs at the time of sampling, and even those without ongoing DMARD therapy might have received immunosuppressive treatment previously. Conclusions Our study suggests that survivin regulates the inflammatory and destructive process inside the joints of patients with RA. Indeed, high levels of extracellular survivin are associated with chronic erosive arthritis, indicating poor prognosis. In contrast, antibodies against survivin are characteristic of the patients with the non-erosive, benign course of RA. Our findings on survivin expression and autoimmunity to this molecule provide new insight regarding the role of apoptosis in RA. Abbreviations BSA = bovine serum albumin; DMARD = disease-modifying anti-rheumatic drug; ELISA = enzyme-linked immunosorbent assay; ERA = erosive rheumatoid arthritis group; FACS = fluorescence-activated cell sorting; FCS = foetal calf serum; FITC = fluorescein isothiocyanate; IAP = inhibitor of apoptosis proteins; IL = interleukin; MTX = methotrexate; NRA = non-erosive rheumatoid arthritis group; PBMC = peripheral blood mononuclear cells; PBS = phosphate-buffered saline; PHA = phytohaemagglutinine; RA = rheumatoid arthritis; RF = rheumatoid factor; TNF-α = tumour necrosis factor alpha; WBC = white blood cell. Competing interests The author(s) declare that they have no competing interests. Authors' contributions MB contributed to the study design, to the clinical, laboratory and statistical evaluation of material from RA patients, and to preparation of the manuscript. SL performed some of the cell experiments. DB performed ELISA assays, bioassays, and some of the transfection experiments. AT contributed to the conception of the study and the study design, to statistical evaluation of the results, and to preparation of the manuscript.

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1065334

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H 2 O 2 ) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.

Introduction Articular cartilage matrix undergoes substantial structural, molecular, and mechanical changes with ageing, including surface fibrillation, alteration in proteoglycan structure and composition, increased collagen cross-linking, and decreased tensile strength and stiffness [ 1 , 2 ]. Deterioration in chondrocyte function accompanies these changes in the extracellular matrix [ 3 ]. Recently, attention has been given to the suggestion that cartilage ageing and chondrocyte senescence play an important role in the pathogenesis and development of osteoarthritis (OA) [ 4 , 5 ]. Several reports revealed that chondrocyte senescence contributes to the risk for cartilage degeneration by decreasing the ability of chondrocytes to maintain and repair the articular cartilage tissue [ 4 - 6 ]. The mitotic and synthetic activity of chondrocytes decline with advancing donor age [ 5 ]. In addition, human chondrocytes become less responsive to anabolic mechanical stimuli with ageing and exhibit an age-related decline in response to growth factors such as the anabolic cytokine insulin-like growth factor-I [ 6 ]. These findings provide evidence supporting the concept that chondrocyte senescence may be involved in the progression of cartilage degeneration. Telomeres, the terminal guanine-rich sequences of chromosomes, are structures that function in the stabilization of the chromosome during replication by protecting the chromosome end against exonucleases [ 7 , 8 ]. The telomere DNA may function as a timing mechanism that, when reduced to a critical length, signals a cell to stop dividing and to enter cellular senescence [ 7 - 9 ]. More recent reports demonstrated that the telomere length of chondrocytes shortened with donor ageing and that decreased mean telomere length was closely related to the increase in senescence-associated β-galactosidase expression in human chondrocytes, suggesting that chondrocyte senescence, at least in part, participates in the age-related loss of chondrocyte function responsible for deterioration in articular cartilage structure and function [ 10 ]. An understanding of the mechanisms of chondrocyte senescence would be helpful to our efforts to devise new approaches to the prevention and treatment of OA. Mechanical and chemical stresses are thought to induce increased free radical production, consequently leading to oxidative damage to the tissue [ 11 - 14 ]. Oxidative damage not only can initiate apoptosis through caspase activation but also may lead to irreversible growth arrest, similar to replicative senescence [ 11 , 12 , 15 ]. Furthermore, it has been reported that oxygen free radicals (O 2 - and peroxynitrite) directly injure the guanine repeats in the telomere DNA, indicating that oxidative stress directly leads to telomere erosion, regardless of cell active division [ 16 ]. Generally, it is now thought that oxidative stress/antioxidative capacity may be prominent among factors that control telomere length [ 17 - 19 ]. These findings strongly suggest that oxidative stress could induce chondrocyte telomere instability with no requirement for cell division in articular cartilage, leading to chondrocyte senescence. Numerous reports have demonstrated that oxidative damage due to the over-production of nitric oxide (NO) and other reactive oxygen species (ROS) may be involved in the pathogenesis of OA [ 20 - 23 ]. However, because of the highly reactive nature of these oxygen reactive species and their short half-lives, it had been difficult to investigate oxidative damage in vivo [ 24 ]. ROS and NO cannot be directly and accurately measured in a cartilage sample. Recently, a reaction product of ROS and NO, namely nitrotyrosine, was used as evidence of oxidative damage in several ageing tissues [ 25 , 26 ]. Loeser and coworkers [ 26 ] demonstrated that nitrotyrosine is over-expressed in normal cartilage from elder donors and in OA cartilage, suggesting the presence of oxidative damage in ageing and degenerative cartilage. These findings provide evidence to support the concept that oxidative stress in articular cartilage affects chondrocyte function, resulting in changes in cartilage homeostasis that are relevant to cartilage ageing, chondrocyte senescence and the development of OA. Based on the properties of chondrocyte senescence and oxidative stress in OA cartilage, as discussed above, we postulated that oxidative stress induces telomere instability and dysfunction in chondrocytes, subsequently resulting in cartilage ageing and the development of OA through a mechanism involving the acceleration of chondrocyte senescence. It is now thought that oxidative stress/antioxidative capacity is prominent among factors that control telomere length, and hence replicative lifespan [ 17 , 18 ]. To clarify the role of oxidative damage in the pathogenesis of OA, we looked for the presence of oxidative damage in degenerated cartilage from OA patients and examined whether chemical oxidative stress (ROS) affects chondrocyte telomere DNA, replicative lifespan, and function in cultured chondrocytes and in explants of articular cartilage. We also examined the effects of the antioxidative agent ascorbic acid on the oxidative stress induced downregulation of cellular lifespan and function in chondrocytes. Methods Articular cartilage tissue and chondrocyte culture Articular cartilage samples were obtained from OA patients ( n = 9) who had undergone arthoplastic knee surgery (all female, age [mean ± standard deviation] 61.5 ± 5.4 years). The patients had given informed consent, in accordance with the ethical committee of the university. All samples were obtained in accordance with institutional protocol, with review board approval. Donor articular cartilage samples were evaluated macroscopically using a modified Collins scale from 0 to 5, as described previously [ 27 - 29 ]. To obtain sufficient numbers of cells for the experiments, cultured chondrocytes were isolated from macroscopically intact zones of cartilage. Cartilage tissue was cut into small pieces, washed in phosphate-buffered saline (PBS), and digested in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO, USA) containing 1.5 mg/ml collagenase B (Sigma). Digestion was carried out at 37°C overnight on a shaking platform. Cells were centrifuged, washed with PBS, and plated with fresh DMEM. Basically, chondrocytes were cultured in DMEM supplemented with 10% heat-inactivated foetal calf serum, 2 mmol/l l-glutamine, 25 mmol/l HEPES, and 100 units/ml penicillin and streptomycin at 37°C in a humidified 5% CO 2 atmosphere [ 30 ]. To avoid loss of chondrocyte phenotypes during passages, we used cultured chondrocytes only from passages 1–4. In parallel cultures, we checked the cell morphology and potential to produce proteoglycan in order to examine whether chondrocyte phenotype had been maintained during the passage. Data from chondrocyte mass cultures with loss of chondrocyte phenotypes were excluded from the analysis. Chondrocytes were cultured in the presence of an antioxidant (100 μmol/l ascorbic acid-2- O -phosphate [Asc2P; Wako Junyaku, Tokyo, Japan]) or a ROS (H 2 O 2 ) at a concentration of 0.1 μmol/l, which was not cytotoxic to the cells [ 17 ]. We had already investigated the effect of H 2 O 2 (0.1–500.0 μmol/l) on chondrocyte viability in vitro . Concentrations of 0.1–200.0 μmol/l of H 2 O 2 exhibited no inhibitory effects on chondrocyte viability (data not shown). In addition, we had also studied the time course of H 2 O 2 treatment (0.1–100.0 μmol/l) in vitro . Based on our preliminary experiments, in the present study we conducted the cell culture and the organ culture in the presence or absence of H 2 O 2 (0.1 μmol/l). In each culture group, the medium including freshly prepared Asc2P or H 2 O 2 was changed every 2 days. Human chondrocytes were subcultured weekly. At each passage, the total number of collected cells in the dish was determined. Then, 2.5–5.0 × 10 5 cells were transferred to a new dish for the next passage, and the number of attached cells was determined 6 hours after seeding. From each passage, the remaining cells after subculture were stored at -180°C until the analysis of cellular activity, telomere length and telomerase activity was conducted. Oxidative stress in human articular cartilage We compared the degree of oxidative stress (antioxidative potential) of the intact cartilage with that of degenerative cartilage tissue. Cartilage samples from the same donor joint were cut and divided into two groups (the degenerated region group, which exhibited macroscopic changes of OA; and the intact region group, which was macroscopically normal). In these donor matched pairs of articular cartilage samples, antioxidative potential of the tissue was measured using an assay that is based on reduction of Cu 2+ to Cu + and the measurement was conducted according to the manufacturer's instructions (OXIS Health Products, Inc., Portland, OR, USA). This assay measures the total contribution of all antioxidants in the tissue sample. The results of the assay were calculated as mmol/l uric acid equivalents, and expressed as a ratio of antioxidative potential of the degenerating region to that of the corresponding intact region from each donor. Immunohistochemistry For immunostaining of human articular cartilage, paraffin blocks of articular cartilage tissues were prepared using standard histological procedures. Serial sections of paraffin-embedded bone and cartilage tissues were cut and immunostained using an antibody for nitrotyrosine. The sections were deparaffinized and hydrated. Then, the slides were stained using horseradish peroxidase method [ 26 ]. Briefly, the slides were blocked with 3% H 2 O 2 . After blocking nonspecific protein binding with blocking agent (Dako, Carpinteria, CA, USA), the sections were incubated with a monoclonal antibody to nitrotyrosine (1:100 dilution; BIOMOL Research Laboratories Inc., Plymouth Meeting, PA, USA) for 1 hour at room temperature, followed by incubation with biotinylated goat anti-mouse IgG (Dako) for 30 min at room temperature. After washing with PBS, the sections were incubated with streptavidin–horseradish peroxidase complex (LSAB2 kit; Dako) for 30 min at room temperature We used diaminobenzidine (Sigma) as a visible peroxidase reaction product. Sections were counterstained with Mayer's haematoxylin (Sigma). Cells positive and negative for nitrotyrosine were counted in the 20 areas of cartilage at 200× magnification (0.785 mm 2 /field). The level of immunostaining for nitrotyrosine was expressed as a mean number of nitrotyrosine-positive cells per field. Chondrocyte activity Chondrocyte activity was measured as the production of glycosaminoglycan (GAG) by cultured chondrocytes [ 15 ]. After undergoing continuous treatment with ROS or ascorbic acid (initial subculture at the start of the experiment: 1 × 10 5 cells/dish, chondrocytes from passage 2), the cells were collected with trypsin and washed with PBS. Then, chondrocytes (1 × 10 5 cells/dish) were plated in the culture dishes and incubated for 12 hours, and the amount of GAG in the supernatant was measured using a spectrophotometric assay with dimethylmethylene blue (Aldrich Chemical, Milwaukee, WI, USA) [ 31 ]. Determination of the lifespan of cultured chondrocytes The increase in cumulative population doublings at each subculture was calculated based on the number of cells attached and the cell yield at the time of the next subculture. Population zero was the primary culture of human chondrocytes, and the number of each successive generation was calculated using the following formula [ 32 , 33 ]: generation number at the start of the subculture + log 2 ([the number of collected cells at the time of the next subculture]/[the number of attached cells at the start of the subculture]). Senescence was defined as less than one population doubling in 4 weeks. The in vitro lifespan (remaining replicative capacity) was expressed as population doublings up to cellular senescence [ 34 ]. Telomere length of cultured chondrocytes Telomere length was determined using terminal restriction fragment Southern blot analysis, as described previously [ 35 , 36 ]. Genomic DNA from 10 6 chondrocytes from each subculture (initial subculture at the start of the experiment: 1 × 10 6 cells/dish, chondrocytes from passage 3 or 4) was digested with 400 μl DNA extraction buffer (100 mmol/l NaCl, 40 mmol/l Tris [pH 8.0], 20 mmol/l EDTA, and 0.5% SDS) and proteinase K (0.1 mg/ml). Extraction was performed using phenol chloroform. Extracted DNA (5–10 μg) was digested with 10 units of Msp I and Rsa I (Boehringer Mannheim, Indianapolis, IN, USA) for 12–24 hours at 37°C. The integrity of the DNA before digestion and the completeness of digestion were monitored by gel electrophoresis. Electrophoresis of digested genomic DNA was performed in 0.5% agarose gels in 45 mmol/l Tris-borate EDTA buffer (pH 8.0) for a total of 660–700 V-h. After electrophoresis, gels were depurinated in 0.2 N HCl, denatured in 0.5 mol/l NaOH and 1.5 mol/l NaCl, transferred to a nylon membrane using 20× SSC, and dried for 1 hour at 70°C. The telomeric probe (TTAGGG) 3 (Genset, La Jolla, CA, USA) was 5' end-labelled with [α- 32 P]ATP using T4 PNK (Boehringer Mannheim). Prehybridization and hybridization were performed at 50°C using 5× Denhardt's, which was composed of 5× SSC, 0.1 mol/l Na 2 HPO 4 , 0.01 mol/l Na 4 P 2 O 7 , 30 μg/ml salmon sperm DNA, and 0.1 mmol/l ATP. The mean terminal restriction fragment length was determined from densitometric analysis of autoradiograms, as described previously [ 35 ]. Tissue culture of human articular cartilage Procedures for preparing articular cartilage were generally the same as mentioned above. Briefly, articular cartilage was excised in small, full-depth slices (typically 1.0 cm square) from patients with OA ( n = 4) who had undergone arthroplastic knee surgery (all females; ages 61, 65, 67 and 68 years). The cartilage explants were cut, weighed and divided into three groups as follows: control group, antioxidative agent + oxidative stress treated group, and oxidative stress treated group. Control and experimental cartilage explants (site-matched pairs) were placed in individual dishes (diameter 6.0 cm) with 10.0 ml DMEM with 10% foetal bovine serum, 100 units/ml penicillin/streptomycin. The process of harvesting the cartilage tissue resulted in significant catabolic activity that was measurable in the absence of interleukin-1 stimulation, presumably due to secretion of proteases in response to trauma. The contribution of this basal catabolic activity could be minimized by culturing for 24 hours before aspiration of the culture medium, washing with PBS, and adding fresh culture medium [ 37 , 38 ]. For the antioxidative agent + oxidative stress treated group, the cartilage explants were incubated in the culture medium with 100.0 μmol/l Asc2P plus 0.1 μmol/l H 2 O 2 . For the oxidative stress treated group, the explants were incubated in the culture medium in the presence of 0.1 μmol/l H 2 O 2 . For each group, culture medium including freshly prepared Asc2P or H 2 O 2 was changed every day. At the end of each incubation period (48, 72, 96, 120 and 120 hours), the cartilage samples and the culture media were collected and re-weighed for analyses. The cartilage samples were washed with PBS. Some parts of cartilage samples were fixed with 4% paraformaldehyde at 4°C, and then paraffin blocks were prepared using standard histological procedures. For nitrotyrosine staining, the sections were deparaffinized and hydrated, and then were immunostained using antibody for nitrotyrosine in accordance with the method described above. Other cartilage samples and supernatants were stored at -80°C for the determination of GAG concentration and isolated chondrocyte telomere length. Catabolic changes to GAG in cartilage were analyzed by determining the GAG content remaining in cartilage tissue relative to the total amount of GAG in the culture (GAG released into the culture media plus GAG in the tissue) in the presence of the antioxidative agent or ROS [ 2 , 39 ]. GAG contents were measured using a spectrophotometric assay mentioned above. Procedures for cultured chondrocyte preparation from tissue cultured explants and telomere length assay were generally the same as those described above. Statistical analysis Results were expressed as a mean value ± standard deviation. Comparison of the means was performed by analysis of variance. P < 0.05 was considered statistically significant. Results Oxidative damage in human articular cartilage tissues To determine whether oxidative damage was present in OA degenerated cartilage, we measured the antioxidative potential of the intact region and degenerated region isolated from the same articular cartilage tissue of patients who had undergone arthroplastic knee surgery. In the donor-matched pair of intact and degenerated regions from same articular cartilage, the antioxidative potential in the intact region was significantly greater than that in the degenerated region of articular cartilage in the OA patient group ( n = 9; mean percentage antioxidative capacity of degenerative cartilage compared with intact cartilage: 45.5 ± 16.8%), suggesting that degenerated cartilage may exhibit more oxidative damage than an intact region from the same OA cartilage. Presence of nitrotyrosine in articular cartilage from patients with osteoarthritis To clarify the relationship between oxidative damage and development of OA, immunostaining for nitrotyrosine was examined in the donor-matched pair of intact and degenerated articular cartilage sections from the same OA sample. Figure 1 shows a representative example of immunohistochemical staining for nitrotyrosine in the articular cartilage from an OA patient (female, 67 years old). Immunostaining for nitrotyrosine was most apparent in the degenerated regions of articular cartilage that showed histological changes consistent with OA (nine patients; positive cells/field, intact cartilage versus degenerated cartilage: 0.3 ± 0.1 versus 7.4 ± 2.4; P < 0.01). Nine of 10 donor samples with degenerated regions were highly positive for nitrotyrosine. Nitrotyrosine was present both within chondrocytes and in the cartilage matrix, and was seen mainly in the more superficial regions. The degree of immunostaining for nitrotyrosine (number of positive cells/field) correlated with the level of histological change in donor cartilage tissues ( n = 9, r 2 = 0.4671; P < 0.01). In contrast to the immunostaining in the degenerated regions, almost all intact regions isolated from the same articular cartilage were negative for nitrotyrosine, even in superficial and deep zones (Fig. 1 ). In vitro chondrocyte activity under the different oxidative conditions Figure 2 shows that GAG synthesis from cultured chondrocytes decreased gradually in a time dependent manner, regardless of the presence of H 2 O 2 or an antioxidative agent in vitro . The H 2 O 2 treated group showed a significant decrease in proteoglycan production by chondrocytes as compared with the control group at any incubation time. In contrast, in the antioxidative agent group the level of proteoglycan production tended to increase as compared with that in control groups, although no significant differences were observed between control groups and antioxidative agent groups at any incubation time (Fig. 2 ). Chondrocyte replicative potential under the different oxidative conditions To clarify the effect of oxidative stress on the replicative potential of chondrocytes, we analyzed the cellular replicative potential of chondrocytes in the presence of the antioxidative agent or ROS in vitro . As shown in Fig. 3 , the replicative potential of cultured chondrocytes was expressed as the cumulative number of cells dividing at each incubation time. After 20 days of incubation the H 2 O 2 treated group exhibited lesser replicative potential as compared with the control group at any incubation time. In contrast, treatment with the antioxidative agent increased the cellular replicative potential at all incubation times after 20 days (Fig. 3 ). During the 4 weeks after a 50- to 60-day incubation, the cumulative population doubling levels of all groups reached a plateau, indicating that the cultured chondrocytes in each group reached the limit of their ability to divide, namely cellular senescence, after about 8 weeks of incubation. The mean lifespan to cellular senescence was 23 population doublings in the antioxidative agent treated group, 18 population doublings in the control group, and 14 population doublings in the ROS-treated group (Fig. 3 ). Chondrocyte telomere length under the different oxidative conditions To clarify the effect of oxidative stress on the telomeric instability in chondrocytes, we analyzed the telomere length of chondrocytes in the presence of an antioxidative agent or ROS in vitro (Fig. 4a ). After five to six population doublings, telomere lengths of chondrocytes were shorter in H 2 O 2 treated groups than in control groups at any level of population doubling. Treatment with an antioxidative agent resulted in a tendency of chondrocyte telomere length to elongate ( n = 9; Fig. 4b ). Immunohistochemical staining for nitrotyrosine of human articular cartilage cultured under different oxidative conditions To examine the influence of an antioxidative agent or ROS in human articular cartilage, immunohistochemical staining for nitrotyrosine was evaluated in cartilage samples that were treated with an antioxidative agent or ROS (H 2 O 2 ) in organ culture. Cartilage from an OA patient was cut and divided into three groups as follows: control group, antioxidative agent (Asc2P) treated group, and H 2 O 2 treated group. After a 48-hour incubation in explant culture, OA articular cartilage in both the control group and the H 2 O 2 treated group exhibited positive immunostaining for nitrotyrosine (Fig. 5a ). The degree of nitrotyrosine staining was higher in the H 2 O 2 treated group than in the control group (Fig. 5b ). In contrast to these two groups, articular cartilage treated with the antioxidative agent showed less staining for nitrotyrosine (Fig. 5b ). Catabolic changes to articular cartilage matrix under different oxidative conditions in organ culture To investigate whether oxidative stress resulted in catabolic changes to the articular cartilage matrix, we examined the amount of GAG remaining in cartilage tissue and that was released into the culture medium in organ culture in the presence of an antioxidative agent or ROS. Catabolic changes to proteoglycan in the tissue were quantified as the percentage of proteoglycan remaining in the cartilage relative to total amount in the culture medium plus cartilage. During culture, the amount of proteoglycan remaining in the cartilage tissue in the control group and H 2 O 2 -treated group decreased gradually in a timedependent manner. After 72 hours of incubation, the percentage of proteoglycan remaining in the cartilage tissue was significantly lower in the H 2 O 2 treated group than in the control group. In contrast, the antioxidative agent (Asc2P) treated group exhibited a tendency to maintain tissue proteoglycan even in the presence of H 2 O 2 during the incubation period we studied in organ culture (Fig. 6 ). Telomere length of chondrocytes from human articular cartilage explants cultured under different oxidative conditions To clarify the effect of oxidative stress on chondrocyte telomeric instability in the cartilage, we analyzed the telomere length of chondrocytes that were isolated from cartilage explants cultured in the presence of an antioxidative agent (Asc2P) or ROS (H 2 O 2 ) in vitro . After 144 hours of incubation, the telomere length of chondrocytes was significantly shorter in H 2 O 2 treated groups (lane 4 in Fig. 7a,b ) than in control group (lane 2 in Fig. 7b ). Treatment with an antioxidative agent showed a tendency to maintain chondrocyte telomere length (lane 3 in Fig. 7 ). Discussion The present study clearly demonstrates for the first time that oxidative stress affects chondrocyte telomeric DNA, cellular replicative lifespan, chondrocyte function, and cartilage matrix proteoglycan structure and composition in vitro and in vivo . These findings are consistent with a large body of data showing that reactive oxidative species, such as NO and ROS, are important in the pathogenesis of OA [ 11 - 16 ]. More recently, a suggestion that chondrocyte senescence may contribute to the risk for cartilage degeneration by decreasing the ability of the cells to maintain and to repair cartilage tissue has attracted attention [ 3 - 6 ]. Age-dependent changes in articular cartilage increase the risk for joint deterioration that causes the clinical syndrome of OA. However, the exact mechanism of chondrocyte senescence remains unclear. Our findings, demonstrating the oxidative stress (ROS) induced telomere erosion and replicative senescence in chondrocytes, suggest the involvement of oxidative stress in both the progression of cartilage ageing (chondrocyte senescence) and the development of OA. Our results also show the presence of oxidative damage in degenerated cartilage from OA patients. Chondrocytes have been shown to be capable of producing ROS and NO [ 15 , 20 , 40 ]. In the present study, stronger staining for nitrotyrosine, a marker of oxidative stress, was observed in degenerating regions as compared with intact regions from the same articular cartilage samples. In addition, the degree of immunostaining was correlated with the level of histological change in articular cartilage. These findings suggest that local accumulation of proteins altered by the reaction between ROS and NO may be important in the pathogenesis of OA. Oxidative damage in cartilage may affect chondrocyte function, resulting in changes in cartilage homeostasis that are relevant to cartilage ageing and the development of OA. We also measured the antioxidative potential of articular cartilage tissue using an assay based on reduction in Cu 2+ to Cu + by the combined action of all antioxidants present in the cartilage sample. Numerous reports have demonstrated that hypoxia is suitable for chondrocyte proliferation in vitro [ 41 - 43 ]. During chondrocyte differentiation, hypoxia may promote the process, although the exact mechanisms of chondrocyte differentiation have not been investigated to date. In addition, there is a general consensus that tissue oxygen partial pressures within articular cartilage decrease with increasing depth from the cartilage surface to deep layers [ 38 , 44 , 45 ]. Oxygen gradients do indeed exist in joint articular cartilage. These findings suggest that hypoxia may be required for homeostasis and maintenance of articular cartilage as well as chondrocyte cell growth and differentiation. During the development of OA, mechanical and chemical stresses may affect cellular adaptation to hypoxia, consequently leading to oxidative damage and changes in the microenvironment due to oxidative damage, resulting in the downregulation of chondrocyte synthesis. Indeed, our results revealed that antioxidative potential was significantly lower in degenerating regions than in intact regions from the same articular cartilage sample in OA. To clarify the involvement of oxidative damage in the development of OA, we focused on chondrocyte telomere instability. Cumulative cell damage from oxidative stress provides an alternative explanation for cellular senescence. Oxygen free radicals directly damage guanine repeats in telomeric DNA, resulting in telomere erosion regardless of cell division [ 16 - 19 ]. DNA single strand damage by oxygen free radicals results in telomere shortening during DNA replication. Oxidative stress increases the telomere shortening rate by up to one order of magnitude [ 46 ]. From these findings, we postulated that oxidative stress directly induces chondrocyte telomere instability in OA cartilage tissue, resulting in chondrocyte senescence with no requirement for cell division. Our results, demonstrating chondrocyte telomere shortening in the presence of H 2 O 2 , at a noncytotoxic concentration, supports this hypothesis. In addition to oxidative stress-induced telomere shortening, chondrocytes under chemical oxidative stress showed lower replicative lifespan and proteoglycan production as compared with normal chondrocytes in vitro . These findings also indicate that oxidative stress affects chondrocyte viability, and replicative potential and function, as well as telomere erosion. We investigated catabolic changes to articular cartilage matrix under different oxidative conditions in tissue culture. The degree of immunostaining for nitrotyrosine was significantly higher in ROS (H 2 O 2 ) treated cartilage tissues than in control cartilage tissues that were derived from the same articular cartilage. In addition, the GAG released to the medium was increased in the presence of ROS, suggesting that oxidative damage induces catabolic changes to cartilage matrix proteoglycan in articular cartilage. These observations led us to the hypothesis that oxidative stress may induce catabolic changes in cartilage matrix, consequently leading to the development of OA. This hypothesis is supported by the results of the present study, demonstrating that treatment of articular cartilage with the antioxidative agent ascorbic acid resulted in less immunopositivity for nitrotyrosine and maintenance of GAG content in articular cartilage in tissue culture. Interestingly, treatment of cultured cartilage with an antioxidative agent not only inhibited GAG loss but also maintained telomere length of chondrocytes from cultured cartilage in contrast to data obtained from cultured cartilage under normal or ROS-treated conditions. These findings may very well indicate the role played by endogenous oxidative agents in catabolic changes to cartilage matrix proteoglycan and telomere length. This is an important observation and will validate the hypothesis that oxidative agents play a role in situ in chondrocytes and in cartilage changes in OA. These results also support the concept that antioxidative agents may prevent oxidative stress-induced chondrocyte dysfunction and degeneration in cartilage. The findings of the present study suggest that cumulative oxidative stress leads to a decrease in antioxidative capacity in articular cartilage, resulting in chondrocyte telomere shortening, regardless of cell proliferation. Oxidative stress may be closely involved in telomere erosion, cellular senescence in chondrocytes and resultant cartilage ageing. Conclusion This study provides insight into the involvement of oxidative stress in the pathogenesis of OA from the viewpoint of oxidative stress induced genomic instability, especially telomere erosion, and chondrocyte senescence. Our findings clearly show the presence of oxidative stress in degenerating cartilage, and the resultant telomere erosion and dysfunction of chondrocytes in vitro and in vivo , suggesting a role for oxidative stress in the development of OA. Also, our results suggest that antioxidative agents are effective in preventing and overcoming oxidative stress induced cartilage degeneration. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage. Abbreviations Asc2P = ascorbic acid-2- O -phosphate; DMEM = Dulbecco's modified Eagle's medium; GAG = glycosaminoglycan; NO = nitric oxide; OA = osteoarthritis; PBS = phosphate-buffered saline; ROS = reactive oxygen species. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KY carried out in vitro studies (cell culture and organ culture), participated in the design of the study, conducted sequence alignment and drafted the manuscript. NvT carried out the immunoassays. HN, KH-M, TK and KN conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript

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1065335

Defective CD4+CD25+ regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4 + CD25 + T reg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of T reg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4 + CD25 + T cells in the central and peripheral lymphoid tissues. In addition, CD4 + CD25 + T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro . However, after immunisation with CII in CFA, the suppressive activity of CD4 + CD25 + T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for T reg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both T reg cells and accessory cells. Our results demonstrate that the decrease in T reg cell activity in CIA is counter-regulated by endogenous IFN-γ.

Introduction The adaptive immune system uses various potent effector mechanisms for the elimination of foreign pathogens. Because these mechanisms are potentially damaging to the host, an essential feature of the immune system is its ability to distinguish self from non-self antigens and to develop tolerance to the former. With regard to T cell tolerance, the immune system has evolved several strategies. Most autoreactive T cells are eliminated during (primary) maturation in the thymus, a process described as negative selection, resulting in central T cell tolerance. Autoreactive T cells that escape negative selection will nevertheless be prevented from being activated as they are confronted with auto-antigen in the periphery. Several mechanisms have been proposed to account for this peripheral tolerance. One of those is suppression by a subset of T cells that express both CD4 and CD25. Evidence for the important role of these cells is overwhelming [ 1 ]. For example, when CD4 + T cells isolated from peripheral lymphoid tissues of normal mice are depleted of CD4 + CD25 + T cells and injected into nu / nu mice, the recipients develop a high incidence of organ-specific autoimmune disease [ 2 ]. Co-transfer of the CD4 + CD25 + population prevents the induction of disease. CD4 + CD25 - and CD4 + CD25 + T cells are therefore often designated as, respectively, T eff and T reg cells. CD4 + CD25 + T reg cells are generated in the thymus. Their development is directed by relatively high-avidity interactions between the TCR and self-peptide ligands [ 3 - 5 ]. The CD4 + CD25 + T reg cell population constitutes 5 to 10% of the mature CD4 + cell population in the adult thymus and the peripheral lymphoid tissue and blood. In vitro , CD4 + CD25 + T reg cells inhibit polyclonal T cell activation [ 6 , 7 ]. The suppression is mediated by a cytokine-independent, cell contact-dependent mechanism that requires activation of the CD4 + CD25 + cells via the TCR with specific antigen [ 8 ]. However, once stimulated, they are competent to suppress in an antigen-independent manner. Although the exact mechanism by which T reg cells exert their regulatory function is still unknown, there are indications that interaction of transforming growth factor-β (TGF-β) with its receptor [ 9 - 11 ], inhibition of IL-2 production [ 6 ] or downregulation of co-stimulatory molecules on antigen-presenting cells [ 12 ] could be involved. T reg cells have proved to be important in various animal models of autoimmune diseases. Administration of anti-CD25 antibody in vivo induces organ-localised autoimmune diseases [ 13 ]. Inoculation of CD4 + T cells depleted of CD25 + cells in nu/nu mice results in autoimmune diseases such as gastritis, thyroiditis and insulitis [ 2 ]. Thus, transfer of T reg cells prevents autoimmune gastritis after neonatal thymectomy, and inhibits gastritis induced by H/K ATPase-reactive effector T cells [ 14 ]. MBP-specific CD25 + CD4 + T cells prevent spontaneous autoimmune encephalomyelitis in TCR-transgenic mice deficient in the recombination activating gene RAG-1 [ 15 ]. Similarly, CD4 + CD25 + T reg cells suppress central nervous system inflammation during active experimental autoimmune encephalomyelitis [ 16 ]. Collagen-induced arthritis (CIA) is a well-described animal model for rheumatoid arthritis. The disease is induced in genetically susceptible DBA/1 mice by immunisation with collagen type II (CII), and both T cell and B cell autoimmune responses are required for its development [ 17 - 19 ]. IFN-γ receptor knock-out (IFN-γR KO) mice have been found to suffer an accelerated and more severe form of CIA [ 20 - 23 ]. Moreover, knocking-out of the IFN-γ gene makes genetically resistant strains of mice susceptible to CIA [ 24 , 25 ]. These data indicate that deletion of the IFN-γ response somehow disrupts an endogenous protective mechanism against CIA. Morgan and colleagues [ 26 ] have recently demonstrated that CD25 + T reg cells are important in the pathogenesis of CIA. In the present study we confirmed the importance of T reg cells in the pathogenesis of CIA by rendering wild-type DBA/1 mice deficient in T reg cells by depleting anti-CD25 antibodies. Anti-CD25-treated mice developed a significantly more severe arthritis, comparable to the disease course in IFN-γR KO mice. Thus, we proposed that the higher susceptibility of IFN-γR KO DBA/1 mice to CIA might be ascribed to defects in the production (differentiation and homeostasis) or function of these CD4 + CD25 + T reg cells. We therefore determined the numbers of T reg cells in central and peripheral lymphoid organs of IFN-γR KO and wild-type mice. We further investigated whether T reg cells of IFN-γR KO mice have defects in the ability to suppress TCR-induced in vitro proliferation of CD4 + CD25 - T eff cells. Materials and methods Mice and experimental conditions The generation and the basic characteristics of the mutant mouse strain (129/Sv/Ev) with a disruption in the gene coding for the α-chain of the IFN-γ receptor (IFN-γR KO) have been described [ 27 ]. These IFN-γR KO mice were backcrossed with DBA/1 wild-type mice for 10 generations to obtain the DBA/1 IFN-γR KO mice used in the present study. The homozygous IFN-γR KO mice were identified by PCR as described [ 23 ]. Wild-type and IFN-γR KO DBA/1 mice were bred in the Experimental Animal Centre of the University of Leuven. The experiments were performed in mice 6 to 10 weeks old, but in each experiment the mutant and wild-type mice were age-matched within 5-day limits. The male : female ratio was kept between 0.8 and 1.3 in each experiment group, unless otherwise mentioned. All animal experiments were approved by the local ethical committee (University of Leuven). Induction and clinical assessment of arthritis Native chicken CII (Sigma-Aldrich, St Louis, MO, USA) was dissolved at 2 mg/ml in PBS containing 0.1 M acetic acid by stirring overnight at 6°C and emulsified in an equal volume of complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA) with added heat-killed Mycobacterium butyricum (0.5 mg/ml). IFN-γR KO and wild-type mice were sensitised with a single intradermal injection at the base of the tail with 100 μl of the emulsion on day 0. From day 0 after immunisation, mice were examined for signs of arthritis five times a week. The disease severity was recorded with the following scoring system for each limb: score 0, normal; score 1, redness and/or swelling in one joint; score 2, redness and/or swelling in more than one joint; score 3, redness and/or swelling in the entire paw; score 4, deformity and/or ankylosis. Media, reagents and antibodies All cells were grown in RPMI 1640 (Bio Whittaker Europe, Verviers, Belgium), supplemented with 10% heat-inactivated FCS (Gibco, Paisley, UK), penicillin (100 IU/ml; Continental Pharma, Brussel, Belgium), streptomycin (100 μg/ml; Continental Pharma), 2 mM L-glutamine, 10 mM Hepes (Gibco), 0.1 mM nonessential amino acids (ICN, Asse Relegem, Belgium), 1 mM sodium pyruvate (Gibco) and 50 μM 2-mercaptoethanol (Fluka, AG, Switzerland). Anti-CD25 IL-2Rα monoclonal antibody was produced by hybridoma PC61 in an INTEGRA CELLine CL1000 (Elscolab, Kruibeke, Belgium) and is a rat IgG1 antibody. The hybridoma supernatant was purified by Protein G-Sepharose chromatography (Amersham Biosciences, Roosendaal, The Netherlands) for administration in vivo . The hamster monoclonal antibody, directed against the mouse CD3 complex, was prepared from the culture supernatant of 145-2C11 hybridoma cells [ 28 ]. The antibodies were purified by affinity chromatography with Protein A-Sepharose (Amersham Biosciences). Batches of anti-CD3 antibody were tested for endotoxin content with the Limulus amebocyte lysate QCL-1000 kit (Bio Whittaker) and were found to contain less than 3 ng/ml endotoxin. Cell purification Lymph nodes (axillary, inguinal and mesenteric) and spleens were harvested from mice 6 to 8 weeks old. Lymph nodes and spleens were gently cut into small pieces and passed through cell strainers (Becton Dickinson Labware, Franklin Lakes, NJ, USA). Red blood cells were lysed by two consecutive incubations (5 and 3 min at 37°C) of the suspension in NH 4 Cl (0.83% in 0.01 M Tris-HCl, pH 7.2). Remaining cells were washed, resuspended in cold PBS and counted. Lymph node preparations were then enriched for CD4 + T cells with the Mouse T cell CD4 Subset Column Kit (R&D systems, Abingdon, UK). To purify CD4 + CD25 + and CD4 + CD25 - cells, the enriched CD4 + T cells were incubated for 20 min at 4°C with FITC-conjugated anti-CD25 and phycoerythrin (PE)-conjugated anti-CD4 antibodies (10 μg per 10 8 cells) in PBS containing 2% FCS. They were sorted by flow cytometry on a FACS Vantage (Becton Dickinson, San Jose, CA, USA). The resultant purity of the CD4 + CD25 - population was 99%, whereas the purity of the CD4 + CD25 + population varied from 96% to 99%. Alternatively, CD4 + T cells were labelled with PE-conjugated anti-CD25 monoclonal antibody, followed by incubation with magnetic-activated cell sorting (MACS) anti-PE beads (CD25 Microbead Kit; Miltenyi Biotec, Bergisch Gladbach, Germany). CD4 + CD25 + T cells were selected on an LS column in a magnetic field and the flow-through was collected as CD4 + CD25 - T cells. After removal of the column from the magnetic field, CD4 + CD25 + T cells were flushed out by a plunger. The purity of the CD4 + CD25 - population was 99% and the purity of the CD4 + CD25 + population varied from 90% to 95%. T cell-depleted spleen suspensions were prepared by MACS (Miltenyi Biotec) and used as accessory cells (ACs). For MACS separation, the cell suspension was magnetically labelled with CD90 (Thy1.2) microbeads and passed through a CS separation column, placed in a magnetic field. The unlabelled CD90 - cells ran through. Flow cytometry Single-cell suspensions (5 × 10 5 cells) were incubated for 15 min with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (CD16/CD32; BD Biosciences Pharmingen, San Diego, CA, USA). Cells were washed with PBS containing 2% FCS and stained with the indicated FITC-conjugated antibodies (0.5 μg) for 30 min, washed twice and incubated for 30 min with the indicated PE- or biotin-conjugated antibodies. For the biotin-conjugated antibodies, a third staining step with streptavidin conjugated with peridinin chlorophyll a protein (PerCP) was performed. After washing, propidium iodide (Sigma-Aldrich) was added at a final concentration of 4 μg/ml to distinguish dead cells from living cells. Biotin-conjugated anti-CD25 (7D4), FITC-conjugated anti-CD25 (7D4), FITC-conjugated CD69 (H1.2F3), PE-conjugated anti-CD4 (RM4-5) and PerCP-conjugated streptavidin were purchased from BD Biosciences Pharmingen. FITC-conjugated anti-CD62L (MEL-14) and anti-CD44-FITC (IM7.8.1) were from CALTAG Laboratories (Burlingame, CA, USA). For intracellular staining with anti-CTLA-4-PE (UC10-4F10-11; BD Biosciences Pharmingen), 10 6 cells were first labelled with anti-CD25-FITC as described above. Then, cells were fixed, permeabilised and stained with anti-CTLA-4-PE using the Cytofix/Cytoperm™ Kit (BD Biosciences Pharmingen) according to the recommendations of the manufacturers. Flow-cytometric analysis was performed on a FACScan flow cytometer with Cell Quest software (Becton Dickinson). Proliferation assays CD4 + CD25 - cells (5 × 10 4 per well) were cultured in U-bottomed 96-well plates (200 μl) with ACs (5 × 10 4 per well, 30 Gy γ-irradiated or treated with mitomycin-C (Sigma-Aldrich)), 3 μg/ml anti-CD3 and the indicated numbers of CD4 + CD25 + cells for 48 hours at 37°C in 7% CO 2 . Cultures were pulsed for the last 16 hours with 1 μCi of [ 3 H]TdR and harvested. The suppressive activity of the T reg cells can be presented by plotting the percentage of inhibition (100 × (Radioactivity in condition without T reg cells – Radioactivity in condition with T reg cells)/Radioactivity in condition without T reg cells) against the number of T reg cells. Antibody administration DBA/1 mice were immunised with CII in CFA; 13 days after immunisation, the mice were treated every second day with 0.25 mg of anti-CD25 (PC61) or control IgG antibodies, for 4 weeks (injected intraperitoneally). Histological examination Forelimbs and hindlimbs were fixed in 10% formalin and decalcified with formic acid (31.5% (v/v) formic acid and 13% (w/v) sodium citrate). The paraffin sections were stained with haematoxylin and eosin. Measurement of serum anti-CII antibodies Blood samples were taken from the orbital sinus and were allowed to clot at room temperature for about 1 hour, and at 4°C overnight. Individual sera were tested by ELISA for antibodies directed against chicken CII. In brief, ELISA plates (Maxisorb; Nunc, Wiesbaden, Germany) were coated overnight at 4°C with native CII (1 μg/ml; 100 μl per well) in coating buffer (50 mM Tris-HCl, pH 8.5, 0.154 mM NaCl), followed by incubation for 2 hours with blocking buffer (50 mM Tris-HCl, pH 7.4, 0.154 mM NaCl and 0.1% caseine) to saturate non-specific binding sites. Serial twofold dilutions of the sera in assay buffer (50 mM Tris-HCl, pH 7.4, 154 mM NaCl and 0.05% Tween 20) were added and incubated for 2 hours at room temperature. The plates were then incubated for 2 hours with peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Finally, the substrate 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) in reaction buffer (100 mM sodium acetate/citric acid, pH 4.9) was added for a 10 min incubation and absorbance was determined at 450 nm. Plates were washed five times between each step with PBS containing 0.05% Tween 20. A serial twofold dilution series of a purified standard was included to permit a calculation of the antibody content of each sample. The standard was purified by affinity chromatography from pooled sera obtained from various arthritic wild-type and IFN-γR KO mice. Quantitative RT-PCR Isolated CD4 + CD25 + and CD4 + CD25 - cells were pelleted and directly used for total RNA isolation, using the Micro-to-Midi Total RNA Purification System (Invitrogen Life Technologies, Carlsbad, CA, USA). Total RNA (1 μg) was used for random primed cDNA synthesis with RAV-2 reverse transcriptase (Amersham, Aylesbury, Bucks., UK). The reaction mixture was incubated for 80 min at 42°C and the reverse transcriptase was inactivated by incubating the cDNA samples for 5 min at 95°C. The cDNA samples were then subjected to real-time quantitative PCR, performed in the ABI prism 7700 sequence detector (Applied Biosystems, Foster City, CA) as previously described [ 29 ]. The sequences of the forward (-FW) and reverse (-RV) primers and probes (-TP) for β-actin and Foxp3 were as follows: β-actin-FW, AGA GGG AAA TCG TGC GTG AC; β-actin-RV, CAA TAG TGA TGA CCT GGC CG T; β-actin-TP, CAC TGC CGC ATC CTC TTC CTC CC; Foxp3-FW, CCC AGG AAA GAC AGC AAC CTT; Foxp3-RV, TTC TCA CAA CCA GGC CAC TTG; Foxp3-TP, ATC CTA CCC ACT GCT GGC AAA TGG AGT C; TGF-β-FW, TGA CGT CAC TGG AGT TGT ACG G; TGF-β-RV, GGT TCA TGT CAT GGA TGG TGC; TGF-β-TP, TTC AGC GCT CAC TGC TCT TGT GAC AG. Probes were dual-labelled with 5'-FAM and 3'-TAMRA. All primers and probes were designed with the assistance of the computer program Primer Express (AB) and were purchased from Eurogentec (Seraing, Belgium). The 5'-nuclease activity of the Taq polymerase was used to cleave a nonextendable dual-labelled fluorogenic probe. Fluorescent emission was measured continuously during the PCR reaction. PCR amplifications were performed in a total volume of 25 μl containing 5 μl of cDNA, 12.5 μl of Universal PCR Master Mix, no AmpErase UNG (AB), each primer at 100 to 300 nM, and the corresponding detection probe at 200 nM. Each PCR amplification was performed in triplicate wells under the following conditions: 94°C for 10 min, followed by 40 or 45 cycles at 94°C for 15 s and 60°C for 1 min. cDNA plasmid standards, consisting of purified plasmid DNA specific for each individual target, were used to quantify the target gene in the unknown samples, as described [ 29 ]. All results were normalised to β-actin and/or hypoxanthine–guanine phosphoribosyltransferase (HPRT) to compensate for differences in the amount of cDNA in all samples. Results were similar whether β-actin or HPRT was used as the housekeeping gene. Results Effect of treatment in vivo with depleting anti-CD25 antibodies on the development of CIA in wild-type DBA/1 mice In a first set of experiments we tested the importance of T reg cells in the pathogenesis of CIA by rendering wild-type mice deficient in T reg cells by treating the mice with depleting anti-CD25 antibody. Starting from day 11 or 13 after immunisation with CII in CFA, wild-type DBA/1 mice were treated every second day with anti-CD25 antibodies or control IgG. In a first experiment, female mice were chosen because these are only moderately sensitive to CIA [ 30 , 31 ], so that we would be able to detect both increased and decreased disease severity after CD25 + cell depletion. Blood samples were taken at intervals to confirm the depletion of the CD25 + population (Fig. 1a ). In control-treated mice, the development of arthritis (day of onset, incidence and mean limb score) was reminiscent of our previously reported findings in which mice received a single immunisation with CII in CFA [ 20 ]. In contrast, mice treated with the anti-CD25 antibodies developed a significantly more severe arthritis with a higher incidence and earlier onset than those receiving control IgG1 (Fig. 1b ). In fact, the disease course in antibody-treated mice was very similar to that of IFN-γR KO mice [ 20 - 22 ]. The results were confirmed in an additional experiment with female mice. A third experiment was also performed on male animals. The data are plotted in Fig. 1c . Here again, anti-CD25-treated mice developed a higher incidence and a more severe form of arthritis than control-treated mice, whereas the onset of arthritis was not significantly earlier (Fig. 1d ). The data from the three experiments were pooled and the percentages of limbs with the different scores from only arthritic mice in the two groups are shown in Fig. 1d . It can be seen that, at an early time point (day 27 after immunisation), the highest scores of arthritis (scores 3 and 4) were already present in anti-CD25-injected mice, but not yet in their control counterparts. On day 40 after immunisation, mice treated with anti-CD25 developed more limbs with a maximum score of 4 than control-treated mice. The mean limb score on the two days for the two groups are indicated and are significantly different ( P < 0.05, Mann–Whitney U -test). The mean number of involved limbs, ± SEM, on day 40 was 2.8 ± 0.2 and 2.2 ± 0.2 for the treated and control mice, respectively ( P = 0.07; Mann–Whitney U -test). Representative pictures of the most severe case of arthritis of anti-CD25-injected and control mice on day 25 after immunisation are shown in Fig. 1e and Fig. 1f , respectively. To ensure that the more severe form of arthritis in the anti-CD25-treated mice was not merely due to oedema, some mice were killed at day 42 for histological evaluation. The presence of hyperplasia and infiltration of immunocompetent cells in the synovium, pannus formation and osteoclast-like multinucleated giant cells confirmed the authenticity of arthritis (Fig. 1g ). On day 35 after immunisation, the titres of collagen-specific antibodies in the sera were determined. No differences in antibody levels in sera of mice treated with anti-CD25 or control IgG could be detected (data not shown). Number and phenotype of CD4 + CD25 + T reg cells in IFN-γR KO and wild-type mice To test whether T reg cells might be less numerous in IFN-γR KO than in wild-type mice – because this might explain the differences in susceptibility to CIA – we counted CD4 + CD25 + cells in thymus, lymph nodes and spleen by flow cytometry. IFN-γR KO and wild-type mice were immunised with CII in CFA on day 0. Thymocytes, splenocytes and lymph node cells were obtained on day 21, a time point at which the difference in severity of arthritis between the two groups of mice is most pronounced [ 20 - 22 ]. Groups of naive IFN-γR KO and wild-type mice were also included. A typical CD4/CD25 staining pattern of thymocytes and lymph node cells from IFN-γR KO and wild-type mice is shown in Fig. 2 ; percentages of CD4 + CD25 + and CD4 + CD25 - cells are indicated. It can be seen that IFN-γR KO mice did not have smaller proportions of CD4 + CD25 + cells in the thymus and lymph nodes. Immunised mice, whether wild-type or IFN-γR KO, had rather lower proportions of total CD4 + cells than naive counterparts (for example 31% versus 50% in wild types). However, the real numbers of CD4 + cells per organ were in fact higher after immunisation and did not differ in IFN-γR KO from those in wild-type mice. In fact, the lower percentages of CD4 + cells after immunisation were due to a still larger expansion of the myelopoietic population, a well-recognised phenomenon arising from the use of CFA [ 22 , 32 ]. When over a total of six experiments (Table 1 ) the numbers of CD4 + CD25 + cells were expressed as fractions of total CD4 + cell numbers, it appeared that spleens and lymph nodes of IFN-γR KO mice, naive as well as immunised ones, contained slightly higher percentages of CD4 + CD25 + cells. In spleens and lymph nodes of wild-type mice, 5 to 10% of the CD4 + T cells were CD25 + , conforming to previously published figures obtained in other mouse strains. Thymuses contained lower percentages of CD4 + CD25 + cells. A possible explanation might be that thymic CD4 + T cell populations contain not only CD4 + CD8 - but also CD4 + CD8 + cells, the latter being mostly CD25 - . In the peripheral lymphoid organs of IFN-γR KO mice, the percentage of CD4 + CD25 + cells was higher (7 to 14%) than in the wild-type mice (Table 1 ). Because CD25 is expressed not only by T reg cells but also by other recently activated T cells, the slightly higher proportion of CD4 + CD25 + cells in IFN-γR KO mice is not synonymous with a higher proportion of T reg cells. In fact, even a lower proportion of such cells cannot be excluded. We therefore compared the CD4 + CD25 + T cells from IFN-γR KO and wild-type DBA/1 mice for expression of various other activation markers. Figure 3a,b shows flow-cytometric expression patterns of CD69, CD62L, CD44 and cytolytic T lymphocyte-associated antigen (CTLA-4) in CD4 + CD25 + T cells from naive and immunised IFN-γR KO and wild-type mice. No major differences in expression levels of these activation markers could be detected between CD4 + CD25 + T cells from IFN-γR KO mice and those from wild-type mice, whether naive or immunised. Thus, this analysis did not provide evidence for different proportions of any cell type, including T reg cells. A specific marker for T reg cells is Foxp3. We determined mRNA for this marker by quantitative PCR in CD4 + CD25 + and CD4 + CD25 - cells, sorted from the lymph node cells of naive or immunised IFN-γR KO and wild-type DBA/1 mice at day 21. In CD4 + CD25 - cells Foxp3 mRNA levels were extremely low (less than 6), and not different between one group of mice and the other. CD4 + CD25 + cells, in contrast, displayed high expression levels. In cells from naive IFN-γR KO and wild-type mice, levels were comparable. However, CD4 + CD25 + T cells of immunised IFN-γR KO mice contained levels of Foxp3 that were one-third of those of wild-type mice (Fig. 3c ). This lower expression level might be indicative of a smaller proportion of T reg cells in the sorted CD4 + CD25 + cell population or of a lower expression level per cell. To distinguish between these alternatives, a tagging anti-Foxp3 antibody would be needed. Thus, after immunisation, IFN-γR KO mice possessed a slightly higher percentage of CD4 + CD25 + cells than wild-type mice. However, the actual T reg cells present in this population might be considerably less numerous or might be qualitatively different so as to express less Foxp3. Reduced suppressive activity of CD4 + CD25 + T reg cells in arthritic IFN-γR KO mice To characterise the CD4 + CD25 + T reg cells functionally, we measured their ability to suppress the anti-CD3-induced proliferation of CD4 + CD25 - T eff cells in vitro . The experiments were performed with CD4 + CD25 + cells, CD4 + CD25 - cells and ACs. T reg suppressive activity was presented by plotting the percentage of inhibition against the number of T reg cells. As shown in Fig. 4a,c , the patterns of inhibition in naive IFN-gR KO and wild-type mice were very similar: in both cases 2 × 10 4 purified CD4+CD25+ cells were able to inhibit more than 90% of the proliferative response of 5 × 104 T eff cells. This result indicates that IFN-γ is not required for T reg cells to be able to suppress anti-CD3-induced in vitro proliferation. In a separate set of seven experiments we investigated the suppressive effect of CD4 + CD25 + cells from mice that had been immunised with CII in CFA. IFN-γR KO and wild-type DBA/1 mice were immunised on day 0, and CD4 + CD25 + cells, T eff cells and ACs were isolated on day 21 after immunisation. The data of the individual experiments are plotted in Fig. 4b and the means of the seven experiments are shown in Fig. 4c . It can be seen that the capacity to suppress TCR-triggered proliferation of T eff cells was significantly lower in CD4 + CD25 + cells isolated from immunised mice than in those of naive animals. Indeed, to obtain 40% inhibition of proliferation, 4.5 × 10 3 CD4 + CD25 + cells from immunised wild-type mice were required, in comparison with only 1.5 × 10 3 CD4 + CD25 + cells from naive wild-type mice. Moreover, CD4 + CD25 + cells from immunised IFN-γR KO mice were significantly less suppressive than those of immunised wild-type mice: 10 4 CD4 + CD25 + cells were necessary to decrease T eff cell proliferation by 40%. In an additional experiment we verified whether the deficit in inhibition by CD4 + CD25 + cells from immunised IFN-γR KO mice could be corrected by adding excess CD4 + CD25 + cells. However, with 2 × 10 4 and 4 × 10 4 CD4 + CD25 + cells the inhibition on T cell proliferation was 64.6% and 65.8%, respectively, indicating that a plateau level of suppressive activity had been reached. Normal levels of TGF-β in IFN-γR KO and wild-type mice Several studies have shown the critical role of TGF-β in the induction of Foxp3 and the activity of T reg cells [ 10 , 33 , 34 ]. Because IFN-γ and TGF-β act antagonistically with each other (reviewed in [ 35 ]), it is possible that TGF-β is upregulated in wild-type mice as a homeostatic response to IFN-γ produced by their activated T cells, and similarly in IFN-γR KO mice the decreased Foxp3 levels and the decreased suppressive activity of T reg cells might be due to inadequate amounts of TGF-β produced in the co-cultures or in vivo in mice. We therefore analysed the expression of TGF-β by quantitative PCR in T reg cells as well as in co-cultures and in spleens of naive and immunised mice. The following results were obtained. First, the levels of TGF-β from the sorted CD4 + CD25 + cells from immunised IFN-γR KO mice were not different from those of wild-type mice (normalised TGF-β mRNA levels were 179 ± 16 and 193 ± 22, respectively; mean ± SEM for three measurements). Second, because TGF-β might be produced by ACs (or T eff cells), quantitative PCR was performed on cells obtained from co-cultures (T reg plus T eff plus ACs) from immunised IFN-γR KO and wild-type mice. It was found that the levels of TGF-β were even increased in IFN-γR KO cells in comparison with wild-type cells (2,184 versus 1,574, respectively, in the condition of 2 × 10 4 T reg cells, in a pool of eight mice). Third, the TGF-β levels were also analysed ex vivo ; that is, in spleen tissue from IFN-γR KO and wild-type mice at day 21 after immunisation (thus at a time point at which T reg , T eff and ACs were isolated). Here again, the TGF-β levels were found to be slightly increased in spleens from IFN-γR KO mice (816 ± 129 and 633 ± 40 for IFN-γR KO and wild-type mice, respectively). If these results are taken together, the defective activity of T reg cells from arthritic IFN-γR KO mice (in comparison with those from wild-type animals) seems not to be associated with a defective TGF-β production. It was notable that the TGF-β levels were higher in immunised mice than in their naive counterparts (for example, 633 ± 40 and 205 ± 19 for immunised and naive wild-type mice, respectively). These data suggest that the differences in suppressive activity of T reg cells from immunised versus naive mice cannot be explained by differences in the TGF-β production. T reg cells from immunised IFN-γR KO mice have the capacity to inhibit proliferation responses We next investigated whether the lower capacity of CD4 + CD25 + cells from IFN-γR KO mice to downregulate proliferation responses is due to an intrinsic defect or to an altered activity of surrounding ACs and T eff cells. We measured the inhibition of anti-CD3-induced proliferation in co-cultures differently reconstituted of CD4 + CD25 + , CD4 + CD25 - and ACs, derived either from the same or from different immunised wild-type or immunised IFN-γR KO mice. The combinations tested are indicated in Fig. 5 . As expected, when all cells in the reconstituted co-cultures were of IFN-γR KO mouse origin, suppressive activity was less than when all cells were of wild-type origin. In co-cultures of mixed composition, suppressive activity of IFN-γR KO-derived CD4 + CD25 + cells was less than that of the wild type only when ACs were from IFN-γR KO origin, but not when they were of wild-type origin. However, such ACs of IFN-γR KO mice were unable to reduce the suppressive effect of wild-type T reg cells against wild-type or IFN-γR KO (data not shown) T eff cells. These data demonstrate that the defect in inhibiting CD4 + CD25 - T eff cells acquired the presence of T reg cells from immunised IFN-γR KO mice in combination with their autologous ACs. Discussion We and others have previously demonstrated that IFN-γ(R) KO mice show an accelerated and more severe from of arthritis than their wild-type counterparts, indicating that endogenous IFN-γ acts as a protective factor in CIA [ 20 , 21 , 24 , 25 ]. Because CIA has been defined as a Th1-driven disease (reviewed in [ 17 ]), the protective effect of IFN-γ in CIA constitutes an enigma that compromises the Th1/Th2 paradigm as a basis for explaining the regulation of autoimmune diseases. A clue to the enigma seemed to be the use of CFA in the induction procedure of CIA. In the absence of IFN-γ, CFA induces an extensive extramedullary myelopoiesis that goes together with an even more pronounced Th1 cytokine profile than in wild-type counterparts [ 22 , 36 ]. The data suggest that IFN-γ can, under certain circumstances, be a strong Th2 inducer, a finding that has recently been confirmed by others [ 37 ]. Here, we tested the hypothesis that this protective action of IFN-γ is due to a stimulatory effect on T reg cells. Specifically, we addressed the following two questions. Are T reg cells important in modulating CIA? And, because we found that depletion of T reg cells in wild-type mice increased the severity of CIA, can the higher susceptibility of IFN-γR KO mice to CIA be explained by defects in the number or function of their T reg cells? As to the first question, we found that administration of a T reg cell-depleting anti-CD25 antibody to wild-type DBA/1 mice after CFA-assisted immunisation with CII resulted in accelerated and more severe arthritis. In fact, the disease course in these mice was comparable to that in IFN-γR KO mice [ 20 - 23 ]. The actual depletion of T reg cells was monitored by flow cytometry, and the authenticity of arthritis was verified histopathologically. These results are in line with those of Morgan and colleagues [ 26 ], who showed that the administration of depleting anti-CD25 antibody before immunisation (days –28, –24, –21 and –14) hastened the onset of severe CIA. Because in our experiments antibodies were administered starting from day 11 or day 13 after immunisation, we can conclude that T reg cells are important in the pathogenesis of CIA, not only in the immunisation phase but also in the effector phase. In contrast to the findings of Morgan and colleagues [ 26 ], the accelerated and more severe course of arthritis was, in our experiments, not accompanied by a higher concentration of anti-collagen II antibodies, possibly due to the different regimen of anti-CD25 treatment. Indirect evidence for the involvement of T reg cells in the pathogenesis of CIA comes from data of Min and colleagues [ 38 ]. They found that the immune tolerance induced by oral feeding of CII before induction of CIA was mediated by IL-10-producing CD4 + CD25 + T cells. Notably, in proteoglycan-induced arthritis, another model of autoimmune arthritis, it has been shown that CD4 + CD25 + T reg cells might not have a critical role [ 39 ]. This might result from the use of a different auto-antigen. To address the second question, we compared CD4 + CD25 + cell numbers and T reg cell function in IFN-γR KO DBA/1 mice with those in wild-type mice. According to our hypothesis we expected numbers of T reg cells in IFN-γR KO mice to be lower. Counter to this expectation, in each of the six experiments done, we found a trend for a higher proportion of CD4 + CD25 + T cells in the total CD4 + cell population. This was true for thymic, splenic and lymph node CD4 + cells, in both naive and immunised mice. Analysis of all data as one set revealed a significant difference of about 30% and 20% in naive and immunised mice, respectively. CD25 is not an exclusive marker of T reg cells: especially in immunised mice, part of the CD4 + CD25 + population might be effector rather than regulatory T cells [ 40 , 41 ]. Therefore, to exclude the possibility that we were comparing two completely different populations, we performed additional flow-cytometric characterisation studies on pre-sorted CD4 + CD25 + cells. Expression of CD44, CD69, CTLA-4 and CD62L in CD4 + CD25 + cells from IFN-γR KO mice did not differ from expression in cells from corresponding wild-type mice, whether naive or immunised. However, because T reg cells display an activated phenotype, activation markers might not be adequate to distinguish T reg cells from activated T eff cells. According to Fontenot and colleagues [ 42 ] a specific marker for T reg cells is Foxp3, because it is highly expressed in CD4 + CD25 + T reg cells and is virtually undetectable in both resting and activated T eff cells. We examined Foxp3 expression by determining mRNA levels with PCR. After immunisation, CD4 + CD25 + cells contained lower levels of Foxp3 mRNA than those of their naive counterparts. Moreover, mRNA levels in immunised IFN-γR KO mice were less than one-third of those in their wild-type counterparts, indicating that IFN-γR KO mice have a smaller number of T reg cells or that expression of Foxp3 in each T reg cell is lower. Recently, Bruder and colleagues [ 43 ] have shown linked expression of neuropilin-1 and Foxp3, thereby identifying neuropilin-1 as a specific surface marker for CD4 + CD25 + T reg cells able to distinguish them from both naive and recently activated CD4 + CD25 + non-regulatory T cells. Nishibori and colleagues [ 44 ] demonstrated impaired development of T reg cells in naive signal transduction and activators of transcription (STAT)-1-deficient mice, associated with an increased susceptibility to autoimmune disease. Because IFN-γ is among the strongest activators of STAT-1, these observations seem to conflict with ours. However, several cytokines, other than IFN-γ, can also activate STAT-1, including IFN-α, IFN-β, IL-6, IL-9, IL-11, oncostatin M, leukaemia inhibitory factor and the chemokines RANTES and macrophage inflammatory protein 1α [ 45 , 46 ]. To determine whether overall T reg cell activity would be lower in IFN-γR KO mice, we co-cultured increasing numbers of CD4 + CD25 + T cells with fixed numbers of CD4 + CD25 - T eff cells and ACs in the presence of anti-CD3 antibody. We observed a dose-dependent inhibition of the proliferative responses by CD4 + CD25 + T reg cells. By estimating numbers of CD4 + CD25 + cells required to attain a selected level of suppression, we could compare suppressive activity in the different groups of mice. In naive mice, the inhibition curves were almost identical, whether the T reg cells were derived from wild-type or IFN-γR KO mice, indicating that endogenous IFN-γ is not an important regulator of the function of constitutive CD4 + CD25 + T reg cells. In co-cultures of cells from immunised wild-type mice, the T reg suppressive capacity was about one-third of that in those from corresponding naive mice, and a further halving was noted in co-cultures of cells from immunised IFN-γR KO mice. The observation that immunisation renders T reg cells less suppressive is in line with results of Pasare and Medzhitov [ 47 ], who found that microbial triggering of the Toll-like receptor (TLR) pathway by lipopolysaccharide or CpG, which are ligands for TLR4 and TLR9, respectively, blocked the suppressive effect of CD4 + CD25 + T reg cells. Because mycobacteria also contain TLR ligands, immunisation with CFA can be expected to affect T reg cell activity similarly. The decrease in suppressive activity that takes place after TLR4 or TLR9 triggering was found to be dependent on IL-6 production [ 47 ]. It might therefore be of interest to note that in our experiments, IL-6 production was enhanced after exposure to CFA-assisted immunisation, and this effect was even more pronounced in IFN-γR KO mice (P Matthys, unpublished data). This could provide an explanation for the fact that CD25 + T reg cells are totally functional before immunisation but lose (part of) their function after immunisation. However, the most important observation is the lower T reg suppressive capacity in IFN-γR KO than in wild-type mice after CFA-assisted immunisation, because this supports our hypothesis that the protective effect of endogenous IFN-γ against CIA could be mediated in part by its stimulatory effect on T reg cells. Because the disease is barely detectable in wild-type mice on day 21 after immunisation, we investigated whether the decreased suppressive activity in immunised wild-type mice was further downregulated at a later time point (namely, day 35 after immunisation, when most of the animals show symptoms of arthritis). However, suppressive activity was not further downregulated to the level seen in homogeneous IFN-γR KO co-cultures, but was comparable to that seen in co-cultures from immunised wild-type mice on day 21 after immunisation (maximal inhibition 60%; data not shown). This indicates that the low suppressive activity as evident in immunised IFN-γR KO mice is restricted to conditions under which IFN-γ is abrogated. The implication is that the CIA immunisation schedule induces a decrease in T reg activity and that endogenous IFN-γ largely counteracts this decrease. It therefore becomes important to know by what mechanism, direct or indirect, IFN-γ influences T reg cell function. Addition of anti-IFN-γ antibody to the co-cultures failed to affect suppressive activity (data not shown), indicating that the relevant IFN-γ effect takes place in vivo before sampling of the T cells. To examine the role of the different cell components, we tested suppressive activity in mixed co-cultures. CD4 + CD25 + cells from immunised IFN-γR KO mice, confronted with T eff cells and ACs from immunised wild-type mice, were not less suppressive than wild-type CD4 + CD25 + confronted with wild-type or IFN-γR KO T eff cells and ACs. This suggests that lower levels of suppression in homogeneous IFN-γR KO cultures result in part from the presence of IFN-γR KO-derived ACs. And, indeed, when CD4 + CD25 + and T eff cells from immunised IFN-γR KO mice were co-cultured with ACs from immunised wild-type mice, suppressive activity was not inhibited. Finally, ACs from IFN-γR KO mice by themselves were unable to downregulate the activity of wild-type T reg cells acting on wild-type T eff cells. We therefore conclude that the in vivo effect of endogenous IFN-γ that accounts for the greater suppressive activity in wild-type mice than in IFN-γR KO mice concerns reprogramming of both ACs and T reg cells. Because CD4 + CD25 + T cells from immunised IFN-γR KO mice were not less suppressive than those of immunised wild-type mice in co-cultures with T eff cells and ACs from immunised wild-type mice, we can refute the proposition that the lower expression of Foxp3 in the CD4 + CD25 + population from immunised IFN-γR KO mice is due to a smaller proportion of T reg cells and a larger number of activated T eff cells. Indeed, if the CD4 + CD25 + population from immunised IFN-γR KO mice contained a higher proportion of activated T eff cells, suppression by these CD4 + CD25 + cells should be lower, irrespective of the origin of the T eff cells and ACs. Another argument is that the addition of more CD4 + CD25 + cells failed to improve suppression in co-cultures of cells from immunised IFN-γR KO mice. Our data are therefore more in line with the proposition of a lower Foxp3 expression level per cell. Expression of Foxp3 could be downregulated by the interaction of T reg cells with ACs. ACs might be source of TGF-β, which has been described to convert naive T cells into CD25 + suppressor cells by inducing Foxp3 expression [ 48 ]. Because IFN-γ and TGF-β act antagonistically with each other, the low levels of Foxp3 in arthritic IFN-γR KO mice might be due to inadequate amounts of TGF-β produced by ACs or other cells. However, quantitative PCR performed on isolated T reg cells, on cells obtained from co-cultures (T reg plus T eff plus ACs) and on splenocytes from immunised IFN-γR KO and wild-type mice does not support the concept that the defective activity of T reg cells in vitro or in vivo is due to defects in the production of TGF-β. ACs have also been shown to be able to reverse suppression by CD4 + CD25 + cells through the GITR/GITR-ligand system [ 49 ]. GITR (glucocorticoid-induced tumour necrosis factor receptor) is expressed on CD4 + CD25 + T cells; GITR-ligand is initially upregulated on activated APCs. It remains to be determined whether this process involves a downregulation of Foxp3 expression. This or a similar mechanism might take place during the interaction of T reg cells and ACs from immunised IFN-γR KO mice. Co-cultures with ACs of immunised wild-type mice might possibly normalise Foxp3 expression in the T reg cells of immunised IFN-γR KO mice, together with their T reg suppressive activity. Conclusions In conclusion, our experiments support a pathogenesis model that ascribes an important role to T reg cells as moderators of the disease course in CIA. In particular we show that T reg cells fulfil this role not only during the induction phase but also during the effector phase of the autoimmune response. Furthermore, we were able to refine the model by showing that, after the immunisation with CII in CFA, T reg cells lose part of their suppressive potential. This effect is more pronounced in IFN-γR KO than in wild-type mice, indicating that, in this system, IFN-γ acts as an upregulator of T reg activity, which might be part of the explanation for the well-known protective effect of endogenous IFN-γ. Finally, we present evidence that the mechanism underlying the effect of IFN-γ on T reg cell activity is exerted in part via ACs. Abbreviations ACs = accessory cells; CFA = complete Freund's adjuvant; CIA = collagen-induced arthritis; CII = collagen type II; CTLA = cytolytic T lymphocyte-associated antigen; ELISA = enzyme-linked immunosorbent assay; fetal calf serum = FCS; FITC = fluorescein isothiocyanate; GITR = glucocorticoid-induced tumour necrosis factor receptor; IFN-γ = interferon-γ; IFN-γR KO = interferon-γ receptor knock-out; IL = interleukin; MACS = magnetic-activated cell sorting; PBS = phosphate-buffered saline; RT-PCR = reverse transcriptase polymerase chain reaction; STAT = signal transduction and activators of transcription; T eff = effector T; TGF = transforming growth factor; TLR = Toll-like receptor; T reg = regulatory T. Competing interests The author(s) declare that they have no competing interests. Authors' contributions BDK, HK and TM performed the CIA induction and evaluation. HK, MVB and GL performed the cell purification. DB performed the quantitative PCR. TM and HK performed the flow cytometry. BDK and HK did the in vitro experiments. HK, GL and PM designed the study. All authors participated in the interpretation of the data. HK, AB, GL and PM prepared the manuscript. All authors read and approved the final manuscript.

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1065336

Infiltration of the synovial membrane with macrophage subsets and polymorphonuclear cells reflects global disease activity in spondyloarthropathy

Considering the relation between synovial inflammation and global disease activity in rheumatoid arthritis (RA) and the distinct but heterogeneous histology of spondyloarthropathy (SpA) synovitis, the present study analyzed whether histopathological features of synovium reflect specific phenotypes and/or global disease activity in SpA. Synovial biopsies obtained from 99 SpA and 86 RA patients with active knee synovitis were analyzed for 15 histological and immunohistochemical markers. Correlations with swollen joint count, serum C-reactive protein concentrations, and erythrocyte sedimentation rate were analyzed using classical and multiparameter statistics. SpA synovitis was characterized by higher vascularity and infiltration with CD163 + macrophages and polymorphonuclear leukocytes (PMNs) and by lower values for lining-layer hyperplasia, lymphoid aggregates, CD1a + cells, intracellular citrullinated proteins, and MHC–HC gp39 complexes than RA synovitis. Unsupervised clustering of the SpA samples based on synovial features identified two separate clusters that both contained different SpA subtypes but were significantly differentiated by concentration of C-reactive protein and erythrocyte sedimentation rate. Global disease activity in SpA correlated significantly with lining-layer hyperplasia as well as with inflammatory infiltration with macrophages, especially the CD163 + subset, and with PMNs. Accordingly, supervised clustering using these synovial parameters identified a cluster of 20 SpA patients with significantly higher disease activity, and this finding was confirmed in an independent SpA cohort. However, multiparameter models based on synovial histopathology were relatively poor predictors of disease activity in individual patients. In conclusion, these data indicate that inflammatory infiltration of the synovium with CD163 + macrophages and PMNs as well as lining-layer hyperplasia reflect global disease activity in SpA, independently of the SpA subtype. These data support a prominent role for innate immune cells in SpA synovitis and warrant further evaluation of synovial histopathology as a surrogate marker in early-phase therapeutic trials in SpA.

Introduction Whereas classical analysis of synovial tissue in chronic inflammatory arthritis suggested that synovitis is a nonspecific phenomenon, a number of studies using new molecular tools and synovial biopsies obtained during active disease indicated clear histopathological differences between spondyloarthropathy (SpA) and rheumatoid arthritis (RA), which are the two most frequent forms of chronic autoimmune arthritis [ 1 - 4 ]. These differences were explored as a diagnostic tool in undifferentiated arthritis [ 5 , 6 ], and highly disease-specific markers as well as less pronounced synovial features turned out to be useful in multiparameter classification models [ 7 , 8 ]. Since some of these features are pathophysiologically related to specific disease mechanisms [ 3 , 4 ], it is tempting to hypothesize that histopathological characteristics of inflamed synovium directly reflect the disease process. In RA, it has been shown that synovial features may help to distinguish different subgroups corresponding to distinct pathogenetic mechanisms and clinical phenotypes. Indeed, three main histological types of synovitis can be distinguished in RA: one type characterized by follicular organization with high numbers of B cells and plasma cells, another type with diffuse infiltration by essentially T lymphocytes and macrophages, and a third, granulomatous type, which is less frequent [ 9 ]. The different histological types are stable over time within one individual, are linked with different cellular and molecular disease pathways, as evidenced, for example, by abundant IL-10 in the follicular type, and are related to phenotypic differences such as seronegativity in the diffuse type and extra-articular manifestations in the granulomatous type [ 10 , 11 ]. Besides distinguishing subtypes within one disease, some of these features may also reflect global disease activity and thus be valuable candidates for evaluation as surrogate markers in trials of new, targeted therapies in autoimmune arthritis. Synovial macrophages have been shown to be related to scores for local disease activity as well as to articular damage in RA [ 12 , 13 ]. Sublining macrophages, but also T cells and plasma cells, were increased in clinically involved versus clinically uninvolved knee joints [ 14 ], while sublining macrophages were also increased in joints in active RA compared with joints in end-stage disease [ 15 ]. Taken together, these various findings strongly suggest that the number of sublining macrophages is a good reflection of active disease processes in RA. This interpretation was further validated by demonstrating that synovial sublining macrophages can be used as surrogate marker for global disease activity in clinical trials evaluating antirheumatic therapy in RA [ 16 ]. In contrast with the situation with RA, these issues have not yet been fully assessed in SpA. We have previously demonstrated a correlation between synovial histology and local disease activity in SpA [ 1 ] and the absence of manifest differences in synovial histopathology between psoriatic arthritis (PsA) and other SpA subtypes. Considering the data in RA synovitis, the increase of specific macrophage subsets and polymorphonuclear leukocytes (PMNs) in SpA synovium compared with RA [ 2 ], and the strong and rapid reduction of synovial macrophages, T lymphocytes, and PMNs during treatment with anti-tumor-necrosis-factor (TNF)-α in SpA [ 17 , 18 ], the objective of the present study was to analyze in more detail whether histopathological features of the synovial membrane reflect specific phenotypes and/or global disease activity in SpA. Materials and methods Patients and samples The study included 99 SpA patients fulfilling the criteria of the European Spondyloarthropathy Study Group [ 19 ]. One cohort consisted of 82 patients, including 19 with ankylosing spondylitis (AS), 33 with PsA, 24 with undifferentiated SpA (USpA), 4 with SpA associated with inflammatory bowel disease, and 2 with reactive arthritis. Since we had previously found no major differences between these SpA subgroups for the synovial histopathology markers used in the present study, we considered them collectively as having SpA (unpublished data). All patients had active disease at the time of inclusion, as evidenced by a mean swollen joint count (SJC) of 3.5 ± 4.1 (mean ± standard deviation), a mean serum C-reactive protein (CRP) concentration of 33 ± 45 mg/L, and a mean erythrocyte sedimentation rate (ESR) of 28 ± 24 mm/hour. The mean duration of disease was 5.5 ± 5.4 years, and 23 of the 82 patients were being treated with a disease-modifying antirheumatic drug (DMARD); none of the patients were being treated with corticosteroids. All patients had at least one swollen knee joint, from which synovial biopsies were sampled by needle arthroscopy. As an independent validation group, a second cohort of 17 SpA patients (4 with AS, 5 with PsA, 8 with USpA) fulfilling the same inclusion criteria was included in the study. This group had a mean SJC of 5.5 ± 5.4, a mean serum CRP of 38 ± 48 mg/L, and a mean ESR of 31 ± 27 mm/hour. None of these patients was receiving DMARDs or corticosteroids. The mean duration of disease in this group was 10.8 ± 10.2 years. For the control group, we included 86 patients fulfilling the American College of Rheumatology criteria for RA [ 20 ]. As for the SpA cohort, all RA patients had at least one swollen knee joint and had active disease, with a mean SJC of 9.2 ± 6.6, a mean serum CRP of 58 ± 67 mg/L, and a mean ESR of 41 ± 27 mm/hour. The mean duration of disease was 6.0 ± 7.5 years. Fourteen patients were receiving DMARDs, 5 patients were receiving corticosteroids, and 21 patients were receiving both. In all the patients, synovial tissue biopsies (16 from each person) were obtained by needle arthroscopy of a clinically involved knee joint, as described previously [ 21 ]. All patients gave their written, informed consent before inclusion in the study, which was approved by the Ethics Committee of the Ghent University Hospital. Synovial histopathology Synovial biopsies were fixed, stained, and scored as described previously [ 1 - 4 ]. Briefly, eight paraffin-embedded biopsies were stained with H&E for histological analysis, including mean thickness of the synovial lining-layer, vascularity of the sublining layer, infiltration of the sublining layer, and the presence of lymphoid aggregates, plasma cells, and PMNs. A separate analysis in 93 samples showed that the evaluation of the number of blood vessels and the number of plasma cells on H&E staining correlated well with staining for, respectively, the endothelial marker CD146 ( r = 0.436; P < 0.0001) and the plasma cell marker CD138 ( r = 0.621; P < 0.0001). The remaining eight biopsies were embedded in tissue-freezing medium and used for immunohistochemistry with the following antibodies: anti-CD1a (interdigitating dendritic cells, mouse, monoclonal; Dako, Glostrup, Denmark), anti-CD3 (T cells, mouse, monoclonal; Dako), anti-CD20 (B cells, mouse, monoclonal; Dako), anti-CD68 (monocytes and macrophages, mouse, monoclonal, clone EBM11; Dako), anti-CD163 (scavenger receptor expressed on mature tissue macrophages, mouse, monoclonal, clone Ber-MAC3; Dako), anti-L-citrulline (citrullinated peptides, rabbit, polyclonal; Biogenesis, Poole, UK), and mAb 12A (detecting MHC class II–HC gp39 peptide complexes, mouse, monoclonal; NV Organon, Oss, Netherlands). Parallel sections were stained with irrelevant origin-, isotype-, and concentration-matched antibody as negative control. Stained sections were coded and analyzed by two independent observers, who were blinded to the diagnosis and clinical data. Due to the low number of positive cells in each sample for anticitrulline, anti-CD1a, and mAb 12A staining, these parameters as well as lymphoid aggregates were scored as present or absent. For all other parameters, the analysis included all areas of the biopsies, and a global score was given for each parameter, using a semiquantitative 4-point scale: 0 represented the lowest and 3 the highest level of expression [ 1 - 4 ]. As some histological markers are more abundant than others, the scoring system was calibrated for each marker separately by examining a representative number of samples. In case of discordant scores between the two observers, the mean of the two scores was used. Since anti-CD68 and anti-CD163 staining, which recognizes a particular subset of the CD68 + macrophages [ 2 ], was observed in both the synovial lining layer and the synovial sublining layer, these markers were scored separately in the two compartments. An overview of the 15 synovial parameters is given in Table 1 . Statistics The histopathological features of the synovial membrane in SpA and RA were compared using the Mann–Whitney U test for semiquantitative parameters and the χ 2 test for dichotomous parameters. Histopathological subgroups were identified by clustering analysis (within-group average linkage with Pearson correlation) using SPSS version 12.0 software (SSPS Inc, Chicago, IL, USA). Correlations between semiquantitative histological parameters and clinical disease activity markers (SJC, CRP, ESR) were calculated using the Spearman ρ test. For dichotomous histological markers, the clincal disease activity parameters of the positive and negative groups were compared using an unpaired Student's t -test. A P value of less than 0.05 was considered statistically significant. The relation between the 15 histological parameters and the 3 clinical parameters was also analyzed by SAM (significance analysis of microarray) software, a statistical analysis model that was specifically developed for multiparameter datasets [ 22 ] (see also ). Measuring the relation between changes in the input parameter (which are here the 15 histological features) and changes in the response variable (SJC, CRP, and ESR), the software assigns a score (expressed as a value d) reflecting the strength of the observed differences. To assess the significance of this relationship, a value q is calculated by permutations of the measurements to estimate the percentage of parameters identified by chance, the false discovery rate (FDR). Using SJC, CRP, or ESR as quantitative response parameters, d>2 (indicating that the strength of the association between the histological input parameter and the disease activity outcome parameter was at least twice the expected value) and q<0.10 (corresponding to an α error of less than 10% in classical statistics or, in other words, indicating that the observed associations had a 90% chance of being real) were considered significant. Finally, histological parameters that not only are correlated with disease activity but also contribute significantly to the prediction of the disease activity in individual samples were identified by PAM (predictive analysis of microarray) software [ 23 ]. Whereas SAM is intended to identify significant differences between groups of samples, PAM is intended to identify those parameters that are most useful in predicting the outcome (in this case, disease activity) in individual samples and to combine those parameters in an optimal multiparameter algorithm to classify single samples or patients. Results Comparative histopathology of SpA and RA The scores for the histopathological features of the synovial membrane in SpA and RA are given in Table 2 . There was significantly higher vascularity ( P = 0.013), lining CD163 ( P < 0.001), and sublining CD163 ( P = 0.003) in SpA than in RA. There was also a trend towards an increase of PMNs in SpA ( P = 0.062). In contrast, there was a significantly lower score in SpA versus RA for lining-layer thickness ( P = 0.032), CD1a ( P = 0.009), lymphoid aggregates ( P = 0.029), anticitrulline staining ( P < 0.001), and mAb 12A staining ( P < 0.001). In contrast with CD163, no differences were found for the pan-macrophage marker CD68 in the lining or sublining layer. The number of plasma cells, which were found in 32 of the 82 SpA samples and 40 of the 86 RA samples, was also not different between the two diseases. Although the mean age of the patients was higher in the RA cohort (56.2 ± 14.9 years) than in the SpA cohort (42.6 ± 13.3 years), none of the differentiating parameters was related to age, excluding the possibility that the difference in age could have induced a systematic bias in the comparison. These findings, which are illustrated in Fig. 1 , are in agreement with previous observations [ 1 - 4 ] and indicate that the patient cohorts used in the present study are representative of the full-blown SpA or RA synovial histopathology. Histopathological heterogeneity within SpA With the exception of anticitrulline and mAb 12A staining, which were found almost exclusively in RA, all investigated histopathological parameters showed a wide range of scores within the SpA group, reflecting wide interindividual variability. Therefore, we next tried to identify specific SpA subgroups by combining the different histological features in a multiparameter model using clustering analysis. Unsupervised analysis yielded two main clusters within SpA, consisting of 39 and 43 samples (Fig. 2 ). Although there were slightly more AS samples in cluster 2, the different SpA subtypes were found both in cluster 1 (4 AS, 14 USpA, 16 PsA, 4 inflammatory bowel disease, 1 reactive arthritis) and in cluster 2 (15 AS, 10 USpA, 17 PsA, 1 reactive arthritis), confirming that synovial histopathology is not basically different between SpA subtypes. The mean duration of disease (5.4 ± 5.0 versus 5.7 ± 5.9 years, respectively), the use of DMARDs (in 10 of 39 versus 13 of 43), and the mean SJC (3.1 ± 3.3 versus 3.8 ± 4.7, respectively) were not different between cluster 1 and cluster 2. In contrast, both serum CRP concentrations (14 ± 12 mg/L versus 51 ± 56 mg/L; P < 0.001) and ESR (19 ± 17 mm/hour versus 35 ± 28 mm/hour; P = 0.003) were significantly lower in cluster 1 than in cluster 2 (Fig. 3 ), indicating that this unsupervised classification based on the synovial histopathology reflects the global disease activity. In contrast, a similar analysis of the RA cohort yielded five separate clusters, without significant differences in SJC, serum CRP concentrations, or ESR between the clusters (data not shown). When the two clusters of the SpA cohort were compared, the cluster with higher CRP and ESR was found to show a significant increase of the following histological parameters: vascularity ( P = 0.001), inflammatory infiltration ( P < 0.001), lymphoid aggregates ( P = 0.027), plasma cells ( P = 0.001), PMNs ( P < 0.001), CD3 + lymphocytes ( P < 0.001), and CD20 + lymphocytes ( P = 0.007). Relation between synovial histopathology and disease activity in SpA Since these data suggest that synovial histopathology reflects global disease activity in SpA, we further analyzed the correlation of individual histological parameters with SJC, CRP, and ESR in the SpA cohort. In order to minimize the risk of false-positive results, only the parameters identified by both classical statistics and SAM analysis were considered as significant. As shown in more detail in Table 3 (top), lining-layer thickness, sublining CD68, and sublining CD163 were weakly but significantly correlated with the SJC in SpA. CRP concentrations correlated significantly with inflammatory infiltration, PMNs, and sublining CD163. Finally, ESR correlated with lining-layer thickness, inflammatory infiltration, PMNs, and sublining CD163. Thus, it appears that global disease activity in SpA is essentially associated with inflammatory infiltration with macrophages – especially the CD163 + subset – and PMNs as well as with lining-layer hyperplasia. Although some of the correlations were relatively weak, the number of CD163 + macrophages in the sublining appeared to be consistently correlated with the three different measures of disease activity, whereas inflammatory infiltration and PMNs showed a stronger correlation with the systemic inflammatory parameters. For comparison, we performed the same analysis in the RA control group. As shown in more detail in Table 3 (bottom), no histological parameters were significantly correlated with the SJC. Serum CRP concentrations were significantly associated with CD3 and mAb 12A staining. ESR was correlated with CD3 as well as with sublining CD68 and anticitrulline staining. Globally, the correlations were weaker and less consistent in RA than in SpA. Supervised clustering in relation with disease activity in SpA On the basis of the previous findings, we redefined two separate clusters within the SpA cohort based not on all synovial features, but on the five synovial characteristics that were significantly associated with disease activity: lining-layer hyperplasia, inflammatory infiltration, PMNs, sublining CD68, and sublining CD163. Cluster 1 ( n = 62) was characterized by significantly lower SJC (3.0 ± 3.2 versus 5.2 ± 5.8; P = 0.037), serum CRP concentrations (23 ± 32 mg/L versus 66 ± 63 mg/L; P < 0.001), and ESR (21 ± 19 mm/hour versus 48 ± 28 mm/hour; P < 0.001) than cluster 2 ( n = 20) (Fig. 4a ). Again, there were no significant differences between the two clusters with regard to the SpA subtypes (12 AS, 21 USpA, 24 PsA, 3 inflammatory bowel disease, and 2 reactive arthritis in cluster 1 versus 7 AS, 3 USpA, 9 PsA, and 1 inflammatory bowel disease in cluster 2), indicating the absence of a relation between histopathology and disease phenotypes. Moreover, there was no difference in DMARD treatment (5 of 20 versus 18 of 62) or disease duration (4.0 ± 5.3 versus 5.8 ± 5.5 years) between the two clusters. In other words, the five previously defined histological parameters were able to identify a subgroup of SpA patients with high disease activity independently of the SpA subtype, treatment, and disease duration, thereby confirming that synovial histopathology reflects not only local inflammation but also global disease activity in SpA. In contrast, a similar analysis of the RA cohort using CD3, sublining CD68, anticitrulline, and mAb 12A as input parameters yielded two clusters (with respectively n = 41 and n = 45 samples) that were not different with regard to SJC (9.1 ± 6.3 versus 9.2 ± 7.0), serum CRP concentrations (52 ± 43 mg/L versus 64 ± 82 mg/L), or ESR (42 ± 27 mm/hour versus 40 ± 28 mm/hour). Confirmation of the supervised clustering analysis on an independent SpA cohort To confirm these findings, we applied the same supervised clustering analysis based on the five previously defined histological parameters to an independent cohort of 17 SpA patients, none of whom were being treated with DMARDs. Two clusters were identified: cluster 1, consisting of 7 patients (1 with AS, 2 with PsA, 4 with USpA) and cluster 2, consisting of 10 patients (3 with AS, 3 with PsA, 4 with USpA). The mean disease duration in the two clusters was similar (10.7 ± 6.9 versus 10.8 ± 12.4 years, respectively). However, the two clusters were again significantly different with regard to SJC (9.3 ± 6.6 versus 2.9 ± 2.3; P = 0.012), serum CRP concentrations (75 ± 55 versus 13 ± 18 mg/L; P = 0.004), and ESR (55 ± 18 versus 15 ± 20 mm/hour; P = 0.001) (Fig. 4b ). Thus, this analysis in an independent validation cohort confirms that well-defined synovial histopathological features in SpA reflect global disease activity independently of SpA subtype, disease duration, and treatment. Prediction of global disease activity in individual SpA samples Since the previous data provided evidence that lining-layer hyperplasia and infiltration with PMNs and macrophage subsets are directly related to the global disease activity in SpA, we next investigated whether synovial histopathology could be a valuable surrogate marker for the prediction of disease activity in individual patients rather than in a patient cohort. Using PAM to classify the 82 SpA patients into tertiles (low, middle, and high disease activity) for respectively SJC, CRP, and ESR, the same histological parameters were identified as having the largest contribution to the predictive algorithms: inflammatory infiltration, sublining CD163, PMNs, sublining CD68, and lining CD68. However, the positive predictive value of these models was relatively poor, as shown by the correct classification of only 51%, 55%, and 49% of the samples for SJC, CRP, and ESR, respectively, compared with an a priori chance of 33%. Similarly, PAM analysis using histopathological parameters was not able to make a good prediction of samples belonging to the highest quartile for disease activity (data not shown). These data indicate that although the previously identified histological parameters are related to disease activity, the wide interindividual variability does not allow a robust prediction in single patients. Discussion It has previously been shown that the synovial histopathology in inflammatory arthritis is dependent on both the disease background and the local disease activity [ 1 , 2 , 12 , 15 ]. When focusing on SpA patients with active synovitis of the investigated joint, however, we still observe a large heterogeneity in synovial features. In an attempt to translate these histological findings into clinically relevant patterns, we assessed whether this heterogeneity was related to the fact that SpA consists of different subtypes with distinct phenotypes. Confirming a recent report in which we found no significant differences between PsA on the one hand and AS and USpA on the other hand (unpublished data), both unsupervised and supervised clustering analysis of the present data indicated that the synovial heterogeneity is not basically associated with specific SpA phenotypes. In contrast, the present study reveals that this heterogeneity directly reflects the global disease activity in SpA. Considering that there are no validated global disease parameters for SpA as a whole, the present study used SJC, serum CRP concentrations, and ESR as clinical outcomes. Although these parameters are not elevated in all patients with active SpA, several studies have shown that peripheral arthritis as well as systemic inflammatory parameters are characteristics of severe disease [ 24 , 25 ]. In this context, the present finding that local synovial features are correlated with systemic inflammatory parameters such as CRP and ESR strengthens the concept that peripheral synovitis, although not present in all SpA patients, contributes significantly to disease severity. Further analysis revealed that not all synovial features were correlated with disease activity. Both classical statistics and SAM analysis demonstrated a correlation with lining-layer hyperplasia and, more consistently, inflammatory infiltration by CD163 + macrophages and PMNs. We previously shown that these CD163 + macrophages, but not the overall number of CD68 + macrophages, are increased in SpA synovitis and play a specific role in the disease pathogenesis [ 2 , 26 ]. In contrast, neither synovial hypervascularity, which is clearly increased in SpA versus RA and contributes to diagnostic classification [ 1 , 7 ], nor lymphocyte-related characteristics (CD3, CD20, plasma cells, lymphoid aggregates) were associated with SJC, CRP, or ESR. This was further confirmed by a supervised clustering analysis that could even better identify a high-disease-activity group on the basis of only five synovial features, both in the first SpA cohort that was used to identify these features and, most importantly, in a completely independent SpA cohort. Both the previous reports and the present study pointed towards the increased presence of CD163 + macrophages and PMNs in SpA versus RA synovitis [ 2 , 27 ]. Moreover, in the RA control group the global disease activity parameters correlated not with these features, but with lymphocyte-related characteristics such as CD3 and putative B- and T-cell autoantigens in RA (intracellular citrullinated proteins and MHC–HC gp39 complexes) [ 3 , 4 ]. Although certainly not excluding a secondary effector function for lymphocytes in SpA or for macrophages in RA synovitis, these findings point towards distinct pathogenetic mechanisms in the two diseases and fit well with the hypothesis that SpA synovitis is primarly driven by innate immune cells with secondary alterations in lymphocyte activation and functions [ 28 ]. Independently of these pathogenetic considerations, the present data also raise the question of the value of synovial histopathology as a biomarker in SpA. Despite the fact that lining-layer hyperplasia and inflammatory infiltration with CD163 + macrophages and PMNs reflected the global disease activity when cohorts of patients were analyzed, multiparameter models based on synovial histopathology turned out to be relatively poor predictors of disease severity in individual patients. This finding is not totally unexpected in view of the wide variability of individual values for both histology and disease activity and the broad overlap between different clusters in the previous analyses. Several factors could play a role in this wide individual variability. Firstly, treatment with DMARDs might be a confounding factor. However, the facts that most SpA patients of the first cohort had been given no treatment at all or were being treated exclusively with nonsteroidal antirheumatic drugs, that the clustering analysis was not influenced by treatment, and that the clustering was confirmed in an independent cohort without DMARD treatment are in accord with previous data showing that DMARDs did not bias the synovial histopathology in patients with persistent, refractory peripheral synovitis (unpublished data). Moreover, even after exclusion of the DMARD-treated patients, the prediction models performed poorly (data not shown). Secondly, the present study used a semiquantitatve scoring system for the histopathology, whereas the previously mentioned RA studies used digital image analysis [ 16 ]. Whereas semiquantitative scoring has been shown in multiple studies to be robust and reproducible, it is less sensitive to change than digital image analysis and might thus underestimate small variations [ 29 ]. Thirdly, recent data obtained with microarrays indicated clearly that setting up profiles using multiple parameters can compensate for the relative lack of precision and the variability of individual parameters [ 30 ]. As we have already demonstrated the added value of combining different histopathological features in multiparameter models for diagnostic classification of inflammatory arthritis [ 7 , 8 ], the same might apply to the use of synovial histology as a surrogate marker for global disease activity. In this context, early–phase, randomized clinical trials in SpA might be of particular interest for the use of synovial histopathology as a biomarker. With the availibility of powerful new treatments such as TNF-α blockers it becomes increasingly important to obtain as much paraclinical and biological information as possible in small patient cohorts early in the clinical development of new drugs. Moreover, in such trials, the emphasis is on groups with uniform treatment schedules rather than on individual patients, and different biological measurements (such as histology, mRNA expression levels, serum protein concentrations) can be combined in multiparameter algorithms, thus overcoming the previously mentioned caveats. In RA, it has recently been demonstrated that the number of sublining CD68 + macrophages is a sensitive surrogate marker for response to therapy [ 16 ], even if this feature was only found to correlate with ESR in RA in the present cross-sectional study and was clearly less robust than the previously discussed SpA parameters. Since the correlations between global disease activity and synovial histopathology of a single joint were consistently stronger in SpA than in RA and since previous studies showed a histopathological response to targeted therapies in SpA and more specifically a decrease of macrophage subsets and PMNs [ 17 , 18 , 31 - 33 ], the data presented here warrant further prospective and longitudinal analysis of synovial histopathology as a surrogate marker in the evaluation of new, targeted therapies for SpA. Conclusion The data presented indicate that inflammatory infiltration of the synovium with CD163 + macrophages and PMNs as well as lining-layer hyperplasia reflect global disease activity in SpA, independently of the SpA subtype. These data support a prominent role for innate immune cells in SpA synovitis and warrant further evaluation of synovial histopathology as a surrogate marker in early-phase therapeutic trials in SpA. Abbreviations AS = ankylosing spondylitis; CRP = C-reactive protein; DMARD = disease-modifying antirheumatic drug; ESR = erythrocyte sedimentation rate; H & E = hematoxylin and eosin; IL = interleukin; mAb = monoclonal antibody; MHC = major histocompatibility complex; PAM = predictive analysis of microarray; PMN = polymorphonuclear leukocyte; PsA = psoriatic arthritis; RA = rheumatoid arthritis; SAM = significance analysis of microarray; SJC = swollen joint count; SpA = spondyloarthropathy; TNF = tumor necrosis factor; USpA = undifferentiated spondyloarthropathy. Competing interests Annemieke M Boots is employed by Organon NV, Oss, The Netherlands. Authors' contributions DB, EMV, and FDK designed the study. DB, EK, and LDR sample and analyzed the synovial tissues. HM selected the patients. DB collected and analyzed the data. mAb 12A was provided by AMB. DB, LDR, and AMB prepared the manuscript, and EMV, HM, and FDK reviewed it. All authors read and approved the final manuscript.

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1065337

Local IL-13 gene transfer prior to immune-complex arthritis inhibits chondrocyte death and matrix-metalloproteinase-mediated cartilage matrix degradation despite enhanced joint inflammation

During immune-complex-mediated arthritis (ICA), severe cartilage destruction is mediated by Fcγ receptors (FcγRs) (mainly FcγRI), cytokines (e.g. IL-1), and enzymes (matrix metalloproteinases (MMPs)). IL-13, a T helper 2 (Th2) cytokine abundantly found in synovial fluid of patients with rheumatoid arthritis, has been shown to reduce joint inflammation and bone destruction during experimental arthritis. However, the effect on severe cartilage destruction has not been studied in detail. We have now investigated the role of IL-13 in chondrocyte death and MMP-mediated cartilage damage during ICA. IL-13 was locally overexpressed in knee joints after injection of an adenovirus encoding IL-13 (AxCAhIL-13), 1 day before the onset of arthritis; injection of AxCANI (an empty adenoviral construct) was used as a control. IL-13 significantly increased the amount of inflammatory cells in the synovial lining and the joint cavity, by 30% to 60% at day 3 after the onset of ICA. Despite the enhanced inflammatory response, chondrocyte death was diminished by two-thirds at days 3 and 7. The mRNA level of FcγRI, a receptor shown to be crucial in the induction of chondrocyte death, was significantly down-regulated in synovium. Furthermore, MMP-mediated cartilage damage, measured as neoepitope (VDIPEN) expression using immunolocalization, was halved. In contrast, mRNA levels of MMP-3, -9, -12, and -13 were significantly higher and IL-1 protein, which induces production of latent MMPs, was increased fivefold by IL-13. This study demonstrates that IL-13 overexpression during ICA diminished both chondrocyte death and MMP-mediated VDIPEN expression, even though joint inflammation was enhanced.

Introduction One of the main pathological features of rheumatoid arthritis is marked destruction of cartilage [ 1 ]. This destruction starts with reversible proteoglycan depletion, which is followed by irreversible cartilage degradation defined as chondrocyte death and breakdown of collagen type II, eventually leading to matrix erosion. The latter is mainly induced by matrix metalloproteinases (MMPs), which generate specific cleavage sites within matrix molecules [ 2 , 3 ]. MMPs are secreted in an inactive form by IL-1-stimulated chondrocytes, synovial macrophages, and fibroblasts [ 4 - 6 ]. Activation of MMPs is still poorly understood, but MMP activity is primarily found in experimental immune-complex (IC)-dependent arthritis models. Immunoglobulin G (IgG)-containing ICs can activate macrophages upon recognition by Fcγ receptors (FcγRs). Three classes of murine FcγR can be distinguished: FcγRI, II, and III. Triggering FcγRI and III activates cellular responses, whereas FcγRII is an inhibitory receptor [ 7 ]. Previous studies have showed that activating FcγRI and III are crucial in induction of severe cartilage destruction, since chondrocyte death and MMP-mediated cartilage damage were absent in FcγR-deficient mice after induction of immune-complex-mediated arthritis (ICA) [ 8 ]. Furthermore, cartilage damage is aggravated by local overexpression of the proinflammatory T helper (Th)1 cytokine IFNγ [ 9 ]. This increase in cartilage destruction was observed only in IC-dependent arthritis models [ 9 ]. FcγRI was found to be crucial in the induction of chondrocyte death, whereas both FcγRI and III mediated MMP-mediated expression of VDIPEN [ 9 ]. Since the Th1 cytokine IFNγ worsens the arthritic response by up-regulation of the activating FcγRs, overexpression of a Th2 cytokine during arthritis might be protective, because of down-regulation of these receptors. In earlier studies, we found that adenoviral overexpression of IL-4 resulted in reduced MMP-mediated cartilage damage and chondrocyte death during ICA and arthritis induced by collagen type II [ 10 , 11 ]. IL-4 is regarded as a potent anti-inflammatory cytokine by direct inhibition of proinflammatory cytokines such as IFNγ, IL-1, and tumor necrosis factor α [ 12 ]. However, IL-4 protein and mRNA are hardly detected in synovial fluid and synovium of rheumatoid arthritis patients [ 13 ]. In contrast, IL-13 is expressed in rheumatoid arthritis synovial fluid and synovial fluid macrophages and resembles many functions of IL-4 [ 14 , 15 ]. Systemic overexpression of IL-13 in collagen-type-II-induced arthritis and local overexpression of IL-13 in rat adjuvant-induced arthritis reduced joint inflammation and bone destruction [ 16 , 17 ]. However, the effect of IL-13 on cartilage destruction was not investigated in detail in these studies and remains to be elucidated. In the present study, we investigated whether IL-13 influences the development of chondrocyte death and MMP-mediated VDIPEN expression in ICA. Subsequently, regulation of FcγR, MMP, and IL-1 expression by IL-13 was studied, as these are important mediators in severe cartilage damage. The present study demonstrates that overexpression of IL-13 in arthritic knee joints reduces chondrocyte death and MMP-mediated VDIPEN expression despite enhanced joint inflammation. Injection of an adenovirus encoding for IL-13 diminished chondrocyte death, which correlated with down-regulation of FcγRI expression in the synovium. Reduction of MMP-mediated VDIPEN expression was not reflected by MMP mRNA and IL-1 concentrations, as these were increased. Materials and methods Animals C57Bl/6 male mice (10 to 12 weeks old) were purchased from Elevage-Janvier (Le Genest Saint Isle, France). Mice were fed a standard diet and tap water ad libitum. Ethical approval was obtained from the research ethics committee of the Central Animal Facility in Nijmegen. Local gene transfer of IL-13 The recombinant adenovirus encoding human IL-13 (AxCAhIL-13) was generated as described before [ 17 - 19 ] and an empty adenoviral construct (AxCANI) was used as control virus. AxCAhIL-13 or AxCANI (1.10 7 plaque-forming units) was injected intra-articularly in naive knee joints. Patellae with adjacent synovium were dissected in a standardized manner [ 20 ] and synovial biopsies were taken with a biopsy punch (diameter of 3 mm). Total RNA was extracted in 1 ml TRIzol reagent and used for quantitative PCR as described below. AxCAhIL-13 or AxCANI was injected intra-articularly 1 day before the induction of arthritis. Induction of immune-complex-mediated arthritis Rabbit polyclonal antibodies directed against lysozyme were injected intravenously into mice. ICA was then passively induced by injecting 3 μg lysozyme coupled to poly-L-lysine in 6 μl pyrogen-free saline into the knee joints. Histology of arthritic knee joints Total knee joints were dissected at days 3 and 7 after the onset of arthritis. Joints were decalcified, dehydrated, and embedded in paraffin. Tissue sections (7 μm) were stained with hematoxylin and eosin. Histopathological changes were scored in two ways. Inflammation was graded on a scale from 0 (no inflammation) to 3 (severely inflamed joint) as influx of inflammatory cells in synovium and joint cavity. Chondrocyte death was scored as the amount of empty lacunae expressed as a percentage of the total number of cells within the cartilage layers. Immunohistochemical detection of macrophages and polymorphonuclear neutrophils (PMNs) Macrophages were detected using a specific antibody against F4/80, a murine macrophage membrane antigen [ 21 ]. PMNs were visualized using NIMPR14, a specific rat anti-mouse monoclonal antibody [ 22 ]. Primary antibodies were detected using rabbit anti-rat IgG and avidin–horseradish peroxidase conjugate. Finally, sections were counterstained with hematoxylin. Macrophage and PMN subsets were quantitatively measured using an image analysis system. The inflammatory cell mass was selected by hand and the amount of positive features present in this area was displayed using a computer imaging system. Three sections of each knee joint were measured and the mean was calculated. We report the amount of positive features per 100,000 μm 2 inflammatory cell mass in the synovium. Immunohistochemical VDIPEN staining Sections were digested with proteinase-free chondroitinase ABC (0.25 units/ml in 0.1 M Tris/HCl, pH 8.0; Sigma, Zwijndrecht, The Netherlands) to remove the side chains of proteoglycans followed by incubation with affinity-purified rabbit anti-VDIPEN IgG [ 23 ]. The primary antibody was detected using biotinylated goat anti-rabbit IgG, and avidin–streptravidin–peroxidase (Elite kit; Vector, Burlingame, CA, USA). Counterstaining was done with orange G (2%). Areas of immunostaining were expressed as a percentage of the total cartilage surface. Quantitative detection of FcγR and MMP mRNA using RT-PCR Specific mRNA levels for FcγRI, II, and III and MMP-3, -9, -12, -13, and -14 were detected using the ABI/PRISM 7000 Sequence Detection System (ABI/PE; Foster City, CA, USA). Briefly, 1 μg of synovial RNA was used for RT-PCR. mRNA was reverse transcribed to cDNA using oligodT primers. cDNA (1/100) was used in one PCR amplification. PCR was performed in SYBR Green Master Mix using the following amplification protocol: 2 min at 50°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C with data collection in the last 30 s. Message for murine FcγRI, II, and III and MMP-3, -9, -12, -13, and -14 was amplified using the primers listed in Table 1 (Biolegio, Malden, The Netherlands) at a final concentration of 300 nmol/l. Relative quantification of the PCR signals was performed by comparing the cycle threshold value (Ct) of the FcγR and MMP genes in the different samples after correction of the GAPDH content for each individual sample. Determination of cytokine and chemokine concentrations To determine concentrations of IL-13, IL-1β, KC (a mouse homologue for human growth-related protein), and macrophage inflammatory protein 1α in patella washouts, synovial specimens were isolated in a standard manner [ 20 ] and incubated in 200 μl RPMI 1640 medium (GIBCO BRL, Breda, The Netherlands) for 1 hour at room temperature. Cytokine and chemokine concentrations were determined using the BioPlex ® system from BioRad (Hercules, CA, USA) for the Luminex ® multi-analyte system and expressed as pg/ml. Statistical analysis Differences between experimental groups were tested for significance using the Mann–Whitney U test. P values <0.05 were considered statistically significant. Results Local IL-13 expression in naive knee joints using adenoviral gene transfer The expression of IL-13 was determined in synovial washouts at days 1, 2, 3, and 7 after injection of the AxCAhIL-13 virus. IL-13 reached a concentration of 0.4 ng/ml after 24 hours. Values increased to 2 ng/ml at day 2 and remained high up to 7 days after injection (Fig. 1a ). IL-13 was not detected after injection of AxCANI. We next investigated whether injection of the adenoviral IL-13 construct causes joint inflammation by itself. Using histology, we found that IL-13 overexpression in naive knee joints did not recruit inflammatory cells at day 1, 2, 3, or 7 (Fig. 1c ). Injection of AxCANI resulted in minor cell influx in the synovial lining and joint cavity (Fig. 1b ), which was not detectable from day 2 onwards. IL-13 overexpression during ICA enhances joint inflammation and alters the composition of the cell mass To investigate whether IL-13 overexpression ameliorated the arthritic response, we injected AxCAhIL-13 1 day before ICA induction. Joint inflammation was studied 3 and 7 days after arthritis onset. IL-13 overexpression significantly increased the inflammatory cell mass in joint cavity and synovium, by 60% and 30%, respectively, 3 days after arthritis induction (Fig. 2a ). After 7 days, joint inflammation seemed to normalize in the IL-13 group (Fig. 2b ). To further investigate inflammatory cell types attracted by IL-13, PMNs and macrophages were detected using specific NIMPR14 and F4/80 antibodies respectively using immunolocalization. At day 3, the amount of PMNs and macrophages was not markedly altered by IL-13 (Fig. 3a and 3B ). At day 7, however, the amount of PMNs in the synovial lining was 10 times higher (Fig. 3a ), whereas the amount of macrophages in the IL-13 group was half that in the mice without IL-13 (Fig. 3b ). KC concentration in synovial washouts is augmented by IL-13 A possible mechanism by which IL-13 can increase joint inflammation in the presence of ICs is elevation of chemokine production. To investigate this, synovial washouts were done on days 3 and 7, and the chemokines KC (chemotactic for neutrophils) and macrophage inflammatory protein1α (chemotactic for macrophages) were measured. Local IL-13 overexpression increased KC concentrations 4- and 18-fold, respectively, at days 3 and 7 after arthritis induction, which correlates with the high amount of PMNs (Table 2 ). Macrophage inflammatory protein1α concentrations at day 3 were comparable between the control and IL-13 groups. At day 7, macrophage inflammatory protein1α expression was slightly increased by IL-13 (Table 2 ). IL-13 strongly inhibits chondrocyte death during ICA: down-regulation of FcγRI Because IL-13 enhanced the inflammatory response, we next investigated the effect of IL-13 overexpression on cartilage destruction. A characteristic feature of irreversible cartilage damage is chondrocyte death; this was scored as the percentage of empty lacunae relative to the total amount of chondrocytes present in various cartilage layers in the knee joint. Three days after ICA induction, chondrocyte death, expressed as the mean for six cartilage layers in the knee joint, was very low in the IL-13 group (5%) and significantly less than in the control arthritic knee joints, which showed 25% chondrocyte death (Fig. 4a ). At day 7, chondrocyte death was even more significantly reduced (65%) in comparison with the control group (Fig. 4a ). In a previous study, we found that FcγRI is the dominant receptor mediating chondrocyte death during ICA [ 9 ]. We speculated that the decreased chondrocyte death might be caused by down-regulation of FcγRI by IL-13. For that reason, we determined the effect of IL-13 on mRNA levels of all three classes of FcγRs in synovium. Cycle values of FcγRI, II, and III in synovium of arthritic knee joints injected with AxCANI were subtracted from cycle values of FcγRs after AxCAhIL-13 injection. Interestingly, FcγRI mRNA level was decreased by IL-13 at day 3 after ICA induction (ΔCt = 2), and was still slightly down-regulated at day 7 (ΔCt = 0.5). In contrast, FcγRII and FcγRIII were up-regulated by IL-13, at both days 3 and 7 after ICA induction (Fig. 4b ). IL-13 increases IL-1 production and MMP mRNA levels in the arthritic knee joint Cartilage matrix degradation is largely mediated by MMPs. Production of latent MMPs is mainly regulated by IL-1 and this cytokine has been shown to be crucial in the generation of MMP-mediated neoepitopes [ 23 ]. The production of IL-1 was determined in synovial washouts of arthritic knee joints at both days 3 and 7. At day 3, IL-1 concentration was between 450 and 500 pg/ml in both the control and the IL-13 group. However, at day 7, the IL-1 concentration was reduced in the control group but remained high in the IL-13 group (control 54 pg/ml vs IL-13 255 pg/ml). This sustained IL-1 production at day 7 may result in high concentrations of MMPs in synovium. Levels of MMP-3, -9, -12, -13, and -14 mRNA were detected by quantitative PCR. MMP-12 mRNA levels were increased 10-fold and 8-fold by IL-13 at days 3 and 7, respectively, after the onset of ICA. At day 7, mRNA levels of MMP-3, -9, and -13 were also significantly increased in the IL-13 group (Table 3 ). MMP-mediated VDIPEN expression is reduced by IL-13 overexpression Increased IL-1 and MMP concentrations may induce enhanced MMP-mediated proteoglycan degradation and this was further investigated by detection of VDIPEN neoepitope expression in the cartilage. In the control group, 35% of the cartilage surface expressed VDIPEN neoepitopes after 3 days (Fig. 5 ). Injection with AxCAhIL13 reduced VDIPEN expression by 43%, as only 20% VDIPEN expression was found in the IL-13 group. The inhibitory effect of IL-13 was still present at day 7 after arthritis induction, as only 10% VDIPEN expression was found in the IL-13 group compared to 25% in the control group (Fig. 5 ). Discussion In the present study, we have shown that local gene transfer of IL-13 reduced severe cartilage destruction defined as chondrocyte death and MMP-mediated aggrecan damage during ICA. Local IL-13 overexpression during IC-dependent arthritis enhanced joint inflammation. To exclude the possibility that IL-13 itself induces influx of inflammatory cells, as is found when IL-13 is overexpressed in the lung [ 24 , 25 ], AxCAhIL-13 was injected in naive knee joints. We observed that IL-13 overexpression in the knee joint did not recruit inflammatory cells. This observation indicates that overexpression of IL-13 induces elevated joint inflammation in combination with IC triggering. In our IC-dependent arthritis model, we showed that joint inflammation is determined by activating FcγRIII [ 26 ]. In the present study, we find that IL-13 increased expression of FcγRIII within the synovium, which is not in line with the study showing that IL-13 decreases FcγRIII expression on human monocytes [ 27 ]. However, regulation of FcγR expression on mouse macrophages by IL-13 has not been described. IL-13 has high similarity with IL-4, which can increase FcγRIII expression on murine mast cells [ 28 ]. Binding of IC to FcγRIII on macrophage lining cells leads to activation, resulting in elevated influx of inflammatory cells. We further found that overexpression of IL-13 in arthritic knee joints particularly increased the amount of PMNs. This is in line with earlier studies in which it was shown that stimulation of FcγRIII induces release of PMN attracting chemokines as IL-8, resulting in neutrophil accumulation [ 29 - 31 ]. The proinflammatory action of IL-13 found in the present study seems to be dependent on costimulation with ICs to trigger arthritis onset, since local overexpression of IL-13 during T-cell-mediated rat adjuvant-induced arthritis diminishes joint inflammation [ 17 ]. In the latter model, ICs do not play a role. Whether IL-13 decreases or enhances joint inflammation may also be dependent on systemic or local overexpression. Systemic overexpression of IL-13 during collagen-type-II-induced arthritis, in which FcγRIII is also required for arthritis development [ 32 ], decreased joint inflammation [ 16 ]. An explanation may be that systemic overexpression of IL-13 hampers the development of the immune response by induction of isotype switching to the nonarthritogenic IgG4 and IgE [ 33 , 34 ], thereby ameliorating the arthritic response. Induction of immunity is hardly affected by local overexpression, as was shown when injection of AdIL-4 (adenovirus expressing IL-4) in knee joints during arthritis induced by collagen type II markedly increased the amount of inflammatory cells [ 11 ]. Cartilage destruction during ICA is mostly related to joint inflammation. Despite the enhanced influx of inflammatory cells, however, a significant reduction of chondrocyte death was induced by IL-13. Chondrocyte death may be the result of increased production of oxygen radicals, as reactive oxygen species can mediate apoptosis [ 35 ]. In a previous study, we showed that there is a prominent role for FcγRI mediating chondrocyte death during ICA. In FcγRI-deficient mice, chondrocyte death was almost absent. When the Th1 cytokine IFNγ was overexpressed, a significant increase in chondrocyte death was observed, which was dependent on FcγRI [ 9 ]. Stimulation of FcγRI leads to production of oxygen radicals via NADPH-oxidase [ 36 ]. In the present study, we find that in knee joints injected with AxCAhIL-13, FcγRI expression remained low, whereas in knee joints injected with control virus, FcγRI expression level was enhanced in the synovium. The decrease in chondrocyte death might be due to a reduced FcγRI concentration. Moreover, it has been shown that IL-13 itself down-regulates production of oxygen radicals by inflammatory cells, since IL-13 can inhibit protein-kinase-C-triggered respiratory burst in monocytes [ 37 ]. The inhibiting effect of IL-13 on oxygen radical production seemed to be monocyte-dependent, as no reduction was found in PMNs [ 38 ]. In addition, IL-13 also reduced MMP-mediated VDIPEN neoepitope expression. It has been reported that IL-13 diminishes the breakdown of collagen and proteoglycans from bovine cartilage, by regulation of MMP expression [ 39 ]. Several mechanisms may inhibit MMP-mediated cartilage destruction, as regulation of MMPs occurs at three different levels: MMP synthesis, activation of latent enzyme, and MMP inhibition. IL-1 is a prominent cytokine controlling the production of latent MMPs [ 40 ], and diminished production of IL-1 might reduce MMP-mediated cartilage damage. We found, however, that IL-13 overexpression in arthritic knee joints strongly increased IL-1β concentrations. IL-13 is described as an anti-inflammatory cytokine, which in general reduces IL-1β production [ 14 , 27 , 41 ]. However, the effect of IL-13 on IL-1 production by IC-stimulated macrophages has not been described to date. In addition to macrophages, fibroblasts and PMNs are also present in the knee joint at day 7 after the onset of arthritis. The sustained production of IL-1 by IL-13 may indeed stimulate MMP production, as reflected by enhanced MMP-3, -9, -12, and -13 mRNA levels 7 days after ICA induction in AxCAhIL-13-injected arthritic knee joints. MMP-12 mRNA level was already increased at day 3 after the onset of arthritis. It has been shown that MMP-12 expression is IL-13-dependent and that MMP-12 is a critical downstream mediator and regulator of IL-13-induced responses [ 42 , 43 ]. Furthermore, IL-13 induction of MMP-2, -9, and -13 is at least partly mediated by MMP-12 [ 43 ], indicating that MMP-12 may be a crucial enzyme inducing MMP-mediated cartilage damage. Furthermore, IL-13 might interfere at the level of activation of MMPs. MMPs are secreted in a latent form and activation occurs after cleavage of a propeptide. Factors that activate latent MMPs are still unknown. However, MMP-mediated VDIPEN expression is mainly found in IC-dependent arthritis models, in which FcγRs are of utmost importance. Down-regulation of the activating FcγRs might reduce VDIPEN expression. Indeed, we found that IL-13 strongly diminished FcγRI expression in synovium. Another mechanism involved in activation of MMPs is production of oxygen radicals. As mentioned above, stimulation of FcγRI results in assembly of the NADPH-oxidase complex, which produces oxygen radicals [ 36 ]. Additionally, oxygen metabolites can be converted into H 2 O 2 , which can activate latent proMMPs [ 44 , 45 ]. Taken together, decreased FcγRI expression reduces the production of oxygen radicals, which apart from chondrocyte protection may also result in diminished MMP-mediated VDIPEN expression. Conclusion The present study shows that IL-13 is a potent cytokine that protects the cartilage matrix against degradation during ICA. In addition, these results indicate that regulation of the expression of FcγR, particularly FcγRI, might be involved in this process. Therefore, modulation of FcγRI by Th2 cytokines seems to be a promising therapeutic tool diminishing cartilage damage in rheumatoid arthritis. Abbreviations AxCAhIL-13 = adenovirus encoding interleukin-13; AxCANI = adenovirus encoding no gene; Ct = cycle threshold; FcγR = Fcγ receptor; IC = immune complex; ICA = immune-complex-mediated arthritis; IFNγ = interferon γ; IgG = immunoglobulin G; IL = interleukin; KC = mouse homologue for human IL-8; MMP = matrix metalloproteinase; NADPH = reduced nicotinamide adenine dinucleotide phosphate; PMN = polymorphonuclear neutrophil; RT-PCR = reverse transcriptase polymerase chain reaction; Th, T helper. Competing interests The author(s) declare that they have no competing interests. Authors' contributions KN designed the experimental design of the study, carried out the experiments, and drafted the manuscript. PL participated in the experimental design of the study and preparation of the manuscript. AH participated in the animal studies. AS participated in isolation of mRNA and performing PCRs. AK provided the adenoviruses and participated in the preparation of the manuscript. TR participated in the preparation of the manuscript. WB participated in the design of the study and preparation of the manuscript. All authors read and approved the final manuscript.

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1065704

Molecular Insights into Human Brain Evolution

As a species, we pride ourselves on the uniqueness of our brain. But comparisons with other species may tell us how our unique brains evolved

As a species, we pride ourselves on the uniqueness of our brain. Relative to our body size, the human brain is bigger than that of any other animal. It may also contain unique structures and patterns of organisation that presumably underlie our intelligence and ability to manipulate our environment. But how did our unique brain originate, and under what selective pressures did it evolve? Some of the answers may lie in the genetic differences that researchers are now uncovering between us and our closest relatives. What Is So Different about the Human Brain? When we compare our brain to those of other animals, the first thing that strikes us is its size. Human brains weigh on average 1,300 grams; a squirrel brain weighs six grams. Some of this difference is because, as larger animals, we need more brain to run our bodies. However, the brains of our nearest relatives, the great apes, weigh only 300–500 grams, even though their body size is similar to ours ( Figure 1 ). “Humans sit on the top of the pile when it comes to relative brain size”, notes geneticist Bruce Lahn (University of Chicago, Illinois, United States) (see Box 1 ). Box 1. Nothing like a Whale Just how unique is human brain evolution? Neuroscientist Lori Marino (Emory University, Atlanta, Georgia, United States) and her colleagues have used computed tomography to estimate the body and brain size of 36 fossil whale species and have compared these data with those for modern toothed whales. Relative to body size, whales and dolphins have the next biggest brains to us, bigger even than chimpanzees, and, says Marino, “there have been dolphins swimming in the oceans with huge brains for more than 15 million years. We are really the new kids on the block.” Like in humans and other primates, the neocortex in whale brains is huge, but its structure is very different to that of our neocortex. Whales have been independent of other lineages for about 60 million years, notes Marino, and haven't shared a common ancestor with primates for 94 million years. “Nevertheless, during evolution, whales have converged upon very similar capacities and behaviours to those of primates, including a highly developed social structure, which tells us that there is more than one way to evolve a complex intelligence.” Figure 1 Comparison of a Human and a Chimpanzee Brain Scale bar = 1 cm (Image: Todd Preuss, Yerkes Primate Research Center) Throughout mammalian and primate evolution, there has been a gradual increase in brain size, superimposed with “spikes” of fast growth such as the tripling in human brain size that occurred about 1.5 million years ago, 4 million years after the human lineage diverged from that of the great apes. “Even in the ape lineage, the brain has been expanding but along the human lineage it has really taken off”, says Lahn. In addition, over time, different parts of our brain have increased in size at different rates. The cerebral cortex has expanded more than other areas, and within the cortex, some areas have expanded differentially while others have lagged behind. “Humans sit on the top of the pile when it comes to relative brain size.” Paleoanthropologist Ralph Holloway (Columbia University, New York, United States) uses endocasts to look for macroscopic differences in the brains of our human ancestors. “We fill human fossil skulls with vulcanised rubber and once it has set, we pull it out of the large hole at the base of the skull and the rubber snaps back into the shape of the skull”, Holloway explains. Endocasts are particularly useful for comparing brain sizes, but they also provide information on when the asymmetries that are present in our brain first appeared. These often reflect cerebral specialisation, and Holloway believes that some of the asymmetries he sees in human fossil skulls may indicate when our ancestors acquired language. More details about how the shape of our brain differs from that of our closest living relatives are emerging from the work of neuroscientist Karl Zilles (Institute of Medicine, Research Center Jülich, Germany). He prepares magnetic resonance images of monkey, ape, and human brains and then uses a nonlinear elastic algorithm to transform one brain into another ( Figure 2 ). “We know what forces we have to apply to the images to do this”, he explains, “which tells us which areas of the brain have changed most during primate evolution”. Zilles and his colleagues also are currently using molecular imaging techniques to update the existing maps of the different areas within our brains. Until we have this information, it is hard to make meaningful comparisons between our brain and that of chimpanzees. Already, Zilles has discovered that there is much more interindividual variation in human brain organisation than anyone suspected. This means, says Zilles, “that a general statement like ‘the neocortex is bigger in human brains than in ape brains’ actually tells us very little. It gives us the general direction that evolution has taken but not whether an ape brain is different because of its sensory, motor, or association areas.” Figure 2 Magnetic Resonance Imaging of Brains Three-dimensional reconstruction of a reference bonobo (pygmy chimpanzee) brain (A) and a reference human brain (B) after magnetic resonance imaging and normalisation of absolute brain sizes. The virtual bonobo brain has been transformed into the virtual human brain using an elastic deformation algorithm. The local deformation vectors are colour-coded and projected onto the virtual human brain (C). The most dramatic changes in brain shape occur in (1) the ventro-orbital prefrontal cortex, (2) the ventral stream of the visual cortex, and (3) the hypothalamic neuroendocrine region. (Image: Karl Zilles, Hartmut Mohlberg, and Peter Pieperhoff, Research Center Jülich) Scientists are also using other techniques to investigate more subtle changes in the organisation of the human brain compared to the brains of other mammals and primates. Indeed, says Holloway, the reorganisation of the brain during evolution has been at least important as its increase in size. Neurobiologist John Allman (California Institute of Technology, Pasadena, California, United States) and his collaborators, for instance, have discovered that a special type of large spindle-shaped neuron, first described in the early 20th century by Constantin von Economo, is unique to apes and humans and much more numerous in the latter. These neurons are found in brain areas that are implicated in decision making in uncertain situations so, Allman speculates, they may help humans to interact rapidly in complex social situations. Costs and Benefits A bigger, more complex brain may have advantages over a small brain in terms of computing power, but brain expansion has costs. For one thing, a big brain is a metabolic drain on our bodies. Indeed, some people argue that, because the brain is one of the most metabolically expensive tissues in our body, our brains could only have expanded in response to an improved diet. Another cost that goes along with a big brain is the need to reorganise its wiring. “As brain size increases, several problems are created”, explains systems neurobiologist Jon Kaas (Vanderbilt University, Nashville, Tennessee, United States). “The most serious is the increased time it takes to get information from one place to another.” One solution is to make the axons of the neurons bigger but this increases brain size again and the problem escalates. Another solution is to do things locally: only connect those parts of the brain that have to be connected, and avoid the need for communication between hemispheres by making different sides of the brain do different things. A big brain can also be made more efficient by organising it into more subdivisions, “rather like splitting a company into departments”, says Kaas. Overall, he concludes, because a bigger brain per se would not work, brain reorganisation and size increase probably occurred in parallel during human brain evolution. The end result is that the human brain is not just a scaled-up version of a mammal brain or even of an ape brain. For natural selection to work, the costs of brain evolution must be outweighed by the advantages gained in terms of fitness. For many years, explains ecological psychologist Robin Dunbar (University of Liverpool, United Kingdom), “people thought that the ability to hunt or forage better was what drove the evolution of our brains. But a better diet had to come before we could grow a bigger brain.” Dunbar believes instead that brain evolution in primates and more generally in mammals “has been driven by the need to manage social relationships, and in primates, in particular, to coordinate coherence in social groups through time and space”. More complex social interactions, he says, mean that individuals are better able to pool resources to solve problems like finding food, and so they survive better. This theory, says Dunbar, is supported by a correlation between social group size and neocortex size across primates and modern humans. Furthermore, during primate brain evolution, the trend has been to add more material to the front than the back of the brain. The front of the brain is where information from the rest of the brain is interpreted, and the capacity to interpret information underlies social interactions, says Dunbar. The number of problem-solving cognitive tasks you can do may well depend on how much frontal lobe volume you have and how it is organised. Just think of how few moves you can run a chess game into the future with a 1980s personal computer compared to a 21st century mainframe machine, he suggests. The human brain is not just a scaled-up version of a mammal brain or even of an ape brain. The Genetics of Human Brain Evolution Selective pressures like those considered by Dunbar and, before him, by scientists like Holloway work on the raw material of random gene mutations, and molecular biologists now have some clues to the gene changes that may underlie brain evolution. Take brain size, for example ( Figure 3 ). Research groups, including those led by Lahn, neurologist Christopher Walsh (Harvard Medical School, Boston, Massachusetts, United States), and clinical geneticist Geoffrey Woods (University of Leeds, United Kingdom), wondered whether the genes that cause microcephaly, an inherited human disorder in which brain size is greatly reduced, might include genes involved in human brain evolution. In 2002, mutations in the genes ASPM (abnormal spindle-like microcephaly associated) and microcephalin were identified as two causes of microcephaly. Three groups have since reported that both these genes have been under selective pressure during primate evolution. ASPM encodes a protein involved in spindle formation, so it is tempting to think that changes in its sequence might result in an increased rate of cell division and hence brain size. But, cautions Walsh, “we really have no idea yet how or even if ASPM is involved in brain evolution”. Figure 3 Primate Brain Sizes These skulls are from the Harvard Museum of Comparative Zoology. (Image: Christopher Walsh, Harvard Medical School) Both Lahn and Walsh believe that ASPM and microcephalin may be only the tip of the iceberg when it comes to genes that have helped to shape our brains. For example, Walsh has recently reported that deletion of a gene called Nde1 produces mice with very small brains. “Our experiments indicate that the loss of Nde1 causes neurons to mature prematurely. This stops them dividing so the mice end up with small brains”, explains Walsh, who is now investigating whether human NDE1 variants have been positively selected during human evolution. Lahn is also searching for additional candidate genes that might help to explain how our brains evolved. In a recent Cell paper, Lahn and his colleagues identify several hundred genes that are involved in nervous system biology and show that, as a group, there are significantly higher rates of protein evolution in these genes in primates than in rodents. Protein evolution rates are particularly high in the lineage leading from ancestral primates to humans, notes Lahn, “so some of these genes may regulate brain size and behaviour”. However, he warns, as with ASPM and microcephalin , “definitive proof for this will only come from functional studies, which are difficult to do”. Enter Glutamate Dehydrogenase For one gene, evidence for an effect on brain function may be emerging. Geneticist Henrik Kaessmann (University of Lausanne, Switzerland) studies the origin of new genes in primates, in particular genes that arise when a DNA copy of an mRNA transcribed from an existing gene is integrated back into the genome. Usually this new “retrocopy” is not expressed, but if the DNA inserts near an active promoter, it can become a transcribable “retrogene”. This is the origin of GLUD2 , a retrogene derived from GLUD1 , which encodes glutamate dehydrogenase. GLUD2 , which first appeared 18–23 million years ago in hominoids, probably immediately picked up a brain-specific promoter and then over the next few million years acquired two critical amino acid changes, explains Kaessmann. These allow GLUD2 -encoded glutamate dehydrogenase to work better in the brain than the GLUD1 -encoded enzyme. Because glutamate dehydrogenase recycles the neurotransmitter glutamate, the presence of GLUD2 may permit a higher neurotransmitter turnover and greater neuronal activity in hominoid brains than is possible in monkey brains, which lack GLUD2 , suggests Kaessmann. Gene Expression Kaessmann plans to search his extensive database of retrocopies in the human genome for other functional genes that could, like GLUD2 , be implicated in brain evolution. By contrast, evolutionary neurobiologist Todd Preuss (Yerkes Primate Research Center, Emory University, Atlanta, Georgia, United States) hopes to identify genes involved in human brain evolution by comparing gene expression patterns in different primates. Preuss, who began training as a paleoanthropologist before turning to neuroscience, has been comparing post-mortem human and chimpanzee brains since the mid 1990s, believing that “if we want to understand human brain evolution, we really have to compare humans with chimpanzees, our nearest relatives”, even though chimp brains have been evolving separately from ours for 5–7 million years. But, warns Preuss, “we have to do these studies now. There are few chimps left and if we lose the opportunity to study them and their brains, we will lose forever a fundamental source of insight into our own species.” To begin with, Preuss used staining techniques that exploit antibodies to examine the neural components of chimpanzee and human brains. Then in 1998, he was asked to collaborate in a microarray project. “My antibody approach was very labour intensive so I jumped at the opportunity to screen 10,000 genes at once”, he says. Preuss and his collaborators now know that more than 100 genes are differentially expressed in chimpanzee and human brains. “Importantly, when we go back into tissue with probes for these gene products, in some cases there are remarkably different spatial patterns of expression in humans, chimps, and macaques”, notes Preuss. “We don't know yet what these differences mean in terms of functional organisation in these different brains but our results open up new and exciting vistas”, particularly since many of the differentially expressed genes have not previously been considered as being potentially involved in brain evolution. The microarray data produced by Preuss and other researchers also indicate that many of the gene expression changes that have occurred during brain evolution involve gene upregulation. For example, there is increased expression of genes involved in metabolism, synaptic organisation, and synaptic function. “All told, it seems that the human brain may be more dynamic than ape or monkey brains”, says Preuss. “The human brain seems to be running hot in all sorts of ways.” Scratching at the Surface As far as understanding how our brains evolved, more questions remain than have been answered. One problem is that we don't really know enough about how our brains differ from those of other mammals and primates, although work by Zilles and others is helping here. We also know very little about how the areas of our brain are physically linked up, and we need to understand that before we can see how we differ from our nearest relatives. And as far as identifying the gene changes that were selected during evolution, although we have several candidates, we don't know how or if these gene variants affect our cognitive abilities. It is one thing, concludes Dunbar, to identify genetic or anatomic differences between human and ape brains, but quite another to know what they mean in terms of actual cognitive processes.

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1065705

The Immune Epitope Database and Analysis Resource: From Vision to Blueprint

A planned repository of immune epitope data with associated analysis tools should be a boon to vaccine development

Introduction Recent concerns about bioterrorism and emerging diseases have led to a new focus on the development of vaccines and drugs targeting infectious pathogens. An important component of vaccine development is the characterization of immune responses (to vaccination, for example, or following infection in experimental settings) by evaluating the epitopes recognized by antigen-specific receptors of the immune system (antibodies and/or T cell receptors (TCRs)) [ 1 ]. In recent years, different groups have followed different approaches to the discovery of immune epitopes, and various assay types have been used to generate data for the purpose of epitope definition or validation. We believe that research in this area could be greatly facilitated by a comprehensive knowledge center: a repository of immune epitope data with associated analysis tools. Our goal is the creation of the Immune Epitope Database and Analysis Resource (IEDB). The IEDB is sponsored by the National Institute for Allergy and Infectious Diseases (NIAID). It will host data relating to both B cell and T cell epitopes from infectious pathogens, as well as experimental and self-antigens (RTP-NIH-NIAID-DAIT-03/31; www.niaid.nih.gov/contract/archive ). Priority will be placed on those epitopes considered to be potential bioterrorism threats, and emerging diseases as defined by NIAID (so-called category A–C pathogens; see: http://www2.niaid.nih.gov/Biodefense/bandc_priority.htm ). As a corollary to the IEDB effort, NIAID has also launched a large-scale antibody and T cell epitope discovery program aimed at generating epitope data and analysis resources to be included in the IEDB. Other data sources to be integrated into the IEDB are publications in peer-reviewed journals, published patents or patent applications, and direct submissions from institutions or companies. Everyone who contributes data or analysis resources to the database will be cited, either by authorship or by other acknowledgment of their contributions. The involvement of the scientific community in the design of the scope and capability of the IEDB will be crucial to the success of the project. The IEDB will be produced in a manner that encourages the incorporation of data and analytical tools derived by research labs at-large. With this paper, we hope to inform the scientific community of our effort and to solicit feedback while the project is still in a design stage. We envision that this resource center will be freely available on the Internet, with a prototype operational in the fourth quarter of 2005. Once the project is online, forms for direct feedback and online submission of data and tools will be provided. Yearly conferences to present data relating to epitope identification and the IEDB itself will be organized, and a newsletter will be published quarterly. Defining the Scope of the IEDB Each scientific approach generates a set of epitope data, specific to itself, which must be integrated into a general representation of epitope information. In a programmatic sense, we believe that selecting data that fit one particular epitope definition or experimental bias is not our prerogative and would be unwise. Rather, we have opted to define a comprehensive, all-inclusive representation of information that separates epitope features into intrinsic and extrinsic features. Intrinsic features are those determined by the sequence and structure of an epitope, while extrinsic features are context-dependent attributes determined by the experimental or natural environment. This immunological perspective will be an organizing principle behind the IEDB. Intrinsic Versus Extrinsic Features of an Epitope At the level of T cell epitopes, intrinsic features included in the IEDB are: the molecular structure of the epitope, the binding affinity for different MHC receptors, and the affinity of MHC/ epitope complexes for TCRs of defined sequence. Likewise, at the level of B cell epitopes, intrinsic features include the epitope's molecular structure and binding affinity for antibody molecules of defined sequence. These features are unequivocally specified and are singularly associated with a given epitope structure or epitope/receptor combination. Other features—such as immunogenicity, or whether an epitope is naturally processed—are not intrinsically associated with a given molecular structure of an epitope alone, but rather are context-dependent (i.e., extrinsic). Context information includes, for example, the species of the host in which a response was found, the assay utilized to measure responses, and the dose and route of administration. Likewise, the yield of a given epitope following proteasomal cleavage of a complex protein precursor is dictated by the overall sequence of the protein in which the epitope is contained. Also, the T cell and B cell responses to an epitope are heavily influenced by previous exposure of the immune system to the same or a related antigen. Collectively, these examples show that to meaningfully capture the immunogenicity of an epitope, the context in which it occurs must be described as well. The IEDB Classes Formalizing the above considerations, we defined the main classes of the IEDB data as Reference, Epitope, Binding, and Context ( Figure 1 ). These classes represent the top level in the data hierarchy used to store epitope information in the IEDB. The class Reference defines one of three possible sources of data, namely literature, patent, and direct submissions. The Epitope class is subdivided into two categories: Epitope Structure, which specifies the molecular structure of an epitope itself, and Epitope Source, which identifies the pathogen/protein in which the Epitope is present. The Binding class captures intrinsic information relating to how the structure specified in the “Epitope” class interacts with well-defined receptors of the immune system such as MHC molecules or antibodies and TCRs of defined sequence. The Context class is organized into three subclasses, including T cell immune responses, naturally processed peptides, and B cell immune responses. Table 1 is an example how the main features of a T cell epitope described in [ 2 ] would be displayed in the IEDB. Many more fields exist that are left blank because they are not appropriate for this particular epitope (such as antibody binding data) or are unknown (such as MHC binding data). Figure 1 Main Classes in the IEDB Table 1 Sample Epitope Entry in the IEDB A Scientific Approach for the Development of the Analysis Resource Our proposal includes the establishment and maintenance of an Analysis Resource of online tools for the Immune Epitope Database. Because this resource must be useful to the entire community, it is important that the tools provided cover a broad range of research areas relating to epitope discovery and analysis, and that no particular scientific “school” has priority. To identify tool candidates, we have generated a list of existing tools of interest through extensive literature searches and expert input. This will be periodically revised, taking advantage of input from the scientific community and NIAID. The current list of candidate tools comprises an extensive menu of prediction tools for the identification of novel antibody and T cell epitopes from genome and protein sequences. At the level of antibody epitope predictions, standard methods of predicting which regions in a protein are likely to be on the surface will be provided, such as hydrophilicity analysis. Tools that use various methods for prediction of MHC binding will also be provided, along with tools predicting proteasomal processing and TAP transport of T cell epitopes. We will also provide analytical tool resources to assist in vaccine discovery and development. These are designed to project population coverage of epitopes in different ethnicities, to project the degree of cross-reactivity within sets of different MHC molecules, and to assess the degree of conservancy of an epitope in various isolates of the same pathogen, both in related pathogens, and in potential hosts. Finally, tools to visualize data will be provided, such as those that display antibody antigen interactions where 3D structural information is available. We also hope that collection of consistently annotated data in the IEDB will allow the development of new, “context-sensitive,” tools. In deciding how many tools should be hosted in the IEDB, a balance has to be achieved between discriminating too much, which may leave user demands unaddressed, and discriminating too little, hosting so many tools that the collection becomes overly redundant and unmanageable. To facilitate an objective and transparent choice of which predictive tools should be hosted, the predictions of all candidate tools will periodically be evaluated. Most importantly, we plan to make all evaluations publicly available through the IEDB website, and we will encourage all different scientific groups to participate by submitting tools and evaluating data. Such prediction “contests” have had a tremendous positive impact in the field of tool evaluation and prediction of protein structure [ 3 , 4 ]. To the best of our knowledge, this would represent the first attempt at a rigorous and comprehensive evaluation of prediction tools found on immune responses. Conclusions We envision a future in which the development of the Immune Epitope Database and Analysis Resource will help researchers throughout the world quickly access relevant information for evaluation of immune responses, assisting them in the development of prophylactic/therapeutic approaches against new and old, emerging and reemerging diseases.

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1065706

Chromosome Cohesion: A Cycle of Holding Together and Falling Apart

When a cell prepares to divide, the chromosomes need to separate at just the right moment. Regulating the cohesion of chromosomes is key to achieving this

All organisms have mechanisms to ensure that dividing cells produce new cells with the proper number of chromosomes. The dividing cell closely monitors that chromosomes are copied exactly once and then distributed correctly to daughter cells. After replication, the chromosomes (now comprising two chromatids) align at the center of the cell, and are attached to a structure known as the spindle apparatus. A key point of attachment is the centromere, a characteristic constriction carried by each chromosome. The spindle, which is composed of microtubules, pulls the chromatids apart so that two complete sets of chromosomes are gathered together at each pole of the cell, which can then divide. Cohesion between chromosome copies, which keeps the chromatids together until just the right time, therefore plays a critical part in this process. Chromosome cohesion is established during S phase (when the chromosomes are replicated) and is then dissolved completely in metaphase to allow sister chromatids to come apart. The dissolution of cohesion is highly regulated; human cell lines that have defects in the regulation of cohesion show the hallmarks of cancer cells [ 1 ]. Furthermore, it has been suggested that the abnormal karyotypes that result in diseases such as Down syndrome are the result of the improper dissolution of chromosome cohesion [ 2 ]. Finally, mutation of a factor required to load cohesin—the protein complex responsible for chromosome cohesion—onto chromosomes appears to cause Cornelia de Lange syndrome, a clinically heterogeneous developmental disorder that may include facial dysmorphia, upper-extremity malformations, hirsutism, cardiac defects, growth and cognitive retardation, and gastrointestinal disorders [ 3 , 4 , 5 ]. Cohesion serves at least three roles in the cell with respect to accurate genome transmission. First, cohesion close to the centromere facilitates bi-orientation of chromosomes, such that each chromosome becomes attached to the two poles of the spindle [ 6 ]. Second, it prevents the splitting of chromosomes until all bipolar attachments are made [ 6 ]. The function of cohesion at the centromere is presumably to oppose the force of microtubules, which pull the chromosomes to opposite spindle poles; this force is not exerted along the chromosome arms, which means that cohesion at centromeres and along arms is functionally distinct. Third, cohesion along chromosome arms may be essential for proper chromosome condensation [ 7 , 8 ], although the function of cohesion at chromosome arms is something of a mystery. Differences between Arms and Centromeres Cohesion in eukaryotic cells is mediated by a multi-subunit protein complex called cohesin. Cohesin consists of four proteins: Smc1, Smc3, Scc1/Mcd1 (also known as kleisin), and Scc3 (SA2). The Smc (structural maintenance of chromosomes) proteins form intramolecular coiled coils that have been observed in the electron microscope to form a V shape with sides that are 50 nm long [ 9 ]. The cohesin complex has been proposed to form a ring structure that encircles sister chromatids [ 10 ]. Alternately, two rings may snap sisters together via interactions between the coiled coils of the Smc proteins [ 11 ]. All members of the cohesin complex are essential in budding yeast, Saccharomyces cerevisiae , since mutation results in the precocious dissociation of sister chromatids. Functional orthologs of these proteins exist in all eukaryotes. There are at least two types of cohesin sites: (1) cohesin associated with the centromere and the nearby pericentric domain, and (2) cohesin associated with chromosome arms [ 12 , 13 , 14 , 15 ]. In S. cerevisiae , cohesin at centromeric and pericentric domains is spread over a broad region (up to 50 kb), large quantities of the protein complex are bound, and binding is not affected by the natural transcriptional and coding status of the regions that are occupied. By contrast, binding sites in arms tend to be much smaller (about 1 kb)—at least in S. cerevisiae , where they have been most extensively characterized—and of lower intensity, and are spaced at approximately every 11 kb (see Figure 1 ). Cohesin in arms localizes to regions lacking transcription in yeast [ 12 , 16 , 17 ]. This reinforces the view that there may be functional differences in arm and pericentric cohesion and perhaps different mechanisms to load cohesin, as has been proposed for cohesin on meiotic chromosomes for S. pombe [ 18 ]. A unifying feature of all cohesin-binding sites in S. cerevisiae is high AT (adenine and thymine) content [ 12 , 15 ]. Figure 1 Cohesin Sites for Sister Chromatids of Chromosome I in S. cerevisiae Cohesin sites (red ovals) are concentrated at the centromere/pericentric region (where the two chromatids are “pinched”), but also occur along the arms of the chromatids. Another important difference between cohesin binding along arms and at centromeres is that the arm sites do not appear to be dependent on a DNA consensus sequence, whereas binding to pericentric regions requires specific centromere sequence [ 13 , 14 ]. The S. cerevisiae centromere sequence is composed of three DNA elements (CDEI, CDEII, and CDEIII). Studies of cohesion at the centromere reveal that as little as 100 bp (a portion of CDEII and the entire CDEIII) are required to direct cohesion [ 13 , 14 , 19 ]. Mutations in the protein Ndc10 have also been shown to affect cohesin deposition at centromeres. Ndc10 forms part of a structure known as the kinetochore, which forms around the centromere and is responsible for the attachment to the spindle; establishment and maintenance of cohesion at pericentric regions may therefore rely on both the centromere sequence and kinetochore function [ 13 , 20 ]. Presumably both arm and pericentric cohesion are important for chromosome dynamics, but the functional differences between the two are not well understood. Cohesion—It's Just a Phase Cohesion can be divided into four phases that occur during the cell cycle ( Figure 2 ): (1) deposition in G1 (the gap in the cell cycle before S phase), (2) establishment in S phase, (3) maintenance in G2 (the gap between S and mitosis), and (4) dissolution in mitosis. During G1, Scc2 and Scc4 are responsible for loading cohesin onto unreplicated double-stranded DNA [ 21 ]. Then, during S phase, several proteins are involved in establishment of cohesion between replicated chromatids. Eco1 and Chl1 are required for establishing cohesion but not for maintenance [ 22 , 23 , 24 ]. The associations between cohesion and DNA replication have led to a model whereby cohesion is established coincident with the passage of the replication fork [ 25 ]. This requires an alternative replication factor C (RF-C) complex [ 26 , 27 , 28 ] and may require the origin recognition complex (ORC) [ 29 ]. Cohesion is maintained during G2 by the cohesin complex, and is eventually dissolved in mitosis to allow sister chromatids to separate. Figure 2 Behavior of Cohesin during the Cell Cycle One cohesin complex is depicted at each site for the sake of simplicity; at the centromere especially there are likely to be many complexes. Cohesion is represented as occurring via the “encircling” model; other models have been proposed. The dissolution of cohesion is regulated by at least two mechanisms. First, subunits of the complex may be phosphorylated, which facilitates their removal. In S. cerevisiae and human cells, phosphorylation of Scc1/Mcd1 by Polo kinase makes it a better substrate for proteolysis [ 30 , 31 , 32 ]. In this issue of PLoS Biology , one of two related papers exploring the regulation of cohesin in vertebrates shows that phosphorylation of Scc3 (SA2) by Polo-like kinase is apparently sufficient to allow dissociation from chromosome arms, which occurs during prophase [ 32 ]. In Xenopus extracts, phosphorylation of cohesin also depends on Polo-like kinase, and this phosphorylation reduces the ability of cohesin to bind to chromatin [ 8 ]. The second mechanism that can facilitate the dissolution of cohesin is proteolysis; this may be particularly important at centromeres. The Scc1/Mcd1 component of the cohesin complex is cleaved by a separase (Esp1) whose activity is held in check by a securin (Pds1) until separation at the metaphase-to-anaphase transition [ 33 , 34 ]. Separase is a cysteine protease that cleaves Scc1/Mcd1, presumably resulting in the cohesin complex falling apart and being unable to hold sister chromatids together. Scc1/Mcd1 at pericentric regions is protected from phosphorylation during prophase—and therefore dissociation from chromosomes is prevented—by proteins known as shugoshins [ 35 , 36 , 37 ]. In the second paper on cohesin in this issue of PLoS Biology , McGuinness et al. show that shugoshin specifically protects Scc3 (SA2) at the centromere, so that centromeric cohesion is preserved until the chromosomes are ready to separate [ 35 ]. Vertebrate shugoshin has been shown to have a strong microtubule-binding domain [ 36 ] and is found at the kinetochore [ 37 ]. Recent evidence suggests that shugoshin in S. cerevisiae may sense tension between sister chromatids, acting as part of a spindle checkpoint that monitors whether chromosomes are properly aligned on the mitotic spindle [ 38 ]. It is currently unclear why the cell has two mechanisms to dissociate cohesin from chromosomes, although it is interesting to speculate that this could be related to different functions of cohesin at chromosome arms versus pericentric domains. For instance, cohesin in chromosome arms may help to organize or condense chromosomes, whereas cohesin at centromeres may be more directly involved in chromosome bi-orientation at the spindle and segregation. These functions may be important during different phases of the cell cycle. A Link between Chromatin and Cohesin Several results suggest that transcription and cohesin binding are incompatible. In Drosophila , one of the components that loads cohesin (Nipped-B or Scc2) has also been shown to be required for long-range promoter–enhancer interactions [ 39 , 40 ]. One model proposed to explain this result is that cohesin can prevent long-range promoter–enhancer interactions and that removal of cohesin can restore these interactions and allow transcription to occur [ 41 ]. In this model, Nipped-B or Scc2 can act as both a loading factor and an unloading factor for cohesin. This model further speculates that rather than Cornelia de Lange syndrome stemming from a cohesin loading defect, the failure to unload cohesin from regions that need to be transcribed leads to transcriptional defects that cause the syndrome. In S. cerevisiae it has been shown that driving transcription through a centromere via an inducible promoter prevents cohesin from associating and results in chromosome missegregation and cell death [ 13 ]. Cohesin is found at the boundaries of the HMR locus, the right telomere of Chromosome III, and the RDN1 array, all regions of silent chromatin [ 16 ]. Cohesin localizes to intergenic regions where transcription is converging [ 12 , 17 ]. Since transcription and chromatin configuration are intimately related, it is possible that chromatin may play an important role in the localization of cohesin. Indeed, the chromatin remodeling complex RSC (remodels the structure of chromatin) has been shown to be important for establishment of cohesin binding [ 42 ], and another study suggests RSC is particularly important for cohesin association with chromosome arms [ 43 ]. The chromatin remodeling complex ISWI (SNF2h) has been shown to be essential for cohesin to localize to Alu repeats (certain DNA sequences) in human cells [ 44 ]. The possibility also exists that cohesin itself may influence transcriptional status and act as a transcriptional boundary [ 39 , 40 , 45 ]. The preferential location of cohesin in heterochromatin in pericentric regions in S. pombe also supports the idea that chromatin modification/structure is a key determinant of cohesin localization [ 46 , 47 ]. It is interesting to speculate that chromatin differences and transcriptional differences between chromosome arms and centric regions will turn out to be related to different mechanisms for loading and removal of cohesin from these regions. While one of the primary roles for chromosome cohesion in bi-orientation and mitotic chromosome segregation is well-established, the complexities of the regulation of cohesion are still being discovered. Cohesin may be involved in multiple ways in chromosome dynamics. Future studies focusing on the differences between cohesion at chromosome arms versus pericentric domains and the link between cohesion and transcription will likely yield very interesting insights into the function of the cohesin complex in the maintenance of genome integrity.

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1065707

Funding the Way to Open Access

The Wellcome Trust - the UK's largest non-governmental funder of biomedical research - is taking action to ensure the work it supports is available to all

Imagine this scenario. You're the director of one of the world's largest medical research charities, and you receive notification from one of your funded investigators in Africa reporting some exciting progress toward the development of a vaccine for malaria. The work has just been published, so you log onto the Web to do a quick keyword search, and a link to the article is brought up on your screen. Then imagine the frustration when you click on the link to read the message, “Access Denied—access to this journal is restricted to registered institutional and individual subscribers.” And there's the rub: this actually happened to the Director of the Wellcome Trust. Prior to this, the committee that advises the Wellcome Trust Library were already asking whether the Trust should adopt a formal position on the continually increasing prices of journal subscriptions and the problems this trend was causing research libraries. These events encouraged the Trust to investigate the publication of scientific research, to see if there was anything research-funding organisations could be doing to stimulate change in what appears to be a failing market. As it turns out, there is quite a lot. I now believe it is the funders of research—charities, governments, and other publicly funded bodies such as national research agencies—who hold the purse strings that can untie scientific discoveries from a publishing market that is no longer serving the community as well as it could. That is why today the Trust is a leading advocate for enabling free access to research literature through support for new publishing models, such as that of the Public Library of Science, and the establishment of publicly accessible repositories, working in partnership with the United States National Institutes of Health–funded PubMed Central [ 1 ]. It is worth noting that the Trust is not a novice in seeking better ways to disseminate research findings. The fact that the sequence of the human genome is an openly accessible work is due in large measure to the Trust's determination that this information be in the public domain and not hidden behind commercial subscriptions. As a consequence of that insistence, we believe, these data are a more widely used and valuable resource. “Trust-funded researchers will have to deposit an electronic version of their manuscripts in PMC to be made available for free via the Internet within 6 months of publication.” The Trust began its investigation of the scientific publishing sector by commissioning two pieces of research: one to inform itself of the economics of the publishing sector, and a second to explore whether there were alternative business models out there that could enable research to have the quality assurance it needs (peer review) whilst being available for free, using the Web as the medium of publication. The Economics of Publishing The first Trust-commissioned study described how scientific research publishing has traditionally worked and why it can be described, in economic terms, as a failing market [ 2 ]. Essentially, the producers (researchers as authors) and the consumers (researchers as readers) are isolated from any of the costs within the system. Researchers give away the copyright to their work, for free, to the publishers, who organise the peer review and copyedit the article. The publishers then sell it to libraries at prices that range from enough to cover their costs through to some pretty high profits—some over 30%. These profits escape from an otherwise self-contained financial cycle to satisfy shareholders or run learned societies; unlike typical publishing relationships, none are returned directly to the author (the researcher who wrote the piece) or even to the consulting experts (the researchers who provided the peer review). At the same time, researchers as readers access the material, if they are able to do so, through their employing institution, either using the library or—more typically now—via the Internet through the institution's subscription. To the researcher this access appears free, effectively creating a market system that has no pressures from the producers or consumers to change. One consequence of this is that publishers have been able to increase subscription prices well above inflation; the United Kingdom has seen subscription rates rise by more than 200% in the last ten years (Blackwell's periodical price indexes; [ 3 ]). The money used to fund UK libraries is all public money, and over 90% of the funds paying for research in the UK university system is either government or charitable [ 4 ]—so in a sense the people who are paying for the research cannot access its findings without paying an additional fee. Access denied at The Journal of Infectious Diseases This then begs the question of what alternatives there are to this traditional system, now that the Internet has become the researcher's tool of choice for searching and accessing the literature. The second piece of research commissioned by the Trust looked at different business models for research publishing, in order to address this question [ 5 ]. It compared open-access journals, which often levy a charge to publish but provide the journal for free, and the majority of the traditional models, which take the research for free but charge readers to read it. This study convinced the Trust that the best way forward to improve access to research findings would be through open access to scientific research articles. This essentially means two things: first, that the copyright holder or holders must grant to the public a free, irrevocable, perpetual license to use, copy, distribute, and make derivative works of their research article, in any medium for any purpose (excepting those that constitute plagiarism or other dishonest acts, of course); and second, that a digital copy must be deposited in an open public archival repository (for example, the US National Library of Medicine's PubMed Central). Whilst a debate continues as to the most appropriate route to achieve open access to all research literature, it is important to bear in mind that the publication and the archiving of research articles are intrinsically linked. Both aspects of open access need to be explored and experimented with, and the Trust is actively pursuing solutions for the problems of both. Alternative Business Models The findings of the second report seem to have caused quite a controversy—particularly in the suggestion that moving wholesale to an open-access publishing model might produce savings of up to 30% [ 6 ]. One common misinterpretation of this conclusion is that any such savings would be due solely to discontinuing the printed versions of publications that are freely available online. This is incorrect. In fact, if savings are to be made in an open-access model, they will largely be found in the variable costs of journal production—since an open-access journal will not have to cover the costs of subscription management, licence negotiations, or sales, and little is required for marketing and distribution. In a comparison included in the report, an article in a good- to high-quality journal produced in the subscription model is estimated to cost US$2,750. The equivalent cost under an author-side payment model is estimated as US$1,950—a comparable saving of 30% on the costs, and a saving of 90% when the variable costs are compared. It must be remembered that cost does not equate to price, so to these figures, regardless of the mode of publication, must be added overhead expenses and, of course, profit. However, if a truly competitive market is created—where payments are directed to publishers not by third parties but by those directly involved in the scientific enterprise, who could easily compare the varying article processing charges of different open-access publishers—then the actual savings might well be substantially higher. At its essence though, the open-access debate is not about economics, it is about access. That is why the Trust has been in discussion with the US National Library of Medicine about the possibility of creating a UK PubMed Central (UKPMC) as a publicly accessible repository for Trust-funded research. UK PubMed Central The proposal is that a UKPMC will be run as a proper electronic library: it will collect, collate, and archive whole journals and be developed to receive single articles as well. Submission will be as straightforward as attaching a document to an email. UKPMC will be able to accept manuscripts in any format, including Microsoft Word, and it will be the responsibility of UKPMC to convert the files it receives into extensible markup language (XML) to enable the appropriate document type definition (DTD) to be assigned. UKPMC will also correct the structural, content, and consistency errors that occur when converting text for digital preservation, and provide the conversion process to print a “clear” PDF version of included articles to those users who download them. This is a process well used by the National Library of Medicine, and the one most suited for the long-term, digital preservation of articles. And once articles are in a digital format they can be searched and used in different ways. For example, genome sequence data, chemical compounds, or protein structures embedded within an article can be searched for in other articles and linked directly to genome or structural databases uncovering new genetic markers, drug uses, or protein functions. The articles themselves become live research material greatly improving the efficacy of the research itself. For a funder, having all its research in one format, “under one roof”, and searchable will improve the efficiency of strategy setting—for example, setting funding priorities—assessing the outputs of the funded research, and even gaining an insight into the impact of the work. As grants management becomes more electronic, there can be a direct link between original research proposals and the research outputs. For a medical charity like the Trust, I believe it is our duty to actively encourage the most efficient processes available to maximise the likelihood that the research we fund will have the greatest possible health benefit. That is why the Trust will be making it a requirement of its grant conditions that Trust-funded researchers deposit an electronic version of their manuscripts in UKPMC to be made available for free via the Internet within 6 months of publication. The delay means that this is not open access in the truest sense. However, the Trust considers that the development of a PubMed Central portal in the UK offers the best next step in the transition towards a situation where all high-quality peer-reviewed research is available for free via the Internet, whilst leaving all publishers room for manoeuvre in this changing market.

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1065708

Microfauna–Macrofauna Interaction in the Seafloor: Lessons from the Tubeworm

New research and techniques are beginning to provide intriguing clues into the complex relationships that tubeworms form with other species at hydrothermal vents and deep-sea cold seeps

Since their discovery in the 1970s and 1980s, giant tubeworms at hydrothermal vents and cold seeps have fascinated biologists and laymen alike—not only for their alien morphology ( Figure 1 ), but also for epitomizing the perfect animal–microbe symbiosis. They are among the biggest worms on this planet—some over 3 m long—yet they do not eat other organisms. Tubeworms thrive independently of photosynthetic production [ 1 ]. They have even lost their entire digestive tract. One of the most exciting findings in early tubeworm research was the discovery that the worm's food is delivered by bacterial symbionts [ 2 ]. The chemoautotrophic symbionts live intracellularly in a specialized worm tissue called the trophosome. They are sulfide oxidizers, using the free energy yield from the oxidation of sulfide with oxygen to fix carbon dioxide with their bacterial Rubisco enzyme. In exchange for providing nutrition for the worm, the symbionts are sheltered from grazing, but most importantly, they receive a steady source of sulfide and oxygen via the highly adapted blood circulation system of the worm. (I will never forget how horrified I was as a young student by the amounts of almost human-like blood flowing into my lab dish while dissecting tubeworms to analyze trophosome enzyme activity.) Tubeworm blood physiology, in particular the hemoglobin molecules, are tailored specifically to the needs of the symbionts. However, the host metabolism in itself is not different from that of many other animals, the main source of energy being aerobic respiration of carbohydrates. In other words, tubeworms and their symbionts need oxygen as an electron acceptor—so, after all, they are dependent on photosynthesis, the main oxygen-producing process on earth. Figure 1 Vestimentiferan Tubeworms (A) Close-up photograph of the symbiotic vestimentiferan tubeworm Lamellibrachia luymesi from a cold seep at 550 m depth in the Gulf of Mexico. The tubes of the worms are stained with a blue chitin stain to determine their growth rates. Approximately 14 mo of growth is shown by the staining here. (Photo: Charles Fisher) (B) Close-up photograph of the base of an aggregation of the symbiotic vestimentiferan tubeworm L. luymesi from a cold seep at 550 m depth in the Gulf of Mexico. Also shown in the sediments around the base are orange bacterial mats of the sulfide-oxidizing bacteria Beggiotoa spp. and empty shells of various clams and snails, which are also common inhabitants of the seeps. (Photo: Ian MacDonald) Classification of Host and Symbiont With their strange morphology, vent tubeworms were first classified as a novel phylum, Vestimentifera [ 3 ]. Recently they have been regrouped together with the pogonophoran tubeworms ( Figure 2 ) into a family of annelid polychaetes called the Siboglinidae [ 4 , 5 ]. Vestimentiferan tubeworms of hydrothermal vents grow on chimneys and other hard substrates in the vicinity of active vents, which emit reduced compounds like hydrogen and sulfide [ 6 ]. Vestimentiferan tubeworms living at cold hydrocarbon seeps, i.e., the lamellibrachids and escarpids, are adapted to a sedimentary environment, with a substantial part of the body and tube of many species extending into the mud. All vestimentiferan tubeworms found today at vents, seeps, and a few other reduced submarine habitats harbor sulfide-oxidizing endosymbionts in their trophosome. These symbionts belong to bacteria of the gamma-proteobacteria clade and are phylogenetically related to each other [ 7 ]. (For the only known exception see [ 8 ].) Figure 2 Pogonophoran Tubeworms Being Sampled at the Haakon Mosby Mud Volcano (Source: AWI/IFREMER expedition RV POLARSTERN/VICTOR 6000 in 2003) Tubeworm Mysteries The study of tubeworms is now in its fourth decade, and there are still many fascinating problems to be solved. One of the most interesting—but also most difficult—questions in tubeworm symbiosis is how this obligate and highly integrated interaction between microbes and animals evolved. How can a worm evolve into a perfect home for chemosynthetic bacteria? What are the main evolutionary steps towards this symbiosis, and in which order did they occur? Another intriguing problem is how the worms acquire their endosymbionts, which appear to be taken up from the environment—but so far have not been detected as free-living forms. How does the host recognize its specific symbiont from the vast diversity of gamma-proteobacteria and sulfide oxidizers in the environment? Furthermore, how do tubeworms populate new vents, seeps, and other reducing environments emerging from the ever-changing ocean floor—how do their larvae migrate and settle, and what determines the distribution and lifetime of tubeworm populations in the different mid-ocean ridge and continental margin habitats? Although these questions are still to be answered, new research and techniques are beginning to provide intriguing clues. Seep Vestimentifera and Their Energy Source At some seeps the vestimentiferan tubeworms are so abundant that they form a special habitat that is attractive for a host of other marine species [ 9 ]. Seep vestimentiferans are usually thinner, have slower growth rates, and have greater longevity than their vent relatives [ 10 ]. For example, a 2-m-long Lamellibrachia luymesi individual is estimated to be more than 200 y old and hence represents the longest-lived animal on earth [ 11 , 12 ]. At seeps, geological processes causing fluid and gas seepage can last hundreds to millions of years, whereas hydrothermal vents often have a lifespan on the order of decades. Vent tubeworm colonies will die when their chimneys stop venting, i.e., delivering sulfide, so they are adapted to a rapidly changing environment, as typified by their fast growth and high reproduction. Like vent vestimentifera, seep vestmentifera also depend on the availability of sulfide in their direct vicinity, but they are sessile, and anchor on hard substrates such as carbonates. Individual aggregations at seeps can consist of hundreds to thousands of worms, requiring sulfide fluxes of half a mole per day—and this for more than 200 y [ 12 ]. So an ecological problem that has always intrigued biologists and geochemists alike is how these tubeworms obtain their energy over the long term. Because vent and seep vestimentifera depend on sulfide-oxidizing symbionts, their distribution is limited to habitats with high sulfide fluxes lasting for at least a few reproductive cycles. However, at cold seeps, unlike hydrothermal vents, most of the chemical energy occurs in the form of hydrocarbons. Cold seeps are characterized by high fluxes of methane, higher hydrocarbons (such as ethane, propane, butane), and/or petroleum from deep subsurface reservoirs. Often the source fluids and gases do not contain much sulfide, because there are no high-temperature seawater–rock interactions involved in their formation, as there are at vents. Some pogonophoran tubeworms at seeps have teamed with methane-oxidizing symbionts to profit from the high availability of hydrocarbons, but seep vestimentiferans do not appear to be able to directly tap this resource. However, seep vestimentiferans are still capable of producing enormous biomass over many years with the help of their sulfide-oxidizing symbionts. So where does the supply of sulfide come from at seeps that enables such large aggregations to be maintained for so long? Only recently was it realized that anaerobic microbial processes, namely, the oxidation of hydrocarbons with sulfate, could produce astonishingly high fluxes of sulfide in cold seep settings [ 13 , 14 ]. At methane seeps, methanotrophic microbial communities inhabiting the surface sediments oxidize methane with sulfate, which results in very high sulfide fluxes [ 13 ]. If the seepage consists of other hydrocarbons such as petroleum, their degradation with sulfate supports an even higher production of sulfide [ 14 ]. In some seep sediments, sulfide concentrations can reach 25 mM in subsurface sediments (5–10 cm below the sediment surface). Such concentrations are not known from tubeworm habitats at hydrothermal vents. However, the zones of high hydrocarbon turnover and sulfide flux at seeps are often limited to only a few centimeters below the seafloor, depending on hydrocarbon flows and the rate of sulfate transport from the bottom water into the sediments. Sulfate is crucial because the free-living hydrocarbon-degrading microbes in seep sediments depend on this electron acceptor for an energy yield. Without sulfate to fuel the oxidation of hydrocarbons, sulfide production stops, even if there is still an enormous reservoir of hydrocarbon available. How might tubeworms, sulfide-oxidizing symbionts, and benthic hydrocarbon degraders overcome these limitations? Ménage à Trois—A Model Solution Cordes et al. [ 15 ] have now provided an answer to how the stability of sulfide production is maintained over such long periods and how the worms optimize sulfide uptake. Seep vestimentifera have specific adaptations to their habitat. A main adaptation is the subsurface part of the lamellibrachids called a “root.” The tubeworm root appears to have a special function in the energy cycle of the organism—as in plant roots. Several authors have proposed that the worm roots are not only important in sulfide uptake, but generally in geochemical engineering of the sediments in the direct environment [ 16 , 17 , 18 ]. Obviously such hypotheses are very difficult to test—today it is still hardly possible to measure gas, petroleum, and sulfide fluxes in the seafloor in situ at depth, especially below tubeworm aggregations. But it is also not possible to recover whole aggregations of worms and to keep them alive in the lab for biochemical and biogeochemical measurements—this would require simulation of seepage under pressure. Instead, Cordes et al. [ 12 , 15 ] have used geochemical and biological modeling to solve the intriguing question of seep vestimentiferan longevity and how they might also interact with free-living anaerobic microbes to increase sulfide availability. To explain the persistence of the large tubeworm colonies in the Gulf of Mexico, Cordes et al. suggest a broader mutualistic interaction between the tubeworm, its endosymbiont, and benthic hydrocarbon-degrading and sulfide-producing microbes. Seep tubeworms take up sulfide from the sulfide-rich subsurface sediment zones through the roots, but, crucially, they may also release sulfate through the roots as a byproduct of sulfide oxidation by the tubeworm's endosymbiont. Sulfate may also be ventilated through the tube into the sediments. Since anaerobic microbial communities in subsurface hydrocarbon-rich sediments are limited by sulfate influx, any additional supply of sulfate enhances their production of sulfide. Furthermore, the removal of sulfide by the worm will thermodynamically favor anaerobic hydrocarbon oxidation coupled to sulfate reduction. Hence, the tubeworm roots may provide an excellent habitat for anaerobic hydrocarbon oxidizers. For example, Cordes et al. predict in their model that nearly all of the sulfate released through the root will be utilized by benthic microbes for anaerobic hydrocarbon degradation in the direct vicinity of the worm. This process could provide 60% of the sulfide needed by a tubeworm aggregation to persist for 80 y. Hence, it may even be concluded that tubeworms farm anaerobic hydrocarbon degraders to provide a steady supply of sulfide to their endosymbionts. Especially at petroleum seeps, this would guarantee a lifelong energy source and help explain the extraordinary longevity of the worms. The mutual benefit arising from the association of sulfide oxidizers, sulfate reducers, and a host worm is known to be exploited by the oligochaete Olavius algarvensis [ 19 ]. In this very effective “ménage à trois” the sulfate reducer has even become an endosymbiont of the worm. Interestingly, some of our recent studies at the methane seeps of Hydrate Ridge (Cascadia margin) also show that certain populations of anaerobic methane oxidizers are specifically associated with seep organisms—such as the symbiotic clam Calyptogena and the giant filamentous sulfide oxidizer Beggiatoa [ 20 ]. But many more examples may be out there, of bacterial and archaeal populations specifically growing in the “rhizosphere” of benthic organisms, potentially profiting from bioturbation, bioirrigation, fecal deposits, and exudates. The association and interaction between benthic fauna and sedimentary microorganisms is a very interesting field of study, although inevitably still very speculative. So far it has been limited by a lack of appropriate technologies, not only for in situ biogeochemical and biological measurements, but also for quantitative investigation of specific functional microbial populations. Some insight can be provided by clever environmental modeling approaches—such as the one developed by Cordes et al., but ultimately the models need empirical verification. Only very recently has it become possible to combine visually targeted sampling ( Figure 2 ) and high-resolution measurements of geochemical gradients with molecular tools for the identification of microbes, such as 16S rDNA and organic-biomarker-based techniques. For the study of continental margin and deep-sea ecosystems, this requires the availability of underwater vehicles ( Figure 3 ) as well as multidisciplinary research platforms and extensive, highly detailed lab work—so this is very expensive research. Yet this is the future, if we want to determine whether such an intriguing ménage à trois as proposed by Cordes et al. accounts for the presence and longevity of these extraordinary tubeworms, and possibly also other chemosynthetic symbioses, forming some of the most fascinating marine ecosystems at continental margins. Figure 3 Harbor Branch Oceanographic Institution's Submersible “Johnson SeaLink” (Source: Gulf of Mexico Cruise SJ0107)

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1065709

Expressing the Big Picture

A recent conference on neuroesthetics brought artists and scientists together to study empathy

The Fourth International Conference on Neuroesthetics was not a large event, but it was an unusual one. Held on a single day in the basement auditorium of the Berkeley Art Museum at the University of California at Berkeley, it brought together a typically motley collection of intellectuals who would willingly give over a sunny Saturday to an opportunity to learn from a panel of distinguished speakers. This was not the unusual part. Nor was it unusual that the meeting was touted as an interdisciplinary event, bringing together the best and brightest of different fields. These days, and perhaps it has always been the case, interdisciplinarity is the rule rather than the exception of innovative science. What set this meeting apart was the fluid progression from art to science, in content as well as in style. The artists were more or less scientific, the scientists more or less artistic. The topic was empathy (“Empathy in the Brain and in Art”)—more particularly, man's (and not just man's) ability to recognize and respond to the expressions of others. What do we respond to in an expression and what are the mechanisms in the brain that underlie these responses? And as the primatologist Frans de Waal (Emory University) highlighted, how much of our empathic natures do we share with our ape cousins? In a slide presentation of her work and sources of inspiration, portrait photographer Judy Dater clearly captured with great sensitivity an infinite variety of poignant expressions. However, when asked, she could not clearly articulate the choices she had made in posing and photographing her subjects, could not give dimensions to the criteria she was using. In contrast, the performance artist Leonard Pitt had clearly made a science out of expression. His physical demonstrations with Balinese masks, carved into iconic images of happiness, sadness, or anger, gave the audience insight into the variety of subtle expression that could be attributed to the mask with simple postural adjustments. Happiness melted into melancholy, sadness into ennui. “It's not about moving,” he observed, “it's about not moving.” The psychologist Paul Ekman (University of California at San Francisco) brought the official stamp of academia to his science of expression, documenting in the language of training-dependent effects on recognition the subtle range of expressions and microexpressions we can identify. For a practical example, he showed a clip from testimony in the O. J. Simpson trial of a moment in which the infamous “houseguest” Kato Kaelin was caught out in a lie. A fleeting hostile look crossed his otherwise carefully schooled features: invisible until pointed out, unmistakable after. Where the artist and psychologist show us the richness of the human behavioral repertoire, the neuroscientist tries to break behaviors down into manageable, testable predictions of the associated brain activity. In contrast to the feasts of expression presented by other speakers, the faces representative of basic emotions used by the cognitive neuroscientist Ray Dolan (University College London) to study the neural activity engendered by expressions seemed almost too caricatured to be meaningful. But Dolan, introducing his subject through the portraiture of American colonial artist Gilbert Stewart, deconstructed the information we derive from the expressions of others into five categories—familiarity, identity, emotion, intentionality, and character—and was able to describe neural activity associated with carefully constructed experiments to probe each of these facets. The Fourth International Conference on Neuroesthetics, "Empathy in the Brain and in Art," took place on 15 January 2005 at the University of California at Berkeley. Further information can be found at http://plaisir.berkeley.edu/ . Physiologist Vittorio Gallese (University of Parma) prompted many nods of satisfaction from the audience with his findings of activity in areas of the brain controlling movement when people simply watched the actions of others (see also the Research Article by Iacoboni et al. in this issue of PLoS Biology [DOI: 10.1371/journal. pbio.0030079 ]). Susan Langer, in her book Mind: An Essay on Human Feeling , has defined empathy as the direct physical reaction inherent in the perception of others, an involuntary breach of individual separateness, and to see the neural resonance, to see that the same activity patterns were being recreated in actor and observer, was to give substance to the intuition of empathy. Themed meetings, particularly when the theme does not conform to one discipline, are hard to pull off. It can be nearly impossible to convince successful professionals on the lecture circuit to modify the presentation of their own work to support such a theme. In that respect, this meeting was no different from many—some speakers were hard-pressed to conform to the theme, and it is not clear that many attendees learned information of practical value to their work from speakers across disciplines. However, it is not often that scientists have the luxury of stepping back and appreciating the context of their work in quite this way. It is not, for instance, usually appropriate to begin a paper on an apoptotic signaling pathway with a philosophical digression into the nature of Death. The abstract dimensions that the visual neuroscientist Alice O'Toole (University of Texas at Dallas) gave to facial characteristics are supposed to shed light on how we instantaneously recognize the friend we have not seen in 30 years. The electrophysiological signals in the brain that neurophysiologist Aina Puce (West Virginia University) described when we view simple movements is ultimately meant to explain how we identify with the subtle shrugging of shoulders that can transmute insouciance into insecurity. By reducing the problem to its simplest, most controlled form, scientists hope to shed light on the complexities of life. Auditory physiologists are supposed to tell us how we hear. And yet it will be a long time before they can explain “music heard so deeply that it is not heard at all, but you are the music while the music lasts” (T. S. Eliot, as quoted by the conference organizer, Semir Zeki [University College London]). But the richness of the goal makes the journey all the more rewarding.

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1065718

Correction: Dictyostelium Myosin Bipolar Thick Filament Formation: Importance of Charge and Specific Domains of the Myosin Rod

10.1371/journal.pbio.0020356 In PLoS Biology, volume 2, issue 11. Figure 3C should have appeared as shown below. The GFP-AD-Cterm (3x Thr) and GFP-AD-Cterm (3x Asp) constructs are slightly less soluble than their headless counterparts. This may be due to the fact that they are somewhat more prone to aggregation over time than the headless proteins. The change does not affect the conclusions of the paper.

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1069645

The Stability of a Stochastic CaMKII Switch: Dependence on the Number of Enzyme Molecules and Protein Turnover

Molecular switches have been implicated in the storage of information in biological systems. For small structures such as synapses, these switches are composed of only a few molecules and stochastic fluctuations are therefore of importance. Such fluctuations could potentially lead to spontaneous switch reset that would limit the lifetime of information storage. We have analyzed a model of the calcium/calmodulin-dependent protein kinase II (CaMKII) switch implicated in long-term memory in the nervous system. The bistability of this switch arises from autocatalytic autophosphorylation of CaMKII, a reaction that is countered by a saturable phosphatase-1-mediated dephosphorylation. We sought to understand the factors that control switch stability and to determine the functional relationship between stability and the number of molecules involved. Using Monte Carlo simulations, we found that the lifetime of states of the switch increase exponentially with the number of CaMKII holoenzymes. Switch stability requires a balance between the kinase and phosphatase rates, and the kinase rate must remain high relative to the rate of protein turnover. Thus, a critical limit on switch stability is set by the observed turnover rate (one per 30 h on average). Our computational results show that, depending on the timescale of fluctuations in enzyme numbers, for a switch composed of about 15 CaMKII holoenzymes, the stable persistent activation can span from a few years to a human lifetime.

Introduction Molecular switches have been implicated in many types of cell-biological processes including the storage of decisions about cell fate [ 1 ], genetic control [ 2 ], and memory storage in the brain [ 3 ]. The mechanisms of such switches generally depend on some kind of autocatalytic process. If a switch is composed of a small number of molecules, stochastic fluctuations are significant and a deterministic description is not sufficient [ 4 ]. Because of the dynamic interaction of opposing reactions, such fluctuations can spontaneously reset the state of a switch. Reset events of this kind impose a temporal limit on the usefulness of the switch for information storage. It is thus crucial to understand the factors that control switch stability and to develop quantitative insight into how the stability required for a particular biological process could be achieved. The stability problem of switches has so far been studied primarily in relation to genetic switches [ 5 , 6 , 7 , 8 , 9 ]. The problem of switch stability is of particular relevance to synaptic function [ 10 , 11 ] since memory is thought to be encoded by changes in synaptic strength [ 12 ] and because there are indications that synaptic strength is controlled by molecular switches [ 13 , 14 , 15 ]. By a molecular switch, we mean a molecule or a small group of molecules that can undergo a persistent change in state. In our definition, the change in state occurs in a discrete rather than a smoothly graded way. Clearly, spontaneous reset of a synaptic switch that encodes memory would be problematic because it would lead to loss of the stored memory. The fact that at least some memories persist for a human lifetime indicates that storage processes of extraordinary stability are present. The mechanisms that underlie synaptic information storage are beginning to be elucidated [ 16 ]. It has been demonstrated that brief periods of strong stimulation can lead to an increase in the strength of synapses, a process termed long-term potentiation (LTP) [ 17 ]. In vivo studies show that LTP can persist for at least a year [ 18 ]. The initiation of LTP is caused by activation of N -methyl- D -aspartate (NMDA) channels and elevation of intracellular calcium (Ca 2+ ) concentration [ 19 , 20 ]. There is general agreement that the resulting activation of calcium/calmodulin-dependent protein kinase II (CaMKII) plays a critical role in LTP (reviewed in [ 3 ]). CaMKII activation is persistent [ 21 ], is required for LTP [ 22 , 23 , 24 ], and is sufficient by itself to produce potentiation [ 25 ]. Genetic modification of CaMKII that prevents its sustained activation prevents long-term memory, as defined in behavioral tests [ 24 ]. The possibility that CaMKII is a synaptic memory molecule is further strengthened by the finding that it has autocatalytic properties that would allow it to function as a molecular switch [ 10 , 26 , 27 ]. Although CaMKII is required for long-term synaptic modification and is therefore a strong candidate as a memory molecule, whether evidence that its persistent activation is necessary for the maintenance of LTP remains an open question [23]. In a previous analysis of the CaMKII switch, Zhabotinsky and Lisman [ 28 , 29 ] proposed a model that incorporated many key biochemical properties of CaMKII holoenzymes and the phosphatase-1 (PP1) enzymes that dephosphorylate them [ 30 , 31 ]. It was shown that an interplay between autophosphorylation of CaMKII holoenzymes and dephosphorylation by PP1 molecules can give rise to two stable states of phosphorylation at basal levels of free Ca 2+ . Therefore, a transient input of high Ca 2+ (such as during the stimulation protocol used in LTP induction) can switch the system from an unphosphorylated (DOWN) state to a persistent, highly phosphorylated (UP) state. Such a persistent change in activation of CaMKII following LTP induction could underlie the persistent change in synaptic strength. In these previous modeling efforts, chemical reactions were described deterministically by the law of mass action, precluding estimates of the switch stability. The need for considering the limits on stability imposed by stochastic fluctuations is made more urgent by recent measurements showing that the number of CaMKII holoenzymes in the postsynaptic density (PSD) of a single synapse is relatively small [ 32 ]. For a typical PSD, there are about 30 holoenzymes [ 32 ]. In the absence of a theory that relates switch stability to the number of switch molecules, the implications of this finding are unclear. The current work addresses this issue using Monte Carlo simulations of the stochastic chemical reactions in the CaMKII/PP1 system. This approach allows us to estimate quantitatively the stability (lifetime) of a CaMKII switch and its dependence on the number of molecules. Our results thus provide new information about the potential for the CaMKII switch within the PSD to store long-term memories. A second goal of our work is to analyze the impact of molecular turnover on switch stability. Because biological switches are themselves composed of molecules that are unstable, turnover must occur. Such turnover is likely to have a detrimental effect on switch stability [ 33 ]. However, turnover need not necessarily lead to switch reset since new molecules may adopt a state that is dependent on the state of the other molecules in the switch [ 33 , 34 ]. Specifically, when the CaMKII switch is in the UP state, the PP1 activity should be saturated. This saturation reduces the effectiveness of the phosphatase so that when a phosphorylated holoenyzme is replaced by a newly synthesized unphosphorylated one, the new holoenzyme will become phosphorylated as a result of the autophosphorylation even at basal Ca 2+ levels. This can restore the state of the switch that was present before the turnover event. Direct measurements show that the CaMKII at synapses turns over about once per day [ 35 ], a timescale much shorter than synaptic memory. However, no theory has been developed for any type of molecular switch that allows an estimation of how this turnover quantitatively affects stability. Here we examine this issue with regard to the CaMKII switch. Our findings reveal general principles with implications for other kinds of molecular switches. Results Autocatalysis Leads to Bistability To understand the effect of stochastic fluctuations in molecular switches, we have implemented simulations of the CaMKII/PP1 switch model [ 28 , 29 ]. In this implementation, reactions are modeled stochastically using Monte Carlo methods and the number of CaMKII and PP1 molecules that are individually considered is comparable to the numbers contained within the PSD at single synapses. A CaMKII holoenzyme is composed of two rings, each with six kinase subunits. Each subunit has a single phosphorylation site at Thr286/287 that, when phosphorylated, makes the subunit active even when Ca 2+ /calmodulin is no longer bound. Autophosphorylation of the site on a given “substrate” subunit proceeds if two necessary conditions are fulfilled [ 36 ]. Ca 2+ /calmodulin must bind to the “substrate” subunit in order to reveal its Thr286/287 site. Also, the counterclockwise neighboring “catalyst” subunit must be active. Hence, the initial autophosphorylation necessary to switch a ring “on” requires the binding of two molecules of Ca 2+ /calmodulin. Subsequent phosphorylation of other subunits within a ring is faster (see Figure 1 A) since the phosphorylated subunit is constitutively active without Ca 2+ /calmodulin. Thus, only a single Ca 2+ /calmodulin is required to phosphorylate a “substrate” subunit if its counterclockwise “catalyst” neighbor is already phosphorylated. (Note that our results are unaffected by the direction of autophosphorylation, but based on geometric considerations, we assume it is asymmetric [ 27 , 37 ].) At the resting Ca 2+ concentration, with our standard parameters, the initial autophosphorylation occurs at an average rate of one per 3.5 h per unphosphorylated ring, while the further phosphorylation steps occur at approximately one per 4 min per available “substrate” subunit. We assume that the molecules of PP1 held in the PSD can dephosphorylate any of the sites on any of the holoenzymes in the PSD. Furthermore, PP1 becomes saturated when the kinase becomes hyperphosphorylated [ 29 ]. Figure 1 CaMKII Autophosphorylation and PP1 Saturation Lead to Bistability in the Phosphorylation States of CaMKII at Resting Ca 2+ (A) Phosphorylation of the first subunit is slow at resting Ca 2+ because of the requirement for two Ca 2+ /calmodulin molecules (see text). Subsequent phosphorylation is faster because only one Ca 2+ /calmodulin is required. (B) Schematic figure indicating how bistability arises from the dependence of phosphorylation and dephosphorylation rates on the number of subunits phosphorylated. Stable states are at the left (DOWN) and right (UP) intersections of the two curves. The middle crossing is unstable. The greater the area of the shaded region between a stable steady state and the unstable steady state, the harder it is for fluctuations to destabilize that stable steady state (the larger their basins of attraction). (C) A 2-s pulse of high Ca 2+ switches the system (with 16 holoenzymes) from a low state of phosphorylation to a higher state within the basin of attraction of the UP state (see [D]). Phosphorylation fraction is Sp tot /12 N CaMK . (D) After the end of the Ca 2+ pulse, it can take tens of minutes for the system to reach the dynamic equilibrium in the UP state. (E) The UP state is stable for many years in a system with 16 holoenzymes. Figure 1 B indicates schematically how the total rates of autophosphorylation and dephosphorylation lead to two stable states at the resting Ca 2+ level. The curves in the figure show how these reactions vary as a function of the total number of sites phosphorylated. The intersection points (where dephosphorylation balances phosphorylation) define three steady states, of which the left and right ones are stable and the middle one is unstable. Switching can occur if phosphorylation of the system is forced (either by a transient signal or by a spontaneous fluctuation) far enough away from one stable value that it passes the unstable value; the system will then fall into the basin of attraction of the second stable point. Once in this basin, the intrinsic dynamics of the system set the timescale of drift to the second stable state. The model presented here includes stochastic turnover of CaMKII holoenzymes with an average lifetime of 30 h that is independent of phosphorylation level, as experimentally determined [ 35 ]. We assume, except where stated otherwise, that once a holoenzyme is removed, it is immediately replaced by an unphosphorylated holoenzyme. If the switch is in the DOWN state, the newly inserted holoenzyme is likely to stay off: any spontaneous phosphorylation will be countered by dephosphorylation, which removes a subunit in approximately 5 min, on average. However, if the switch is in the UP state, the phosphatase is so saturated by other phosphorylated holoenzymes, that the average time for a newly inserted subunit to be dephosphorylated is almost 1 h. Hence, the newly inserted holoenzyme can turn on as a result of the activation of the kinase by basal Ca 2+ levels [ 28 , 29 ], since the time for it to be phosphorylated is significantly less than the time for turnover. Thus, the UP state of the switch is stable, in spite of turnover. Parameters for the model were constrained according to the references cited in Table 1 . Where the constraints allowed a range of variation, we chose values of parameters so that the system would be bistable and have approximately equal lifetimes of the UP and DOWN states (see below). We required that our standard system have equal numbers of CaMKII holoenzymes and PP1 molecules, as their concentrations are known to be similar, but adjusted the less well-determined Hill constants, Κ H1 and Κ H2 , to maximize system lifetime (see below). While our model is more sensitive to the value of Κ H1 than any other parameter, bistability still exists in a significant range (10% around its optimal value) when all other parameters are fixed. Compensatory covariation of other parameters maintains the system's bistability at fixed calcium when Κ H1 varies by more than a factor of three. Our study aims to test whether a plausible set of kinetic parameters enables a molecular switch, with a small number of participating molecules, to be stable in spite of fluctuations. Table 1 Parameters Used in the Model PKA, cAMP-dependent protein kinase Two critical requirements exist for the switch to function. First, the initial (P0 to P1) phosphorylation step must be significantly slower than further phosphorylation steps. This is true as the Hill constant for Ca 2+ activation of CaMKII [ 38 , 39 ] is significantly greater than the average Ca 2+ concentration in the physiological resting state (0.7 μM versus 0.1 μM in most of this paper). Second, the phosphatase activity must saturate so the rate of dephosphorylation per phosphorylated subunit is significantly slower in the UP state than in the DOWN state. This is achieved as the Michaelis constant, Κ M , of PP1 is much lower than the concentration of CaMKII subunits (0.4 μM versus 400 μM in this paper, though we also test the model with a Κ M of 10 μM [ 40 ]). Thus, according to the latest experimental data, the two critical requirements for a functioning switch are met. Figure 1 C and 1 D (with 16 holoenzymes simulated) show that a large, 2-s-long Ca 2+ elevation of the kind that may occur during LTP induction [ 41 ] can switch the system from the unphosphorylated DOWN state to a highly phosphorylated, persistent UP state. The system drifts toward the UP state even after Ca 2+ falls to its basal level ( Figure 1 D). Such a drift has been observed experimentally [ 42 ]. During the drift period, which can take tens of minutes, the system would be more vulnerable to depotentiation. In this particular example (with 16 holoenzymes), the stable UP state is reached in under an hour ( Figure 1 D). As seen in Figure 1 E, the resulting UP state remains stable for at least 10 y. Spontaneous Transitions between the Baseline and Memory States We examined the distribution of times between spontaneous switching events as a measure of the stability of memory storage. Figure 2 A shows that the total phosphorylation level of a small system with eight holoenzymes is only stable on the time scale of months, not years; sporadically, fluctuations cause the system to change from one state to the other, as indicated by the random switching of phosphorylation level. Analysis reveals that the times spent in either the DOWN or UP state (i.e., the lifetimes) are distributed exponentially ( Figure 2 B) (apart from brief transition times). Such an exponential distribution [ 43 ] indicates that the probability of transition per unit time is constant for a given state. The exponential distribution of lifetimes has a characteristic time constant that equals the average lifetime in the state (and the inverse of the probability of transition per unit time). Figure 2 Switch Stability Is a Trade-Off between Lifetimes of UP and DOWN States (A) Spontaneous switching between UP and DOWN states in a system with eight CaMKII holoenzymes. (B) The distribution of lifetimes between switching events is exponential, as demonstrated by the straight line fit for lifetimes of the UP state on a semi-logarithmic scale. (C and D) Removing one PP1 enzyme (seven instead of eight) (C) leads to a longer lifetime for the UP state but shorter lifetime for the DOWN state; whereas adding one PP1 enzyme (nine instead of eight) (D) yields the opposite effect. (E) Dependence of average lifetimes of the UP state (squares) and DOWN state (circles) as a function of the number of PP1 enzymes (with eight CaMKII holoenzymes). Filled blue symbols correspond to data points from (A), (C), and (D). Lines are approximate, analytic results (based on Materials and Methods and [ 51 ]). The optimal lifetime of the switch is defined by the intersection point of the two curves, at which the lifetimes of the UP and DOWN states are equal. Blue indicates reference parameters. Red indicates k 1 = 0.75 s −1 , one-half of the standard value. The lifetime does depend on kinetic parameters, but maximum stability is approximately the same, albeit with a different number of PP1 molecules. The overall stability of the system is dependent on the lifetimes of both the UP and DOWN states. In general, any change in the system that increases the rate of phosphorylation tends to increase the lifetime of the UP state while reducing the lifetime of the DOWN state. The opposite is true for an increase in dephosphorylation rate. This tradeoff between lifetimes of the two states can be demonstrated by varying the number of phosphatase enzymes in the system while all other parameters are fixed. As seen in Figure 2 C, a reduction in the amount of phosphatase resulted in an increased lifetime of the UP state, but destabilization of the DOWN state; in contrast, increasing the amount of phosphatase had the opposite effect ( Figure 2 D). Figure 2 E (blue) shows the lifetimes averaged over 400 transitions, as a function of the number of phosphatase enzymes. Again, the lifetime of the UP state decreases, and the lifetime of the DOWN state increases with the number of PP1 molecules. We define the “system's lifetime” as the smaller of the two lifetimes, because we assume that a random potentiation of a synapse is as equally undesirable as a random depotentiation; a spontaneous transition from either state would be detrimental for memory. It follows that the system is optimal at the crossing of the two curves ( Figure 2 E), where the two lifetimes are equal (so that neither lifetime is too small). Such an optimum corresponds to a balance between the processes of phosphorylation and dephosphorylation. We therefore define the phosphatase concentration at which the two curves cross to achieve balance as the optimal concentration. In the simulations described above, we set the optimal phosphatase concentration equal to the concentration of the CaMKII holoenzymes (R. J. Colbran, personal communication; see Table 1 ). We adjusted the less well-constrained parameters to achieve this. To see how sensitive the system's lifetime is to these particular choices, we simulated the system with a different value of the phosphorylation rate constant for the kinase, changing k 1 from 1.5 s −1 to 0.75 s −1 . Figure 2 E (red) shows that the optimal phosphatase concentration is also reduced, but at this concentration the system lifetime remains as high as in the original system (the intersection of the two curves is shifted but remains at the same lifetime). We also tested the system's robustness to a 25-fold larger value, 10 μM, for Κ M . With an increased optimal phosphatase concentration, the system of 20 holoenzymes was stable for over 10 y. Thus, achieving long lifetimes does not require a specific level of enzyme activity, but does require an appropriate balance between phosphorylation and dephosphorylation rates. See Discussion for how this might be achieved. Stability Increases Exponentially with the Number of Molecules We next considered how the system's lifetime varies with the number of holoenzymes (while PP1 varies in proportion, as does the system's volume). Figure 3 A and 3 B show the behavior of the model switch with four and 16 holoenzymes, respectively. We found ( Figure 3 C) that the system stability increases exponentially with the number of holoenzymes (i.e., the system size). As a result of this exponential dependence, the lifetime of the system almost doubles for each additional holoenzyme in the PSD. These simulations show that a switch made of only four holoenzymes can only be expected to have stability on the order of days to weeks, whereas increasing the system to 16 holoenzymes could result in a switch that is stable for a human lifetime. Figure 3 Stability of the Switch Increases Exponentially with System Size (Number of CaMKII Holoenzymes and PP1 Molecules) (A) Spontaneous transitions in a system with four CaMKII holoenzymes and four PP1 enzymes. Phosphorylation fraction is Sp tot /12 N CaMK . (B) System with 16 holoenzymes and 16 PP1 enzymes, with four times the volume of (A). Note different timescales between (A) and (B). Phosphorylation fraction is Sp tot /12 N CaMK . (C) The switch's lifetime increases exponentially with system size. Numbers of all species scale together with system volume. Circles are data points, line is a linear fit, indicating an exponential dependence, because the ordinate is in logarithmic scale. The red asterisks indicate data points where we included the PP1–I1P fluctuations explicitly. Protein Turnover Limits Memory Lifetime In order to understand more deeply the cause of spontaneous switching, we examined what was occurring in the switch during the period preceding switching events. The two examples in Figure 4 A and 4 B show the total instantaneous level of phosphorylation of the system during the time preceding a spontaneous transition to the DOWN state ( Figure 4 A is for a system of four holoenzymes, Figure 4 B for a system of 16). Holoenzyme turnover events are evident in these traces, because a turnover event causes an abrupt drop in the level of phosphorylation (marked by red arrows). In Figure 4 A, four turnover events occur in a 3-h period prior to a downward switching event, and in Figure 4 B, six turnover events occur in the same length of time ( t = −7 h to −4 h), after which intrinsic dynamics take over to complete the downward transition. Based on the average time for turnover of 30 h per holoenzyme, one would expect a turnover event every 7.5 h in a system with four holoenzymes, and every 1.9 h in a system with 16 holoenzymes. Hence, the figures indicate that high numbers of turnover events occur in the periods before a transition, as expected if turnover initiates switching to the DOWN state (as explained on the next page). Such high amounts of turnover result from the stochastic nature of the turnover process, and occurred prior to such spontaneous transitions in all the traces we examined. Hence, protein turnover, and, in particular, its stochastic nature, strongly affects the system's ability to store information. Figure 4 The Rate of Protein Turnover Limits the Maximal Lifetime of the System and Leads to a Minimal Rate of Energy-Consuming Activity (A and B) Stochastic changes in total phosphorylation during a transition from the UP to DOWN state, with turnover events marked by arrows, in a system with (A) four CaMKII holoenzymes and (B) 16 CaMKII holoenzymes. Red arrows indicate turnover events, which cause an abrupt drop in the level of phosphorylation. (C) Log–log plot of lifetime of the switch as a function of turnover rate for the system with eight CaMKII holoenzymes (the red asterisk marks 30-h turnover, used as standard in this paper). (D) The rate for an individual ring of subunits to switch off as a function of the total number of rings that are on (shown here for a system with eight CaMKII holoenzymes). As more rings are turned on, the phosphatase activity saturates and the equilibrium level of phosphorylation per ring increases. As a result, the switching-off rate for rings in the UP state for the system approaches the turnover rate (dashed line), because the probability of total ring dephosphorylation by PP1 becomes small. (E) Dynamic equilibrium between turnover (vertical solid black lines) and switching on of rings (colored step-like lines) when the system is in the UP state. At the time of the first turnover, five rings are already unphosphorylated by prior turnover. The system is stable because the rate of rings switching on matches the rate of turnover of phosphorylated rings (each turnover event can result in the loss of zero, one, or two phosphorylated rings). A system with 20 holoenzymes is shown. We next investigated how the rate of protein turnover, ν T , affects the switch's stability. We found that an increase in the rate of protein turnover has little effect on the lifetime of the DOWN state, but dramatically reduces the lifetime of the UP state. Again, this is to be expected if turnover is responsible for initiating a switch DOWN in the system. Since the system is optimal when the two lifetimes are similar, in a series of simulations where we used different rates for protein turnover, we also adjusted the amount of PP1 to return to an optimal system (where UP and DOWN state lifetimes are similar). Hence, we can plot in Figure 4 C the optimal lifetime of the switch as a function of average time for protein turnover. For turnover times of less than 1 mo, the optimal system stability is strongly dependent on the rate of turnover, suggesting that protein turnover is a limiting factor in the stability of the switch. Indeed, making the turnover rate very fast (hourly) can cause the system to lose all bistability. In the UP state of our system, protein turnover replaces phosphorylated holoenzymes with unphosphorylated ones and is thus effectively acting like a phosphatase. It was therefore of interest to compare this effective phosphatase activity to the rate of dephosphorylation produced by the phosphatase itself. We proceeded by calculating the total rate for individual phosphorylated rings to switch off, that is, to become an unphosphorylated ring in state P0. Such a switching-off rate is the sum of the turnover rate and the rate for dephosphorylation by phosphatase. Our approximate calculation is accurate when the switching-on and switching-off rates for a ring are much slower than the rates for individual subunits to be phosphorylated or dephosphorylated, as it assumes a ring has time to reach all configurations of phosphorylation before it switches off (see Materials and Methods ). Given that assumption, we obtained the proportion of time a ring that is on spends in each configuration of phosphorylation. The unphosphorylated state, P0, where a ring is off, can be reached either by turnover, or by dephosphorylation from the state with a single phosphorylated site, P1. Hence, the average rate at which a ring switches off ( Figure 4 D) is given by ν 3 Ρ Ρ 1 + ν T , where ν 3 is the rate per phosphorylated subunit of phosphatase activity ( ν 3 of equation 8 ), Ρ Ρ 1 is the proportion of time a ring that is on spends in the configuration P1 and ν T is the protein turnover rate. Note that as the number of rings switched on increases, so the total phosphorylation of the system increases, causing both ν 3 and Ρ Ρ 1 to decrease. As is evident in Figure 4 D, when more than half the rings in the system are on, the rate of rings switching off becomes identical to the turnover rate, ν T , itself (horizontal dashed line in Figure 4 D). Hence, with a 30-h turnover rate and the optimal concentration of phosphatase, the phosphatase is unable to switch a ring off in the UP state; loss of phosphorylated rings is purely due to turnover. We next sought to visualize how the system remains in the UP state even while turnover is causing the replacement of approximately two-thirds of the phosphorylated holoenzymes with unphosphorylated ones during a 30-h period (on average). In Figure 4 E, we present a snapshot of a few hours of activity to show the time course for turnover and rephosphorylation of individual rings. The system in Figure 4 E contains 20 holoenzymes, so the UP state is stable for many decades. Turnover events are marked by vertical lines. Unphosphorylated rings that become phosphorylated are indicated by the colored step-like lines, where each step indicates the phosphorylation of a subunit. It should be noted that at any one time, even though the system is in the UP state, a number of rings are unphosphorylated because of previous turnover. The rate of switching on for rings is proportional to the number of rings off. On average, the total rate of switching on matches the rate of phosphorylated rings lost by turnover. This dynamic equilibrium between the switching on of rings and turnover determines the average number of unphosphorylated rings at any time. In the system of 20 holoenzymes (and therefore 40 rings), the number of unphosphorylated rings typically varies from four to eight. The number is five before the first turnover event on Figure 4 E and reaches a maximum of nine following the two closely spaced turnover events to the right in Figure 4 E. The figure also illustrates how once one subunit is phosphorylated on a ring, the others rapidly follow. Effect of Fluctuations in Reactant Concentrations In our simulations thus far, we have not considered noise in the signaling pathways that control the kinase and phosphatase reactions. A careful analysis of this issue requires an understanding of the signals that lead to bidirectional synaptic modification, as well as a consideration of the noise reduction mechanisms; both are beyond the scope of this paper. Nevertheless, we wanted to determine whether the switch we have modeled could tolerate reasonable noise levels in its input. In this class of models, reaction rates are nonlinear in Ca 2+ concentration, so fluctuations in Ca 2+ concentration affect the mean reaction rate, as well as providing additional noise about the mean rate. Moreover, the functional dependence on Ca 2+ is not the same for all reaction steps. In particular, fluctuations in Ca 2+ concentration increase the average rate of the slow initial (P0 to P1) phosphorylation step, which requires two Ca 2+ /calmodulins, to a greater extent than any other reaction steps. The change in relative reaction rates means that, in principle, large enough fluctuations can destroy the bistability altogether, whatever the system size. This class of switch has no absolute protection against Ca 2+ fluctuations—indeed, we require in our model that a strong enough change in Ca 2+ , as occurs during LTP, leads to a switch from the DOWN to the UP state (see Figure 1 C). However, the system should be robust to smaller, realistic fluctuations that occur in the absence of LTP. Figure 5 A (blue squares) shows that the system with our standard parameters (but with a lower, 0.07 μM, baseline) is able to tolerate the moderate fluctuations that might arise from Ca 2+ influx through NMDA-receptor-mediated channels (0.1 μM amplitude with 100 ms decay time; [ 44 ]) due to spontaneous presynaptic action potentials (a 0.5-Hz Poisson train) with only modest reduction in stability. Although the lifetime increases less steeply with system size than in the case without fluctuations ( Figure 5 A, black circles), extrapolation suggests that a system with 20 holoenzymes would be sufficient to have a lifetime of 100 y. If we use larger fluctuations, of amplitude 1.0 μM, it is important to adjust parameters to compensate for the change in average activity produced by fluctuations. With such an adjustment (see Materials and Methods ) the system with 1.0-μM fluctuations in free Ca 2+ concentration (with 100 ms decay time, above a 0.1 - μM baseline in a 0.5-Hz Poisson train) is slightly more stable than the original system without fluctuating Ca 2+ ( Figure 5 A, red triangles). We conclude that plausible levels of Ca 2+ fluctuations in spines do not necessarily compromise switch stability. Figure 5 Switch Stability in the Presence of Spontaneous Fluctuations in Free Calcium Concentration and the Total Number of Enzymes (A) Effect of Ca 2+ fluctuations on stability (lifetime) as a function of number of CaMKII holoenzymes. Circles indicate the original system without Ca 2+ fluctuations. Squares indicate the original system with free Ca 2+ fluctuations of amplitude 0.1 μM and baseline 0.07 μM. Triangles indicate adjusted system with free Ca 2+ fluctuations of amplitude 1.0 μM and baseline 0.1 μM. The adjusted system has alternative parameters, such that N PP 1 = N CaMK /2, k 1 = 6 s −1 , k 2 = 7 s −1 , and K H 1 = 4.0 μM. The ordinate is in logarithmic scale. (B) Effect of fluctuations in the number of PP1 molecules on the lifetime of UP states (squares/solid line) and DOWN states (circles/dashed line) (16 holoenzymes). The timescale on the abcissa is the average time for the number of PP1 molecules to increase or decrease by one. Red indicates 12 < N PP 1 < 20. Blue indicates 8 < N PP 1 < 24. The ordinate is in logarithmic scale. (C) Lifetime of UP states (squares/solid line) and DOWN states (circles/dashed line) when the number of holoenzymes and PP1 molecules fluctuate in the respective ranges 14 < N CaMK < 18 and 8 < N PP 1 < 24. The timescale for PP1 fluctuations varies along the abcissa. The timescale for CaMKII fluctuations is fixed by the turnover rate (30 h per holoenzyme). The ordinate is in logarithmic scale. In our simulations so far, we have assumed fixed numbers of enzyme molecules, but in principle these numbers may fluctuate with time, potentially compromising stability. We implemented several sets of simulations to address this issue. In each set, we carried out a number of simulations corresponding to a range of timescales for fluctuations of PP1. Specifically, we varied the average time between random steps of plus one or minus one in the number of PP1 molecules in the PSD, and plotted this time as the x-axis in Figure 5 B and 5 C. It should be noted that the total time the system spends away from its optimum is usually the sum of many such time steps. In the first set of simulations, with the number of CaMKII holoenzymes held fixed, we assumed the number of PP1 molecules could fluctuate by plus or minus 25%, so in the particular case of 16 holoenzymes, the number of PP1 molecules varies between 12 and 20. The resulting lifetimes for the UP and DOWN states are given in Figure 5 B in red. The lifetime decreases as the average time between changes in PP1 increases. This is because slow fluctuations lead to long periods of time when the system is far from optimal. In contrast, if the fluctuations are rapid, the system may not have time to make a transition even if the system loses bistability temporarily. Still, even with a change every 6 h, the system with 16 holoenzymes is stable for over about 20 y. Second, we increased the amplitude of fluctuations in PP1 to plus or minus 50% (a variation of a factor of three, from eight to 24 in this case). The simulation results in Figure 5 B (blue) indicate that such an increase in amplitude of fluctuation causes the average lifetime for the system to decrease. Again, if the fluctuations are relatively rapid, they do not seriously degrade the switch. It is somewhat remarkable that when the number of PP1 molecules varies by a factor of three over a timescale of tens of minutes, the switch lifetime still averages over 10 y. Third, we introduced slow fluctuations in the number of CaMKII holoenzymes, assuming stochastic insertion of holoenzymes as well as stochastic turnover. Since the timescale of removal is set at 30 h per holoenzyme [ 35 ], the average rate of insertion is fixed (at 16 every 30 h) to ensure the appropriate average of 16 holoenzymes within the PSD. As above, the simulations covered a range of timescales for the fluctuations of PP1, whose number could vary between 8 and 24. We found that variations in the number of holoenzymes are more deleterious than variations in PP1 alone. In particular, loss of holoenzymes from the PSD destabilizes the UP state. This is because the effect seen above, of a nonoptimal CaMKII to PP1 ratio, is exacerbated by a reduction in switch size when holoenzymes are lost (cf. Figure 3 C). Figure 5 C indicates the resulting lifetimes when the number of CaMKII holoenzymes varied between 14 and 18. Without PP1 fluctuations (equivalent to a time step of zero) the DOWN state is little affected by these slow fluctuations in the number of holoenzymes (circles/dashed line), but stability of the UP state is greatly reduced (squares/solid line). Including slow PP1 fluctuations of ± 50% reduces the lifetimes of both UP and DOWN states to below 10 y. Although a system averaging 20 holoenzymes would be more stable, we conclude that during turnover, a holoenzyme removed from the PSD needs to be replaced relatively rapidly—on a timescale of minutes, not hours—to avoid degradation of the switch. Moreover, if CaMKII were not anchored, but able to freely diffuse in and out of the PSD, fluctuations in the number of holoenzymes present would be increased, and bistability would not be possible [ 45 , 46 ]. Discussion In this paper, we have considered the stability against fluctuations of a bistable switch based on the interaction of CaMKII and PP1 in the PSD. Although a deterministic model of such a switch has been presented before [ 28 , 29 ], it was not previously possible to assess quantitatively the potentiality of the switch for long-term information storage, because the rate of spontaneous reset was not known. Given the small number of CaMKII molecules at synapses [ 32 ], stochastic fluctuations in the reactions must necessarily lead to switch reset on some timescale. Our results show that that this timescale depends crucially on the number of molecules that make up the switch (see Figure 3 C). Indeed, this dependence is highly nonlinear, scaling exponentially with the number of molecules involved. We have shown that this timescale can exceed human lifetimes when the number of holoenzymes is greater than 15 (see Figure 3 B), provided the parameters of the system are in an optimal range. A substantially smaller number of holoenzymes, such as four, would result in spontaneous transitions on a timescale of a week (see Figure 3 A). One interesting possibility is that initially a small number of CaMKII holoenzymes is sufficient for the immediate information storage and with time, during an initial consolidation period [ 47 , 48 ], a larger number of holoenzymes accumulate at the PSD, allowing for more permanent memory formation (see Figure 3 C). Our general conclusion is that relatively small groups of CaMKII molecules, such as are found in the PSD (where the average is 30 holoenzymes) can function as highly stable switches and could potentially subserve information storage for very long periods. The exponential dependence of lifetime on system size is consistent with general theoretical considerations [ 11 ]. This is because the switch can be described as a biochemical system with a “double well” effective energy potential in which two minima are separated by a barrier. The fluctuations in the reactions generate noise-driven hopping over the barrier in a manner analogous to thermally driven hopping over a real potential barrier [ 11 , 49 , 50 , 51 ]. The effective barrier height is proportional to the system size [ 49 ], and it is well-known that with a constant noise source, the time for transitions across a barrier increases exponentially with barrier height [ 51 , 52 ]. Intuitively, a transition from the UP to DOWN state is triggered when a critical number of CaMKII rings (proportional to the system's size N ) are dephosphorylated at the same time. In terms of probability, the decrease in likelihood with N is the same effect as the increase in expected number of coin tosses necessary to obtain N heads in a row, each with a probability p = 1/2. The probability for N consecutive heads is p N , and the expected time (number of coin tosses) it takes before this happens is (1/ p ) N = 2 N , which grows exponentially with N . By analogy, the larger the system, the more rings of CaMKII have to turn off randomly without recovery before they are able to cause a switch in the whole system. If the dephosphorylation events do not occur “in a row,” switching off a critical number of CaMKII rings within a short time interval, the opposing reactions (autophosphorylation) have time to counteract and turn rings on, allowing the natural dynamics to drive the system back to the UP equilibrium state. An additional finding is that the molecular turnover of CaMKII strongly limits switch stability (see Figure 4 ). If turnover were absent, switch stability could be an order of magnitude higher (see Figure 4 C). Our analysis shows an interesting set of relationships between the rates of phosphatase and kinase activities and the rate of protein turnover with regards to their effect on switch stability. In principle, lowering the basal phosphatase and kinase rates in proportion increases switch stability. Such lowering of the dephosphorylation rate has a second desirable feature of lowering energy consumption. This is because the UP state consists of what biochemists call a “futile cycle,” in which the rate of ATP-utilizing phosphorylation equals the rate of dephosphorylation. Although minimizing energy utilization dictates that the system be “cooled” (lowering the phosphatase and kinase rates), our results show that there are limits to how much cooling is effective and that this limit is set by the protein turnover rate. Specifically, if cooling sets the phosphatase and kinase rates too low, the system cannot regain steady-state values after a molecular turnover event (newly inserted unphosphorylated kinase molecules will not become fully phosphorylated, as they do in Figure 4 E). In this case, unphosphorylated kinase molecules will accumulate, leading inexorably toward the threshold for reset to the DOWN state. Our results suggest that the stability of a switch depends sensitively on a balance of phosphorylation and dephosphorylation rates. Hence we assessed how changes in the ratio of PP1 to CaMKII affect the lifetimes of states of the switch (see Figure 2 E). We find that short-term fluctuations in the ratio, on a timescale of tens of minutes, do not significantly degrade the switch (see Figure 5 B). Slower fluctuations are more deleterious, particularly as stability of the UP state is compromised if the number of CaMKII holoenzymes becomes too low (see Figure 5 C). In contrast to its robustness to short-term fluctuations, our system requires the long-term average ratio of activities is constrained (see Figure 2 E). Hence, if all other parameters and concentrations are fixed, the ratio of numbers of PP1 molecules to CaMKII molecules should lie in a narrow optimal range (see Figure 2 E). It will thus be of interest to see whether special mechanisms exist to stabilize the ratio of PP1 to CaMKII in the long term. Promoting the necessary fixed ratio of PP1 to CaMKII may be one of the functions of the scaffolding proteins that hold CaMKII and PP1 within the PSD structure [ 53 , 54 , 55 , 56 , 57 , 58 ]. Moreover, our results (see Figure 5 A) indicate that moderate Ca 2+ fluctuations are tolerable on short timescales, but we find the average level must be tightly regulated over the long term (to within several percent; data not shown). Since the kinase activity depends on the level of free calmodulin, it may further be expected that free calmodulin is regulated over long timescales. This may be an important function of the known calmodulin buffers [ 59 , 60 ]. In the absence of control mechanisms, the stability of the switch would be greatly reduced. Several limitations of our study should be noted. While we addressed the effect of Ca 2+ fluctuations (see Figure 5 A), we did not include the detailed reaction steps of Ca 2+ binding to calmodulin in our model, but used the steady-state values for reaction rates based on Ca 2+ /calmodulin. These steady-state rates may not be reached during rapid changes in concentration. Hence, the phosphorylation rates may not vary with Ca 2+ precisely as we have modeled, in which case other parameters or concentrations would have to be altered to maintain an optimal system. However, including calmodulin-binding steps, and removing the assumption of excess, freely available calmodulin, would reduce the effect of sharp, brief rises in free Ca 2+ . Hence, the influx of Ca 2+ necessary to cause LTP could be greater than presented here (see Figure 1 C), and the system may be stable to larger Ca 2+ fluctuations than those we include. Quantitative measurements of spontaneous Ca 2+ fluctuations in vivo will be needed to assess whether the switch stability is robust against realistic fluctuations. A second type of simplification that we have made is likely to lead to an underestimate of stability. We have assumed that the CaMKII molecules bound in the PSD are operating with the same kinetic constants measured in free solution. However, some or all of the CaMKII in the PSD may be bound to NMDA receptors [ 61 , 62 ]. This binding increases the rate of autophosphorylation of the first site, allowing it to occur with only one calmodulin bound rather than two. If such binding to NMDA receptors occurs significantly only after an LTP event, when the system is in the UP state, the effect of protein turnover will be reduced, because unphosphorylated rings would become rapidly phosphorylated before the whole system has time to switch to the DOWN state. While the switch we have described could be stable for a human lifetime, long-term information storage may not require stability of this magnitude. One possibility is that such long-term stability is not solely a property of the switch, but emerges from interaction between the switch and an attractor network created by memory-specific synaptic connections. According to this idea [ 47 , 48 ], reactivation of the attractor, perhaps during sleep, may serve to refresh the memory by setting switches back to their correct state. For such a system to work, average switch stability need only be larger than the time between reactivations of the attractor. Such reactivations appear to be important in the early stages of memory, when consolidation is important [ 47 , 63 ]. However, we know little quantitatively of how frequently such reactivation processes take place. Moreover, the role of reactivation in long-term maintenance of a memory trace, after initial consolidation, remains unclear. Advances in this direction would enhance our understanding of the interplay of molecular and network properties in determining the overall stability of memory in the brain. Materials and Methods The model We treat each ring of six subunits of CaMKII as an independent entity [ 37 , 64 , 65 , 66 ]. Each subunit can be in one of two states: either phosphorylated or unphosphorylated. The set of possible configurations among the six subunits results in 14 distinguishable states for a ring, labeled here by the number of phosphorylated subunits: P0, P1, P2 (three configurations for P2 because the two phosphorylated subunits can be either neighboring, or separated by either one or two unphosphorylated subunits), P3 (four configurations), P4 (three configurations), P5, and P6. The different configurations have different multiplicities, which are counted when calculating rates of reactions that change configurations. Phosphorylation of the first subunit of a ring (P0 to P1) requires the binding of two activated calmodulins, so is slow at resting Ca 2+ concentrations (see Figure 1 A). Once a single subunit is phosphorylated, it can catalyze the phosphorylation of neighboring subunits in a directional manner [ 27 ], so that further phosphorylation steps are faster at resting Ca 2+ concentrations (see Figure 1 A). We refer to a ring in the unphosphorylated state, P0, as off, and a ring with any other level of phosphorylation as on. Once a ring is on, we do not include further slow steps for that ring in the calculations, because the faster, directional steps dominate. Taking a Hill coefficient of three [ 40 , 67 ], we have for the initial phosphorylation rate per subunit: and for the phosphorylation of a clockwise neighboring subunit: The dephosphorylation occurs through the Michaelis–Menten scheme: with Michaelis constant, , and where Sp denotes a phosphorylated subunit while S denotes an unphosphorylated one. In the simulations presented here, we assume k + = k 2 / K M and set k − to zero, but we find the results are not noticeably influenced by the relative values of k − and k + at fixed k 2 and K M . The phosphatase is deactivated by phosphorylated inhibitor-1 (I1P), a noncompetitive antagonist [ 68 , 69 , 70 ]. We follow the formulation of Zhabotinsky [ 28 ], assuming the level of free inhibitor-1 (I1) is constant (at 0.1 μM) in the PSD owing to free exchange of I1 with the larger cell volume. Such free exchange with the larger cell is important, as the number of free I1 molecules in the PSD is less than one on average. Even the spine itself can contain fewer I1 molecules than there are PP1 molecules in the PSD. However, the rapid and strong binding of PP1 to the inhibitor means that the PSD acts as a sink of free I1, and the total concentration of all I1 in the PSD is significantly greater than that of free I1. Importantly, I1 exchanges between the PSD and spine volume vary rapidly, and between the spine and parent dendrite with τ ≤ 1 s, a timescale much faster than that of the phosphatase and kinase reactions. I1 is phosphorylated by cAMP-dependent protein kinase and dephosphorylated by calcineurin. The rate of dephosphorylation of I1P by calcineurin increases with Ca 2+ , with a Hill coefficient of three [ 71 ], because calcineurin requires Ca 2+ /calmodulin to activate. Hence I1 is less phosphorylated and the phosphatase is less inhibited at higher Ca 2+ concentrations. These reaction steps (modeled by Zhabotinsky [ 28 ]) are assumed to be fast, so we can write down the stationary level of free I1P concentration as and the fraction, f e , of phosphatase that is free of inhibitor and hence active as with K I = k 4 /k 3 . In the majority of results presented here, we do not simulate the reaction of phosphatase inhibition stochastically. Since the reaction occurs on a timescale faster than other reaction steps, we can use the quasi-steady-state assumption and use only its equilibrium values [ 72 ]. We did test this assumption by carrying out simulations that included both a stochastic step for phosphorylation of I1 (instead of equation 4 ) and for inhibition of PP1 (instead of equation 5 ). Simulating such fast processes (two to three orders of magnitude faster than other reactions) means a huge increase in the total number of reactions per unit time and hence a corresponding increase in the computer time required. The results for the three systems we tested (red asterisks in Figure 3 C) showed no significant difference from the results without such fast fluctuations. Hence, we have confidence in our use of the equilibrium values for other data points. Turnover occurs at a rate, ν T , and acts equivalently to a non-saturating dephosphorylation process, as we assume all holoenzymes are replaced by unphosphorylated ones, and any attached PP1 is released back to the system. It is possible to calculate analytically ν 3 , the rate of dephosphorylation by PP1 per phosphorylated subunit. Note that from Figure 1 B, we require ν 3 to decrease at high phosphorylation so that the total concentration of subunits dephosphorylated per unit time, ν 3 ·[ Sp tot ], saturates (we have written the total concentration of phosphorylated subunits, [ Sp tot ] = [ Sp ] + [ PP 1. Sp ]). To write the rate of dephosphorylation on the right hand side of equation 3 as ν 3 ·[ Sp tot ], we assume the intermediate product, [ PP 1. Sp ], is at steady state. We combine equation 3 with the other two ways that [ PP 1. Sp ] can change. That is, noncompetitive inhibition leads to the reaction while turnover leads to So we calculate an effective dephosphorylation rate constant, ν 3 , from ν 3 ·[ Sp tot ] = k 2 [ PP 1. Sp ] assuming d [ PP 1. Sp ]/ dt = 0. The result is, writing [ E 0 ] = N PP 1 N A / vol, where the concentration of phosphorylated subunits without phosphatase attached, [ Sp ], is given by As expected, the rate, ν 3 , decreases significantly for [ Sp tot ] > K M such that at large total phosphorylation in the UP state, the product is a constant (see Figure 1 B). In the limit of negligible phosphorylation, , equation 9 simplifies to give hence, from equation 8 , In the limit of high phosphorylation (where ), equation 9 becomes leading to Our derivation differs from the standard Michaelis–Menten approach because we cannot assume that at all times, so [ Sp ] ≠ [ Sp tot ]. Monte Carlo simulations We conducted Monte Carlo simulations of this model, in which all the microscopic configurations of CaMKII holoenzymes were counted and chemical reactions between these states were simulated as stochastic Markov processes. We used the algorithm of Gillespie [ 51 , 73 ], which can be summarized as follows. We identify all the possible configurations of rings. There are 56 in total, because for a configuration with a given number, n, of phosphate groups, the number of PP1 bound can vary from 0 to n (assuming n < N PP 1 ). So for each of the 14 configurations of phosphate groups, the number of configurations including enzymes is multiplied by n + 1. For example, a ring with two subunits phosphorylated (P2) can have up to two PP1 enzymes attached. As there are three distinguishable configurations for two phosphorylated subunits on a ring, and each configuration can have zero, one, or two PP1 enzymes attached, we include nine separate configurations for P2. We have the following numbers of configurations: P0 (1), P1 (1 × 2), P2 (3 × 3), P3 (4 × 4), P4 (3 × 5), P5 (1 × 6), and P6 (1 × 7), to obtain 56 in total. We do not explicitly count the different relative positions of PP1 bound to phosphate groups, but just select one of the phosphorylated subunits at random when the dephosphorylation occurs. At any point in time, the system is in a state that is defined by the number of rings in each configuration, denoted by { N i }, i = 1, 2,…, 56. The numbers of rings in each configuration { N i } determine the rates of each of the possible reaction steps, { r j }. A reaction step takes a ring from one of the 56 configurations to another. With the three types of reaction steps (phosphorylation, PP1 binding, or dephosphorylation) able to occur from most of the configurations, in total the system has 144 distinguishable reaction steps. However, most of the rates are zero at any particular instance. Protein turnover is treated stochastically like any other process. Turnover results in two randomly chosen rings (i.e., one holoenzyme) being replaced by two rings in the state P0 (totally unphosphorylated). The fundamental assumption behind the simulations is that any particular rate, r j ( t ), depends only on the present configuration, not on the history of the system. This makes the reaction scheme a Markov process. The key step that is necessary when dealing with small numbers of molecules is to recall that the deterministic rate is the macroscopic average of a stochastic process, such that the probability, P j dt of reaction step j in a small time interval, dt, is given by that is, the rate is simply the probability of occurrence per unit time, and, given the Markov assumption, we now have a Poisson process. Gillespie's algorithm proceeds by first summing the rates of all reaction steps to obtain the average rate, R T , for any change in configuration: The distribution of times elapsed before a reaction step follows an exponential decay, which is a standard result, easily verified as it satisfies the necessary requirement: that is, the probability of reaction in time step from τ to τ + dτ equals R ( τ ) dτ multiplied by the probability that a reaction did not happen before τ . Once the time for the next reaction step is randomly selected, a second random selection is taken to decide which particular reaction occurs. The relative probabilities, p j , of each reaction step are simply proportional to their rates, p j = r j / R T . Given the time and type of the next reaction step, the total time for the system is advanced by τ , and the quantities, { N i }, in relevant configurations are updated, as are the reaction rates for the affected reaction steps. The process now repeats itself with a new set of reaction rates. Total phosphorylation is monitored, and thresholds for determining a switch to the UP state or a switch to the DOWN state are set according to the equilibrium values, but typically if the total falls to below 10% phosphorylation, we record a transition to the DOWN state, and if it spontaneously rises to 70% phosphorylation, we record a transition to the UP state. It should be noted that the time it takes for the system to switch between UP and DOWN states is on the order of hours, which gives an overestimate of the lifetime of a state when in reality the system is “in transition.” However, such an error on the order of hours is insignificant (compared to many years for typical average lifetimes) except in the most unstable systems presented here. Simulations begin with either 0% or 100% phosphorylation, but within a very short time (at least compared to the lifetimes of UP and DOWN states) the system settles near the equilibrium values for UP or DOWN states, respectively. Switching-off rate calculations We define a ring in the unphosphorylated state, P0, as off, and one with any subunits phosphorylated as on. We calculate the rate for rings to switch off by assuming that all rings in the system that are on reach a dynamic equilibrium in their phosphorylation levels, in the time between switching-on and switching-off events for rings. This approximation, known as separation of timescales, is valid when the individual subunit phosphorylation and dephosphorylation rates are much faster than the rates for rings to switch on and off (as evident in Figure 4 E). For a given amount of total phosphorylation, we know exactly the average activity of the phosphatase. Knowing this activity means average rates can be calculated for all reactions. The different average rates determine the rate of change from one configuration of a holoenzyme to another, so knowing the average rates allows us to obtain numerically the relative amounts of time, Ρ i , spent in each configuration, i . Hence we can calculate when the system has a given total level of phosphorylation, Sp tot , what is the average phosphorylation level per ring, , where P i is the phosphorylation of configuration i . The total level of phosphorylation, Sp tot , when a given number of rings are on, N on , is given by Since Sp tot is proportional to P av and P av depends on Sp tot , we iterate the equations to find a self-consistent solution for Sp tot and P av for each value of N on . To find the switching-off rate per ring, plotted in Figure 4 D, for each number of rings switched on, the total phosphorylation, Sp tot , is calculated as above. The value of Sp tot determines the phosphatase activity per subunit, ν 3 , which affects the proportion of time spent by a ring in each configuration. Notably, the lower ν 3 is, the more phosphorylated are the on rings, and the lower is Ρ P 1 . The rate for a ring to switch off is the sum of the turnover rate, ν T , and rate of dephosphorylation by PP1 of holoenzymes with only a single phosphorylated subunit, ν 3 Ρ P 1 . Ca 2+ influx and fluctuations For the LTP induction protocol, we use a burst of Ca 2+ influx constituted by a Poisson train of Ca 2+ pulses at 100 Hz. Each pulse is a 0.1-μM step increase, followed by a 100 ms exponential decay in free Ca 2+ concentration. In the study of the effects of background Ca 2+ fluctuations, we assume Ca 2+ entry through NMDA receptors occurs as a random Poisson train with an average rate of 0.5 Hz. We model each Ca 2+ influx as a step rise of 0.1 or 1.0 μM, followed by exponential decay with a time constant of 100 ms ([ 44 ], assuming a membrane potential of −50 mV and a spine volume of 0.1 μm 3 ; peak [Ca 2+ ] rise is 0.14 μM per presynaptic spike). We reduce the base level of Ca 2+ to 0.07 μM in the 0.1 - μM amplitude simulations, keeping all other parameters the same, to maintain an approximate balance between phosphorylation and dephosphorylation rates. We assume a refractory period of 2 ms for the presynaptic neuron, so that no two Ca 2+ influxes occur within such a short interval (hence the train of Ca 2+ inputs is not quite Poisson, but includes a 2-ms negative correlation). If the fluctuation amplitude is too large (for example, if it is doubled from 0.1 μM to 0.2 μM in this case) and the model parameters are fixed, then bistability is completely lost, because the fluctuations actually change the average effective kinetic rates of reaction steps and bring them outside of the range for bistability of the system. Hence, in the simulations with amplitude 1.0 μM, we maintain a base Ca 2+ level of 0.1 μM and adjust other parameters (see legend of Figure 5 ) to maintain an appropriate balance between the different reaction rates. For example, we include a higher Κ H 1 to maintain low rates for the initial phosphorylation step. With the alternative parameters, the system is only bistable if the Ca 2+ fluctuations are present. Notice that the number of free Ca 2+ ions in the PSD is on average less than one. However, Ca 2+ acts through Ca 2+ -bound calmodulin (which can be at a higher concentration), and the exchange of both free Ca 2+ and calmodulin between postsynaptic density and the spine is much more rapid than both the fluctuations considered here and typical CaMKII reaction steps. Hence, we can neglect such strong, but fast, “shot” noise. Exchange of free Ca 2+ or calmodulin between the dendritic shaft and spine will cause significant additional fluctuations, but only if it is on a slower timescale than the dissociation steps between Ca 2+ and calmodulin, or Ca 2+ /calmodulin and CaMKII. Future work, including on these specific binding reaction steps [ 74 ], is necessary to clarify more precisely the dynamical effects of Ca 2+ changes on the system. Parameters Parameters for the standard system are given in Table 1 . In figures where one parameter varies, all others are fixed according to the table, unless otherwise stated. Figures where the number of holoenzymes changes also include a proportionally changed volume and number of phosphatases, to maintain fixed concentrations. The standard concentration of PP1 is equal to that of CaMKII holoenzymes, at 20 molecules per 10 6 nm 3 or 33 μM. Our maximal system with 20 holoenzymes is slightly smaller than an average synapse of 30 holoenzymes [ 32 ]. Hence, the volume is smaller than average, corresponding to a cylindrical PSD of diameter 250 nm (compared to an average diameter of 350 nm in [ 32 ]), assuming the holoenzymes are predominantly in a single layer of a little over 20 nm thickness, to give a volume of 10 6 nm 3 for the domain of reactions. For those parameters in the table without experimental references, we chose values that were in a reasonable range given the values for similar chemical reactions. We picked simple values that would work well for our system. For example, a low Michaelis constant is beneficial because the range of bistability increases [ 28 ]. In general, variation of any one parameter, without alteration of the others, leads to an effect like that shown in Figure 2 , where number of phosphatases is varied. If, for example, the activity of calcineurin is higher (e.g, giving ν Ca N = 2.0 instead of ν Ca N =1.0), then the amount of I1P is halved and the amount of uninhibited PP1 increases. Hence, the stability of the UP state decreases while stability of the DOWN state increases. However, a system with a lower overall PP1 concentration would work as well as the original system. So, modification of individual parameters does degrade the system, reducing the lifetime of one state relative to the other. Nevertheless, if the cell is able to maintain concentrations of species in an optimal range determined by the actual value of parameters, then the best results shown here can be achieved (cf. [ 28 ]).

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1069646

The Wolbachia Genome of Brugia malayi: Endosymbiont Evolution within a Human Pathogenic Nematode

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia 's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi ( w Bm) with the Wolbachia endosymbiont of Drosophila melanogaster ( w Mel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to w Mel, w Bm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of w Bm in contrast to the parasitic lifestyle of w Mel. The smaller genome size of w Bm, relative to w Mel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.

Introduction Over 1 billion people in more than 90 countries are at risk from filarial nematode infections, and 150 million people are infected. The parasitic nematodes are insect-borne and are responsible for lymphatic or cutaneous filariasis, leading to medical conditions including elephantiasis or onchocerciasis (African river blindness). Lymphatic filariasis is caused predominantly by Wuchereria bancrofti and Brugia malayi and affects 120 million individuals, a third of whom show disfigurement, while onchocerciasis, caused by Onchocerca volvulus, affects 18 million people of whom 500,000 have visual impairment and 270,000 are blind [ 1 , 2 ]. Within these filarial parasites are intracellular bacteria that were first observed almost 30 y ago [ 3 , 4 , 5 , 6 ]. The establishment in 1994 of a Filarial Genome Project funded by the World Health Organization (WHO/Tropical Disease Research/United Nations Development Programme/World Bank) contributed to the rediscovery of these endosymbiotic bacteria. In the analysis of cDNA libraries generated from different life cycle stages of B. malayi, the presence of rare non- Escherichia-coli- like, alpha-proteobacterial sequences implicated the occurrence of endobacterial DNA [ 7 ]. Phylogenetic analyses subsequently identified the bacteria as Wolbachia [ 8 ]. These endosymbionts have now been found in the vast majority of filarial nematode species, with notable exceptions [ 3 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 ]. Wolbachia appear to be absent in nonfilarial nematodes [ 20 ]. In nematodes that contain Wolbachia and which have been well examined, the bacteria are located in the lateral chords (invaginations of the body wall hypodermis that project into the body cavity) in both sexes. They are also localized in oocytes but not in the male reproductive tract. The endosymbionts appear to be present in 100% of individuals within a population, when that species contains them, suggesting that they are required for worm fertility and survival [ 10 , 21 , 22 ]. They are therefore potential therapeutic targets for filariasis control. Certain antialpha proteobacterial agents, most notably tetracycline and doxycycline, but also rifampicin and azithromycin, show inhibitory effects on parasitic nematode development and fertility [ 13 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. After antibiotic treatment, immunogold staining, using Wolbachia- specific cell-surface probes, shows the absence of Wolbachia in the female reproductive tract and the degeneration of embryos, while Wolbachia remain in the lateral chords, albeit in reduced numbers [ 34 ]. Genchi et al. [ 35 ] have also shown that Wolbachia are present at 1000X lower frequencies after antibiotic treatment and can still be detected by PCR from female hypodermis tissues, but not from female reproductive tissue. No antibiotic effects are observed in filarial nematodes that do not harbor Wolbachia, nor are they observed with other antibiotics (e.g., penicillin, gentamicin, ciprofloxacin, or erythromycin), suggesting that these effects correlate with Wolbachia presence [ 11 , 12 , 13 , 36 , 37 ]. Human trials using doxycycline, undertaken in Ghana, have shown that this antibiotic interferes with embryogenesis in adult female filariae with a concomitant depletion of Wolbachia from both adults and microfilariae (first stage larvae) of O. volvulus and W. bancrofti [ 38 , 39 , 40 , 41 , 42 ]. Thus, as in animal models, Wolbachia appears to be a therapeutic target for human filarial parasitic infections. The use of anti- Wolbachia chemotherapy against filarial parasites has initiated a novel approach for filarial disease control and eradication. Previous strategies for elimination of filariasis have included vector control in the presence or absence of antiparasitic drugs [ 43 , 44 , 45 , 46 , 47 ]. Diethylcarbamazine, albendazole, and ivermectin have been the most recent drugs of choice for prevention of filarial infections, but they have little effect on adult worms. Thus repeated doses in endemic areas are required to eliminate infections that can arise again within months of treatment [ 39 , 44 , 48 ]. In addition, the possibility of drug resistance, as observed with intestinal helminths in animals is a concern [ 49 , 50 , 51 ]. No new therapeutics have been developed in over 20 y, and there is a need for better drugs that permanently sterilize or kill adult worms. Wolbachia play a role in the host immunological response to filarial parasite invasion. Infection by filarial parasites results in B-cell proliferation and the generation of antibodies directed toward parasite- and Wolbachia -specific antigens, including those to Wolbachia surface protein, heat shock protein, aspartate aminotransferase, and Htr serine protease [ 11 , 52 , 53 , 54 , 55 , 56 , 57 ]. Other Wolbachia -specific molecules also play roles in the immune response to filarial infections including the release of stimulatory and modulatory factors from neutrophils and monocytes, which may be related to Wolbachia release upon worm death [ 58 , 59 , 60 , 61 ]. One component of the host immune response appears to mimic a lipopolysaccharide (LPS)-like response, typically observed as a host immune response to Gram-negative bacteria (such as the alpha-proteobacterial Wolbachia ) [ 22 , 58 , 62 , 63 , 64 , 65 ]. Further, LPS-like products of Wolbachia appear to be involved in the eye inflammation observed in African river blindness. Leukocytes (neutrophils and eosinophils) infiltrate the cornea as a result of microfilarial invasion and death within the eye, leading to a loss of corneal transparency [ 66 ]. LPS-like molecules are implicated in this process due to activation of the toll-like receptor 4 (TLR4) pathway by Wolbachia [ 61 , 67 ]. Release of filarial worm-associated molecules, especially after drug treatments that cause worm death in the host, leads to pathogenesis (“Mazzotti Reaction”) [ 68 , 69 , 70 , 71 , 72 ], and Wolbachia has been associated with chronic and acute infection states of filariasis (reviewed in [ 59 ]). Repetitive exposures to LPS-like molecules due to release of Wolbachia following death of microfilaria are thought to induce chronic inflammation events giving rise to immune tolerance [ 65 , 73 ], as hyporesponsiveness occurs with increasing parasite load [ 74 , 75 , 76 ]. Wolbachia endosymbionts can be separated into six supergroups based upon 16S rRNA, Wolbachia surface protein, and fts Z phylogenetics [ 8 , 11 , 15 , 77 , 78 , 79 , 80 , 81 , 82 ]. Four supergroups contain Wolbachia from arthropods while supergroup C contains Wolbachia from the nematodes O. volvulus and Dirofilaria immitis, and supergroup D contains Wolbachia from B. malayi, W. bancrofti, and Litomosoides sigmodontis [ 11 , 82 ]. In nematodes, the evolution of Wolbachia parallels the phylogenetics of their hosts, while in the other supergroups, horizontal transmission appears to have occurred [ 11 , 14 , 15 , 79 , 82 ]. The closest bacterial relatives to the Wolbachia are in the Order Rickettsiales, including Rickettsia, Ehrlichia, Cowdria, and Anaplasma, all parasites of mammals that require arthropod vectors for transmission [ 83 , 84 ]. Up to 70% of all insect species appear to harbor Wolbachia [ 85 , 86 , 87 ]. While parasitic and maternally inherited in insects, they appear not to be required for host survival. But when present in appropriate genetic backgrounds, they confer developmental effects leading to sex ratio disturbances, feminization of genetic males, parthenogenesis, cytoplasmic incompatibilities and/or reciprocal-cross sterility [ 79 , 88 , 89 , 90 ]. It has been suggested that endosymbionts, including Wolbachia, might be of medical importance and used for insect vector control to deliver antiparasitic products to recipient hosts [ 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 ]. For these reasons, a genome project was initiated and completed on the Wolbachia endosymbiont of Drosophila melanogaster ( w Mel) [ 103 ]. Identification of Wolbachia in parasitic nematodes, their role in pathogenesis, their potential as a target for development of antifilarial therapeutics, and their widespread occurrence in arthropods triggered a meeting held in 1999 to initiate a consortium of Wolbachia researchers [ 104 , 105 ]. Three additional meetings have been held (see http://www.wolbachia.sols.uq.edu.au/index.html , and eight additional Wolbachia genomes responsible for diverse phenotypes are being sequenced. We report the second complete genome sequence of Wolbachia and the first from a parasitic nematode, B. malayi ( W. pipientis, BruMal TRS strain; Wolbachia endosymbiont of B. malayi [ w Bm]). We also describe a comparative analysis of reductive evolution in different lineages of endosymbiotic bacteria, a major evolutionary trend in all intracellular parasites and symbionts. Features of the w Bm genome are presented as a systematic comparison to w Mel and Rickettsia spp., the closest fully sequenced relatives of w Bm and more distant intracellular parasites and symbionts of the gamma-proteobacterial lineage, such as Buchnera (aphid endosymbiont), Blochmannia (ant endosymbiont), and Wigglesworthia (tsetse fly endosymbiont) [ 106 , 107 , 108 , 109 , 110 , 111 , 112 ]. We also delineate the metabolic pathways that might account for the mutualistic relationship between Wolbachia and its nematode host. Results/Discussion Genome Properties and General Comparison with the Genomes of Other Parasites and Endosymbionts The genome of w Bm is represented by a single circular chromosome consisting of 1,080,084 nucleotides and is 34% G+C. The size agrees with the 1.1 Mb length previously determined by both pulsed-field gel electrophoresis and restriction mapping [ 113 , 114 ]. The origin of replication (oriC) was tentatively mapped immediately upstream of the hem E gene on the basis of GC- and AT-skew analyses [ 115 ] ( Figure 1 ). The genome of w Bm has an extremely low density of predicted functional genes compared to all other bacteria, with the exceptions of R. prowazekii ( Table 1 ) and Mycobacterium leprae. Both Wolbachia spp. and Rickettsia spp. have undergone considerable gene loss in many metabolic pathways, relative to other alpha-proteobacteria ( Table 2 ). A comparison of predicted functional genes in w Bm and Rickettsia spp. reveals a large core set that is conserved among these genomes, as well as smaller sets unique to each genome ( Figure 2 ). In contrast, nearly all observed pseudogenes are unique to each genome ( Figure 2 ), suggesting substantial independent genome degradation. Wolbachia ( w Bm) and R. conorii contain, in addition to many demonstrable pseudogenes, a considerable number of short open reading frames (ORFs), which have no detectable orthologs in current protein databases but are recognized as probable genes by gene prediction programs. However, most of these sequences, which comprise approximately 5% of the total predicted gene number in w Bm, are likely to be fragmented genes as well ( Table 1 ). Figure 1 Genogram of the Complete Circular Genome of w Bm The scale indicates coordinates in kilobase pairs (kbp) with the putative origin of replication positioned at 0 kbp. The outermost ring indicates the GC-skew over all bases in the forward strand using a window size of 40 kbp and a step size of 1 kbp. Positive and negative skew are shaded gold and blue, respectively. Features are shown as paired rings separated by a circular baseline. In each pair, the outer and inner rings represent the forward and reverse DNA strands, respectively. Working inward from the scale, the features displayed are as follows: identified genes and their broad functional classification (multihued, as listed); tRNA (blue)/rRNA (red) genes; putative pseudogenes (green); repeated sequences (red) and transposon-related repeats (blue). Figure 2 Venn Diagram Showing Comparison of Conserved and Unique Genes and Pseudogenes in w Bm (Wolbachia from B. malayi), Rickettsia prowazekii, Rickettsia conorii, and in w Bm and w Mel (among Those Assigned to COGs) (A) Predicted functional protein-coding genes. (B) Pseudogenes. (C) Combined results for comparison between wBm and w Mel. G, intact gene; P, pseudogene. Table 1 Comparison of Genome Features of Proteobacterial Endosymbionts and Endoparasites a Independent estimates obtained during this work. w Bm, Wolbachia from B. malayi; w Mel, Wolbachia from Drosophila melanogaster; R. conorii, Rickettsia conorii; R. prowazekii, Rickettsia prowazekii; B. aphidicola, Buchnera aphidicola; B. floridanus, Blochmannia floridanus; W. glossinidia, Wigglesworthia glossinidia. IS, insection element sequence Table 2 Gene Loss and Decay in Wolbachia and Rickettsia Gene conservation and loss were determined with respect to the set of 1,177 genes that are represented by confidently identifiable orthologs in all free-living alpha-proteobacteria. For each category, the first number indicates retained genes, the second number indicates lost genes, and the third number indicates pseudogenes. The sum of these numbers equals the total number of genes in this category in the alpha-proteobacterial core set. w Bm, Wolbachia from B. malayi; w Mel, Wolbachia from Drosophila melanogaster; R. conorii, Rickettsia conorii; R. prowazekii, Rickettsia prowazekii. The w Bm genome contains one copy of each of the ribosomal RNA genes (16S, 23S, and 5S), which do not form an operon, as also observed in w Mel and Rickettsia but in contrast to most other bacteria, and 34 tRNA genes that include cognates for all amino acids. Probable biological function was assigned to 558 (approximately 70%) of the 806 protein coding genes; a more general prediction of biochemical function was made for an additional 49 ORFs. Most of the predicted genes (617, 76%) could be included in clusters of orthologous groups of proteins (COGs) with orthologs not only in w Mel and Rickettsia but also in more distant organisms. A lack of flagellar, fimbrial or pili genes indicates that w Bm is probably nonmotile ( Table 2 ). However, some intracellular pathogens, including spotted fever group Rickettsia, exploit a different motility mechanism that makes use of the host cell actin polymerization to promote bacterial locomotion. Actin-based motility of Rickettsia depends upon activation of the host Arp2/3 complex by the WASP family protein RickA [ 116 , 117 ]. A gene coding for WASP family protein (Wbm0076) was identified in w Bm suggesting that it might be able to employ actin polymerization for locomotion and cell-to-cell spread. Informational and Regulatory Systems Comparison with an obligatory gene set characteristic for free-living alpha-proteobacteria ( Table 2 ) shows that both Wolbachia spp. and Rickettsia have retained an almost intact gene set for translational processes (greater than 84%). Several RNA metabolism genes are among the few shared losses, including tRNA and rRNA modification enzymes (LasT, RsmC, Sun, TrmA, CspR) and even pseudouridine synthase, TruB (pseudogenes in both lineages). TruB is present in all gamma-proteobacterial endosymbionts but absent in other parasites and endosymbionts, including Mycoplasma, Chlamydia, and spirochetes. It is likely that the lack of these modifications affects reading frame maintenance and translation efficiency in both Wolbachia spp. and in Rickettsia. Further reduction of genes involved in RNA modification occurs specifically in w Bm and in w Mel, which have lost several genes involved in queuosine biosynthesis (COG0809, COG603, COG702, COG0602, COG0780) [ 118 ] and 16S rRNA uridine-516 pseudouridylate synthase. The absence of RNA methylase (COG1189) highlights the loss of RNA modification systems, which is a general trend in evolution of endosymbionts among various lineages [ 119 ]. Although w Bm retains most of the genes for DNA replication and repair, the loss of several genes present in other alpha-proteobacteria (except w Mel) is notable. These include the chi subunit of DNA polymerase III (HolC), chromosome partitioning proteins ParB and ParA, repair ATPase (RecN), exonuclease VII (XseAB), and the RNA processing enzyme RNase PH (Rph). Both Wolbachia spp. and Rickettsia have a complete repertoire of UV-excision (UVR-ABCD-mediated), recombinational synaptic (RecA/RecFOR-mediated), and postsynaptic (RuvABC-mediated) DNA repair pathways. In contrast, Buchnera and Blochmannia are devoid of conventional homologous recombination and uvr pathways, although they encode a putative phrB familyphotolyase [ 107 , 109 , 110 , 112 , 120 , 121 ]. Wolbachia, Rickettsia, Buchnera, and Wigglesworthia all encode enzymatic machinery to counter the deleterious effects of various types of base oxidative damage, which could be important for defense against mutagenic metabolic by-products in the intracellular environment [ 103 , 108 , 109 , 119 , 122 ]. Many proteins categorized as being involved in protein fate in the two Wolbachia spp. and Rickettsia spp. (CcmF, CcmB, CcmH, CcmE, CcmC, Cox11, CtaA), but which are absent in the genomes of gamma-proteobacterial endosymbionts, are involved in biogenesis of cytochrome c oxidase and c-type cytochromes typical of alpha-proteobacterial aerobic respiratory chains. Respiratory chains of gamma-proteobacterial endosymbionts employ quinol oxidase rather than cytochrome c oxidase. A major loss of transcriptional regulators likely occurred in the common ancestor of Wolbachia and Rickettsia spp. ( Table 2 ). Only a few of these genes have been additionally lost in the w Bm lineage, including those from COG1396, COG1959, COG1329, COG1678, and COG1475. This is a general trend in evolution of endosymbionts and parasites [ 118 , 122 , 123 ], suggesting that most of their genes are likely constitutively expressed. Those few regulators found in w Bm that are not present in other alpha-proteobacteria, including two Xre-like regulators (COG5606), may be of interest for future experimental characterization. Similarly, most genes implicated in signal transduction systems are absent in both Wolbachia and Rickettsia spp. Several regulatory proteins that remain in the genome are involved in various stress responses (Wbm0660, MerR/SoxR family; Wbm0707, cold shock protein; Wbm0494, stress response morphogen; Wbm0061, TypA-like GTPase) or in cell cycle regulation (Wbm0184, PleD-like regulator; Wbm0596, cell cycle transcriptional regulator CtrA). Metabolic Capabilities of w Bm are Key to Understanding its Interaction with the Host One of the roles of w Bm as an obligate endosymbiont may be to provide its host with essential metabolites. Although w Bm has retained more metabolic genes than Rickettsia spp., its biosynthetic capabilities appear to be rather limited. Unlike Buchnera spp. [ 107 , 109 , 112 , 122 , 123 ], w Bm is able to make only one amino acid— meso -diaminopimelate ( meso -DAP), a major peptidoglycan constituent. In most bacteria, it is produced as an intermediate in the pathway of lysine biosynthesis. Similar to Rickettsia spp. [ 122 ], w Bm lacks meso -DAP decarboxylase (LysA, COG0019), necessary for lysine biosynthesis, such that the biochemical pathway ends with meso -DAP. Complete pathways for de novo biosynthesis of purines and pyrimidines are found in w Bm, as opposed to Rickettsia and many other endosymbionts and parasites, including Buchnera, Blochmannia, Mycoplasma, and Chlamydia ( Table 3 ). The general trend for nucleotide biosynthesis pathways to be lost in these organisms appears to be independent of the presence of ADP/ATP translocase (COG3202) (present only in Rickettsia and Chlamydia ), which facilitates the uptake of nucleotide-triphosphates from the hosts. This observation suggests that w Bm produces nucleotides not only for internal consumption but also for supplementation of the nucleotide pool of the host ( Figure 3 ) when needed, such as during oogenesis and embryogenesis, where the requirement for DNA synthesis is likely very high [ 124 ]. Figure 3 Metabolic Pathways Retained in wBm Pathways shared by Wolbachia and Rickettsia are shown with black arrows. Pathways present in Wolbachia but not in Rickettsia are shown with green arrows. Numbering alongside pathway arrows reflects enzyme annotation, a table of which is available at http://tools.neb.com/wolbachia/ . Table 3 Differential Loss of Functionality and Differentially Preserved Functionality, if Only a Few Compared Alpha- and Gamma-Proteobacterial Parasite/Symbiont Genomes Have Lost or Preserved This Functionality w Bm, Wolbachia from B. malayi ; w Mel, Wolbachia from Drosophila melanogaster; R. conorii, Rickettsia conorii; R. prowazekii, Rickettsia prowazekii; B. aphidicola, Buchnera aphidicola; B. floridanus, Blochmannia floridanus; W. glossinidia, Wigglesworthia glossinidia. All genes required for biosynthesis of fatty acids and all but one gene for biosynthesis of phospholipids (phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine) are present in the w Bm genome. The absent gene in phospholipid biosynthesis is glycerol-3-phosphate acyltransferase (COG2937), which catalyzes the transfer of the first fatty acid to glycerol-3-phosphate. However, a “fatty acid/phospholipid biosynthesis enzyme” PlsX is present, which can complement the absence of glycerol-3-phosphate acyltransferase in E. coli [ 125 ]. All but one gene for biosynthesis of isoprenoids has been found in the genome. This absent gene is 1-deoxy-D-xylulose-5-phosphate synthase (COG1154), an essential gene in the nonmevalonate pathway. It is possible that this biochemical function could be complemented by a transketolase or transaldolase, two highly promiscuous enzymes encoded by the w Bm genome or, alternatively, 1-deoxy-D-xylulose-5-phosphate must be supplied by the host. Unlike Rickettsia, w Bm contains all the enzymes for the biosynthesis of riboflavin and flavin adenine dinucleotide ( Figure 3 ). w Bm could be an important source of these essential coenzymes for the host nematode. No genes for riboflavin biosynthesis have been detected in the ongoing B. malayi genome data (9X coverage) [ 126 ]. Similar to most other endosymbionts, w Bm lacks complete pathways for de novo biosynthesis of other vitamins and cofactors such as Coenzyme A, NAD, biotin, lipoic acid, ubiquinone, folate, and pyridoxal phosphate, retaining only a few genes for the finals steps in some of these pathways. These incomplete pathways may make w Bm dependent upon the supply of those precursors from the host. Heme serves as a prosthetic group of cytochromes, catalase and peroxidase, and may be another metabolite provided by w Bm to B. malayi. w Bm has all but one gene for heme biosynthesis and has maintained all genes for maturation of c-type cytochromes. The absent gene in the heme biosynthesis pathway encodes protoporphyrinogen oxidase, a gene not identified in many alpha-proteobacteria. It is likely that these bacteria contain a functional form of protoporphyrinogen oxidase, which is not yet known, or that the missing function is complemented by another gene function, as in E. coli [ 127 ]. Heme could play an important role in filarial reproduction and development. It is possible that molting and reproduction are regulated by ecdysteroid-like hormones, since the insect hormones ecdysone and 20-hydroxyecdysone and their inhibitors affect molting and microfilarial release in D. immitis and B. pahangi [ 128 , 129 ]. In Drosophila, five enzymatic reactions in the pathway of ecdysteroid biosynthesis are catalyzed by microsomal and mitochondrial cytochrome P450 mono-oxygenases [ 130 ]. If similar enzymes participate in the pathway of biosynthesis of filarial steroid hormones, heme depletion caused by elimination of w Bm could result in a decreased activity of these enzymes, which might account for the effects on nematode viability, larval development, and reproductive output observed following antibiotic treatment of filarial parasites. There is currently no evidence of heme biosynthesis enzymes in B. malayi (analysis of the draft genome sequence of B. malayi does not identify any genes for heme biosynthesis [ 126 ]). These enzymatic activities have been detected in Setaria digitata, a cattle filarial parasite, which is devoid of typical cytochrome systems, yet has heme-containing enzymes, such as microsomal cytochrome P450, catalase, and peroxidase [ 131 ]. It is not known whether S. digitata contains Wolbachia and whether heme biosynthesis detected in this worm is due to the presence of endosymbiotic bacteria. However the closely related filarial parasites, S. equina, S. tundra, and S. labiatopapillosa are devoid of endosymbiotic Wolbachia [ 15 , 16 ]; perhaps they have retained the genes for heme biosynthesis. Genes for biosynthesis of glutathione are present in the w Bm genome (Wbm0556; Wbm0721). Two physiological roles of glutathione in bacteria are known: one is detoxification of methylglyoxal [ 132 ], and the other is protection against oxidative stress through activation of the glutathione peroxidase–glutathione reductase system [ 133 , 134 ]. Methylglyoxal is accumulated in phosphate-limited environments, such as those encountered by Salmonella inside macrophages [ 132 ]. It is possible that w Bm encounters phosphate-limited conditions inside the host and therefore needs glutathione as a quencher of methylglyoxal. This view is supported by the presence of the gene encoding the Kef-type potassium efflux system, a participant in methylglyoxal detoxification through acidification of cytosol [ 132 ]. However, no homologs of E. coli glo A– glo B genes responsible for glutathione-dependent methylglyoxal detoxification were found in the genome. Glutathione peroxidase is also absent, hence the physiological role of glutathione in w Bm is unclear. Although genes for glutathione biosynthesis are present in the B. malayi genome, it is possible that w Bm provides glutathione to the host, since the latter needs high levels of this essential metabolite for protection against oxidative stress [ 135 ] and detoxification [ 136 ]. Intermediates for these biosynthetic pathways are likely derived from gluconeogenesis, the nonoxidative pentose phosphate shunt, and the tricarboxylic acid (TCA) cycle. Glycolytic enzymes encoded by w Bm probably function in a gluconeogenesis pathway ( Figure 3 ), since the genes coding for two enzymes catalyzing irreversible glycolytic reactions, 6-phosphofructokinase and pyruvate kinase, are absent. Instead, the gluconeogenic enzyme fructose-1,6-bisphosphatase (Wbm0132) and pyruvate-phosphate dikinase (Wbm0209), which functions predominantly in gluconeogenesis in bacteria, are present suggesting that the pathway functions as gluconeogenesis, albeit ending with fructose-6-phosphate rather than glucose-6-phosphate. While fructose-6-phosphate is necessary for biosynthesis of the peptidoglycan components N-acetylglucosamine and N-acetylmuramate, no enzymes capable of utilizing glucose-6-phosphate as a substrate are encoded in the w Bm genome. It is reasonable to suggest that the most likely growth substrates for w Bm would be those compounds that are highly abundant in the worm. In adult B. malayi, B. pahangi, and Dipetalonema viteae (Acanthocheilonema viteae), these include the excretory metabolites lactate and succinate, which are the principal products of glucose utilization under both aerobic and anaerobic conditions, and a disaccharide trehalose, which is used by the worms as a storage compound [ 137 , 138 ]. Nuclear magnetic resonance studies of adult B. malayi identified phosphoenolpyruvate as the major energy reservoir [ 139 ]. However, w Bm is not predicted to be able to utilize lactate due to the absence of genes coding for lactate dehydrogenases and is likely unable to grow on sugars, as evidenced by the lack of genes encoding sugar transporters or sugar kinases. Thus, the most likely growth substrates for w Bm are pyruvate and TCA cycle intermediates derived from amino acids, with enzymes present for amino acid degradation, a pyruvate dehydrogenase complex, a complete TCA cycle, and a respiratory chain typical of alpha-proteobacteria ( Figure 3 ). Amino acids are likely imported from the extracellular environment where they are obtained by proteolysis of host proteins by proteases and peptidases. Indeed, the genome of w Bm encodes a variety of proteases, including predicted metallopeptidases (at least seven Zn-dependent proteases of four distinct families compared to only one in Rickettsia ) (Wbm0055, Wbm0153, Wbm0221, Wbm0311, Wbm0419, Wbm0418, Wbm0742). In addition, two Na + /alanine symporters were found (Wbm0197, Wbm0424), which are absent in Rickettsia. Cell Wall Structure A dramatic case of lineage-specific gene loss in both Wolbachia spp. includes approximately 20 genes for enzymes of cell-envelope LPS biosynthesis. It has been reported that soluble endotoxin-like products of Wolbachia endosymbionts of filarial nematodes, including B. malayi, B. pahangi, L. sigmodontis, O. volvulus, and D. immitis, contribute to the immunology and pathogenesis of filarial diseases through induction of potent inflammatory responses, including production of tumor necrosis factor alpha, interleukin-1-beta, and nitric oxide by macrophages [ 22 , 58 , 59 , 60 , 71 , 72 , 140 , 141 ]. Chemokine and cytokine responses to the sterile extracts of Brugia and Onchocerca were dependent on signaling through TLR4 and could be blocked by neutralizing antibodies to CD14 and by the antagonistic lipid A analogs, indicating that the inflammatory response was induced by an LPS-like molecule. Recently the major surface protein of Wolbachia spp. was implicated as the inducer of the immune response acting in a TLR2- and TLR4-dependent manner [ 141 ]. However, it is not clear whether this protein is the only Wolbachia -specific molecule eliciting a TLR4-dependent innate immune response. Analysis of the w Bm genome indicates that, like Ehrlichia chaffeensis and Anaplasma phagocytophilum [ 142 ], it lacks homologs of the genes responsible for biosynthesis of lipid A. Although lipid A structure can vary in different bacteria, it always consists of a polysaccharide backbone carrying fatty acid residues. The only predicted genes belonging to the glycosyltransferase family were those participating in peptidoglycan biosynthesis, and one glycosyltransferase pseudogene is present. Similarly, the only genes from the acyltransferase family are those participating in fatty acid and phospholipid biosynthesis. Thus, it is unlikely that the cell wall of w Bm contains LPS-like molecules. This idea is supported by the absence of the gene products responsible for maintaining the outer membrane structure in Gram-negative bacteria, such as TolQ, TolR, TolA, and TolB. Several lines of evidence suggest that the structure of the w Bm peptidoglycan is very unusual, and peptidoglycan derivatives might be responsible in part for the observed inflammatory responses. First, although all the genes necessary for biosynthesis of lipid II are present in the w Bm genome, there are no homologs of alanine and glutamate racemases responsible for synthesis of pentapeptide components D-alanine and D-glutamate. While the genomes of Rickettsia spp. contain L-alanine racemase that could catalyze racemization of both alanine and glutamate, the only amino acid racemase present in the genomes of both Wolbachia is meso -DAP epimerase (Wbm0518), an enzyme catalyzing interconversions of LL- and meso- isomers of diaminopimelate. It is possible that meso -DAP epimerase is able to catalyze racemization of alanine and glutamate, although this activity has never been experimentally demonstrated. Alternatively, instead of the usual D-isomers, w Bm peptidoglycan might contain L-isomers of alanine and glutamate. Second, Gram-negative bacteria (including Rickettsia spp.) usually contain two monofunctional transpeptidases. One of them, FtsI (also known as PBP3), is localized to the septal ring and is required for peptidoglycan biosynthesis in the division septum, while the other, PBP2, is localized preferentially to the lateral cell wall [ 143 ]. FtsI and PBP2 are recruited to the sites of their action by two membrane proteins, FtsW and RodA, respectively. In the w Bm genome, only functional orthologs of E. coli RodA and PBP2 were found; the orthologs of FtsW–FtsI are disrupted by multiple frameshifts. Third, genomes of bacteria that have peptidoglycan in their cell wall usually contain at least one gene coding for a high molecular weight penicillin-binding protein responsible for cross-linking of the murein sacculus. The transpeptidase and transglycosylase domains of this protein catalyze transpeptidation and transglycosylation of the murein precursors, respectively, to form the carbohydrate backbone of murein and the interstrand peptide linkages. No homologs of bifunctional transpeptidase/transglycosylase or monofunctional biosynthetic transglycosylase were found in the genomes of Wolbachia spp., although they are present in the Rickettsial genomes. The homolog of lytic transglycosylase, which is responsible for hydrolysis of the carbohydrate backbone during bacterial growth and division, is also absent from the genomes of both Wolbachia spp. Thus, their peptidoglycan can be cross-linked by the interstrand peptide linkages, but the carbohydrate backbone is not polymerized. These observations suggest that peptidoglycan of w Bm has some features in common with the peptidoglycan-derived cytotoxin produced by Neisseria gonorrhoeae and Bordetella pertussis [ 144 , 145 ] and that muramyl peptides derived from w Bm peptidoglycan could elicit the inflammatory response contributing to the pathogenesis of filarial infection. Other Host Interaction Systems As expected, functional Type IV secretion genes were found in the w Bm genome, including two operons: Wbm0793–Wbm0798 and Wbm0279–Wbm0283. These systems are indispensable for successful persistence of endosymbionts within their hosts [ 146 ]. Similar genes have been observed in the sequence of w Mel [ 103 ]. A role in the adaptation to the intracellular existence seems likely for several genes that are present in w Bm, w Mel, and Rickettsia. Thus, w Bm encodes five ankyrin-repeat-containing proteins and, in addition, has at least seven related pseudogenes, while w Mel contains 23 ankyrin -repeat-containing genes. Rickettsia contains two or three functional ankyrin-repeat genes (and probably one pseudogene) [ 147 ]. In eukaryotes, ankyrins connect cell membranes, including membranes of endosymbionts to the cytoskeleton [ 148 ], while in bacteria the function of ankyrin-like proteins remains largely unknown. One physiological function of bacterial ankyrin-like proteins was demonstrated in Pseudomonas aeruginosa, where ankyrin repeat AnkB is essential for optimal activity of periplasmic catalase, probably serving as a protective scaffold in the periplasm [ 149 ]. Another ankyrin-repeat protein, AnkA from E. phagocytophila, was detected in association with chromatin in infected cells, suggesting its possible role in regulation of host cell gene expression [ 150 ]. Another interesting protein is a member of the WASP family and is conserved in Rickettsia and w Bm (Wbm0076). Eukaryotic homologs of these proteins are suppressors of the cAMP receptor and regulate the formation of actin filaments [ 151 ]. The genes for an ankyrin-repeat protein and a WASP protein might have been acquired from a eukaryotic host by the common ancestor of Rickettsia and Wolbachia and could have contributed to the evolution of the intracellular lifestyle of these bacteria. w Bm also encodes several proteins with large nonglobular or transmembrane regions or internal repeats, orthologs of which are present also in the w Mel genome (Wbm0010, Wbm0304, Wbm0362, Wbm0749, and others). These proteins are likely to be surface proteins interacting with host cell structures. Further Comparisons of w Bm and w Mel One of the most striking characteristics of the w Mel genome is a large amount of repetitive DNA and mobile genetic elements, including three prophages, altogether comprising more that 14% of genomic DNA (and about 134 ORFs). Despite the abundance of repeats in the w Bm genome (5.4%) ( Figure 4 ), the percentage of repetitive DNA in w Bm is considerably less than in w Mel. This may reflect a stronger selection in w Bm for repeat loss and, as no prophages were identified in the w Bm genome, little exposure to foreign DNA. No plasmid maintenance genes were identified in the w Bm genome. Figure 4 Organization of Direct and Palindromic Repeats in w Bm Circles represent the complete genomic sequence of w Bm. Repeats were identified using the REPuter program [ 182 ] and are connected by line segments. Direct repeats are shown in the graphs in the top row, while palindromic repeats are shown in the lower row of graphs. The left column graphs display repeats of 50 to 500 bp in length. The rightmost graphs display repeats of greater than 500 bp in length. Comparison of the repetitive elements between these two genomes suggests the invasion of mobile genetic elements occurred after the divergence of the two Wolbachia along the w Mel branch, or that the majority of the transposons and phages were eliminated (degraded) specifically in the w Bm lineage. There is a similarly large difference in the amount of repetitive DNA in the two Rickettsia species ( Table 1 ). While an appropriate outgroup would be useful in both comparisons, the apparent degradation of repetitive DNA in Buchnera spp. [ 111 , 112 , 152 , 153 , 154 , 155 ] suggests the specific elimination of nonessential DNA is a result of reduced selection on gene functions no longer necessary in the host cells in Wolbachia spp. [ 156 ]. The large number of repeats and an apparently active system of DNA recombination suggest that extensive genome shuffling within w Bm and w Mel has eliminated colinearity between their genomes ( Figure 5 ). Frequent rearrangements in Wolbachia might be expected, given the exceptionally high levels of repeated DNA and mobile elements and the presence of several prophages in w Mel. It has been suggested that the surprisingly high percentage of repetitive DNA in w Mel might reflect a lack of selection for its elimination [ 103 ]. An alternative hypothesis might be that in Wolbachia there is a selective benefit to systems that maintain genetic diversity and that a high percentage of repeats may contribute to genome plasticity, as has been suggested for Helicobacter [ 157 ]. It has been suggested that the presence of a high level of repetitive DNA in w Mel, relative to w Bm, might reflect recurrent exposures to mobile elements and bacteriophages, as a result of its parasitic lifestyle [ 156 , 158 ]. Figure 5 Absence of Gene Order Colinearity between w Bm and Rickettsia and Disruption of Gene Colinearity between w Bm and w Mel Each dot represents a pair of probable orthologs defined as reciprocal BLAST best hits with E-value less than 0.001. (A) Genome dot-plot comparison of w Bm (Wolbachia from B. malayi) and Rpro (R. prowazekii). (B) Genome dot-plot comparison of w Bm (Wolbachia from B. malayi) and Rcon (R. conorii). (C) Genome dot-plot comparison of Rpro (R. prowazekii) and Rcon (R. conorii). (D) Genome dot-plot comparison of w Bm and w Mel. Comparative analysis of the genes assigned to COGs in both w Mel and w Bm shows that the genome of w Bm is more reduced ( Figure 2 ; Table 2 ). In total, 696 individual proteins from wBm have an ortholog in the w Mel genome; 84 such proteins are not assigned to COGs, and a considerable fraction of them are specific for only these two genomes. At least half of these predicted genes are larger than 100 amino acids, and orthologs have a similar length and presumably encode functional proteins. One of the important differences between the two Wolbachia for which genomes are available is that w Bm is apparently a mutualistic symbiont of its host, while w Mel is parasitic. The smaller size of the w Bm genome might be related to this difference. w Mel likely has to retain genes required for infecting host cells and avoiding host defense systems, whereas wBm may have lost many of these genes, as has been seen in organelles and other mutualistic symbionts such as the Buchnera symbionts of aphids. Despite there being considerably fewer predicted genes in w Bm ( Table 1 ), the metabolic capabilities of w Mel and w Bm are very similar. Unlike w Bm, w Mel has retained some enzymes for folate and pyridoxal phosphate biosynthesis, two subunits of cytochrome bd-type quinol oxidase, and a few additional enzymes for amino acid utilization (proline dehydrogenase and threonine aldolase). Among the genes unique to w Bm, there are two extracellular metallo-peptidases (Wbm0384, Wbm0742) that are only distantly related to counterparts in the w Mel genome. These results suggest a basic common strategy used by w Bm and w Mel during the evolution of their host symbiosis. In the case of w Bm, the basis of the interaction may be to provide essential vitamin cofactors, heme biosynthesis intermediates, and nucleotides while requiring amino acids and perhaps other nutrients supplied by the host. Both Wolbachia have lost a considerable number of membrane biogenesis genes that make them apparently unable to synthesize lipid A, the usual component of proteobacterial membranes. However, a few differences do exist. For example, in w Mel there is a predicted gene belonging to the family of GDSL-like lipases (WD1297), similar to the major secreted phospholipase of Legionella pneumophila [ 159 ], which also has phospholipid-cholesterol acyltransferase activity. Its ortholog in w Bm is disrupted by a frameshift (Wbm0354 corresponds to the C-terminal portion of the gene). However, it is still possible that, similar to E. chaffeensis and A. phagocytophilum [ 160 ], w Bm and w Mel incorporate cholesterol into their cell walls. Furthermore, w Mel retains several genes absent in w Bm that might be involved in cell wall biosynthesis. These include a small gene cluster (WD0611–WD0613) and several other enzymes (WD0620, WD0133, WD0431), suggesting that w Mel might produce peptidoglycan modified with an oligosaccharide chain, while w Bm makes unmodified peptidoglycan. Possible differences in peptidoglycan structure may be additionally predicted by the already mentioned loss of FtsW–FtsI genes in w Bm and their presence in w Mel. These differences may reflect the occurrence of a mutualistic lifestyle ( w Bm) in contrast to a parasitic lifestyle ( w Mel). Somewhat surprisingly, no recent apparent horizontally transferred genes from hosts were found in either Wolbachia genome. Moreover, an aforementioned WASP protein homolog, apparently acquired by a common ancestor of Wolbachia and Rickettsia from an animal host, is disrupted in the w Mel genome (WD0811). However, in w Mel there are two proteins encoded in the region of the prophages (WD0443, WD0633) that have “eukaryotic” OTU-like protease domains with their predicted catalytic residues apparently intact [ 161 ]. Proteases from this family are shown to be involved in ubiquitin pathways [ 162 ]. To our knowledge, this is a rare appearance of these proteases in prokaryotic genomes, although they are present in the genomes of C. pneumoniae [ 161 ] and in a closely related genome, Chlamydophila caviae (CCA00261). Conclusions Comparing the genomes of w Bm and Rickettsia to those of gamma-proteobacterial symbionts points to general similarities and distinctions in the evolution of endosymbionts. The genomes of R. conorii and Wolbachia species contain numerous repeats of various classes that are much more abundant than in the gamma-proteobacterial endosymbionts ( Table 1 ). This correlates with the minimal gene colinearity between the genomes of Wolbachia and Rickettsia [ 103 , 114 , 163 ] ( Figure 5 ). By contrast, gamma-proteobacterial endosymbionts share a variety of operons with one another, and even with free-living relatives, despite the dramatic gene loss. Furthermore, gamma-proteobacterial endosymbionts (with the exception of Wigglesworthia ) have lost crucial genes involved in recombinational repair, whereas almost no gene loss in this functional class was observed in Wolbachia or Rickettsia spp. Active recombination between repeats might have led to both gene loss and genome shuffling in Wolbachia and Rickettsia spp., whereas other mechanisms of genome reduction were probably involved in the evolution of gamma-proteobacterial endosymbionts [ 109 , 120 , 121 , 122 , 123 , 164 ] Comparative genome analysis highlights the different metabolic capabilities that render endosymbionts indispensable to their hosts [ 108 , 119 , 121 ]. For example, Buchnera and Blochmannia retain a nearly complete repertoire of amino acid biosynthesis pathways and supply amino acids to their insect hosts [ 110 , 112 ]. In contrast, w Bm, w Mel, and Wigglesworthia [ 103 , 108 ] have lost nearly all of these pathways but retain the pathways for the biosynthesis of nucleotides and some coenzymes ( Table 3 ). Thus, endosymbiotic organisms in different divisions of proteobacteria independently evolved distinct strategies for symbiont–host interactions. Genomic analysis of the alpha-proteobacterium w Bm, the first sequenced endosymbiont from a human parasitic nematode, provides new insights into the evolution of intracellular bacterial symbiosis and clues to the role of Wolbachia in the mutualistic relationship with the nematode. It is anticipated that continued genome analysis of nematodes and their endosymbionts will provide novel targets for antimicrobials aimed at the elimination of human filarial parasites. Materials and Methods Materials and Methods B. malayi microfilaria worms were purchased from TRS Labs (Athens, GA, United States) for preparation of DNA. Because of the difficulties in obtaining purified Wolbachia DNA from the B. malayi host, bacterial artificial chromosome (BAC) libraries were created [ 114 ]. From these libraries, a minimum tiling path of 21 Wolbachia BACs was created and used for subcloning into plasmid vectors for genomic sequencing. This ordered BAC approach was useful in the assembly phase of the project because of the highly repetitive nature of this genome. For plasmid library generation, equal amounts of BAC DNAs were pooled and 50 μg of DNA from the pool was sheared into 2.0–3.0 kb fragments (HydroShear device, GeneMachines, Genomic Solutions, Ann Arbor, Michigan, United States). Sheared DNA was purified from a 0.7% agarose gel, blunted, and cloned into cleaved, dephosphorylated plasmid vectors. Libraries were generated containing DNA from 1 to 9 BACs. Plasmid DNA was isolated by a modified alkaline lysis protocol. Sequencing reactions were performed at Integrated Genomics (Chicago, Illinois, United States) using the DYEnamic ET Dye Terminator Cycle Sequencing Kit (Amersham Biosciences, Little Chalfont, United Kingdom). Unincorporated dye was removed by isopropanol precipitation as recommended by the manufacturer. Samples were run on MegaBace 1000 (Amersham Biosciences) sequencers; 87% of plasmid sequencing reactions were successful. The genome was sequenced to an average coverage of 10.7X and at 2X minimum coverage (at least once in each direction) and assembled. The sequence was assembled into contigs by using PHRED–PHRAP–CONSED [ 165 , 166 , 167 ], and gaps were initially closed by primer walking (1,766 reactions). Regions considered to be potential frame shifts or sequencing errors after the first round of annotation were resequenced from direct genomic PCR products. The completed sequence was used to identify homologous sequences in the independent ongoing B. malayi sequence project (TIGR parasites genome database: http://www.tigr.org/tdb/e2k1/bma1/ [ 126 ]). The sequence of one BAC had been previously determined [ 163 ]. The final assembly was in full agreement with the BAC physical map [ 114 ]. Integrated Genomics ERGO software [ 168 ] and other software programs [ 169 ] were used for ORF calling, gene identification, and feature recognition. Computational analysis of the genome sequence was performed as previously described. Briefly, the tRNA genes were identified using the tRNA-SCAN program [ 170 ], and the rRNA genes were identified using the BLASTN program [ 171 ]. For the identification of the protein-coding genes, the genome sequence was conceptually translated in six frames to generate potential protein products of ORFs longer than 100 codons. These potential protein sequences were compared to the database of proteins from the COG database using COGNITOR [ 172 ]. After manual verification of the COG assignments, the validated COG members from w Bm were called as protein-coding genes. The COG assignment procedure was repeated with ORFs of greater than 60 codons from the intergenic regions. Additionally, the potential protein sequences were compared to the nonredundant protein sequence database using the BLASTP program [ 171 ] and to a six-frame translation of unfinished microbial genomes using the TBLASTN program [ 171 ], and those sequences that produced hits with E (expectation) values less than 0.01 were added to the protein set after an examination of the alignments. Finally, protein-coding regions were predicted using the GeneMarkS program [ 173 ]. After manual refinement, the genes predicted with these methods in the regions between evolutionarily conserved genes were added to produce the final protein set. Protein function prediction was based primarily on the COG assignments. In addition, searches for conserved domains were performed using the Conserved Domain Database (CDD) search option of BLAST ( http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ) and the SMART system [ 174 ], and in-depth, iterative database searches were performed using thePSI-BLAST program [ 175 ]. The KEGG database [ 176 ] ( http://www.genome.ad.jp/kegg/metabolism.html ) and the Integrated Genomics ERGO database pathway collection [ 168 ] were used, in addition to the COGs, for the reconstruction of metabolic pathways. Paralogous protein families were identified by single-linkage clustering after comparing the predicted protein set to itself using the BLASTP program [ 171 ]. Signal peptides in proteins were predicted using the SignalP program [ 177 ], and transmembrane helices were predicted using the MEMSAT program [ 178 ]. Gene orders in bacterial genomes were compared using the Lamarck program [ 179 ]. Two closely related genome sequences were completed and published since the above comparative analysis was undertaken [ 180 , 181 ]. Supporting Information Data Access DNA sequence, ORF, as well as annotation and positional information tables, are available at the following Web site: http://tools.neb.com/wolbachia/ . Accession Number The genome sequence was deposited in GenBank ( http://www.ncbi.nlm.nih.gov/ ) under accession number AE017321.

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1069647

Memories Are Made of This: Modeling the CaMKII Molecular Switch

We all know that our memories are stored somehow in our brains. But exactly how do we remember the way to our office or what our mother looks like or the date we got married? Scientists attribute our ability to store apparently infinite numbers of memories for decades to long-lasting changes in the electrical, structural, and biochemical properties of neurons. One cellular mechanism proposed to be involved in the storage of memories—long-term potentiation—involves alterations in the strength of messages passed from one neuron to another across structures known as synapses. The initiation of long-term potentiation is caused by activation of n -methyl- d -aspartate receptors on the receiving neuron and a subsequent increase in the intracellular calcium concentration in a region of the neuron that is called the postsynaptic density. The increase in calcium, in turn, activates the calcium/calmodulin-dependent protein kinase II (CaMKII). This enzyme seems to play a critical role in long-term potentiation, and has been proposed as one of the leading candidates to act as the molecular switch that maintains stable synapse-specific cellular changes. To fulfill this role, CaMKII would need to have stable UP and DOWN positions, or states, much like a light switch. Xiao-Jing Wang and colleagues now provide a new analysis that strengthens the argument that CaMKII is a molecular switch involved in the storage of long-term neural changes. The activity of the CaMKII holoenzyme (the complete enzyme consisting of both regulatory and catalytic subunits) is controlled by its autophosphorylation state—the enzyme is able to add phosphate groups to specific amino acids within itself. Previous modeling studies have shown that the interplay between the autocatalytic addition of phosphate groups to CaMKII and the removal of phosphate groups by protein phosphatase-1 (PP1) enzymes produces two stable states of the CaMKII enzyme at basal free calcium levels. The DOWN state is unphosphorylated; the UP state is highly phosphorylated. When there is a transient high input of calcium, as happens when long-term potentiation is induced, the CaMKII enzyme flips from a DOWN state to a persistent UP state. The questions that Wang and colleagues have now asked are what factors affect the stability of the state of this switch, and how many CaMKII holoenzymes are needed to construct a switch that could last a lifetime. These questions are important because a switch that could be spontaneously reset by small, random fluctuations of the conditions within the postsynaptic density would not be useful in maintaining stable long-term changes. The researchers have used a mathematical probabilistic modeling technique known as Monte Carlo simulation, together with the known biochemical and thermodynamic characteristics of CaMKII and PP1, to test how random fluctuations in the chemical reactions involved in the CaMKII/PP1 system change the state of the switch. They report that switch state stability requires a balance between the phosphorylation and dephosphorylation rates of CaMKII, and that the turnover rate of the kinase—the replacement of old molecules with new ones—critically affects switch stability. However, their main finding is that the lifetime of states of the switch increases exponentially with the number of CaMKII holoenzymes that are present. This finding is important because experimental work by other researchers has estimated that there are about 30 CaMKII holoenzymes present in a typical postsynaptic density, and until Wang's team did their modeling it was unclear whether this number of holoenzymes could build a switch stable enough to last a lifetime. In fact, Wang and co-workers estimate that a switch containing as few as 15 holoenzymes can remain activated for longer than a human lifetime. Thus, the researchers conclude, CaMKII switches may indeed play a critical role in preserving our precious memories throughout our lives.

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1069648

And Littler Genomes inside 'Em: Clues to a Parasitic Nematode's Bacterial Partnership

“Great fleas have little fleas upon their backs, to bite 'em. And little fleas have lesser fleas and so on, ad infinitum.”— Augustus DeMorgan, based on a Jonathan Swift poem More than a billion people are at risk for infection with filarial nematodes, parasites that cause elephantiasis, African river blindness, and other debilitating diseases in more than 150 million people worldwide. The nematodes themselves play host to bacteria that live within their cells, but in this case, the relationship is classic mutualism, with each benefiting from the other. Indeed, the Wolbachia bacterium is so crucial to its host nematode that apparently eradicating it with antibiotics severely compromises the nematode's ability to complete its life cycle within its human host. Thus, understanding the details of this symbiosis may help identify new strategies for controlling diseases caused by filarial nematodes. In a new study, Barton Slatko and colleagues present the complete DNA sequence of the Wolbachia pipientis strain within Brugia malayi , a parasitic nematode responsible for lymphatic filariasis, and analyze its genome for clues to the interdependence of the two species. This Wolbachia genome is small, only about a million base pairs, and many metabolically critical genes have degraded through mutation to the point of uselessness. This phenomenon, called reductive evolution, is typical of long-term symbioses, as the two partners increasingly complement one another's biochemical activities, reducing the selection pressure on otherwise lethal mutations. Wolbachia 's translational machinery and DNA repair equipment are largely intact. The bacterium appears to supply nucleotides to its host, as it contains complete pathways for biosynthesis of both purine and pyrimidine nucleotides. This is in contrast to Rickettsia , a close relative of Wolbachia and a mammalian parasite. Slatko and colleagues enumerate a variety of other pathways that have either been degraded or preserved, and highlight patterns in the genome structure through comparisons with both Rickettsia and another Wolbachia strain, found in fruit flies. For example, the two Wolbachia strains appear to have different membrane structures, possibly reflecting their different lifestyles (mutualistic versus parasitic). Over a billion people are at risk for infection by filarial nematodes, parasites that cause elephantiasis (Photo: Dr. Steven A. Williams) Wolbachia can manufacture riboflavin and FAD, which are essential metabolic coenzymes and which do not appear to be made by its host. Conversely, it cannot synthesize amino acids and a variety of other vitamins and cofactors, and probably depends on the nematode to supply them. One discovery of possible significance is the presence in the bacterium of the synthetic pathway for heme—the oxygen-carrying iron component of hemoglobin. The nematode may require heme for synthesis of developmental hormones, so Wolbachia 's heme pathway may be an inviting target for therapy against nematode infection. Since no new antifilarial has been developed in two decades, these results may quickly lead to new therapeutic strategies against these parasites.

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1069649

Vocal Experimentation in the Juvenile Songbird Requires a Basal Ganglia Circuit

Songbirds learn their songs by trial-and-error experimentation, producing highly variable vocal output as juveniles. By comparing their own sounds to the song of a tutor, young songbirds gradually converge to a stable song that can be a remarkably good copy of the tutor song. Here we show that vocal variability in the learning songbird is induced by a basal-ganglia-related circuit, the output of which projects to the motor pathway via the lateral magnocellular nucleus of the nidopallium (LMAN). We found that pharmacological inactivation of LMAN dramatically reduced acoustic and sequence variability in the songs of juvenile zebra finches, doing so in a rapid and reversible manner. In addition, recordings from LMAN neurons projecting to the motor pathway revealed highly variable spiking activity across song renditions, showing that LMAN may act as a source of variability. Lastly, pharmacological blockade of synaptic inputs from LMAN to its target premotor area also reduced song variability. Our results establish that, in the juvenile songbird, the exploratory motor behavior required to learn a complex motor sequence is dependent on a dedicated neural circuit homologous to cortico-basal ganglia circuits in mammals.

Introduction The acquisition of complex motor sequences, such as swinging a golf club or playing the piano, can be thought of as reinforcement learning. This learning process requires the exploration of a range of motor actions and the concomitant evaluation of the resulting performance, reinforcing motor programs that lead to improved outcomes [ 1 ]. Similarly, juvenile songbirds explore a large range of vocalizations by continuously varying their song [ 2 ], utilizing auditory feedback to improve their performance [ 3 ]. Thus, song learning encompasses the two ingredients of reinforcement learning: exploratory motor behavior, and performance evaluation. In the songbird, two main neural pathways are involved in song production and song learning ( Figure 1 A). The “motor pathway” controls the vocal motor program through the hierarchical organization of several premotor nuclei [ 4 ]. A key nucleus in the motor pathway is the robust nucleus of the arcopallium (RA), which projects to brainstem nuclei controlling the vocal and respiratory muscles [ 5 ]. During singing, RA neurons in adult birds generate a highly stereotyped sequence of bursts [ 6 , 7 ], which appear to be driven by precisely timed inputs from a higher premotor vocal area, nucleus HVC [ 8 ]. RA also receives input from the “anterior forebrain pathway” (AFP), a circuit homologous to the basal ganglia thalamo-cortical loops [ 9 , 10 ] that may be involved in controlling motor behavior and stereotypy in mammals [ 11 ]. Lesions of the AFP in juvenile zebra finches have devastating effects on song development, whereas the same manipulations in adults have few short-term consequences for song production [ 12 , 13 ]. Figure 1 Inactivation of LMAN Significantly Reduces Vocal Experimentation, Making the Otherwise Variable Song of the Juvenile Zebra Finch Highly Stereotyped (A) Two major pathways in the vocal control system of the songbird. The motor pathway (gray) includes motor cortex analogs HVC and RA, while the AFP (white), a basal ganglia thalamo-cortical circuit, consists of Area X, the dorsolateral anterior thalamic nucleus (DLM), and LMAN, which, in turn, projects to RA. To inactivate the output of the AFP, injections of TTX and muscimol (red bolus) were made into LMAN. (B) Examples of a juvenile zebra finch song (57 dph) showing large variability in the sequence and acoustic structure of song syllables. (C) Inactivating LMAN with TTX produces an immediate reduction of sequence and acoustic variability, revealing a highly stereotyped song produced by the motor pathway. The song snippets shown in (B) and (C) are from consecutive song bouts, immediately before and 1 h after drug injection. Songs are displayed as spectral derivatives calculated as described [ 36 ]. The frequency range displayed is 0–8.6 kHz. For audio of song bouts before and during LMAN inactivation in this bird, refer to Audios S1 and S2 , and S3 and S4 , respectively. While the critical importance of the AFP for song learning has been established, its specific role remains unknown [ 14 ]. It has been proposed that the AFP may be involved in comparing the auditory feedback of the bird's vocal output with a stored auditory template of the desired song—an evaluation process that could provide a corrective signal to the motor pathway needed for learning [ 15 ]. However, recent results showing that the firing patterns of neurons in the lateral magnocellular nucleus of the nidopallium (LMAN) of adult birds are insensitive to distorted auditory feedback have called this idea into question [ 16 , 17 ]. Here we test the alternative hypothesis that, in juvenile songbirds, LMAN is involved in generating vocal variability [ 18 ]—the other important ingredient of reinforcement learning. Results Our approach was to transiently inactivate LMAN in juvenile zebra finches ( n = 7 birds, see Materials and Methods ), and observe whether and how their songs were affected. Birds were briefly head-restrained, and injections of a sodium channel blocker, tetrodotoxin (TTX, 30 nl, 50 μM), were made in LMAN in both hemispheres, inactivating the nucleus (see Figures S1 and S2 ). After injections, birds were returned to a sound-isolated chamber, where they typically began to sing after 0.5–1.5 h. In all birds probed, LMAN inactivation resulted in an immediate loss of acoustic variability across song renditions. The effect was particularly dramatic in birds at an early stage of song development (approximately 55 d post hatch [dph]) because these birds normally exhibit greater song variability ( Figures 1 B, 1 C, and S3 ; Audios S1–S4 ). To quantify song variability, experiments were carried out in slightly older birds with less sequence and acoustic variability ( n = 6 birds; age range, 59–72 dph) ( Figure 2 ). This allowed us to reliably identify song syllables, the basic acoustic units of zebra finch song, across song renditions ( Figure 2 A). The variability score (V)— a measure reflecting the acoustic variability of a syllable across renditions (see Materials and Methods )—was calculated for all identified syllables before and after TTX injection. Without exception, the syllables showed a highly significant reduction in variability as a consequence of LMAN inactivation ( Figure 2 B; n = 25 syllables; 〈 V 〉 before = 0.46, 〈Δ V 〉 = 0.2; p ave < 0.0001, t -test). In fact, the juvenile song after inactivation was significantly less variable than songs of adult zebra finches singing undirected song (i.e., songs not directed to a female; Figure 2 D; p < 0.001, t -test). LMAN inactivation also eliminated 75% of the difference in mean variability between juvenile song and adult directed song—the most highly stereotyped form of song [ 19 ]. Figure 2 Analysis of the Effect of Bilateral LMAN Inactivation on Song Variability (A) Consecutive renditions of a repeating song motif of 0.5 s duration in a juvenile bird (59 dph) arranged vertically. Note the large variations in acoustic structure within individual syllables before LMAN inactivation (left). Following TTX injection into LMAN, the acoustic variability is dramatically reduced (middle), only to return to the original level by the following day (right). Numbers below each column indicate the variability index (See Materials and Methods section) calculated for the four renditions of the syllables shown. (B) Scatter plot of variability scores before and during LMAN inactivation with TTX (red) and muscimol (blue). Also shown are results for bilateral TTX injection into MMAN (black; see text), and saline injection into LMAN (green). (C) Time course of variability reduction following TTX (red) and muscimol (blue) injections show a time dependence that reflects the known in vivo pharmacology of the respective agents. Data were averaged over four identified syllables and taken from the same bird over consecutive days (dph = 70 and 71; muscimol inactivation followed by TTX inactivation). (D) Distribution of variability scores for all syllables analyzed in the TTX and muscimol experiments (25 unique syllables, six birds) before (black) and during (red) LMAN inactivation in juvenile birds. Shown for comparison are the variability scores for adult zebra finch syllables (18 syllables, 4 birds; undirected song, green; directed song, light blue). Dots represent raw data, while the lines are smoothed running averages. (E) TTX inactivation of LMAN significantly increased syllable sequence stereotypy. Sequence stereotypy scores (see Materials and Methods ) for six birds before (black) and after (red) TTX injections into LMAN. For comparison, the average stereotypy score for adult birds singing directed song was 0.95 ( n = 4 birds). To verify that the loss of variability resulted from silencing LMAN neurons, and not from inactivating fibers of passage near LMAN, a GABA A receptor agonist (muscimol, 30 nl, 25 mM) was injected bilaterally into LMAN ( n = 2 birds; 66 and 70 dph). Again, all syllables showed a dramatic reduction in variability after injection ( n = 8 syllables; 〈 V 〉 before = 0.43, 〈Δ V 〉 = 0.16; p ave < 0.0001, t -test). While the reduction in acoustic variability was similar to that resulting from TTX injections ( Figure 2 B), the duration of the effect of muscimol was substantially shorter than observed for TTX ( Figure 2 C). This difference in temporal profile was in good agreement with the known in vivo pharmacology of TTX and muscimol [ 20 , 21 ], suggesting a direct link between suppression of spiking activity in LMAN and loss of song variability. An additional effect of LMAN inactivation was a significant reduction in sequence variability, a measure of the variability in syllable ordering ( Figure 2 E; p < 0.005, paired t -test; see Materials and Methods ). In fact, the sequential ordering of syllables after TTX injection was comparable in stereotypy to that of adult song. Thus, LMAN activity may influence sequence generation, possibly through an indirect feedback pathway going from RA to HVC, the putative sequence generator [ 6 , 8 , 22 ]. We confirmed that the loss of song variability following injections into LMAN did not result from diffusion of the drugs into the medial magnocellular nucleus of the nidopallium (MMAN), a nucleus approximately 1.25 mm medial from LMAN with projections to HVC. Bilateral injections of TTX into MMAN, done in the same birds in which LMAN injections were previously made, had no significant effect on acoustic variability ( Figure 2 B). We next considered the neural mechanisms by which LMAN affects variability in the motor pathway. One intriguing possibility is that song variability is driven by fast synaptic input from LMAN. If true, then acoustic variability should be accompanied by variability in the firing patterns of RA-projecting LMAN neurons. To test this idea explicitly, we recorded single-unit signals from 29 LMAN neurons in singing juvenile birds ( n = 3 birds; age range, 62–79 dph) ( Figure 3 ). In all, 17 of these were antidromically identified as RA-projecting LMAN neurons (see Materials and Methods ). These neurons exhibited song-related changes in firing rate (spontaneous activity, 12 ± 4 Hz; during singing, 39 ± 6 Hz [mean ± standard deviation]), and generated significantly more bursts during singing ( Figure 3 C). Raster plots of the spike trains aligned to the song motif showed that the patterns of spikes and bursts generated by individual neurons were different each time the bird sang ( Figure 3 A and 3 B). Figure 3 Song-Aligned Firing Patterns of RA-Projecting LMAN Neurons in Singing Juvenile Zebra Finches Are Highly Variable (A) Three successive renditions of a 67-d-old bird's song motif. Displayed under each spectrogram is the simultaneously recorded voltage waveform of an antidromically identified RA-projecting LMAN neuron (verified by collision testing). Average syllable variability for the three motifs is 0.31. Motif alignment was done at the onset (yellow lines) of syllable C. (B) Raster plot showing the spike patterns for 50 consecutive motif renditions for the same cell as in (A). The motifs from (A) are indicated in green. (C) Relative frequency of inter-spike intervals during singing (black) and non-singing (blue) for all the 17 identified projection neurons (units are intervals per second; bin size is 0.04 log units). (D) Distribution of spike-train correlations across all pairs of motifs for the cell in (B) (solid red line). Correlations calculated with random time shifts added to the spike trains have a similar distribution (dashed red line; see Materials and Methods ). Also shown is the correlation distribution for the population of identified projection neurons (solid black line; mean correlation indicated by solid arrowhead), and for the population with random time shifts added (dashed black line). In comparison, spike trains of neurons in premotor nucleus RA of the adult bird are highly stereotyped (from [ 23 ]; mean correlation indicated by open arrowhead). Correlations in the spike trains across different renditions of the motif were small (0.054 ± 0.34 [mean ± standard deviation]) compared to those observed in premotor neurons of adult birds (0.90 ± 0.1) [ 7 ]. We also compared the correlation distributions to those calculated after random time shifts were added to the spike trains (see Materials and Methods ). In general, the correlation distributions of the randomized spike trains were very similar to those calculated for the motif-aligned spike trains ( Figure 3 D), confirming that the firing patterns of LMAN neurons are highly variable. Nevertheless, in 13 out of the 17 identified RA-projecting neurons the correlation distributions were still significantly different from those of the randomly shuffled spike trains ( p < 0.01, Kolmogorov-Smirnov test), suggesting that while LMAN activity is highly variable, it is not completely random with respect to the song. Guided by the neural data, we next tested the hypothesis that LMAN drives song variability by providing excitatory glutamatergic input to RA—which in the zebra finch is mediated almost exclusively by N -methyl- D -aspartate (NMDA)–type receptors [ 24 ]. In contrast, glutamatergic inputs to RA from HVC are mediated by a mixture of NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)–type receptors ( Figure 4 A) [ 25 ]. Thus, if LMAN drives song variability through glutamatergic input to RA, then blocking NMDA receptors should reduce this variability, while sparing the AMPA-mediated drive from HVC. In line with our hypothesis, bilateral injections of the NMDA receptor antagonist 2-amino-5-phosphonovalerate (AP5, 50 nl, 30 mM) into RA significantly reduced acoustic variability in all song syllables examined ( Figure 4 B and 4 C; n = 4 birds; age range, 57–73 dph; 11 syllables; 〈 V 〉 before = 0.47, 〈Δ V 〉 = 0.16; p ave < 0.0001, t -test). The time course of the variability reduction ( Figure 4 D) was consistent with the temporal profile of AP5 effects seen in other in vivo studies [ 26 ]. Figure 4 Bilateral Injections of the NMDA Receptor Antagonist AP5 into RA Significantly Reduced Song Variability (A) Excitatory synaptic inputs to RA from LMAN and HVC are mediated by a different mix of glutamate receptor types (see text). Using AP5 we could block LMAN input while only partially inactivating HVC input. (B) Eight sequential renditions of one song syllable in a juvenile zebra finch (63 dph) before and after AP5 injection into RA. Note the rapid fluctuations in pitch, the appearance of noisy acoustic structure, and variations in syllable duration before injection. The average variability scores (V) before and after injections for the eight shown syllable renditions were 0.50 and 0.25, respectively. (C) Following injection of AP5 into RA, fluctuations in acoustic structure were substantially reduced. Variability scores of 11 syllables in four birds before and after injection of AP5 into RA. (D) Time course of acoustic variability following drug injection averaged over all identifiable syllables for the bird in (B). Given that AP5 has effects beyond blocking LMAN input to RA, it may influence the song in ways other than reducing variability. To examine whether AP5 injections affected the acoustic structure of syllables, we compared the acoustic features of syllables after AP5 injection to the same syllables before injection (average similarity score 78.0, 11 syllables; see Materials and Methods ). In comparison, the average similarity score across renditions of the same syllables prior to injection was 77.7, suggesting that the effect of AP5 injection was largely limited to song variability. Discussion Previous studies have shown that permanent LMAN lesions in the juvenile bird disrupt song learning and result in an impoverished and prematurely stereotyped song [ 12 , 13 ]. Such lesions are known to produce synaptic maturation in RA within a few days [ 27 ], perhaps because of a loss of neurotrophic input from LMAN [ 12 , 13 ]. Because of the long delay from lesioning to singing (often several days), these studies could not address whether increased stereotypy was caused by synaptic reorganization in RA, or by a more immediate mechanism such as the loss of fast synaptic input from LMAN. In our experiments, we observe singing within an hour after injection, and find that LMAN inactivation reduces song variability reversibly and on a short timescale. This observation implies that, in addition to slow neurotrophic effects, LMAN acts on RA rapidly to drive or control song variability, a necessary ingredient of reinforcement learning. Thus, our results suggest that the loss of vocal plasticity following permanent lesions of LMAN may, in part at least, be due to the immediate loss of exploratory behavior. What is the mechanism by which neural activity in LMAN controls motif-to-motif variability in the song? Our experiments tested the hypothesis that fluctuations in the song are driven directly by synaptic input from LMAN [ 25 ]. In this view, the premotor circuit generates a stereotyped song sequence upon which the AFP acts to drive variations. This hypothesis requires that neural activity in LMAN be highly variable across different song motifs, a prediction that was borne out by our recordings in LMAN (see Figure 3 ). In comparison, premotor neurons in adult birds (singing song of comparable stereotypy to our LMAN-inactivated juvenile birds) generate extremely stereotyped, song-locked spike patterns [ 6 , 7, 8 ]. In itself, the result that LMAN neurons are only weakly time-locked to the song may not be surprising. The significance of this observation becomes apparent when considering that these neurons send excitatory projections to the motor pathway, and that they are necessary for the expression of song variability as demonstrated by our inactivation results. Together with the finding that electrical stimulation of LMAN in adult birds can drive transient changes in the song [ 19 ], these observations make LMAN a likely source for the variability in the premotor pathway. Because LMAN input to RA neurons is mediated almost exclusively by NMDA receptors, another strong prediction of our hypothesis was that blockade of NMDA receptors in RA should reduce song variability. Our results from the injection of AP5 into RA confirmed this. However, given the presence of NMDA receptors in the projection from HVC to RA [ 24 ], and perhaps in recurrent connections within RA, blockade of NMDA receptors is likely to have effects on RA circuitry beyond the loss of direct synaptic input from LMAN. Thus, this experiment cannot preclude other hypotheses—for example, that LMAN acts to regulate stochastic processes intrinsic to the premotor circuit, through some yet unknown mechanism. Further support for the idea that LMAN can drive song variations comes from studies in the adult zebra finch. Song-related neural activity in LMAN is variable also in the adult bird, and this variability has been shown to be larger during undirected as compared to directed singing [ 27 , 28 ]. A recent study [ 19 ] linked the increased neural variability in LMAN during undirected singing to an increase in motif-to-motif variability in song features (see also Figure 2 D). How does the role and function of LMAN change as song variability is reduced during learning and finally during song crystallization? To the extent that the variability of LMAN firing patterns in the adult bird during undirected song [ 28 ] is similar to that in the juvenile bird, an essential part of song development may be a reduction of the gain by which LMAN drives RA. This could occur as a result of synaptic changes within RA that weaken input from LMAN and/or strengthen the projections from HVC. While there is evidence that this may indeed occur [ 26 , 29 ], more experiments are needed to establish how the developmental reduction in song variability is related to changes in song circuitry. Reinforcement learning requires that variability in the motor output be accompanied by a mechanism that evaluates the resulting performance. In the songbird, such an evaluation signal could be sent directly to the motor system (e.g., to RA), perhaps via a neuromodulator [ 30, 31 ], to reinforce the states of the motor pathway that lead to a better-than-expected match to the memorized template. A reinforcement signal could also be sent to the AFP to shape or regulate the fluctuations introduced into the motor pathway via LMAN. This would make LMAN more than a simple “noise generator,” allowing it to bias vocal fluctuations in the direction of the desired song. Such bias is suggested by the presence of small but significant correlations in the motif-aligned firing pattern of LMAN neurons (see Figure 3 ). This bias could permit a more efficient exploration of motor space, and even allow LMAN activity to drive plastic changes in the motor circuitry. The exploratory motor behavior exhibited by juvenile songbirds may also provide general insights into how the brain generates fluctuations required for learning. Such fluctuations could be generated within the motor pathway or by brain regions projecting to it, and could result from stochastic processes, such as randomness in synaptic release [ 32 ], noise propagated by summation of irregular patterns of inhibitory postsynaptic potentials and excitatory postsynaptic potentials [ 33 ], or complex collective dynamics of the neuronal network [ 34 ]. Our results strongly suggest that, whatever the detailed biophysical mechanisms, the neural circuits generating these fluctuations are located outside the motor pathway in a specialized pathway involving the basal ganglia. The output of this circuit acts on the motor pathway, allowing the song system to explore the vocal space in a purposeful manner. Whether inducing exploratory motor behavior is a general feature of basal ganglia circuits is an intriguing idea that remains to be explored. Materials and Methods Subjects Subjects were juvenile male zebra finches (54–79 dph). Birds were obtained from the Massachusetts Institute of Technology zebra finch breeding facility (Cambridge, Massachusetts), and from the aviary at the Rockefeller Field Research Station (Millbrook, New York). The care and experimental manipulation of the animals were carried out in accordance with guidelines of the National Institutes of Health and were reviewed and approved by the Massachusetts Institute of Technology Institutional Animal Care and Use Committee. Reversible inactivation Birds underwent a brief surgery to attach to the skull a means of restraining the head during drug injections. The animals were anesthetized with isoflurane (2%) and placed in a stereotaxic apparatus (MyNeuroLab.com, St. Louis, Missouri, United States). Two stainless-steel screws (#0–80 6 mm long) were secured to the skull with dental acrylic. Small holes (approximately 300 μm in diameter) were drilled through the cranium bilaterally over LMAN or MMAN, or RA using stereotaxic coordinates. The holes were covered with a thin layer of Kwik-Kast (World Precision Instruments, Sarasota, Florida, United States). The animals were then placed in a custom sound-isolation chamber where they began to sing prolifically after a few days—typically 200–1,000 song motifs per hour. Inactivation of song control nuclei in the singing bird was carried out by placing the bird, unanesthetized, in a small foam restraint and attaching the head-mounted screws to a metal plate bolted to the stereotaxic apparatus. The Kwik-Kast over the cranial holes was removed, and 30 nl of TTX (50 μM, #T5651, Sigma, St. Louis, Missouri, United States) or muscimol (25 mM, #M1523, Sigma) was injected bilaterally into the brain region of interest using a Nanoject II injector (Drummond Scientific, Broomall, Pennsylvania, United States). The procedure of injecting the birds took approximately 10 min. Experimental confirmation of the physiological effects of TTX injections showed that LMAN was likely completely inactivated after our injections (see Figure S2 ). Regions immediately surrounding LMAN were also affected, and we cannot rule out an indirect contribution from the partial inactivation of these regions. For inactivation of NMDA-mediated synapses in RA, AP5 (#A5282, Sigma) was injected bilaterally into RA (50 nl, 30 mM). The injection site was guided by electrophysiological recordings of spontaneous activity in RA. Injected solutions also contained dye-conjugated dextrans (#D22912, Molecular Probes, Eugene, Oregon, United States). All injection sites were verified by histological examination and were found to be within the target nucleus (see Figure S1 ), except for TTX injections in LMAN in two birds: one in which the LMAN injection site in one hemisphere was found to be approximately 100 μm anterior to the edge of LMAN, the other in which the injections were approximately 200 μm posterior to LMAN, but right in the middle of the fiber tract leading from LMAN to RA. The results from these birds were similar to those from other birds, and were included in the analysis. Chronic neural recordings in LMAN Experiments were timed such that the birds were at an age at which they produced readily identifiable syllable sequences, yet showed variable acoustic syllable structure across song renditions. Recordings were carried out using a motorized microdrive described previously [ 35 ]. Cells were isolated by searching for spontaneous or antidromically evoked spiking activity; units typically had signal-to-noise ratios greater than 10:1. Antidromic identification of RA-projecting LMAN neurons was carried out with a bipolar stimulating electrode implanted in RA using techniques described previously for antidromic identification of RA-projecting HVC neurons [ 8 ]. Neurons exhibiting a short-latency antidromic spike (<5 ms) with a root-mean-squared latency jitter of less than 100 μs (at a stimulation current of approximately 10% above threshold) were counted as identified RA-projecting neurons. Of the 17 antidromically identified neurons in our dataset, ten were further validated with collision tests [ 8 ]. An additional ten putative projection neurons did not respond to RA stimulation with a short-latency spike, but exhibited spike patterns and correlations similar to the identified projection neurons. For the cells in our dataset, we recorded signals for many song motifs (range, 5–133 motifs; mean, 56). Data analysis To assess the effects of drug injections on acoustic variability and average acoustic structure, analysis was done on reliably identifiable song syllables (range, 2–5 per bird; see Figure 2 A for an example). Each data point was derived from 45 pairwise comparisons made across ten consecutive renditions of a given syllable, recorded immediately before and after injection. Acoustic variability was quantified using the Sound Analysis Pro 1.04 software [ 36 ], and pairwise comparisons of the acoustic features of identified syllables were made using the local similarity measure (“accuracy”). This measure is based on pitch, frequency modulation, amplitude modulation, Wiener entropy, and goodness of pitch, and is calculated in 9-ms intervals and averaged over the duration of the syllable; syllables were aligned in time so as to maximize the similarity, allowing for 5% time warping. For the variability measurements, the resulting similarity score (S, ranging from zero to 100) was converted, through a linear remapping, to a variability score (V) by the following formula: 〈 S min 〉 is the average similarity score of randomly chosen pairs of syllables from unrelated birds, which in our finch colony was measured to be 50 ± 12 (mean ± standard deviation, n = 200 pairwise comparisons; comparisons were made across syllables of birds from different fathers). The similarity of identical syllables, S max , is 100 by definition of the similarity measure. Thus, a variability score of one means that syllables are as different as two unrelated syllables would be on average, while variability score of zero means that the syllables are identical. Error bars for V in the figures all denote standard error of the mean. 〈 V 〉 denotes the average variability score across birds and syllables for a given condition. The variability of syllable ordering in a song was quantified using the stereotypy score of Scharff and Nottebohm [ 13 ], excluding the variability in the number of introductory notes and in the end syllable of a song bout. The score is a combination of “sequence linearity,” which addresses the way in which notes are ordered, and “sequence consistency,” a measure of the frequency with which the main motif sequence appears. Complete stereotypy yields a score of one, while a completely random sequencing will have a score close to zero. Stereotypy scores were calculated over ten consecutive song bouts, before and after LMAN injections. For the analysis of the neural recordings in LMAN, we determined the sequence of song syllables most frequently produced by each bird. Motifs that matched this sequence were identified and time-aligned using the onset of one of the syllables. The alignment syllable was chosen for a sharp onset in acoustic power. The relative jitter in the timing of other syllables in the motif was found to be less than 9 ms (root mean squared). Spike times were extracted, and the instantaneous firing rate during each motif rendition was estimated by smoothing the spike train with a Gaussian of half-width 20 ms (to the 1/e points). Correlations were calculated between the firing rate functions for all pairs of smoothed spike trains. Correlations were also calculated for all pairs of spike trains after a random time shift. The shift was circular, such that spikes wrapped around to the beginning of the motif; time shifts were chosen randomly from a uniform distribution with the width of the motif. For each cell the correlation distribution of the time-shifted firing rates was calculated with 100 different ensembles of random shifts. This random shift ensured zero mean correlation while preserving spike statistics. Thus, the distribution of time-shifted correlations provides a zero-correlation baseline with which to compare our results. Supporting Information Figure S1 Histology Confirming the Injection Sites for the LMAN Inactivation Experiments in Figures 1 and 2 (A) A parasaggital Nissl-stained section of a zebra finch brain showing the location of LMAN. (B) Inverted darkfield image showing LMAN in one of the juveniles injected (red markers in [D] and [E]). (C) Combined darkfield and fluorescence image showing the spread of the dye that was co-injected with the drug. (D and E) Estimated injection sites relative to the boundaries of LMAN for all birds in Figures 1 and 2 in the saggital (D) and coronal (E) planes, respectively (individual birds are color coded). (F) Estimated maximum diameter of LMAN in the saggital plane. (G) Estimated lateral extent of LMAN in the coronal plane. The estimates in (F) and (G) are based on the contrast borders seen in the darkfield images (see [B]). Note that fibers from LMAN to RA leave the posterior edge of LMAN. (369 KB PDF). Click here for additional data file. Figure S2 Dose- and Distance-Dependent Effects of TTX Injections in and around LMAN (A) Decrease in acoustic variability (ΔV) approximately 1 h after injection, as a function of location and concentration of TTX injections. Red bars indicate dose response for TTX injections in LMAN ( n = 2 birds; 8 syllables; injection sites for the two birds correspond to the blue and grey markers in Figure S1 ). Blue bars indicate 30-nl saline injections in LMAN ( n = 2 birds; 7 syllables). Green bars indicate 30-nl (50 μM) TTX injections 1.25 mm medial (MMAN, n = 2 birds; 6 syllables) and dorsal (“above,” n = 2; 8 syllables) from the center of LMAN. (B and C) Summary of experiments done to verify the physiological spread of TTX. Experiments were done in anesthetized birds (2% isoflurane). A bipolar stimulating electrode was placed in RA, and a recording electrode in LMAN, producing antidromically evoked activity in LMAN (stimulus pulses, 175 μA, 0.2 ms, 0.5 Hz ). TTX (30 nl, 50 μM) was injected at different distances away from the recording electrode. (B) Examples of recorded signals for TTX injections 400 μm (top) and 1,250 μm (bottom) away from the recording electrode (averaged over 30 stimulus pulses). The baseline stimulus artifact recorded 1 mm above LMAN is shown in the green boxes (left). Signal recorded in LMAN immediately before injection is shown in the black boxes (middle). Signal recorded 1 h after injection is shown in the red boxes (right). (C) Summary of evoked activity 1 h after TTX injections made at different distances away from the recording site. Evoked activity was measured as the root-mean-squared deviation of the signal from the baseline in the interval 1.5–4.5 ms after the stimulation pulse (six birds, two at 400 μm, two at 600 μm, and one each at 800 μm and 1,250 μm). (1.1 MB PDF). Click here for additional data file. Figure S3 Example of a Juvenile Zebra Finch Song (54 dph) Showing a Loss of Sequence and Acoustic Variability following LMAN Inactivation by TTX Injection The song snippets shown are from three consecutive song bouts, immediately before and 1 h after TTX injection. Tutor song is shown for comparison. (1.8 MB PDF). Click here for additional data file. Audio S1 Example of a Song from the Bird in Figure 1 prior to TTX Inactivation of LMAN (Bout 1) (545 KB WAV). Click here for additional data file. Audio S2 Example of a Song from the Bird in Figure 1 prior to TTX Inactivation of LMAN (Bout 2) (455 KB WAV). Click here for additional data file. Audio S3 Example of a Song from the Bird in Figure 1 during TTX Inactivation of LMAN (Bout 1) (430 KB WAV). Click here for additional data file. Audio S4 Example of a Song from the Bird in Figure 1 during TTX Inactivation of LMAN (Bout 2) (360 KB WAV). Click here for additional data file.

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1069650

To a Zebra Finch: How the Brain Cultivates Birdsong

From the “ecstatic sound” of Thomas Hardy's thrush to the “full-throated ease” of Keats's nightingale, the dulcet tones of songbirds have long inspired poetic explorations of the human spirit. Scientists have more recently found inspiration in songbirds, but it is their behavior and not their song that tickles the scientific imagination. Just as the vocal explorations of toddlers reflect the (no doubt) consequential conversations of their elders, the highly variable chirps and warbles of juvenile songbirds echo the precise melodies of the adult songbird. Through trial and error and random forays into harmolodic dissonance, the young bird patterns his performance after a tutor song (usually performed by dad) until he produces a workable facsimile. It is this behavior— known as reinforcement learning—that makes songbirds an ideal model for studying the interplay between experience, brain activity, and learning. Michale Fee's lab studies the neural basis of song learning in the zebra finch, the organism of choice for birdsong researchers. In a new study, Bence Ölveczky, Aaron Andalman, and Fee study just how young songbirds generate the vocal explorations that help the apprentice master its song. Two major neural pathways control zebra finch song. The motor pathway controls vocal outputs through the RA (for robust nucleus of the arcopallium) neuron cluster, which indirectly stimulates vocal and respiratory muscles. When adult birds sing, RA neurons show a signature sequence of bursts during each syllable. Another pathway, called the anterior forebrain pathway (AFP), appears to be critical for song learning. AFP shares characteristics with the mammalian basal ganglia, which regulates movement and motor learning in mammals. The vocal explorations of young zebra finches shed light on the neural basis of learning motor tasks (Photo: Daniel D. Baleckaitis) To explore the nature of the AFP's contributions to song learning, Fee and colleagues recorded brain activity from young zebra finches (54–79 days old) learning to sing. Then they injected young birds with drugs that temporarily blocked activity in a brain region that is part of the AFP called LMAN (lateral magnocellular nucleus of the nidopallium). Zebra finch songs typically contain three to seven syllables—the basic acoustic units of zebra finch songs—that follow a specific sequence. Thirty to 90 minutes after LMAN inactivation, the birds sang with less syllabic variation. This effect was especially dramatic in the youngest birds, which normally exhibit the greatest acoustic variation. LMAN inactivation, the authors note, “eliminated 75% of the difference in mean variability between juvenile song and adult directed song [wooing a mate, for example]—the most stereotyped form of song.” LMAN inactivation also reduced the birds' variation in syllable sequence, which again hewed closer to the orthodoxy of adult song than to the exuberance of youthful experimentation. The authors go on to show that changes in the firing patterns of LMAN neurons projecting into the motor pathway accompany changes in song. That LMAN inactivation reduces song variability quickly and reversibly, the authors argue, indicates that LMAN supports experimental behavior and controls song variability by providing rapid inputs to the motor pathway. This model requires that LMAN neurons show high variability across different song motifs—which is what Fee and colleagues found. As the bird sings, some as yet unknown brain areas must also evaluate the song against a template, modulating the actions of the motor pathway as a conductor might correct a performer's mistakes in note and pitch until she masters the tune. It's thought that birdsong serves multiple purposes—staking a territorial claim, for example, and attracting a mate—though precisely how the song relates to fitness is still an open question. Whether inducing the type of exploratory motor behavior that's so critical to motor learning is a fundamental feature of basal ganglia circuits also remains to be determined. But it does seem clear that these circuits play a significant role in generating the variability that songbirds need in order to acquire the communication skills of their parents—a finding that may shed light on how the brain produces the fluctuations required for learning other tasks. For more on song learning, see the primer by Fernando Nottebohm (DOI: 10.1371/journal.pbio.0030164 , available online May 2005).

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1069657

Creating Locally Relevant Health Information

Lack of health information marginalises the poor and prevents them from making informed decisions. What we need, argues Carter, is to promote health in ways that are accepted by local communities

Background It is an ongoing challenge to share health information with resource-poor communities that is locally relevant and owned by the communities themselves. Too often, new information brought to these settings is seen as coming from “outside” and therefore as having little local relevance. People may look with suspicion at those who bring such information. Many factors—the background, attitudes, clothing, employers, and the language of those who bring information—may have more impact on the way new ideas are received than the actual relevance of the ideas themselves. We have only to consider our own attitudes to politicians for an example of how such factors influence our receptivity to information. When health information from outside the community goes against deeply held beliefs and attitudes about personal and sexual matters, this challenge becomes still greater. Providing opportunities for people to discuss the impact of HIV and AIDS on their communities in a relaxed and open manner is key to enabling people to engage in potentially lifesaving discussion and attitude change. But as positive a step as open discussion is, unless poor people can access and accept the information they need, they will not be able to make informed decisions regarding their lives and future. Ignorance about the impact of HIV and AIDS and how the virus is transmitted is potentially life-threatening; we urgently need to raise awareness in ways that are accepted by local communities. PILLARS: Partnership in Local Language Resources Between 1995 and 1999, Tearfund UK, a Christian community development and disaster relief charity, conducted research into the flow of information at grassroots level in Uganda and Ghana, with the support of the education division of the United Kingdom Department for International Development [ 1 ]. The findings highlighted the importance of small groups in sharing information, the lack of relevant printed materials for the poor, and the need for end users to be involved in the creation of relevant printed information in their own languages. These findings have now been translated into practical action through Partnership in Local Language Resources (PILLARS). PILLARS guides provide small community groups with simple printed information, written in local languages, on community development issues such as nutrition, food security, micro-credit, HIV and AIDS, and community mobilisation (see www.tilz.info/resources ). These guides are not seen by communities as information from an outside entity, but rather as locally consolidated information on relevant issues for groups to discuss in their regular meetings. Rather than acting as passive recipients of information, group members can bring useful experience, knowledge, and insights into the discussion ( Figure 1 ). Figure 1 Participants Discussing a Topic on Leadership Styles during a PILLARS Workshop in Delhi Each guide contains 20–24 topics, with illustrations, text, and discussion questions. Any group that meets on a regular basis can set aside time to read through and discuss one of the topics. A trained leader is not required, though it helps if someone in the group knows how to facilitate discussion. The guides build on existing knowledge and experience shared among group members. Empowering the Community The ultimate goal of PILLARS is to empower community groups in developing nations by building their capacity for collective learning, consensus-building, and subsequent action. Use of the guides restores their right to receive and share information in their own tongue and to participate in the development of their communities. The generation, use, and distribution of information in local languages encourages and gives confidence and value to marginalised groups. The discussion process helps groups manage their own change and engage in local decision-making processes. Guides are now available on nine subjects relating to community development. They are designed for ease of translation into any language using a CD-ROM with design and text files. Pages or illustrations can be contextualised to meet local needs, and participatory bible studies are included at the back of the guides for faith-based groups to use. A further aspect of PILLARS is that development workers can be equipped to translate and write new guides over the course of three workshops. During the first two workshops, participants learn skills in translation, reviewing, and field testing. In the final workshop, participants write their own guide and plan for future sustainability. Training in facilitation skills and participatory techniques equips participants to use the guides. This process has been piloted in southern Sudan, Ethiopia, Nigeria, Burkina Faso, Brazil, and Myanmar. In Myanmar, development workers produced guides in Burmese and then replicated the training with a further 13 language groups, generating considerable energy and empowerment. A facilitator commented: “We have so many languages in our country. Through this programme, people are encouraged to value their culture and to share useful information about development with the community.” In Ethiopia, participants wrote a guide on “harmful traditional practices”. Training has also been conducted there with a refugee community from southern Sudan, helping them plan for repatriation. A recent evaluation, led by Dr Clinton Robinson of PILLARS in Myanmar, Brazil, and Ethiopia, revealed a dearth of written information in the languages of minority groups. Access to information and to the media was generally low. The evaluation found that improving access to simple, relevant and practical information in local languages increased people's self-confidence and their ability to make positive change. It commented on the benefits of the emphasis PILLARS places on collective learning rather than on individual reading. Discussion-Based Learning on HIV and AIDS A new guide, Responding More Effectively to HIV and AIDS , is now available ( Figure 2 ) [ 2 ]. With funding from Development Corporation Ireland, this guide is being translated into a further nine languages: French, Spanish, Portuguese, Hindi, KiSwahili, Amharic, Khmer, Kinyarwandan, and Chinese. Figure 2 A Recent PILLARS Guide The guide first gently challenges misconceptions to ensure that people have the correct facts about HIV/AIDS and how the virus is passed on. Issues raised include traditional practices that might spread HIV, the need for HIV testing and accompanying counselling, and the needs of children who lose their parents from HIV/AIDS. The guide encourages discussion of how to talk about sexual issues with children, with partners, and within faith-based teaching. The burden that can fall upon carers who respond to the needs of families living with HIV/AIDS can be immense, and several topics encourage people to discuss this. The guide also addresses relevant and challenging questions, including who provides the caring, who else could help, what support systems are available, and how the local community can increase its support. Recent advances in antiretroviral therapy and the latest advice regarding breastfeeding by mothers with HIV are also covered. At a recent workshop in Nairobi, staff from Sudan gained facilitation skills and learned techniques to share information effectively. They worked in small groups to develop simple role-plays to introduce topics from the HIV and AIDS guides. Though few of the participants had used this method before, they produced some amazingly powerful role-plays that provided a very effective introduction to the group discussion and learning that followed. The Local Production of Information A number of organisations have developed tools and training to help with the local production of information ( Box 1 ). PILLARS differs from these approaches, both in its focus on providing information for discussion-based learning targeted at grassroots community groups, and in providing technical support and design files to simplify the translation process. Box 1. Tools for the Local Production of Information Agricultural kits produced by the International Institute for Rural Reconstruction ( www.iirr.org ). Shell booklets produced by the Summer Institute of Linguistics ( www.sil.org ), often used as literacy primers in local languages. The REFLECT approach ( www.reflect-action.org ), a method of increasing literacy using participatory techniques. Each literacy circle produces its own learning materials, analysing its own local community and its immediate circumstances. The STAR (Stepping Stones and Reflect) Initiative ( www.healthcomms.org/pdf/STARsummary.pdf ), which provides draft guidelines that support communities or organisations to analyse and tackle issues that affect them, from agriculture to war, in the context of HIV and AIDS. The Quest manual, from Healthlink Worldwide ( www.healthlink.org.uk/consult/quest.html ), a tool to support organisations to develop their capacity to produce effective communication and information resources. PILLARS guides have now been translated into more than 30 different languages and are being used with basic literacy programmes and training workshops, and as discussion-based materials for women's, farmers', and credit groups. They offer a simple, yet potentially very effective method of sharing health messages. The use of print means that such messages can be used over the long term and widely distributed. A free copy of the HIV and AIDS guide for groups in resource-poor nations is available from E-mail: roots@tearfund.org .

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Alas, Poor Yorick: Digging Up the Dead to Make Medical Diagnoses

Is it ethical to dig up famous dead people to make tissue diagnoses?

Preparing for Death How does one prepare for death? Those who have created a public persona must add to any spiritual ponderings about eternity the mundane chore of organizing their literary archives to protect any of life's secrets that seem worth the effort. That task involves choosing what diaries, letters, drafts, and laundry lists to donate to a university or to leave in a closet for legions of biographical ragpickers to quote, misquote, or variously interpret in as yet unimaginable contexts—or to burn. Many well-known figures contemplating their posthumous selves have been foiled in exercising control over their literary remains. Purposefully confounding future biographers, Sigmund Freud burned his early papers and admonished his wife Martha to destroy their love letters. Instead, she bequeathed us this charming insight into the youthful exuberance of the patriarch of psychoanalysis, written in 1884: “Woe to you, my Princess, when I come. I will kiss you quite red and feed you till you are plump. And if you are forward, you shall see who is stronger, a gentle little girl who doesn't eat enough or a big wild man who has cocaine in his body” [ 1 ]. Anaïs Nin, whose voluminous diaries recorded her daily life in exquisite, compulsively recorded detail, had better luck in choreographing her literary afterlife. While alive, she published volumes of carefully edited literary diaries. When someone at a seminar remarked to her that her life seemed more, well, racy than those diaries revealed, she smiled mysteriously and said that after the death of all concerned, “unexpurgated” editions would be published. Several decades later, companion volumes to the literary diaries revealed passionate incest with her father, Joachim Nin, an affair with her analyst, Otto Rank, and successfully bigamous marriages in New York and California. When André Gide revealed that Oscar Wilde had had sexual relations with a young Arab boy in Egypt, Wilde's friend Robert Sherard lamented: “Heavens! The task of shooing hyenas away from the graves of the illustrious dead.” Sherard meant Wilde's literary grave—but what about actual graves? What about history's corpus delicti? The Line between Scientist and Grave Robber How many giants and tyrants unlucky enough to have left body parts or ashes behind when they shuffled off the mortal coil could have imagined what scientists and medical practitioners of the future would do with their physical remains? Here, the line between the scientist and the grave robber blurs, as corpses are exhumed and cremation urns raided to provide organic remnants for any number of curious purposes. Ethical debates about the appropriate care and maintenance of biological relics often begin at the autopsy table. Having removed Albert Einstein's brain, pathologist Thomas Harvey chopped it into 240 pieces and stored it in a cookie jar in his basement, often shipping slabs (mailed in mayonnaise jars) to brain researchers eager to count glia and neurons. Forty years later, Harvey lugged what remained of the brain cross-country to deliver it to Evelyn Einstein, a woman rumored to be the physicist's daughter from an affair with a New York dancer. Dr. Charles Boyd had tried to prove this paternity with his brain-chunk, but Einstein's DNA proved “too denatured to decipher.” Harvey's volunteer driver, Michael Paterniti, described getting his hands in the cookie jar: “I actually feel as if I might puke. The pieces are sealed in celloidin—the pinkish, liver-colored blobs of brain rimmed by gold wax. I pick some out of the plastic container and hand a few to Evelyn. They feel squishy, weigh about the same as very light beach stones. We hold them up like jewelers, marveling at how they seem less like a brain than—what?—some kind of snack food, some kind of energy chunk for genius triathletes” [ 2 ]. Pilferers cannot resist snipping body parts. While Einstein was being autopsied, his ophthalmologist, Dr. Henry Abrams, dropped by and filched Einstein's brown eyes as a keepsake, storing them in a jar in a Philadelphia bank vault. There were rumors that singer Michael Jackson, a collector of body parts, offered Abrams several million dollars for the eyes. Does confidentiality extend beyond the grave? Beethoven's ears were hacked out and soon went missing. René Descartes's middle finger was stolen. (His head was also separated from his body for shipping—a philosopher's in-joke, since Descartes introduced the mind/ body split into Western philosophy.) Napoleon's reputed penis went on a picaresque odyssey of its own, being displayed at the Museum of French Art in New York, auctioned, and finally ending up in the possession of a urologist—or so the story goes. Josef Haydn's head was stolen by phrenologists at his burial. In 2004, Dr. Anunciada Colon presided over the opening of a golden trunk from the 16th century, containing ashes and bone fragments presumed to belong to her ancestor Christopher Columbus, an event chronicled by a television crew. Officials at the Seville Cathedral allowed researchers at the University of Granada to borrow the bones for a DNA study. Being unsuccessful at extracting DNA from pulverized fragments, Professor José A. Lorente loaded the bones in a shoulder bag and flew them to Dallas, Texas, where more sophisticated DNA tests (developed for the victims of the terrorist attack of 9/11) provided a disappointingly short and impure sequence of mitochondrial DNA. Remaining ashes and shards were inelegantly deposited on a metal storage shelf in a lab, in a Styrofoam picnic basket labeled “Colon” in black marker, awaiting better tests [ 3 ]. Vladimir Ilyich Lenin remains the most visible deceased person. His body, or what remains of it since his brain and other organs were removed, has been viewed by the millions who have passed by his open casket in a mausoleum on Moscow's Red Square. A waterproof suit under his uniform holds in the embalming fluid. His hands and head are bathed frequently. His microtomed (31,000 sections) and dyed brain resides down the street from his body at the Moscow Brain Institute, joining the brains of his countrymen Stalin and Tchaikovsky. Many Russians who find Lenin's public resting place a macabre embarrassment think his soul will only rest (and theirs with it) once he goes underground. But who can decree his burial? When I was four, my mother found me exhuming a goldfish we had ceremoniously buried in the garden in a little fish coffin a few days before. How different, I wonder now, was my childish curiosity and wonderment at the mysterious process happening to my no-longer-swimming fish below the earth from that of grown-up exhumers? Consider Gira Fornaciari, who unearthed 49 members of the Medici family to confirm various causes of death, or the committee that had Beethoven and Schubert dug up to transfer them to more secure zinc coffins (borrowing both heads for a bit more measuring, and swiping Schubert's luxuriant, larvae-laden hair while they were at it). Archaeologists have braved curses and biohazards to retrieve mummies from pyramids. Doctors from Japan, however, were not allowed to take DNA from King Tut's mummy to sort out his genealogy; the Egyptian government's supreme council of antiquities, after first agreeing, reversed the decision. A non-invasive x-ray of the mummy suggests a murder plot: King Tut may have been done in by a blow to the back of the skull. Guidelines for Bioethical Research When a committee was convened to decide whether specimens of Lincoln's blood and bones should be tested for DNA to discover whether he suffered from Marfan syndrome, ethicists voted yes but scientists vetoed the plan, claiming that the precious material should not be destroyed in case future tests would prove more effective [ 4 , 5 ]. But what if they were even asking the wrong question? Lincoln once told his biographer and friend William Herndon that he had been infected with syphilis by a prostitute in Beardstown around 1835 [ 6 ]. What if a future test could prove that Lincoln had spoken the truth? Imagine, if you will, a press release from the Armed Forces Institute of Pathology revealing that hot potato about the most beloved of American presidents. The Lincoln testing question spurred bioethicist Lori Andrews and her colleagues at the Chicago Historical Society to join with the Illinois Institute of Technology to review existing ethical issues of biohistorical research. Their conclusion, after studying professional codes from 23 other organizations: none contained guidelines for conducting biohistorical research and analysis [ 7 ]. They recommend genetic testing for “historically significant” questions. But who is to define that loaded phrase? The newly dead are warm, soft, and somehow still human; by contrast, aged corpses and skeletons rising from the cold ground are the stuff of horror films, vampires and ghouls. While fascinating, they also unnerve. Medical examiners in fiction (Kay Scarpetta) and television (Dr. Quincy, Jordan Cavanaugh) capture wide audiences with their gruesome and graphic dissection of putrefied, maggot-ridden corpses, all in the service of solving some medical mystery. Respect for the Dead Does confidentiality extend beyond the grave? Should doctors publish articles in medical journals about diagnoses that were confidential when the patient was alive? Physicians have often raced to put pen to paper and reveal the signs and symptoms of their more illustrious deceased patients. According to Anne Sexton's biographer Diane Wood Middlebrook, who used tapes of hundreds of hours of therapy sessions given to her by Sexton's therapist Dr. Martin Orne, the dead have no rights [ 8 ]. Although Dr. Orne insisted that Sexton had given him permission to do what he thought appropriate with the tapes, his colleagues howled that he had made a travesty of doctor-patient confidentiality, Sexton's wishes be damned. The long-dead are latecomers to the game of lobbying for rights. Who owns their bones? Who is to choose the right test, the right time, the appropriate question to ask? Who gets to decide whether they should be sliced, diced, dyed, pulverized, displayed, x-rayed, photographed, and subjected to the esoteric tests developed for forensic laboratories to reveal secrets they carefully took to their graves or urns? An interdisciplinary committee? The law? The government? Should such decisions be made by bioethicists, scientists, medical examiners, lawyers, archaeologists, descendants of the deceased? Where does simple respect for the dead play into this issue? The answers change over time and from place to place. The quagmire of ethical, legal, moral, and even aesthetic questions that surround the use (and misuse) of leftover body parts can only become more complex and contentious, not less. A word of warning, then, to the famous not-yet-deceased: consider the disposition of your physical remains as carefully as you consider the packaging of your archive. Swear your doctor to posthumous secrecy. Be cremated. And have your ashes scattered to the wind. Is it ethical to remove body parts to make a tissue diagnosis? (Illustration: Margaret Shear, Public Library of Science) Victor McKusick of the Johns Hopkins School of Medicine chaired a committee to decide whether specimens of Lincoln's blood and bones should be tested for Marfan syndrome (Photo: Alexander Gardner, Library of Congress)

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Protecting Children from Environmental Toxins

Lanphear and colleagues argue that the existing requirements in the US for toxicity testing and regulation of pesticides and industrial chemicals are inadequate to safeguard children

Epidemics of overt toxicity following widespread environmental contamination from commercial toxins heralded the discovery of children's enhanced vulnerability to lead, methyl mercury, polychlorinated biphenyls (PCBs), and tobacco [ 1 , 2 , 3 , 4 , 5 ] ( Box 1 ). Over the past three decades, researchers have found that remarkably low-level exposures to these toxins are linked with less overt symptoms of toxicity—intellectual impairments, behavioral problems, spontaneous abortions, or preterm births [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. Moreover, there is emerging evidence that decrements in intellectual abilities and low birth weight linked with lead or tobacco are, for a given increment of exposure, greater at lower levels than those found at higher levels [ 10 , 41 , 42 , 43 ]. Box 1. Poisoning following Widespread Environmental Contamination from Commercial Toxins Lead: One hundred years ago, an epidemic of lead poisoning was described among children who ingested leaded house paint [ 2 , 3 ]. The children developed anemia, encephalopathy, paralysis, and blindness. Methyl Mercury: In the 1950s, in the Japanese fishing village Minamata Bay, which was contaminated with methyl mercury, children developed cerebral palsy, limb defects, and mental retardation [ 4 ]. PCBs: In Taiwan and Japan during the 1960s and 1970s, the ingestion of PCB-contaminated rice bran oil by pregnant women led to fetal wasting and cola-colored, dull, apathetic children [ 5 ]. Tobacco: During the past century, widespread tobacco use has led to an epidemic of undersized, premature babies and children with repeated bouts of wheezing or asthma [ 6 , 7 , 8 , 9 , 10 ]. The consequences of exposure to many other chemicals or mixtures of chemicals, such as insecticides—chemicals oftentimes specifically designed to be toxic—are largely unknown [ 33 , 34 , 35 , 44 ]. Many of these chemicals or their metabolites are routinely found in the blood and body fluids of pregnant women and children [ 45 ]. Children's Vulnerability to Environmental Toxins The developing fetus and young child is particularly vulnerable to certain environmental toxins [ 46 , 47 , 48 , 49 , 50 ]. Critical neurodevelopmental processes occur in the human central nervous system during fetal development and in the first three years of life. These processes include cortical functional differentiation, synaptogenesis, myelination, and programmed apoptosis [ 46 ]. Children's exposure to environmental toxins is insidious. Environmental toxins covertly enter a child's body transplacentally during fetal development or by direct ingestion of house dust, soil, and breastmilk and other dietary sources during early childhood [ 51 , 52 , 53 , 54 , 55 , 56 ]. Our ability to directly measure the actual levels of environmental chemicals in human tissues and body fluids using biologic markers (biomarkers) enables scientists to more effectively link exposures to environmental toxins with disability or disease [ 57 ]. Despite our increased knowledge of the toxicity of environmental chemicals, testing for developmental neurotoxicity (DNT) and reproductive toxicity is rarely done. DNT testing uses animal experiments to provide information on the potential functional and morphologic toxicity to the fetal nervous system that results from the mother's exposure to toxins during pregnancy and lactation. Paradoxically, DNT testing of a chemical is seldom requested, and then typically requested only if there is pre-existing evidence that it is neurotoxic. The Prevalence of Diseases and Disabilities Linked to Environmental Toxins Based on parental reports, one in six United States children has one or more developmental disabilities, from a subtle learning disability to overt behavioral or emotional disorders [ 58 ]. Exposures to environmental toxins have been linked with higher rates of mental retardation, intellectual impairment, and behavioral problems, such as conduct disorder and attention deficit hyperactivity disorder [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 30 , 31 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 59 , 60 , 61 ]. One in ten US babies is born preterm and about 5% have low birth weight [ 62 , 63 ]. Preterm birth, defined as birth at less than 37 weeks of gestation, is a major determinant of infant mortality and morbidity throughout childhood [ 62 , 63 , 64 ]. Exposures to environmental toxins such as lead, tobacco smoke, and DDT have been linked with an increased risk for spontaneous abortion, low birth weight, or preterm birth [ 6 , 9 , 10 , 13 , 14 , 15 , 28 , 32 , 65 , 66 ]. The rate of occurrence for many of these diseases or disabilities has been rising, as has treatment for attention deficit hyperactivity disorder and depression in children [ 62 , 63 , 67 , 68 , 69 , 70 ]. Multiple risk factors, including both genetic and environmental influences, interact in complex and often unknown ways to cause disease and disability in children. But efforts can be undertaken to prevent or reduce environmental exposures linked to disease without full elucidation of the underlying mechanism [ 71 ]. Thus, conducting some sort of test to identify pesticides and industrial chemicals that could cause reproductive or neurobehavioral toxicity before the chemical reaches widespread use is essential to protect pregnant women and children. Origin and Evolution of DNT Tests The process for testing potential developmental neurotoxins in laboratory animals evolved out of a series of tragic epidemics. Widespread use of the drug thalidomide during the 1950s led to an epidemic of phocomelia, an absence or deformity of limbs and other congenital defects in children exposed in utero to the drug [ 72 ]. Subsequently, in 1965, the Food and Drug Administration (FDA) developed the Teratology Guidelines. Because thalidomide induced gross defects in rabbits but not in rats, these guidelines called for toxicity tests in two species. Moreover, these guidelines focused on gross abnormalities; they did not require testing for behavioral or DNT. Following the outbreak of methyl mercury poisoning in Minamata Bay ( Box 1 ), Japan and the United Kingdom added behavioral (DNT) guidelines to their teratology requirements in 1974 and 1975, respectively [ 73 ]. In 1978, the Collaborative Behavioral Teratology Study (CBTS) was conceived to standardize and evaluate methods for DNT testing in the US [ 74 ]. The final report was issued in 1985, and shortly thereafter, Dr. Donald Kennedy, who was then Commissioner of the FDA, supported the adoption of the CBTS recommendations. But the FDA failed to implement these recommendations after Kennedy's departure. Children's exposure to environmental toxins is insidious In 1990, the US Environmental Protection Agency (EPA) identified nine developmental neurobehavioral teratogens for both humans and animals (lead, PCBs, methyl mercury, cocaine, alcohol, phenytoin, heroin, methadone, and ionizing radiation) and developed rules for DNT testing in laboratory animals [ 49 , 50 ]. By 1991, the Developmental Neurotoxicity Test Guidelines (OPPTS 870.6300) had been established for use when submitting chemical data to the EPA [ 49 ]. In 1993, the National Research Council recommended that DNT data be included in the EPA's evaluations of pesticides, which include classes of chemicals specifically designed to be toxic [ 44 ]. The Precarious US Framework for Protecting Children Despite numerous attempts to upgrade the regulatory system, such as the CBTS, the framework to protect children from environmental toxins is precarious. Under current regulations, manufacturers of commercial chemicals (excluding pesticides) are not required to supply any toxicity data before selling their products. Nor are pesticide manufacturers obligated to supply basic premarket toxicity and exposure data necessary to ensure that children will be protected from exposure and potential harm from use of those pesticides. Indeed, the vast majority of chemicals have not been tested for DNT. The most basic toxicity tests in animals are lacking for 75% of the 3,000 highest production volume chemicals—chemicals for which annual production exceeds 1 million pounds per year [ 49 , 75 , 76 , 77 ]. The US EPA has entered into an agreement with the American Chemistry Council, the chemical manufacturer's trade association, to provide basic toxicity screening tests for the high-production-volume chemicals by 2005 ( http://www.epa.gov/chemrtk/volchall.htm ), but this is voluntary. For new pesticides intended for use on food crops—one of the areas in which regulations are most stringent—regulations require only that DNT testing be evaluated for substances already known or suspected of being toxins. Further, neurotoxicity testing need be conducted only in adult animals. The EPA acknowledges that over 140 registered pesticides are neurotoxic (i.e., specifically designed to act against pests by interfering with neurotransmitters or other processes shared by mammals and insects), but the EPA has received DNT testing using validated protocols for only nine pesticides [ 49 , 75 , 76 , 77 ]. There is no general requirement that pesticides or other chemicals be tested for potential DNT prior to their registration and use [ 49 ]. For pesticides—which undergo more premarket testing than other chemicals—the EPA has relied on a tiered system of toxicity testing. The assumption underlying this system is that positive findings on earlier, more basic tests of neurotoxicity in adult animals will “trigger” the EPA to request more extensive testing by manufacturers, including tests in immature animals. Unfortunately, this tiered process has failed to result in appropriate DNT testing. In 1998, an internal EPA Toxicology Working Group concluded that these triggers may not be sufficient to identify all chemicals that have the potential to produce DNT [ 75 ]. Moreover, this tiered system discourages industry from conducting testing in immature animals because the findings could necessitate further costly testing and hinder a chemical from reaching the market. The European Framework: “REACH” In 2001, the European Commission affirmed that the European Union's legislative framework did not provide adequate information about the adverse effects of chemicals on human health, and that when hazards were identified the regulatory agencies were slow to assess risks and to introduce measures to reduce those risks [ 78 ]. Indeed, chemical manufacturers are not required to “prove” that a chemical is safe before marketing it. The European Commission proposed a new regulatory framework for chemicals, REACH (Registration, Evaluation, and Authorization of Chemicals) [ 78 , 79 ] ( Figure 1 ). Figure 1 Flow Chart Summarizing REACH (Registration, Evaluation, and Authorization of Chemicals)—the European Commission's Regulatory Framework for Chemicals (Illustration by Sapna Khandwala, Public Library of Science, adapted from [ 86 ]) Under REACH, chemical manufacturers would have to assume a much greater burden for showing the lack of harm from use of their products. Specifically, REACH would require both European and non-European manufacturers doing business in Europe to submit more extensive toxicity data for about 30,000 chemicals on the market, including reproductive and DNT data for those chemicals produced in highest quantity. Chemicals found to be hazardous would be subject to an authorization procedure to show that they can be used safely or that there are no safer alternatives. This registration process would not guarantee that chemicals are safe, but it is a step in the right direction. The American Chemistry Council has objections to REACH, stating that “the proposed regulation is burdensome, costly, and impractical” ( http://www.accnewsmedia.com/site/page.asp?TRACKID=&VID=1&CID=359&DID=1256 ). The pharmaceutical industry used similar objections to ward off regulations before the thalidomide epidemic ushered in requirements for pharmaceutical agents to undergo extensive premarket testing in clinical trials [ 80 ]. Limitations of Existing Animal Tests for DNT The US EPA has been slower than the EU to adapt to the overwhelming evidence that low-level exposure to environmental toxins can be harmful. The EPA continues to rely heavily on data from animal (toxicity) testing conducted on only a single animal species and in adult animals. Furthermore, EPA guidelines for a general developmental toxicity screening test typically examine only crude toxicological endpoints such as death, body weight, or organ dysfunction. In contrast, the DNT includes tests of locomotor activity, acoustic startle, learning, and memory. But, as currently designed, the existing tests may miss important effects such as mood changes, impulsive behaviors, and attentional problems that in humans have been shown to result from exposures to environmental toxins [ 24 , 27 , 30 , 37 , 40 ]. While these effects might seem subtle, they can seriously interfere with a child's social and emotional well-being. It is also uncertain whether tests conducted under current EPA guidelines will detect subtle deficits in key human skills such as reading. There are other problems with relying principally on adult animals to signal the potential for DNT in humans. The structure and development of the cerebral cortex of animals commonly used in these studies differs markedly from that of humans. A chemical's effects on one type of animal may differ from its effects on other animals and on humans. In the case of thalidomide, high-dose fetal exposure had adverse morphologic effects on rabbits, but not rats; functional effects have only recently been described [ 81 ]. Although there is some concordance of human and animal data for the adverse effects of lead, mercury, and PCBs, intake limits for these compounds established exclusively on the basis of rodent studies have not been sufficiently protective of human health compared with epidemiologic studies [ 47 ]. Indeed, there is compelling evidence from epidemiologic studies of widespread contaminants such as lead, tobacco, and PCBs that human studies are essential to ensure that children are not harmed by low levels of exposure [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. From a scientific standpoint, data from epidemiologic studies represent the “gold standard” for detecting subtle effects of environmental toxins on humans. But epidemiological studies are expensive to mount, difficult to execute, and take years to complete. Using observational studies to disentangle the adverse consequences of a single toxin from other environmental influences and to promulgate regulations is a difficult and painfully slow process. There is also a financial disincentive for chemical registrants to voluntarily fund such studies because a positive epidemiological study could lead to stricter regulations. More importantly, if society continues to rely on epidemiologic studies to evaluate the toxicity of chemicals only after they are marketed, many children will first be harmed. Steps to Protect Children from Environmental Toxins Children must be better protected from both new and existing chemicals that are known or possible toxins [ 49 ]. To protect children from existing toxins, such as lead, mercury, and tobacco, the US EPA and FDA need more authority and resources to regulate and reduce emissions and exposures. Under our current system, efforts to enhance regulations to protect children from confirmed toxins are costly and protracted. Indeed, countless communities across the globe suffer from widespread environmental contamination. If there is any lesson from our experience with environmental toxins, it is that we need to identify environmental chemicals that are toxic before they are marketed or widely disseminated. For new commercial chemicals, toxicity testing in animals should be required before they are marketed. For all new chemicals, including pesticides, extensive premarket testing should be required in multiple animal species of both sexes and at different developmental stages. These tests should be designed to have adequate statistical power to detect subtle differences within the ranges of exposure that occur in human populations. If implemented, these testing requirements would represent a dramatic departure from existing regulations, while providing a powerful incentive for industry to develop less toxic chemicals. Toxicity testing in animals is essential but insufficient to protect pregnant women and children. For one thing, uncertainties about the safety of a chemical for humans will persist even after toxicity testing in animals is successfully completed. One additional safeguard that deserves further debate is whether prevalent environmental chemicals to which children could be exposed should undergo more extensive testing in human trials before they are marketed. If done, these trials should examine exposure, uptake (using biomarkers), and adverse effects among children or other populations only when the product is used as intended. For example, once animal toxicity testing of a residential pesticide is complete (including DNT and reproductive toxicity testing), a pesticide could undergo further testing in the home environment. Using an experimental group and a control group, researchers would compare levels of pesticides found in settled dust, on children's hands, and in their blood, urine, or hair. Children would be followed, when indicated, to ensure that an excess of neurobehavioral problems or other relevant outcomes did not develop among those whose homes were assigned to receive the pesticide application. If such trials were undertaken, families would need to be fully informed about the purpose, potential benefits, and risks of participating. The trials should be conducted by the federal government—or other independent entities that do not have any ties to the chemical industry—and funded by an industry fee or tax. Community representatives would need to be involved in the review and approval of such trials, and ethical standards would need to be established regarding, for example, the role of data safety and monitoring boards. Many families would undoubtedly find it objectionable and would choose not to participate. Indeed, some products might never undergo testing if they failed to offer meaningful benefits to families, in which case the product would either be taken off the market or never reach the market. This type of trial sounds extreme, but it is quite rational when compared to the existing approach of disseminating a potential toxin into children's environments without any human data about exposure, uptake, or toxicity. Furthermore, under our existing system, families are neither informed nor given an option to decline involvement in what ultimately are experiments exposing millions of pregnant women and children to potential toxins. Thus, we need to thoughtfully deliberate about whether these types of trials can be done in an ethical fashion. We also need to have further debate about whether it is ethical to continue to disseminate chemicals of unknown toxicity into children's environments or to allow children to continually be exposed to prevalent toxins, like lead, despite considerable evidence that they are toxic [ 82 ]. Too often, it is left up to a few investigators or community leaders to discover and quantify the adverse effects of toxins, and advocate efforts to reduce children's exposure. Conclusion In contrast with the EU's proposed REACH program, which would require industry to conduct more tests or analyses to demonstrate that high-production chemicals will not cause harm to fetuses or children, the Bush administration has argued—in unison with the American Chemistry Council—that such regulations would harm industry [ 83 , 84 ]. It is time to acknowledge that the existing requirements for toxicity testing and regulations are inadequate to safeguard pregnant women and children. Until a formal regulatory system is developed to effectively screen and identify new and existing chemicals that are toxic to pregnant women and children, we are left to await the next epidemic to warn us about an environmental disaster. Unfortunately, by then we will have once again fouled our nest [ 85 ]. The US framework to protect children from environmental toxins is precarious (Photo: Earl Dotter, http://www.earldotter.com )

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1069660

The Coming of Age of Multicultural Medicine

Meeting the challenge of providing health care for a multicultural population is now a major movement that is impacting health care worldwide

In Stockton, California, a city of 269,000 people nestled in California's largest agricultural valley, residents are reported to speak 100 different languages. Acculturation is difficult in the best of circumstances, but what happens when those people with limited or no proficiency in English have a medical problem? Many United States hospitals are required to provide some manner of interpreter services for people with limited English proficiency—but do those services also bridge the cultural divide? Meeting the challenge of providing health care for a multicultural population is now a major movement that is affecting health care in developed countries, principally the US but also in European countries and Australia. Although the bulk of studies and commentaries on the subject began to appear in the 1990s, the literature dates back much further, to articles written in the 1960s and 1970s by medical anthropologists, sociologists, nurses, mental health professionals, and others. Wake-Up Calls In the US, the first major alert on this problem came in 1985, when the Report of the Secretary's Task Force on Black and Minority Health was issued [ 1 ]. (The “Secretary” was the head of the Department of Health and Human Services (DHHS).) The report painted a bleak picture of the quality of health care afforded to African-Americans and other racial and ethnic minorities. A decade later, reports from the US Institute of Medicine began to appear. Three of the ten reports, which spanned a ten-year period, dealt with the need to greatly diversify the health professions work force—still a somewhat unachieved goal. The most recent, considered a new wakeup call, was the 2003 report Unequal Treatment: Confronting Racial and Ethnic Disparities in Health Care [ 2 ]. It minced few words in describing the problems faced by racial and ethnic minorities who sought health care: “The conditions in which many clinical encounters take place—characterized by high time pressure, cognitive complexity, and pressures for cost-containment—may enhance the likelihood that these processes will result in care poorly matched to minority patients' needs. Minorities may experience a range of other barriers to accessing care, even when insured at the same level as whites, including barriers of language, geography and cultural familiarity” ( Figure 1 ). Figure 1 Minority Groups Face a Disparity in the Quality of Health Care (Illustration: Giovanni Maki, adapted from [ 2 ]). The Institute of Medicine defined “disparities” in health care as racial or ethnic differences in the quality of health care that are not due to access-related factors, clinical needs, patient preferences, or appropriateness of intervention [ 2 ]. Soon afterward, another US government arm, the Agency for Healthcare Research and Quality of the DHHS, issued two other reports: the National Healthcare Disparities Report [ 3 ] and the National Healthcare Quality Report [ 4 ], with annual updates promised. The reports focused on seven clinical conditions, including cancer, diabetes, and mental health, and discussed the quality of care and differences in access to such care for special population groups, including minorities and the disabled. All of these reports make it clear that health care professionals and health systems need to change. In recent years, in order to improve their lives economically or avoid war and/or famine, many people have migrated from less to more developed areas of the world, changing the demographics of the US and a number of other societies. Evidence that they and nonmigrant minorities experience inequities in attaining quality health care is abundant [ 5 ]. Studies also indicate that although genetics is involved in some health-related differences between racial and ethnic groups, such as in the incidence of certain diseases and responses to pharmaceuticals, it is probably not a major factor in explaining health disparities [ 6 ]. The Era of Action A primary result of these reports on health disparities? A truly dizzying array of offices, centers, programs, and initiatives within the main DHHS as well as in some of its major branches such as the National Institutes of Health and the Centers for Disease Control and Prevention, all designed to improve health care for racial and ethnic minorities in one way or another. Some of these programs also fund grants to outside organizations, public and private, and coordinate with state offices of minority health. And there are more activities devoted to reducing health disparities: (1) university-level institutes, offices, and programs, such as those at the UMDNJ-Robert Wood Johnson Medical School and Georgetown University, (2) private foundations, such as the California Endowment, (3) agencies and programs within the various states, such as the very active Ohio Commission on Minority Health, and (4) combinations of groups, such as DiversityRx ( www.diversityrx.org ), an informational organization sponsored by the National Conference of State Legislatures, Resources for Cross-Cultural Health Care, and the Henry J. Kaiser Family Foundation. All these efforts might suggest that there are no problems left to be solved, but this is hardly the case. Providing quality health care to those who differ from a country's majority population in terms of language and culture (and often race) is a mammoth task that does not yield to easy or quick fixes, but rather to consistent and determined efforts at improvement. Cultural Competence The most common term used in this effort is “cultural competence,” essentially defined as a respectful knowledge of and attitude toward people from different cultures that enables health professionals who work with people from another culture to develop and use standard policies and practices that will increase the quality and outcome of their health care. With cultural competence as the centerpiece, social and behavioral scientists have started consulting companies to (1) train health care professionals working in private and public health care settings (hospitals, community clinics, managed health care plans) in cultural competence, and (2) propose as well as study the effects of such changes in these settings. Some hospitals and managed health care plans have developed their own programs; examples that stand out are the M.D. Anderson Hospital in Texas and Kaiser Permanente health plans. In 2000, the M.D. Anderson Hospital established an Office of Institutional Diversity, which emphasizes the use of employees with a variety of backgrounds and experiences to examine cancer and its impact on all kinds of people. Educational forums, employee network groups, and the use of evidence-based hypotheses to design and implement pilot interventions are all part of the effort to improve care of culturally diverse patients. Kaiser Permanente's Institute for Culturally Competent Care selects and coordinates Kaiser Permanente's several Centers of Excellence, which each serve specific populations. For example, a West Los Angeles center focuses on the diagnosis, treatment, and management of conditions prevalent among African-Americans, such as sickle cell disease and prostate cancer. The National Diversity Department emphasizes a diverse workforce and has published a number of providers' handbooks on culturally competent care for specific racial or cultural patient groups, such as Latino patients. Not to Be Left Out Pharmaceutical companies have also discovered multicultural medicine. Many that offer continuing medical education courses to help publicize their new drugs now also offer courses on diseases more prevalent in certain racial and ethnic groups than others (such as diabetes in the Hispanic/ Latino population). These courses include information on how to treat such groups with the company's drugs. Interestingly, in 2004 a clinical trial proved the effectiveness of the first drug specifically designed for the treatment of congestive heart failure in African-Americans [ 7 ]. The drug, a combination of fixed doses of isosorbide and hydralazine, may now be nearing the market. Despite the fact that the Association of Black Cardiologists was a cosponsor of the trial, the trial drew criticism on the basis that it allowed race to interfere with treatment decisions [ 6 ]. A Global Issue The increased diversity of European populations, with the expected stress on entrenched health care systems and on the migrants themselves, has led to Migrant-Friendly Hospitals ( http://www.mfh-eu.net ), a “European initiative to promote health and health literacy of migrants and ethnic minorities” begun in October 2002. With funding from the European Commission and the Austrian Federal Ministry for Education, Science and Culture, a network of 12 pilot hospitals from European Union member states has been implementing and evaluating the effectiveness of three health care models for migrants and minorities. The models are: the improvement of interpreting in clinical communication, the creation and distribution of migrant-friendly information and training in mother and child care, and staff training in cultural competence. Results of the pilot experiences were reported at a final conference in December 2004 and will form the basis of European recommendations on migrant-friendliness as a quality criterion for hospital development and on the role of hospitals in promoting health and health literacy for migrants and ethnic minorities. One of the 12 pilot hospitals mentioned above is the Bradford Hospitals NHS Trust, long active among a number of other hospitals and health projects in the UK that strive to improve services for racial and ethnic minorities in their areas. Australia also has a multicultural society, and The Centre for Culture and Health of the University of New South Wales in Sydney has an active program aimed at increasing cultural competency, both among medical students at the University and in the country's medical community at large ( http://cch.med.unsw.edu.au/ ). The Centre offers graduate certificates and diplomas in public health (culture and health), as well as a Masters in Public Health with a concentration in multicultural health, and a postgraduate research degree. It emphasizes the establishment of partnerships with Area Health Services around New South Wales, grassroots organizations, and governmental organizations. A number of research projects also are underway. There are, for example, intervention strategies designed to reduce risk for cardiovascular disease in various cultural groups, such as the Arabic and Farsi-speaking communities, and studies of cancer among Chinese families in Australia. Conclusion People's basic medical needs do not vary greatly; they can be accommodated with appropriate understanding, awareness, and education. In the end, medicine and health care can only be enhanced and informed by the broadening of cultural awareness. Further Reading on Multicultural Medicine Here are three captivating books that yield knowledge through narrative. The Spirit Catches You and You Fall Down: A Hmong Child, Her American Doctors, and the Collision of Two Cultures by Anne Fadiman (Farrar, Straus and Giroux, 1997). Healing by Heart: Clinical and Ethical Stories of Hmong Families and Western Providers by Kathleen Culhane-Pera and coauthors (Vanderbilt University Press, 2003). Healing Latinos: Realidad y Fantasia , a collection of physician-patient vignettes edited by David E. Hayes-Bautista and the late Roberto Chiprut (Cedars Sinai Health Systems and the UCLA Center for Latino Health, 1998).

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1069661

Recurrent Pleural and Pericardial Effusions Due to Sarcoidosis

A 54-year-old man presented with fever, shortness of breath, and left-sided pleuritic chest pain. His bilateral pleural effusions and pericardial effusion turned out to be due to sarcoidosis

PRESENTATION of CASE A 54-y-old white male presented to the hospital during the winter of 2002 with complaints of fever, shortness of breath, and left-sided pleuritic chest pain of 2 d duration. He was a nonsmoker without any significant family history of pulmonary disease. He was retired, and denied any past exposure to chemicals including beryllium. On physical examination, the patient was febrile with tachycardia and normal blood pressure. Air entry at both lung bases was diminished. The remainder of his history and physical examination was unremarkable. He had been hospitalized twice before for similar complaints during the prior 6 wk. Investigations during the first admission showed mild leukocytosis with normal electrolytes. Chest radiograph at that time showed bilateral pleural effusions and cardiomegaly, while echocardiography showed cardiac tamponade for which he underwent emergency pericardiocentesis. A computed tomography (CT) scan of the chest after the pericardiocentesis showed bilateral pleural effusions without hilar or mediastinal lymphadenopathy. A CT scan of the abdomen and pelvis, to look for any occult malignancy, was normal. Therapeutic thoracentesis was performed to relieve his symptoms. Pleural and pericardial fluid analyses were exudative without malignant cells, and were negative on culture for bacterial, mycobacterial, and fungal organisms. Investigations for HIV, syphilis, rheumatological diseases, hepatitis, and occult malignancies were all negative. The patient improved symptomatically with pericardiocentesis and thoracentesis, and he was discharged with the differential diagnosis of an atypical vasculitic syndrome or a paraneoplastic syndrome. On the second admission, he presented with increasing shortness of breath, and a CT scan of the chest showed recurrent bilateral pleural effusions without pulmonary disease. Therapeutic thoracentesis was performed, relieving his symptoms, and he was discharged. During his third (current) admission, laboratory investigations were unremarkable. Repeat CT scan of the chest showed a left-sided pleural effusion, normal lung parenchyma, a small pericardial effusion, and mediastinal lymph nodes ( Figure 1 ). Video-assisted thoracoscopic pleural biopsy showed nonspecific chronic inflammation. Mediastinal lymph node biopsy showed benign reactive lymph nodes with focal epithelioid, non-caseating granulomas consistent with sarcoidosis. The patient was treated with oral prednisone, 20 mg daily. He remained asymptomatic when seen at his 1-y follow-up, when he was taking 7.5 mg of prednisone daily. Figure 1 CT of the Chest during the Third Hospitalization The CT shows mediastinal lymphadenopathy (pink arrows) and left pleural effusion. DISCUSSION Sarcoidosis is characterized by non-caseating granulomas in affected organs. It primarily affects the lungs. Pleural effusion occurs in 5% of patients and may be the presenting feature of the disease [ 1 ]. Cardiac manifestations include bundle branch block, arrhythmia, congestive heart failure, pericarditis, and cardiomyopathy. Asymptomatic minimal pericardial effusion has been shown to occur in 20% of cases [ 2 , 3 ]. Sarcoidosis presenting with pleural and pericardial effusion is extremely rare, and only one previous case has been reported [ 4 ]. Patients presenting with coincident pleural and pericardial effusions need to be investigated for rheumatological diseases, occult malignancies, and chronic infections such as HIV, tuberculosis, hepatitis, and syphilis. The diagnosis of sarcoidosis requires the presence of clinical and radiographic findings suggestive of sarcoidosis, non-caseating granulomas found on biopsies obtained from one or more sites, and the exclusion of other granuloma-forming diseases [ 5 ]. The differential diagnosis of non-caseating granulomas includes mycobacterial infections, berylliosis, and sarcoidosis. Our patient had clinical and radiological features suggestive of sarcoidosis and had non-caseating granulomas on his mediastinal lymph node biopsy, and we excluded other possible causes of recurrent pleural and pericardial effusions. The optimal management strategy for sarcoidosis still remains uncertain. Asymptomatic pulmonary sarcoidosis is best treated with a wait-and-watch approach [ 6 ]. But steroids remain the mainstay of treatment for systemic sarcoidosis involving the cardiovascular system, nervous system, or eyes, and for cases with progressive pulmonary involvement [ 7 , 8 ]. Earlier institution of steroids in cardiac sarcoidosis may prevent progressive disease and improve outcomes [ 9 , 10 ]. Cytotoxic agents are used as steroid-sparing agents in patients requiring large doses of steroids and who experience serious side effects [ 11 ]. Learning Points • Patients with recurrent pleural and pericardial effusion should be investigated for chronic infections, rheumatological illnesses, and malignancies. • Sarcoidosis should be included in the differential diagnosis of patients presenting with bilateral pleural and pericardial effusion, as early treatment may improve the outcome in cardiac sarcoidosis. • Steroids remain the mainstay of treatment for systemic sarcoidosis involving the cardiovascular system, nervous system, or eyes, and for cases with progressive pulmonary involvement.

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1069662

Antifibrinolytic Agents in Traumatic Haemorrhage

Among trauma patients who survive to reach hospital, exsanguination is a common cause of death. Could anti fibrinolytics reduce the death rate? Only a large randomized controlled trial can answer the question

Introduction This article is an invitation to doctors around the world to participate in the CRASH-2 trial (Clinical Randomisation of an Antifibrinolytic in Significant Haemorrhage), a large, multi-centre, randomised controlled trial of a simple and widely practicable treatment for traumatic hemorrhage. The rationale for the trial and contact details for those who would like to take part are given below. Evidence from randomised controlled trials is essential for improving health care. In the case of widely practicable treatments for common health problems, even modest treatment effects can result in substantial health gains. However, to detect such modest effects requires large multi-centre randomised trials involving hundreds of collaborating health professionals internationally. Many health professionals would be pleased to collaborate in such trials if they knew that they were underway, but at present there is no easy way to bring these trials to their attention. Three years ago, in the context of the CRASH-1 trial (Corticosteroid Randomisation After Significant Head Injury), the trial investigators sent a message to the electronic mailing list of the World Association of Medical Editors, asking them to consider publishing an editorial about the CRASH-1 trial that invited doctors around the world to participate. In response to this request, many medical journals around the world published the CRASH-1 trial editorial in various different languages, and as a result, many more doctors joined the CRASH-1 trial. The trial was completed in May 2004 and involved around 400 hospitals in almost 50 countries, and because of its size provided a reliable answer to an important question (see www.crash.lshtm.ac.uk ). This current article is being published as the result of a similar such request to medical editors in the context of the CRASH-2 trial. A Possible Role for Antifibrinolytics For people at ages five to 45 years, trauma is second only to HIV/AIDS as a cause of death. Each year, worldwide, over three million people die as a result of trauma, many after reaching hospital [ 1 ]. Among trauma patients who do survive to reach hospital, exsanguination is a common cause of death, accounting for nearly half of in-hospital trauma deaths [ 2 ]. Central nervous system injury and multi-organ failure account for most of the remainder, both of which can be exacerbated by severe bleeding [ 3 ]. The haemostatic system helps to maintain the integrity of the circulatory system after severe vascular injury, whether traumatic or surgical in origin [ 4 ]. Major surgery and trauma trigger similar haemostatic responses, and any consequent massive blood loss presents an extreme challenge to the coagulation system. Part of the response to surgery and trauma, in any patient, is stimulation of clot breakdown (fibrinolysis) which may become pathological (hyper-fibrinolysis) in some [ 4 ]. Antifibrinolytic agents have been shown to reduce blood loss in patients with both normal and exaggerated fibrinolytic responses to surgery, and do so without apparently increasing the risk of post-operative complications; most notably there is no increased risk of venous thromboembolism [ 5 ]. Systemic antifibrinolytic agents are widely used in major surgery to prevent fibrinolysis and thus reduce surgical blood loss. A recent systematic review [ 6 ] of randomised controlled trials of antifibrinolytic agents (mainly aprotinin or tranexamic acid) in elective surgical patients identified 89 trials including 8,580 randomised patients (74 trials in cardiac, eight in orthopaedic, four in liver, and three in vascular surgery). The results showed that these treatments reduced the numbers needing transfusion by one third, reduced the volume needed per transfusion by one unit, and halved the need for further surgery to control bleeding. These differences were all highly statistically significant. There was also a statistically non-significant reduction in the risk of death (relative risk = 0.85: 95% confidence interval, 0.63–1.14) in the antifibrinolytic-treated group. Why a Large Trial Is Needed Because the haemostatic abnormalities that occur after injury are similar to those after surgery, it is possible that antifibrinolytic agents might also reduce blood loss, the need for transfusion and mortality following trauma. However, to date there has been only one small randomised controlled trial (70 randomised patients, drug versus placebo: zero versus three deaths) of the effect of antifibrinolytic agents in major trauma [ 7 ]. As a result, there is insufficient evidence to either support or refute a clinically important treatment effect. Systemic antifibrinolytic agents have been used in the management of eye injuries where there is some evidence that they reduce the rate of secondary haemorrhage [ 8 ]. A simple and widely practicable treatment that reduces blood loss following trauma might prevent thousands of premature trauma deaths each year, and secondly, could reduce exposure to the risks of blood transfusion. Blood is a scarce and expensive resource, and major concerns remain about the risk of transfusion-transmitted infection. Trauma is common in parts of the world where the safety of blood transfusion is not assured. A recent study in Uganda estimated the population-attributable fraction of HIV acquisition as a result of blood transfusion to be around 2%, although some estimates are much higher [ 9 , 10 ]. Only 43% of the 191 WHO member states test blood for HIV, Hepatitis C, and Hepatitis B viruses. Every year, unsafe transfusion and injection practices are estimated to account for 8–16 million Hepatitis B infections, 2.3–4.7 million Hepatitis C infections, and 80,000–160,000 HIV infections [ 11 ]. A large randomised trial is therefore needed of the use of a simple, inexpensive, widely practicable antifibrinolytic treatment such as tranexamic acid (aprotinin is considerably more expensive and is a bovine product with consequent risk of allergic reaction and hypothetically transmission of disease), in a wide range of trauma patients, who when they reach hospital are thought to be at risk of major haemorrhage that could significantly affect their chances of survival. A Call to Health Professionals The CRASH-2 trial will be a large, international, placebo-controlled trial of the effects of the early administration of the antifibrinolytic agent tranexamic acid on death, vascular events and transfusion requirements ( http://www.crash2.lshtm.ac.uk ). The trial aims to recruit some 20,000 patients with trauma and will be one of the largest trauma trials ever conducted. However, it will only be possible to conduct such a trial if hundreds of health care professionals worldwide work together to recruit patients to the trial in order to make it a success. If you are interested in recruiting patients, please contact Ian Roberts at the CRASH-2 trial coordinating centre ( Box 1 ). Box 1. Contact Information for the CRASH-2 Trial Ian Roberts CRASH-2 Trial Co-Ordinating Centre London School of Hygiene and Tropical Medicine Keppel Street, London WC1E 7HT Phone: 0207 958 8128 Fax: 0207 299 4663 Web site: www.crash2.lshtm.ac.uk E-mail: Ian.roberts@lshtm.ac.uk A similar version of this article is being published in several medical journals worldwide.

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1069663

Hookworm: “The Great Infection of Mankind”

Over the last five years, there has been increasing recognition of the global health importance of hookworm. New international efforts to control the morbidity of hookworm are in progress

Introduction In 1962, Norman Stoll, the distinguished Rockefeller Institute scientist who helped to establish human parasitology research in North America, described the unique health impact of hookworm as follows [ 1 ]: As it was when I first saw it, so it is now, one of the most evil of infections. Not with dramatic pathology as are filariasis, or schistosomiasis, but with damage silent and insidious. Now that malaria is being pushed back hookworm remains the great infection of mankind. In my view it outranks all other worm infections of man combined…in its production, frequently unrealized, of human misery, debility, and inefficiency in the tropics. Like many other global disease experts who witnessed dramatic reductions in malaria prevalence as a result of DDT spraying during the late 1950s [ 2 ], Stoll did not anticipate malaria's imminent re-emergence in India. However, he articulated with eloquence the magnitude of the disease burden resulting from hookworm infection. He further offered the silent and insidious character of hookworm as a partial explanation for its neglect by the global medical community. This neglect subsequently intensified during the 1970s, 1980s, and 1990s with the omission of hookworm from the list of diseases covered by the World Health Organization's Special Programme for Research and Training in Tropical Hookworm has proven to be extremely difficult to eliminate or eradicate in areas of poverty and poor sanitation. Diseases, as well as from other global health initiatives. Over the last ten years, however, there has been increasing recognition of the global health importance of hookworm. Today, new international efforts to control the morbidity of hookworm and other soil-transmitted helminth infections are in progress ( www.who.int/wormcontrol ). Etiology and Global Distribution Human hookworm infection is caused by blood-feeding nematode parasites of the genus Ancylostoma and the species Necator americanus . Worldwide, N. americanus is the predominant etiology of human hookworm infection, whereas A. duodenale occurs in more scattered focal environments [ 3 ]. These two hookworms, together with the roundworm, Ascaris lumbricoides , and the whipworm, Trichuris trichiura , are often referred to collectively as soil-transmitted helminths (STHs). No international surveillance mechanisms are in place to determine the prevalence and global distribution of hookworm infection. However, based on an extensive search of the literature since 1990, the worldwide number of cases of hookworm was recently estimated to be 740 million people [ 4 ]. The highest prevalence of hookworm occurs in sub-Saharan Africa and eastern Asia ( Figure 1 ). High transmission (defined below) also occurs in other areas of rural poverty in the tropics, including southern China [ 5 ], the Indian subcontinent [ 6 ], and the Americas [ 7 ]. In all regions, there is a striking relationship between hookworm prevalence and low socioeconomic status ( Figure 2 ) [ 4 ]. Hookworm's neglected status partly reflects its concentration among the world's poorest 2.7 billion people who live on less than $2 a day. Figure 1 Global Distribution of Human Hookworm Infection (Illustration: Margaret Shear, Public Library of Science, adapted from [ 4 ]) Figure 2 The Relationship between Poverty and Hookworm Prevalence (Illustration: Margaret Shear, Public Library of Science, adapted from [ 4 ]) Clinical Features, Epidemiology, and Disease Burden Hookworm infection is acquired by invasion of the infective larval stages through the skin ( A. duodenale larvae are also orally infective). Following host entry, the larvae undergo a journey through the vasculature, then the lungs and other tissues, before they enter the gastrointestinal tract and molt twice to become one-centimeter-long adult male and female worms [ 3 ]. The worms mate and the female hookworms produce up to 30,000 eggs per day, which exit the host's body in the feces ( Figure 3 ). Figure 3 Life Cycle of the Human Hookworm N. americanus The BZA anthelminthics albendazole and mebendazole remove adult hookworms from the gastrointestinal tract. In contrast, the Na -ASP-2 Hoookworm Vaccine is designed to target third-stage infective larvae (filariform larvae). Humoral immunity to the vaccine inhibits the entry of larvae into the gastrointestinal tract and thereby prevents their development into blood-feeding adult parasites. (Illustration: Sapna Khandwala, Public Library of Science, adapted from [ 3 ] and [ 33 ]) Because hookworms do not replicate in humans, the morbidity of hookworm is highest among patients that harbor large numbers of adult parasites. Estimates of the intensity of hookworm infection are typically obtained by using quantitative fecal egg counts as a surrogate marker for worm burden. The World Health Organization defines moderate-intensity infections as those with 2,000–3,999 eggs per gram of feces, and heavy-intensity infections as those with 4,000 or more eggs per gram (p. 26 in [ 8 ]). Compared to other STH infections and schistosomiasis, hookworm infection exhibits a unique age-intensity profile—whereas the intensities for the former peak in childhood and adolescence, hookworm intensity usually either steadily rises in intensity with age or plateaus in adulthood [ 3 , 9 ]. The biological basis for this observation is unknown [ 10 ]. Adult hookworms cause morbidity in the host by producing intestinal hemorrhage [ 3 ]. The adult hookworms then ingest the blood, rupture the erythrocytes, and degrade the hemoglobin [ 11 ]. Therefore, the disease attributed to hookworm is silent blood loss leading to iron deficiency anemia and protein malnutrition. There is a correlation between parasite intensity and host intestinal blood loss [ 12 ]; in children, women of reproductive age, and other populations with low iron stores, there is often a correlation between parasite intensity and reductions in host hemoglobin [ 3 , 12 , 13 , 14 , 15 , 16 ]. In children, chronic heavy-intensity infections are associated with growth retardation, as well as intellectual and cognitive impairments; in pregnant women, they are associated with adverse maternal–fetal outcomes [ 3 , 12 , 13 , 14 , 15 , 16 ]. When measured in disability-adjusted life years, the global disease burden from hookworm exceeds all other major tropical infectious diseases with the exception of malaria, leishmaniasis, and lymphatic filariasis (pp. 192–193 in [ 17 ]). In addition, hookworm has been associated with impaired learning, increased absences from school, and decreased future economic productivity [ 18 ]. Therefore, like other neglected diseases, chronic infection with hookworm promotes long-term disability and increases the likelihood that an afflicted population will remain mired in poverty. Hookworm Control Strategies Because of its high transmission potential, hookworm has proven to be extremely difficult to eliminate or eradicate in areas of poverty and poor sanitation [ 19 ]. Indeed, in the absence of comprehensive economic development, the impact of sanitation, footwear, and health education has been minimal [ 19 ]. Control efforts have therefore shifted to reducing morbidity through mass treatment (also known as “deworming”) of affected populations with anthelminthic drugs [ 19 ]. Although benzimidazoles (BZAs) are the most commonly used agents for treating STH infections, levamisole and pyrantel may also be used in some circumstances. Periodic and repeated deworming with BZAs and praziquantel, complemented by basic sanitation and adequate safe water, is considered the most cost-effective means to control the morbidity caused by STH and schistosome infections [ 19 , 20 , 21 , 22 ]. Efforts led by the World Health Organization have focused on annual, twice-yearly, or thrice-yearly doses in schools because the heaviest intensities of STH infections are most commonly encountered in school-age children [ 23 ]. Among the health benefits of periodic deworming of schoolchildren are improvements in iron and hemoglobin status, physical growth and fitness, and cognition [ 20 , 21 , 22 , 23 ]. In addition, there are important externalities, including improvements in education and reduced community-based transmission of ascaris and trichuris infections [ 23 ]. Accordingly, at the 54th World Health Assembly in 2001, a resolution was passed urging member states to attain a minimum target of regular deworming of at least 75% and up to 100% of all at-risk school-age children by 2010 [ 20 , 23 ]. Developing a New Control Tool: The Na -ASP-2 Hookworm Vaccine Deworming satisfies a number of United Nations Millennium Development Goals including those related to poverty reduction, child health, and education. However, there are also several reasons to believe that the school-based deworming programs could have less of an impact on the control of morbidity from hookworm than from other STH and schistosome infections [ 3 ]. As noted above, heavy-intensity hookworm infections are common among both adults and children, so school-based programs would not be expected to have an impact on hookworm transmission in the community [ 24 ]. School-based programs are also not likely to affect either preschool children or pregnant women, despite evidence for the health benefits from BZAs in both populations [ 16 , 25 ]. Finally, a single dose of mebendazole (one of the two major BZAs) has variable efficacy against hookworm [ 26 ], and following treatment, hookworm reinfection to pre-treatment levels can occur within 4–12 months [ 27 ]. This, and the observation that the efficacy of mebendazole against hookworm can diminish with frequent and repeated use, has prompted concerns about the possible emergence of BZA resistance [ 28 ]. As a complementary strategy, the Human Hookworm Vaccine Initiative (HHVI) is developing a safe, efficacious, and cost-effective vaccine, the Na -ASP-2 Hookworm Vaccine, that would provide an additional tool for the control of hookworm [ 29 , 30 ]. The HHVI is a non-profit partnership comprising research, process development, vaccine manufacturing and control, and pre-clinical and clinical testing units at the George Washington University, London School of Hygiene and Tropical Medicine, and Oswaldo Cruz Foundation (FIOCRUZ), and sponsored by the Sabin Vaccine Institute ( www.sabin.org ). The HHVI selected the hookworm larval antigen ASP-2 (ancylostoma secreted protein-2) based on studies that (1) identified the molecule as a protective antigen linked to earlier-generation irradiated infective larval vaccines [ 29 ], (2) determined a relationship between human anti-ASP-2 antibodies and reduced risk of heavy hookworm infection in populations living in hookworm-endemic regions of Brazil and China ([ 30 ]; J. Bethony, A. Loukas, M. J. Smout, S. Brooker, S. Mendez, et al., unpublished data), and (3) confirmed the ability of recombinant ASP-2 to partially protect laboratory animals against larval hookworm challenges [ 30 , 31 , 32 ]. Process development, cGMP manufacture and control, and pre-clinical testing of Na -ASP-2 from N. americanus were completed in 2004 ( Figure 4 ). Pending United States Food and Drug Administration approval, clinical testing of the vaccine will take place in 2005. The Na -ASP-2 Hookworm Vaccine will be developed almost entirely in the non-profit sector. Ultimately, the vaccine will be indicated for the active immunization of susceptible individuals against moderate and heavy necator infection. Vaccination would reduce the number of hookworm infective larvae entering the gastrointestinal tract, thereby reducing the number of adult worms and the fecal egg counts in individuals exposed to the larvae. Figure 4 Scheme for the Development and Quality-Control Testing of the Na -ASP-2 Hookworm Vaccine, and Its Transition from the Laboratory into the Clinic After the selection of ASP-2 from N. americanus ( Na -ASP-2) as the lead candidate antigen based on a series of research and development (R&D) tests—which included immunoepidemiology studies identifying human correlates of immunity to hookworm and confirmatory laboratory animal vaccine trials—the recombinant antigen was expressed in yeast and then developed as a biologic through a well-defined product development strategy (PDS). By following the product development strategy, process development (PD) and manufacturing led to the generation of pilot batches at different scales prior to technology transfer to a cGMP manufacturing facility. Both process development and manufacturing rely on developing assays for the product's identity, color and appearance, purity, immunological recognition, and potency, as well as qualification of the assays for sensitivity, specificity, accuracy, and reproducibility. Each of these processes must maintain a high level of quality control by following a set of policies, protocols, and standard operating procedures. After the manufacturing of a cGMP product and the required pre-clinical animal testing, a clinical development plan (CDP) was generated. Because the Na -ASP-2 Hookworm Vaccine is a product destined for the world's poorest, it is being developed almost exclusively in the non-profit sector, along with government manufacturers in middle-income countries. Hookworm as a Model Because immunization would only affect hookworm larvae and not adult hookworms already residing in the gastrointestinal tract of infected individuals, the first dose of the vaccine would be administered following deworming. Therefore, use of the vaccine could build on the infrastructures developed as part of school-based programs. Given that hookworm afflicts only the world's most impoverished, a major hurdle for the development of the Na -ASP-2 Hookworm Vaccine is its small commercial market. Innovative financing mechanisms must be considered to produce this orphan biologic. Towards that end, the HHVI has partnered with manufacturers in hookworm-endemic middle-income countries that would commit to industrial scale-up of the Na -ASP-2 Hookworm Vaccine pending proof-of-principle for its efficacy. This approach might help to inform the development of business models for the production and distribution of orphan biologics for other neglected diseases.

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1069664

Exercise and Health: Can Biotechnology Confer Similar Benefits?

Education and public policies are largely failing to encourage people to exercise. Could our knowledge of exercise biology lead to pharmaceutical treaments that could confer the same benefits as exercise?

Health Benefits of Physical Activity Regular physical activity has been recognized to confer health benefits since antiquity [ 1 ]. However, for most of humankind, voluntary discretion over whether or not to exercise is a recent phenomenon limited to advanced industrialized societies. A large body of epidemiological literature consistently documents greater longevity in persons who are physically active on a near-daily basis, and reveals inverse relationships between levels of daily exercise and incidence of major chronic disorders such as obesity [ 2 ], hypertension [ 3 ], diabetes [ 4 ], ischemic heart disease, and all causes of mortality [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ]. From a public health perspective, there is little question that even modest increases in daily activities such as walking or stair climbing would have important positive consequences in reducing the burden of illness. However, knowledge of the likely health benefits accruing to the physically active so far has not been a sufficient stimulus to promote sustained changes in behavior for most of the American population. If education and public policies are insufficient to promote behavioral changes to increase physical activity among most people, can advances in biotechnology confer such benefits to individuals unable or unwilling to perform the necessary physical effort? Translating Knowledge of Exercise Biology to Novel Therapeutics Greater knowledge of how cells and tissues are modified in response to recurring bouts of exercise provides a basis for more precise recommendations as to the mode, intensity, and amount of exercise required to produce specific health benefits (e.g., treatment of dyslipidemia [ 13 ], control of body weight [ 14 ], or prevention of diabetes [ 15 ]). In addition, an understanding of the molecular signaling events that drive the beneficial effects of exercise on human physiology could foster the development of novel drugs, devices, or biological agents designed to substitute for exercise. Many individuals who otherwise would develop diabetes or cardiovascular disease would benefit if advances in exercise biology revealed novel measures to promote the favorable effects on insulin sensitivity, lipoprotein metabolism, and blood pressure that are known to accrue through regular physical activity. Physiological Properties of Skeletal Muscle What do we know about basic muscle and exercise biology? The cells that constitute our skeletal muscles are called myofibers—large multinucleated cells that may extend for the full length of individual muscles. There are different types of myofibers, which vary in size and with respect to metabolic and contractile capability [ 16 ] ( Figure 1 ). Skeletal myofibers are innervated by motor neurons that contact each myofiber, and the intensity, duration, and timing of each muscle contraction are determined by the pattern of motor neuron firing. A pattern of occasional intense contractions separated by longer periods of rest is called “phasic,” while a pattern characterized by brief contractions occurring repeatedly over an extended period is called “tonic.” Endurance training regimens like running or cycling employ tonic patterns of contractile work, and it is this form of habitual activity that serves best to reduce risk for obesity, diabetes, hypertension, and heart disease. Figure 1 Specialized Myofibers in a Mammalian Skeletal Muscle A cross-section of the gastrocnemius muscle of a mouse has been stained to detect myoglobin, which is found selectively in slow oxidative and fast oxidative myofibers (stained brown), but not in fast glycolytic myofibers (unstained). Human muscles exhibit a similar mosaic pattern. In response to sustained periods of motor nerve stimulation repeated daily for several weeks, the percentage of myofibers that contain myoglobin is increased, in synchrony with an increased abundance of mitochondria and a shift of myosin subtypes from fast glycolytic to slow or fast oxidative. Dynamics of Muscle Mass Maintenance of normal muscle mass requires some minimal level of ongoing work activity, and building and maintaining muscle mass is most effectively done through phasic contractions. A slow but inexorable loss of muscle mass is a feature of advancing age in human populations [ 17 ]. Loss of muscle mass and strength is an important determinant of injury and disability in the elderly, but even rigorous weight training programs cannot completely counteract this age-related decline that becomes particularly troublesome in the eighth and ninth decades of life. Efforts to develop effective countermeasures to maintain muscle mass in the elderly constitute an active and important area of current research [ 18 , 19 , 20 ]. Although the molecular signaling mechanisms that transduce the effects of phasic patterns of work activity to modify muscle mass are incompletely understood, recent evidence implicates pathways that include the signaling molecules PI3 kinase, Akt, mTOR, S6K, and ERK, the ubiquitin ligases MAFbx and MuRF1, and transcription factors of the FOX superfamily in the control of both catabolic and anabolic processes [ 21 , 22 , 23 , 24 ]. Contractile and Metabolic Properties With respect to variations in contractile and metabolic properties, myofibers are classified on a spectrum between two extremes on the basis of contractile (fast versus slow) and metabolic (glycolytic versus oxidative) properties. At one extreme, the fastest glycolytic fibers have high levels of enzymes that generate ATP via glycolysis but few mitochondria (approximately 1% of cell volume). At the other end of the spectrum, slow oxidative fibers generate force with slower kinetics but are capable of long periods of repeated contraction without fatigue. They are rich in mitochondria (3%–10% of cell volume). Other myofibers, called fast oxidative, are both relatively fast and resistant to fatigue, and are rich in mitochondria (like the slow oxidative fibers). Muscles composed primarily of fast glycolytic fibers are needed for rapid movements (e.g., escape from predators) but fatigue when sustained periods of activity are required (e.g., migration). Most human muscles exhibit a mosaic pattern of different fiber types ( Figure 1 ), with a great deal of variation among individuals, which is influenced at least in part by patterns of use. When we exercise daily, or at least several times weekly, we deliver a stimulus to the specific muscle groups involved in these activities that is sufficient to alter specialized properties of myofibers within these muscles. While habitual physical activity promotes a great variety of physiological adaptations that alter vascular reactivity, cardiac function, adipocyte function, and neurophysiology, adaptive responses of skeletal myofibers confer at least some of the health benefits. Patterning of skeletal muscle fiber composition is initially determined during embryonic development, but can be partially or completely overturned by stimuli applied to fully mature adult myofibers: by hormonal influences (e.g., thyroid hormone), but most importantly by different patterns of motor nerve activity and contractile work. Myofibers that experience phasic patterns of contractile work—brief bursts of activity interspersed within long periods of inactivity—will assume the fast glycolytic phenotype. Myofibers subjected to tonic patterns of work activity—sustained periods of repetitive contraction on a habitual basis—will take on fast oxidative or slow oxidative properties. Under experimental conditions in laboratory animals, it is possible to transform muscles completely from one myofiber phenotype to another in a reversible manner, solely by altering the pattern of neural stimulation. We know that having a high proportion of oxidative muscle fibers conveys health benefits, and the possibility to control fiber composition through therapeutic intervention is promising. Molecular Signaling Pathways At a cellular and molecular level, how does a fast glycolytic myofiber sense a tonic pattern of contractile activity and transduce that information to transform itself into a cell with fast oxidative or slow oxidative properties? We know that such signals must be transduced to the nucleus, activating certain genes and suppressing others, for myofiber plasticity to occur. We know the identities of some of the nuclear transcription factors that carry these signals, and of other proteins that regulate the function of these transcription factors ( Figure 2 ). Figure 2 Molecular Signaling Pathways Link Changes in Contractile Activity to Changes in Gene Expression That Establish Myofiber Diversity A tonic pattern of motor nerve activity promotes changes in intracellular calcium that trigger a variety of intracellular events that modify the function of nuclear transcription factors. The pathway transduced by calcineurin and NFAT is highlighted in larger type. Other signals are received by cell surface receptors to activate similar or parallel signaling events. Signaling proteins that participate in transducing effects of contractile activity to specific genes include ion channels (TRP), scaffolding proteins (Homer), protein phosphatases and protein kinases (calcineurin, CAMK, p38MAPK), DNA-binding transcription factors (shown in red; NFAT, MEF2, PGC-1, ATF2), and endogenous inhibitors (shown in blue; GSK3, HDAC, and MCIP) (inhibitors antagonize gene activation via the pathways indicated, in some cases acting as negative feedback regulators). Quite a variety of intracellular messengers have been proposed to provide the proximate signals in exercising muscles to stimulate activity-dependent gene regulation. This discussion will focus on a signaling cascade mediated by calcineurin, a calcium-regulated protein phosphatase that signals to the nucleus via transcription factors of the nuclear factor of activated T cells (NFAT) family. Upon receipt of the appropriate calcium signal, calcineurin is activated and removes phosphate groups from NFAT, thereby permitting translocation of NFAT to the nucleus. Within the nucleus, NFAT binds DNA and activates transcription (in concert with other transcription factors) of relevant downstream target genes that encode proteins necessary for fast oxidative or slow oxidative myofiber phenotypes. Calcineurin and NFAT proteins are abundant in skeletal myofibers, and several lines of evidence support the viewpoint that the calcineurin–NFAT pathway plays a role in mediating activity-dependent gene regulation in muscle [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. For example, in mice genetically engineered to distinguish the inactive (cytoplasmic) from active (nuclear) forms of NFAT by means of a sensor, it is evident that NFAT is inactive in resting muscles, but activated by tonic patterns of muscle contraction (running or electrical stimulation of the motor nerve) [ 40 ]. Using other genetic manipulations in mice to produce in muscle a form of calcineurin that remains active even in the absence of calcium signals, myofibers are converted from fast glycolytic to fast oxidative or slow oxidative forms [ 41 ]. And in muscles of mice genetically engineered to lack calcineurin, fiber type switching is impaired [ 42 ]. Cellular Memory Muscle contractions are initiated under the influence of the motor nerve by release of calcium from the sarcoplasmic reticulum, which triggers actin–myosin crossbridge cycling ( Figure 3 ). Calcium released via ryanodine receptors is completely sufficient to activate muscle contractions, and the effects are immediate (within milliseconds). It is also sufficient to initiate calcineurin–NFAT signaling to the nucleus, but cannot by itself sustain the signal in a manner necessary to promote myofiber remodeling [ 40 ]. Changes in gene expression evoked by neuromuscular activity are not immediate but require that the stimulus be sustained for an extended period (minutes to hours). Moreover, tonic stimulation of the motor nerve must be repeated daily, or nearly so, over several weeks for the changes in myofiber properties to become fully manifest. We have characterized this requirement for repetition of the activity stimulus over days as a form of “cellular memory.” The effects of the tenth or 20th day of exercise are not the same as the effects of the first day. The myofiber somehow “remembers” not only the pattern of activity it has experienced today, but what has gone on over the preceding days or weeks, such that the changes in abundance of proteins that control contractile function and metabolism accrue over time. Figure 3 Proposed Model for Cellular Memory, Based on Activity-Induced Changes in TRPC3—A Putative Store-Operated Calcium Channel Neural activation triggers muscle contraction by releasing calcium stored within the sarcoplasmic reticulum (SR) through mechanisms that involve channel proteins called dihydropyridine receptors (DHPR) and ryanodine receptors (RYR). Inactive myofibers have a low abundance of TRPC3 channels, and calcium released from SR is not sufficient to maintain the calcium-regulated transcription factor NFAT in the nucleus. Under conditions of tonic activity (training stimulus), TRPC3 channels become more abundant, and are regulated by the scaffold protein Homer, which binds RYR. Under these conditions, the combined effect of calcium entering the cell via TRPC3 channels and exiting the SR via RYR channels maintains NFAT in the nucleus, where it promotes transcription of genes that establish the slow oxidative phenotype in myofibers. Once the slow oxidative phenotype is established (trained myofiber), the continued expression of TRPC3 allows this state to be maintained even with a less intensive tonic activity pattern of neural stimulation. (Figure adapted from [ 40 ].) To explain this cellular memory, we propose that, as the bursts of contractile activity are sustained over time (through a tonic pattern of neural stimulation), a second source of calcium is mobilized from outside of the cell and enters via a class of calcium channels that are called “store-operated” or “non-voltage-dependent.” This second source of calcium is not required for muscle contractions, but is required to sustain calcium-dependent signaling to the nucleus. Phasic patterns of contractile activity do not promote calcium entry via store-operated channels. Tonic patterns of activity, in contrast, would not only promote the mobilization of extracellular calcium but also increase the number of store-operated calcium channels with each bout of exercise. Myofibers would thereby grow progressively more responsive to tonic activity. Consistent with this model, we know that daily running increases the expression of a putative store-operated calcium channel called TRPC3. Moreover, increasing the abundance of TRPC3 in cultured myotubes prolongs the period in which intracellular calcium is elevated following a depolarizing stimulus, sustains the transcription factor NFAT within the nucleus, and augments expression of NFAT-dependent target genes [ 40 ]. A great deal of additional research remains to be done before we have a comprehensive understanding of how habitual physical activity promotes changes in gene expression in skeletal muscles, and in turn improves fitness and reduces risk for diabetes, hypertension, dyslipidemia, and coronary artery disease. However, studies of the relationships between the proteins of calcium metabolism and calcium-regulated signaling pathways—as described here in a simplified manner with respect to TRPC3, calcineurin, and NFAT proteins—are illustrative of progress in this field. Other notable findings point to additional signaling proteins (CAMK, p38MAPK, and AMPK) and transcription factors (PGC-1, MEF2, ATF2, PPARs) active in pathways that intersect with calcineurin–NFAT signaling [ 31 , 43 , 44 , 45 , 46 , 47 , 48 ] (see Figure 2 ). It is encouraging that some of these proteins are attractive targets for drug discovery. Summary and Conclusions Long the province of physiologists who have contributed valuable insights in past decades, exercise science more recently has attracted the attention of molecular biologists, who have recognized the biological interest and medical importance of this field. Biotechnology and pharmaceutical companies also are beginning to take interest. This review has focused on adaptive responses of skeletal muscle to changing patterns of physical activity, and on the role of the calcium–calcineurin–NFAT signaling cascade in controlling gene expression in skeletal myofibers. Further advances in our understanding of signaling mechanisms that govern activity-dependent gene regulation in skeletal muscle could lead to drugs, gene therapy, or devices that can, at least in part, substitute for daily exercise. Although it is unlikely that such technologies would fully recapitulate exercise-induced adaptations that affect other tissues of the body, beneficial effects on work performance and whole-body metabolism have been demonstrated using gene transfer techniques to alter skeletal muscles in animal models. If it proves possible to drive similar effects in skeletal muscles in humans, the interventions capable of providing such effects would almost certainly find broad clinical application.

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1069665

Noninvasive Visualization of the Activated αvβ3 Integrin in Cancer Patients by Positron Emission Tomography and [18F]Galacto-RGD

Background The integrin αvβ3 plays an important role in angiogenesis and tumor cell metastasis, and is currently being evaluated as a target for new therapeutic approaches. Several techniques are being studied to enable noninvasive determination of αvβ3 expression. We developed [ 18 F]Galacto-RGD, a 18 F-labeled glycosylated αvβ3 antagonist, allowing monitoring of αvβ3 expression with positron emission tomography (PET). Methods and Findings Here we show by quantitative analysis of images resulting from a small-animal PET scanner that uptake of [ 18 F]Galacto-RGD in the tumor correlates with αvβ3 expression subsequently determined by Western blot analyses. Moreover, using the A431 human squamous cell carcinoma model we demonstrate that this approach is sensitive enough to visualize αvβ3 expression resulting exclusively from the tumor vasculature. Most important, this study shows, that [ 18 F]Galacto-RGD with PET enables noninvasive quantitative assessment of the αvβ3 expression pattern on tumor and endothelial cells in patients with malignant tumors. Conclusions Molecular imaging with [ 18 F]Galacto-RGD and PET can provide important information for planning and monitoring anti-angiogenic therapies targeting the αvβ3 integrins and can reveal the involvement and role of this integrin in metastatic and angiogenic processes in various diseases.

Introduction Cell–cell and cell–matrix interactions play essential roles in tumor metastasis and angiogenesis. Integrins are one of the main classes of receptors involved in these processes. In addition to having adhesive functions, integrins transduce messages via various signaling pathways and influence proliferation and apoptosis of tumor cells, as well as of activated endothelial cells. One prominent member of this receptor class is the integrin αvβ3. It has been demonstrated that αvβ3 is an important receptor affecting tumor growth, local invasiveness, and metastatic potential [ 1 , 2 ]. This integrin is expressed on various malignant tumors and mediates adhesion of tumor cells on a variety of extracellular matrix proteins, allowing these cells to migrate during invasion and extravasation [ 3 ]. The integrin αvβ3 is also highly expressed on activated endothelial cells during angiogenesis [ 4 ]. In contrast, expression of αvβ3 is weak in resting endothelial cells and most normal organ systems [ 5 ]. On activated endothelial cells, the receptor mediates migration through the basement membrane during formation of the new vessel, which is essential for sufficient nutrient supply of the growing tumor. Inhibition of the αvβ3-mediated cell–matrix interaction has been found to induce apoptosis of activated endothelial cells. Thus, the use of αvβ3 antagonists is currently being evaluated as a strategy for tumor-specific anti-cancer therapies [ 6 , 7 , 8 ]. Owing to the weak expression on non-activated endothelial cells, treatment with αvβ3 antagonists does not affect preexisting blood vessels. Inhibition of blood vessel formation in tumor models using αvβ3 antagonists not only blocks tumor-associated angiogenesis, but in some cases results in tumor regression [ 9 ]. However, αvβ3 antagonists can induce apoptosis not only of activated endothelial cells but also of αvβ3-positive tumor cells [ 10 ], resulting in a direct cytotoxic effect on tumor cells. Moreover, blocking of the receptor expressed on tumor cells can reduce invasiveness and spread of metastases [ 11 ]. Furthermore, αvβ3-binding molecules have been successfully used to “target” a variety of therapeutic agents to the tumor tissue. These include chemotherapeutic agents [ 12 ], cDNA-encoding anti-angiogenic genes [ 13 ], and T lymphocytes [ 14 ]. These encouraging experimental studies have already led to initial clinical trials evaluating the use of αvβ3 antagonists (e.g., vitaxin [ 15 ] and cilengitide [ 16 ]) in patients with various malignant tumors [ 17 , 18 , 19 , 20 ]. Currently available imaging techniques are limited in monitoring treatment with this class of drugs. Anti-tumor activity is generally assessed by determining the percentage of patients in whom a significant reduction in tumor size is achieved during a relatively short period of therapy (“response rate”). Thus, this method may not be applicable for a form of therapy that is aimed at disease stabilization and prevention of metastases. New methods are urgently needed for planning and monitoring treatments targeting the αvβ3 integrin. Based on cyclo(-Arg-Gly-Asp- D Phe-Val-) [ 21 ], a variety of radiolabeled αvβ3 antagonists for single photon emission tomography and positron emission tomography (PET) have been developed (for review see [ 22 , 23 ]). [ 18 F]Galacto-RGD (arginine–glycine–aspartic acid), a glycosylated cyclic pentapeptide, resulted from a consequent tracer optimization [ 24 ] based on the first-generation peptide [ 125 I]-3-iodo- D Tyr 4 -cyclo(-Arg-Gly-Asp- D Tyr-Val-) [ 25 ] and showed high affinity and selectivity for the αvβ3 integrin in vitro, receptor-specific accumulation in αvβ3-positive tumors, and high metabolic stability in a murine tumor model, as well as rapid, predominantly renal elimination [ 26 , 27 ]. Here we describe how [ 18 F]Galacto-RGD allows quantification of αvβ3 expression in vivo, show that tumor-induced angiogenesis can be monitored in a murine tumor model, and for the first time, to our knowledge, demonstrate that this class of tracers can be used in patients for noninvasive determination of αvβ3 expression. Methods Tracer Synthesis Synthesis of the labeling precursor and subsequent 18 F-labeling was carried out as described [ 27 ]. For application in patients, after high-performance liquid chromatography the collected fraction was evaporated to dryness; 0.5 ml of absolute ethanol and 10 ml of phosphate-buffered saline (pH 7.4) were added; and the product was passed through a Millex GV filter (Millipore, Eschborn, Germany). Quality control of the product was carried out according to the demands of the local regulatory authorities. Murine Tumor Models For in vivo evaluation, xenotransplanted human melanoma models (M21 and M21-L) and a human squamous cell carcinoma model (A431) were used. The M21 cell line expressing αvβ3 [ 25 , 28 ] acted as a positive control and the M21-L cell line, a stable variant cell line of M21 failing to transcribe the αv gene, as a negative control [ 29 ]. Cell culture conditions for M21 and M21-L cells are described elsewhere [ 26 ]. Similar protocols were used for A431 cells. The experimental protocol involving animals was approved by the Committee of Veterinarian Medicine of the State of Bavaria; handling of animals was performed according to the standards set by the Committee of Veterinarian Medicine. In order to study the correlation between αvβ3 expression and tumor uptake of [ 18 F]Galacto-RGD, we injected mice subcutaneously with mixtures of M21 and M21-L cells. Pilot experiments had indicated that injection of 1.5 × 10 6 M21 cells leads within 4 wk to the formation of tumors with a diameter of approximately 8 mm. To obtain similarly sized M21-L tumors, it was necessary to inject 6 × 10 6 cells. In order to study tumors with approximately 10%, 25%, 50%, and 75% M21 cells, we injected mice with the following mixtures of M21 and M21-L cells: 1.5 × 10 5 / 5.4 × 10 5 , 3.8 × 10 5 / 4.6 × 10 6 , 7.5 × 10 5 / 3 × 10 6 , and 1.1 × 10 6 / 1.5 × 10 6 . Four weeks after inoculation, nude mice were injected with 7.4 MBq of [ 18 F]Galacto-RGD and scanned at the small-animal PET. Subsequently, tumors and other organs of interest were dissected, immediately counted, cut in two pieces, and frozen for further workup. For experiments with the squamous cell carcinoma model, approximately 10 6 A431 cells were injected subcutaneously in nude mice. Two weeks after inoculation, 7.4 MBq of [ 18 F]Galacto-RGD was injected, and mice were scanned in the animal PET. Animals were sacrificed, and organs of interest were dissected and subsequently weighed and counted or used for immunohistochemical analysis. Immunohistochemistry For immunohistochemical investigation, frozen tumor tissues from mice, as well as from patients, were sectioned (6 μm) and stained using the biotinylated monoclonal anti-αvβ3 antibody LM609 (1:100; Chemicon Europe, Hofheim, Germany). For staining the murine β subunit, a monoclonal hamster anti-mouse antibody (1:10; Chemicon Europe) and a biotinylated mouse anti-hamster IgG secondary antibody (1:200; Chemicon Europe) were used. Sections were processed by peroxidase staining (peroxidase substrate KIT AEC, Vector Laboratories, Burlingame, California, United States). Western Blotting The frozen tumor tissue was homogenized and extracted with lysis buffer (50 mM Hepes (pH 7.5), 150 mM NaCl, 10% Glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM Na 4 P 2 O 7 , 1 mM MSF, 10 μg/ml Aprotinin, 10 μg/ml Leupeptin). Protein concentration was determined according to Bradford [ 30 ] and adjusted to equivalent values using lysis buffer. After SDS-PAGE and transfer, immunoblotting was carried out using a polyclonal rabbit anti-αv antibody (1:500; Chemicon Europe) and a 125 I-labeled polyclonal donkey anti-rabbit IgG antibody (1:400; 477 kBq/μg, Amersham Buchler, Braunschweig, Germany). For analysis, blots were placed on a phosphor screen for 2 d. For readout out, a Molecular Dynamics PhosphorImager 445 SI (Sunnyvale, California, United States) was used. PET Studies with a Small-Animal Scanner PET imaging of tumor-bearing mice was performed using a prototype small-animal positron tomograph (Munich Avalanche Photodiode PET; [ 31 ]). The animal scanner consists of two sectors, comprising three detector modules each, which rotate around the animal for acquisition of complete projections in one transaxial slice (30 angular steps). Each module consists of eight small (3.7 × 3.7 × 12 mm3) lutetium-oxy-orthosilicate crystals read out by arrays of avalanche photodiodes. List mode data are reconstructed using statistical, iterative methods including the spatially dependent line spread function. Reconstructed image resolution is 2.5 mm (full width at half maximum) in a transaxial field of view of 7.5 cm, and the slice thickness is 2 mm. Ninety minutes after the injection of approximately 7.4 MBq of [ 18 F]Galacto-RGD, animals were positioned prone inside the tomograph, and a transaxial slice through the tumor region was measured for 480 s Patient Study The study protocol was approved by the ethics committee of the Klinikum Rechts der Isar ( Protocol S1 ), and each patient gave written and informed consent prior to the study ( Protocol S2 ). Nine patients were scanned (five female and four male; age, 26–75 y), who suffered from either malignant melanoma with lymph node metastasis (stage IIIb; patients 1–3), malignant melanoma with distant metastasis (stage IV; patients 4 and 5), chondrosarcoma (patient 6), soft tissue sarcoma (patient 7), osseous metastasis of renal cell carcinoma (patient 8), or villonodular synovitis of the knee (patient 9). Patient selection was focused on melanoma and sarcoma because there is considerable evidence that these tumor types express αvβ3. Diagnosis prior to scanning was made by biopsy (patients 6–8), by CT (patients 1, 2, and 4–8), by MRI (patient 9), and/or by [ 18 F]fluorodeoxyglucose ([ 18 F]FDG)–PET (patients 1–4 and 8). After scanning, the diagnosis was confirmed by surgery and histopathological examination of the resectioned specimen (patients 1, 2, 5, 6, 8, and 9) or by combined analysis of morphological imaging, [ 18 F]FDG PET, and the patient's clinical data and history (patients 3, 4, and 7). For immunohistochemistry, sampled specimens (patients 1, 2, 5, 6, 8, and 9) were snap frozen in liquid nitrogen and stored at −70 °C until staining was performed. Tissue samples were taken within 1 wk after scanning from the tumor regions with the maximum tracer uptake. Light microscopic evaluation of the density of αvβ3-positve microvessels was performed as described previously [ 32 ]. Briefly, areas with the highest density of αvβ3-positve microvessels were identified using scanning magnification. Subsequently, αvβ3-positve microvessels were counted in three adjacent microscopic fields using a 40× magnifying lens and 10× ocular, corresponding to an area of 0.588 mm 2 . Determination of microvessel density was performed by one senior pathologist (M. S.), who was blinded for the results of the corresponding standardized uptake value (SUV) analysis of tracer accumulation. Then the correlation between the mean values of the vessel counts and the corresponding SUVs was analyzed. PET scanning was performed using an ECAT Exact PET scanner (Siemens-CTI, Knoxville, Tennessee, United States). After injection of 144–200 MBq of [ 18 F]Galacto-RGD, three consecutive emission scans (starting at 7 ± 2.7 min, 37 ± 10.5 min, and 79 ± 18.4 min post injection [p. i.]) from the body stem and, if necessary, from tumor regions outside the body stem were obtained. For one patient, only one scan starting 120 min. p. i. was carried out. Attenuation- and decay-corrected images were reconstructed by using an ordered subsets expectation maximization algorithm. The accumulation of [ 18 F]Galacto-RGD was evaluated by calculating the mean SUV normalized to the patient's body weight according to the following formula [ 33 ]: (measured activity concentration [Bq/ml] × body weight [kg]) / injected activity [Bq]. The axial slice of the lesion with the maximum activity accumulation was chosen by visual estimation, a region of interest with a diameter of 15 mm was selected on the lesion, and the resulting mean SUV was used for further analysis. For lesions smaller than 2 cm in diameter, a region of interest with a diameter of 10 mm was used and the analysis was based on maximum SUV rather than mean SUV, in order to minimize partial volume effects, which could lead to an underestimation of the SUV. Dosimetry calculations are based on the MIRDOSE 3.0 program. Data from six patients were analyzed by selecting regions of interest with a diameter of 1.5 cm on the source organs (lung, liver, spleen, kidneys, muscle, bladder, intestine, and heart [left ventricle]). Activity measurements (in Becquerels per cubic centimeter) were performed for all three consecutive scans (mean time p.i. ± standard deviation, 7 ± 2.7 min, 37 ± 10.5 min, and 79 ± 18.4 min, respectively), using a monoexponential fit for calculation of residence times. The volume of the source organs lung, liver, spleen, and kidneys was measured by CT volumetry (Siemens, Forchheim, Germany) in four patients. For the other source organs in these four patients and all organs in the remaining two patients, standardized volume values of the source organs adapted to the patient's body weight were used. Statistical Methods All quantitative data are expressed as mean +/− one standard deviation. The correlation between quantitative parameters was evaluated by linear regression analysis and calculation of Pearson's correlation coefficient. Statistical significance was tested by using analysis of variance (ANOVA). Results Correlation of Tracer Uptake with αvβ3 Expression We have previously demonstrated, using a murine tumor model in which the tumor cells are either αvβ3-positive (human melanoma M21) or αv-negative (human melanoma M21-L), that [ 18 F]Galacto-RGD shows receptor-specific accumulation in the αvβ3-positive tumor [ 26 ]. Here we studied the correlation of [ 18 F]Galacto-RGD uptake with the level of αvβ3 expression. We injected tumor cell mixtures containing increasing amounts of αvβ3-positive M21 cells subcutaneously into nude mice. Transaxial images of mice 4 wk after cell inoculation and 90 min after tracer injection using a prototype small-animal PET scanner [ 31 ] showed increasing tracer uptake in the tumor corresponding with the percentage of receptor-positive cells ( Figure 1 A and 1 B). Figure 1 Preclinical Evaluation of [ 18 F]Galacto-RGD (A) Transaxial images of nude mice bearing tumors with increasing amounts of αvβ3-positive M21 cells (0% [M21-L], 25%, 75%, and 100% [M21]) 90 min p. i. provided by a prototype small-animal PET scanner show an increasing tracer uptake in the tumor and low background activity. (B) Immunohistochemical staining of tumor tissue sections prepared after PET imaging with an anti-human αvβ3 monoclonal antibody (LM 609) indicate that there is a correlation between tracer uptake and αvβ3 expression. (C) Western blots of the dissected tumors show a band at 25 kDa that corresponds with the mass of the αv subunit under reductive conditions, and indicate the increasing αvβ3 density in the murine tumor model used. (D) The correlation between receptor expression and [ 18 F]Galacto-RGD accumulation is confirmed by quantitative analysis based on the tumor/background ratios and tumor/muscle ratios calculated from the PET images and from the biodistribution studies, respectively, and by the relative αv expression in Western blot analyses. We validated these qualitative results by determining the relative amount of the αv subunit in the dissected tumors through Western blot analysis ( Figure 1 C). These data were correlated with the tumor/background ratios resulting from the quantitative analysis of the PET images ( Figure 1 D), as well as with the tumor/muscle ratios resulting from the biodistribution analysis carried out after the PET study ( Figure 1 D). Both analyses showed a significant correlation between [ 18 F]Galacto-RGD and relative αv expression, thus confirming the qualitative analysis by immunohistochemistry. The systematic difference between tumor/background and tumor/muscle ratios is due to the fact that the region of interest used to define the tumor region in the PET images will always contain not only tumor, but also normal tissues with low [ 18 F]Galacto-RGD uptake, such as muscle and lung. This is due to the limited spatial resolution of the PET scanner, which does not allow a sharp distinction between tumor and normal tissue. Accordingly [ 18 F]Galacto-RGD uptake by the tumor tissue will be underestimated, and the tumor/background ratio will be lower than the tumor/muscle ratio. Furthermore, tissue sampling was performed 30 min after the start of the PET scan. Clearance of radioactivity from the muscle tissue during this time period will also systematically increase the tumor/muscle ratio compared to the tumor/background ratio calculated from the PET images. When correlating the weight of the tumor with the relative αv expression, we found a nonsignificant trend for lower αv expression in larger tumors ( r = 0.34, p = 0.09). This is probably related to the presence of necrotic regions in larger tumors, which do not demonstrate αv expression. Thus, it can be excluded that the positive correlation between αv expression and [ 18 F]Galacto-RGD uptake is due to systematic differences in the size of tumors. Noninvasive Determination of αvβ3 Expression on Endothelial Cells To determine whether PET with [ 18 F]Galacto-RGD allows noninvasive determination of αvβ3 expression on activated endothelial cells, we used A431 tumor xenografts. A431 cells do not express αvβ3, but induce extensive angiogenesis when subcutaneously transplanted into nude mice. [ 34 ]. Immunohistochemical staining of tumor sections using a monoclonal anti-human αvβ3 antibody confirmed that the tumor cells do not express this integrin ( Figure 2 A). In contrast, staining with a polyclonal antibody against the murine β3 subunit demonstrated expression of β3 on endothelial cells of the tumor vessels. Since αIIbβ3, the only further integrin containing a β3 subunit, is mainly expressed on platelets, it can be excluded that staining depends on this receptor. Thus, in this case, staining for the β3 subunit correlates with αvβ3 expression. Figure 2 Noninvasive Monitoring of αvβ3 Expression on the Tumor Vasculature (A) Immunohistochemical staining of tumor section using the anti-αvβ3 monoclonal antibody LM609 demonstrates that squamous cell carcinoma cells of human origin do not express the αvβ3 integrin. In contrast, staining of section with an antibody against the murine β3 subunit indicates that the tumor vasculature is αvβ3-positive. (B) Transaxial images of a nude mouse bearing a human squamous cell carcinoma at the right shoulder (left) acquired at the small-animal PET 90 min after tracer injection show a clearly contrasting tumor. Tracer accumulation in the tumor (right, top image) can be blocked by injecting 18 mg of cyclo(-Arg-Gly-Asp- D Phe-Val-) per kilogram of mouse 10 min prior to tracer injection (right, bottom image), indicating receptor-specific accumulation. Transaxial images of tumor-bearing mice 90 min after injection of [ 18 F]Galacto-RGD showed a contrasting tumor on the right flank of the mouse, reflecting αvβ3-targeted tracer accumulation on endothelial cells of the tumor vasculature ( Figure 2 B). Moreover, we demonstrated receptor-specific tracer accumulation at the tumor site by injecting 18 mg of the pentapeptide cyclo(-Arg-Gly-Asp- D Phe-Val-) per kilogram of mouse 10 min prior to tracer injection. After blocking tracer accumulation, we found approximately 25% of the initial activity in the tumor (0.28 ± 0.05% injected dose per gram versus 1.07 ± 0.33% injected dose per gram). Studies in Humans For the initial evaluation in humans, we imaged nine patients (five with malignant melanomas, two with sarcomas, one with osseous metastasis from renal cell carcinoma, and one with villonodular synovitis) with approximately 185 MBq of [ 18 F]Galacto-RGD. For all patients, rapid, predominantly renal excretion was observed, resulting in fast tracer elimination from blood and low tracer concentration in most of the organs. Besides the kidneys (SUV = 5.5 ± 3.7; 79 min), the highest activity concentration was found in spleen (SUV = 2.5 ± 0.5; 79 min p.i.), liver (SUV = 2.4 ± 0.5; 79 min p.i.), and intestine (SUV = 2.1 ± 0.8; 79 min p.i.). In tumor lesions, tracer accumulation showed great heterogeneity, with SUVs ranging from 1.2 to 10.0. The SUV in the villonodular synovitis was 3.2. The radioactivity was retained in the tumor tissue for more than 60 min ( Table 1 ), whereas in all other organs a decrease of activity concentration was observed over time. Tumor/blood and tumor/muscle ratios 79 min p. i. were 3.8 ± 2.6 and 8.8 ± 6.0, respectively. Although for one melanoma patient multiple lesions were detected by the [ 18 F]FDG scan, which indicates viable tumor cells, no activity accumulation was found using [ 18 F]Galacto-RGD ( Figure 3 A). For other patients, however, similar uptake patterns were observed for [ 18 F]FDG and [ 18 F]Galacto-RGD ( Figure 3 B). The metabolite analysis of blood samples 10, 30, and 120 min p. i. showed in the soluble fractions more than 96% intact tracer ( n = 4) over the whole observation period and confirmed our preclinical data [ 27 ]. An effective radiation dose of 18.0 ± 3.2 μSv/MBq was calculated on the basis of the patient data ( n = 5). The highest absorbed dose was found in the urinary bladder wall (0.20 ± 0.04 mGy/MBq). Figure 3 Comparison of [ 18 F]FDG and [ 18 F]Galacto-RGD Scans Coronal image sections, acquired 60 min p. i. (A) Patient with malignant melanoma stage IV and multiple metastases in liver, skin, and lower abdomen (arrows): marked uptake of [ 18 F]FDG in the lesions (left), but no uptake of [ 18 F]Galacto-RGD (right). (B) Patient with malignant melanoma stage IIIb and a solitary lymph node metastasis in the right axilla (arrow): intense uptake of both [ 18 F]FDG (left) and [ 18 F]Galacto-RGD (right). Table 1 SUVs Determined Approximately 5 min, 35 min, and 75 min Post Injection Values are given as mean ± standard deviation ( n = 8, unless otherwise indicated) a n = 9 b n = 7 c n = 10 Immunohistochemical staining of sections obtained from tumor tissue after surgery using an anti-αvβ3 antibody showed αvβ3 expression on the endothelial cells of the tumor vasculature (6/6), and for two patients expression on the tumor cells as well (2/6) ( Figure 4 ). The density of αvβ3-positive vessels showed wide variation intraindividually and between individual cases. Light microscopic quantification revealed between one (inflammation of the knee due to previous operation) and 35 (soft tissue sarcoma of the knee, same patient) αvβ3-positive vessels per microscopic field. Moreover, in the six cases under analysis, density of immunohistochemically determined αvβ3-positive vessels was significantly associated with tracer accumulation as determined by SUV analysis ( r = 0.94, p = 0.005). Figure 4 Correlation of Tracer Accumulation and αvβ3 Expression (A–C) patient with a soft tissue sarcoma dorsal of the right knee joint. (A) The sagittal section of a [ 18 F]Galacto-RGD PET acquired 170 min p. i. shows circular peripheral tracer uptake in the tumor with variable intensity and a maximum SUV of 10.0 at the apical-dorsal aspect of the tumor (arrow). (B) The image fusion of the [ 18 F]Galacto-RGD PET and the corresponding computed tomography scan after intravenous injection of contrast medium shows that the regions of intense tracer uptake correspond with the enhancing tumor wall, whereas the non-enhancing hypodense center of the tumor shows no tracer uptake. (C) Immunohistochemistry of a peripheral tumor section using the anti-αvβ3 monoclonal antibody LM609 demonstrates intense staining predominantly of tumor vasculature. (D–F) patient with malignant melanoma and a lymph node metastasis in the right axilla. (D) The axial section of a [ 18 F]Galacto-RGD PET acquired 140 min p. i. shows intense focal uptake in the lymph node (arrow). (E) Image fusion of the [ 18 F]Galacto-RGD PET and the corresponding computed tomography scan after intravenous injection of contrast medium. (F) Immunohistochemistry of the lymph node using the anti-αvβ3 monoclonal antibody LM609 demonstrates intense staining predominantly of tumor cells and also blood vessels. Discussion Recently, we demonstrated that radiolabeled RGD peptides allow receptor-specific monitoring of αvβ3 expression in murine tumor models [ 24 , 25 , 26 , 27 , 35 ]. Here we have translated these findings to the clinical setting and for the first time, to our knowledge, demonstrated noninvasive imaging of αvβ3 expression in patients with malignant tumors. Furthermore, we have shown that the activity accumulation in the tumor correlates with the receptor density, determined by immunohistochemistry and Western blotting. This indicates that a noninvasive quantitative determination of αvβ3 expression is feasible. Furthermore, we have demonstrated in a squamous cell carcinoma model that the sensitivity of PET is adequate to image expression of αvβ3 in the tumor vasculature. This indicates that PET with [ 18 F]Galacto-RGD can be applied to study αvβ3 expression during angiogenesis. The correlation between [ 18 F]Galacto-RGD uptake in the tumor and αv expression shows considerable scattering. This is probably due to several factors. As for any imaging probe, tumor uptake of [ 18 F]Galacto-RGD is not only influenced by the expression of the αvβ3 integrin, but also by unspecific factors such as perfusion and vascular permeability. Furthermore, heterogeneous tracer uptake within a tumor, e.g., due to the presence of necrotic areas, will influence the correlation between [ 18 F]Galacto-RGD uptake and αv expression, since separate samples were used for measurements of tracer uptake and quantitative assessment of αv expression. Finally, the present study evalutated [ 18 F]Galacto-RGD uptake only at a fixed time, 90 min p. i. Imaging of the dynamics of [ 18 F]Galacto-RGD accumulation in the tumor tissue and tracer kinetic modeling may allow a better quantitative assessment of αvβ3 expression by PET imaging, and this approach should be evaluated in animal models as well as in patients. Nevertheless, the significant correlation between the uptake of [ 18 F]Galacto-RGD at a fixed time after injection and αvβ3 expression is very important for clinical studies, since it suggests that estimates of αvβ3 expression levels may be obtained from simple whole-body PET scans. It has been shown that the highly bent integrin conformation is physiological and has low affinity for biological ligands, such as fibrinogen and vitronectin. Inside-out and outside-in signaling involve a switchblade-like opening to an extended structure with high affinity for endogenous ligands, as well as integrin antagonists (for overview see [ 36 ]). The inside-out activation is induced by conformational changes in the membrane-proximal regions of the α and β subunit (e.g., by intracellular proteins like talin). Outside-in signaling is triggered by Mn 2+ , which defines by quaternary rearrangements a pathway for communication from the ligand-binding site to the cytoplasmatic proximal segments. However, it is also reported that cyclo(-Arg-Gly-Asp- D Phe-Val-), in addition to binding to the high-affinity conformer, can bind to the low-affinity conformation when used at concentrations far above its dissociation constant, resulting in a similar activation as found for Mn 2+ . The nanomolar concentration used in our radiotracer approach is approximately 10,000-fold lower than that reported for the activation of the low-affinity conformation. Thus, PET with [ 18 F]Galacto-RGD is expected to provide information not only about the expression of αvβ3 but also about the functional status of this integrin. The glycopeptide [ 18 F]Galacto-RGD showed high metabolic stability in patients and rapid, predominantly renal elimination, resulting in good tumor/background ratios and, thus, in high-quality images. Moreover, this finding confirms the general advantage of the glycosylation approach [ 24 , 26 , 37 , 38 , 39 ] in designing peptide-based tracers with favorable imaging properties for clinical applications. Another approach to optimize pharmacokinetics is based on the conjugation of polyethyleneglycol [ 40 , 41 , 42 , 43 , 44 , 45 ]. It has been demonstrated that such polyethyleneglycolated peptides also improve pharmacokinetics and tumor retention. However, a direct comparison of tracers resulting from the different strategies has not yet been carried out. The correlation between regional tracer uptake in the lesion and density of αvβ3-positive vessels confirms that this technique allows not only visualization but also noninvasive quantitative assessment of the integrin expression. Interestingly, our study demonstrated high both inter - and intraindividual variances in tracer accumulation in the different lesions, indicating a great diversity in receptor expression. This finding demonstrates the value of noninvasive techniques for appropriate selection of patients entering clinical trials of αvβ3-targeting therapies. This is further emphasized by the fact that in some cases no [ 18 F]Galacto-RGD uptake was found, despite viable tumor cells being detected via a [ 18 F]FDG scan. Furthermore, PET imaging with [ 18 F]Galacto-RGD can be applied to assess successful blocking of αvβ3 by therapeutic agents, thereby providing essential information for the dose and dose scheduling of αvβ3 antagonists. Further studies are needed to demonstrate the impact of this new technique as a novel prognostic indicator in cancer. However, the first evidence of the prognostic value is given by Gasparini et al. [ 46 ], who found αvβ3 expression in tumor vasculature “hot spots” to be a significant prognostic factor predictive of relapse-free survival in both node-negative and node-positive patients. αvβ3 is also found on endothelial cells during wound healing, in restenosis, in rheumatoid arthritis, and in psoriatic plaques. Thus, radiolabeled RGD peptides may be used to characterize not only malignant tumors but also inflammatory diseases. Most recently, we demonstrated in a murine model for cutaneous delayed-type hypersensitivity reaction that [ 18 F]Galacto-RGD allows noninvasive assessment of αvβ3 expression in inflammatory processes [ 47 ]. Our preliminary data from a villonodular synovitis show that αvβ3 expression on endothelial cells in this lesion can be monitored in patients. Altogether, these findings indicate that [ 18 F]Galacto-RGD might also become a new biomarker for disease activity in inflammatory processes. The primary advantage of PET in imaging molecular processes is its high sensitivity combined with high penetration of the gamma radiation resulting from positron decay. Thus, PET imaging allows quantification of regional radioactivity concentrations in human studies. The optical imaging approach has an even higher sensitivity, but suffers from the low penetration of light in most tissues. This results in a very limited ability to carry out tomographic imaging and to quantify the optical signal. Thus, optical imaging is currently limited to preclinical studies in mice, whereas PET can be performed in preclinical as well as in clinical studies. Magnetic resonance imaging provides high spatial resolution and can combine morphological and functional imaging, but has approximately 1,000-fold lower sensitivity compared with PET. Thus, PET is the most appropriate technique for noninvasive determination of molecular processes in patients at the current time. Obviously, the patient is exposed to high-energy γ-rays during this procedure. However, based on our radiation dose estimates, the effective radiation dose for a [ 18 F]Galacto-RGD scan is in the same range as for a [ 18 F]FDG scan, an approved routine examination in the clinic in many countries [ 48 ]. In preclinical studies, different targeted magnetic resonance contrast agents have been evaluated, using either anti-αvβ3 antibody-conjugated polymerized liposomes [ 49 ] or nanoparticles [ 50 ], or nanoparticles linked with an αvβ3 peptidomimetic antagonist [ 51 ]. In those studies, depending on the contrast agent and animal model used, an average magnetic resonance signal intensity enhancement between approximately 20% and 120% was found, a finding which has not yet been confirmed in clinical studies. In our patient study using [ 18 F]Galacto-RGD and PET, a 9-fold higher activity accumulation, on average, was found in the tumor than in muscle, further indicating the currently superior properties of this radiotracer for molecular imaging. Moreover, recent developments in combining PET with computed tomography or future possibilities to combine PET with magnetic resonance imaging will allow correlation of these processes with the corresponding morphology. To further improve tumor retention of αvβ3 radioligands, multimeric RGD peptides were recently introduced. Our group developed different series of multimeric structures with up to eight RGD units linked via different spacers [ 40 , 41 , 42 ]. These multimeric RGD peptides showed increased binding affinities in vitro and improved tumor accumulation and tumor/background ratios in rodents compared with the monomeric compounds. These data and data from other groups [ 52 , 53 , 54 ] indicate that the multimeric ligand approach may be used for optimization of the performance of peptide-based tracers. However, studies in patients will be necessary to demonstrate the potential of this approach in clinical settings. In summary, this new class of PET tracer may offer insights into molecular processes during tumor development and dissemination in preclinical as well as clinical settings, and will be a helpful tool in planning and controlling novel αvβ3-directed therapies. Supporting Information Protocol S1 Approval of Ethics Committee (1.4 MB PPT). Click here for additional data file. Protocol S2 Patient Consent Form (4.2 MB PPT). Click here for additional data file. Patient Summary Background Tumor cells express many different molecules on their surface. These cell membrane molecules are involved in a variety of different processes, such as those that hold cells together, trigger cell death, or determine whether the tumor spreads. Some of these molecules can be tagged with radiolabeled compounds, called tracers. These tracers can show where these molecules are found and how many there are by methods such as PET and SPECT scans that don't require a biopsy, i.e., are not invasive. These methods can then be used for planning treatment with anti-cancer drugs that bind these molecules What Did the Investigators Do? They induced tumors in mice and injected them with a tracer for one cell surface molecule—an integrin. They showed that the amount of the molecule on the tumor could be measured by the intensity of tracer seen on a PET scan. They also showed that the same molecule was present on the new blood vessels that tumors produce. In a small study of patients with various tumors, including melanomas, the researchers found that the same tracer could be used to measure the expression of the integrin on tumor cells as well as on endothelial cells, such as those found in blood vessels, and hence measure the amount of new vessels in the tumors. What Does This Mean for Patients? This tracer could be useful to determine integrin expression noninvasively, to determine how many new vessels tumors have, to get information for planning anti-cancer therapies targeting integrin, and to study response to anti-cancer drugs. However, this study involved only nine patients, so much more work will need to be done before such a technique is shown to be generally reliable. Where Can I Get More Information? The National Cancer Institute has information on melanomas for patients: http://www.nci.nih.gov/cancertopics/pdq/treatment/melanoma/patient Radiology Info explains PET scanning: http://www.radiologyinfo.org/content/petomography.htm

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Is It Always Unethical to Use a Placebo in a Clinical Trial?

Background to the debate: Placebos are used in trials to conceal whether a treatment is being given or not and hence to control for the psychosomatic effects of offering treatment. Placebo-controlled trials are controversial. Critics of such trials argue that if a proven effective therapy exists, a placebo should not be used. But proponents argue that placebo trials are still crucial to prove the efficacy and safety of many treatments.

Andreas Stang, Hans-Werner Hense, and Karl-Heinz Jöckel's Viewpoint: It Is Unethical When a Beneficial Standard Treatment Exists A better understanding of the aetiology and pathological mechanisms of diseases often results in new ideas for their treatment. It is then necessary to put these ideas to a formal empirical test in a trial setting. The randomized controlled trial (RCT) is the closest that clinical research can get to the experimental situation. In the RCT, patients are assigned at random to an intervention of putative effectiveness, with the aim of minimizing the potential for bias inherent in nonrandomized clinical research settings. The triumphal advance of RCTs is reflected in their prominent role as one of the pillars of evidence-based medicine. Initially, when there is uncertainty about the efficacy of a new treatment, clinical researchers are advised to compare the experimental intervention with a placebo. Placebo-controlled trials serve to show that a specific treatment has a beneficial effect on defined clinical endpoints beyond that attributable to mere administration of the intervention by medical professionals. Thus, the early trials of antihypertensive medications and statins were placebo-controlled and were considered to be proof of their beneficial effects. But what about the next phase? What happens when a treatment for a certain condition, such as hypertension, has been shown to be effective in placebo-controlled RCTs but a newer intervention has been developed for that condition? Let us assume that there is evidence from basic and early clinical trials that the new intervention has a biological effect and has no major side effects in appropriate doses. Should the researchers test it against placebo to prove the superiority of the new treatment? It is arguably unethical to withhold a therapy of proven efficacy from any patient in a research trial just for the purpose of increasing scientific knowledge. Paragraph 29 of the Declaration of Helsinki states: “The benefits, risks, burdens and effectiveness of a new method should be tested against those of the best current prophylactic, diagnostic, and therapeutic methods” [ 1 ]. A note of clarification for paragraph 29 states: “The World Medical Association hereby reaffirms its position that extreme care must be taken in making use of a placebo-controlled trial and that in general this methodology should only be used in the absence of existing proven therapy” [ 1 ]. Rothman and Michels have argued that the declaration should include specific examples showing how placebo trials are unethical: “It might suggest as one such example that even in studies of new analgesics to study relief from pain such as headache, the new remedies should be compared only with existing analgesics, and never with placebo. The example will reinforce the point that this principle is not a blurry boundary” [ 2 ]. Critics of the declaration argue that forbidding placebo trials puts the manufacturers of a new treatment at a scientific and commercial disadvantage. The manufacturers of a new treatment, say the critics, have to prove that their treatment is as good as an existing one, whereas the manufacturers of the existing treatment had to pass a “lesser test” (superiority over placebo) to get their drug on the market. For practitioners, though, the crucial question in evaluating a new treatment is how it compares with the standard available treatment, and not whether it is better than placebo. So the important issue is to decide when it is that we can call a therapy “standard”—that is, when can we speak of an indisputable benefit that would make a currently available treatment's use in a trial control group ethically imperative? Clinical guidelines or recommendations based on high-quality evidence sometimes exist to support use of such a therapy. In situations where no such guidance exists, it is important to assess both the benefits of the therapy (for example, in terms of survival, and relative and absolute risk reduction) and possible harms (including side effects, impaired quality of life, and economic costs). There may be therapies that prolong survival (there is a “gross benefit”) but that cannot be considered to be beneficial because the adverse effects cancel out any survival benefit (there is no “net benefit”). Such therapies cannot be considered “standard” treatment. One framework for grading the quality of evidence and strength of recommendations on any treatment was published last year by the GRADE working group [ 3 ]. The framework stresses the need for judgments based on a formally structured consideration of the balance between benefits and harm, the quality of the evidence, translation of the evidence in specific clinical situations or settings, and the certainty of baseline risks, including resource utilisation. So when is use of a placebo trial unethical? It is unethical if, in accordance with an assessment similar to the one suggested by the GRADE working group (that is, balancing gross and net benefits in a given trial through a transparent and formalised process), therapies other than the experimental one are judged to be beneficial and are available. In the many situations where such a decision is not clear-cut, the use of placebo may be considered ethically appropriate. Erick Turner and Martin Tramèr's Viewpoint: It Can Be Unethical Not to Use Placebo It is generally agreed that placebo is unethical when its use is likely to result in irreversible harm, death, or other serious morbidity. A common argument against placebo is that its use is unnecessary, and therefore unethical, when “proven effective therapy” exists, in which case any new treatment should be tested against this existing treatment. The argument is that if a study drug appears to perform at least as well as a drug that has already been “proven effective”, then the study drug must be effective as well. The problem with this reasoning is that drug efficacy is not a simple all-or-none matter. If a drug with historical evidence of efficacy could be relied upon to be unfailingly effective—and placebo unfailingly ineffective—in all future clinical trials, we would readily admit that placebo is unnecessary and therefore unethical. The reality is that “proven effective therapy”—better called “assumed effective therapy” (AET)—often fails to show superiority to placebo. This is not because these drugs are in fact ineffective, but because the trials in question lack assay sensitivity [ 4 , 5 ]. Assay sensitivity is defined as the ability of a trial to distinguish an effective from an ineffective therapy. Unfortunately, the extent of this problem is poorly appreciated because of publication bias: the tendency for studies that are positive to be published and the tendency of negative and indeterminate studies never to see the light of day [ 6 ]. Thus, the myth of infallible “proven therapy” is sustained. But, like a mirage, it vanishes on closer examination. Khan et al. gained access to unpublished, as well as published, clinical trials data on antidepressants from the Food and Drug Administration (FDA) via the United States Freedom of Information Act [ 7 ]. They obtained the FDA review documents on 51 clinical trials on nine antidepressants approved between 1985 and 2000. Of 92 active treatment arms (all involving doses that were eventually approved), 47 (51%) failed to demonstrate statistical superiority to placebo. Of these, there were seven cases (15%) in which the placebo arm was actually superior to AET. Thus, it can be seen that the phrase “proven effective therapy” should be taken with a grain of salt. Now, what if the FDA had not had the benefit of looking at the placebo arms and relied on an equivalence or noninferiority design [ 8 ] comparing study drug with AET? Khan et al. list 12 flexible-dose studies in which (now-approved) study drug outperformed AET (previously approved antidepressants) [ 7 ]. Many opponents of placebo would argue that each of these 12 trials provides ample evidence for efficacy of the study drug. However, because these trials did include placebo arms, we discover that in four of them (33%), neither AET nor study drug beat placebo. (In fact, in two of these four trials, AET was numerically inferior to placebo.) Therefore, in these four antidepressant trials, the two “active” drugs were not equally effective, but rather equally ineffective. This critical distinction would have been lost without placebo, and it would have been impossible to ascertain that these seemingly positive trials were in fact false positive trials. The problem of assay sensitivity is not confined to antidepressants or even to psychotropic drugs in general. A meta-analysis by Tramèr et al found that, among 52 possible comparisons between the “proven” antiemetic ondansetron and placebo, 19 (37%) failed to show a difference [ 5 ]. Additionally, many drug classes have shown problems with assay sensitivity ( Box 1 ). The potential for reaching erroneous conclusions by omitting placebo also exists outside of drug studies, as in the example regarding the usefulness of prophylactic respiratory physical therapy on pulmonary function after cardiac surgery [ 9 ]; in this specific case, the placebo would be a no intervention control. Box 1. Drug Classes That Have Shown Problems with Assay Sensitivity Analgesics Antiemetics Anxiolytics Antihypertensives Hypnotics Antianginal agents Angiotensin-converting enzyme inhibitors for heart failure Beta-blockers given after myocardial infarction Antihistamines Nonsteroidal asthma prophylaxis Motility-modifying drugs for gastroesophageal reflux disease If we were to rely on equivalence or noninferiority designs in studying drugs for indications for which assay sensitivity cannot be assumed, we would risk approving ineffective drugs. It is conceivable that even placebo itself could be approved under such conditions. According to the Declaration of Helsinki, “Medical research is only justified if there is a reasonable likelihood that the populations in which the research is carried out stand to benefit from the results of the research” [ 1 ]. To approve ineffective drugs based on flawed science and to let them loose on an unsuspecting public would be unethical. This is akin to the phenomenon of hypercorrection, in which, in trying very hard to be grammatically correct, the person ends up being grammatically incorrect [ 10 ]. In this case, by trying very hard to be ethical and adhering too rigidly to the anti-placebo dogma, one can end up being unethical. In order to best serve the public health, we must ensure that our clinical drug trials yield scientifically valid results. Where assay sensitivity can be guaranteed, equivalence or noninferiority trials omitting placebo may be ethically preferable. However, where assay sensitivity cannot be guaranteed—and this problem is probably more widespread than we yet realize—difference-showing superiority studies, usually involving placebo, either as monotherapy or add-on therapy, are ethically preferable. Stang, Hense, and Jöckel's Response to Turner and Tramèr's Viewpoint There is some common agreement between Turner and Tramèr's viewpoint and ours—we agree that it is unethical to use placebo when a proven effective therapy exists. However, we question their very narrow definition of “proven effective therapy”. In their definition, a placebo is unethical when the proven effective therapy “could be relied upon to be unfailingly effective—and placebo unfailingly ineffective—in all future clinical trials”. It is possible to argue that all empirical evidence or knowledge is temporary and uncertain. Replication carries no implication for validity [ 11 ]. Corroborated hypotheses—in this case, about the effectiveness of a drug—merely “survive” and the degree of corroboration depends on the number and “severity” of tests the hypothesis has survived [ 12 ]. We are therefore left with the difficult task of having to evaluate the current evidence of the effectiveness of available treatments in order to decide whether placebo is ethical or not. In other words, we have to make some evaluation on what constitutes “proven effective therapy” based on our current knowledge. The evaluation of current evidence cannot protect us against misinterpretations in the light of future evidence. It appears to us that Turner and Tramèr think that superiority of a new drug to a control drug could only be established if all trials consistently show a statistically significant superiority of the new drug over the control. But studies that evaluate the effectiveness of a new drug may not show identical results for several reasons. Features of the study design, including sample size, dosage, patients' inclusion and exclusion criteria, choice of active control treatment, quality of study conduct, patients' compliance, and other factors can all have an influence on the trial results. Therefore, the proportion of trials showing statistically significant superiority (bullet counting) is an inappropriate indicator of drug superiority and a proportion less than 100% is no indicator of lack of superiority. Several analytical techniques, including meta-analysis and meta-regression, that account for design features are available and provide better insights into the superiority of drugs than bullet counting. Turner and Tramèr's Response to Stang, Hense, and Jöckel's Viewpoint Stang and colleagues quote from part of a clarification to the Declaration of Helsinki. But the clarification continues: “a placebo-controlled trial may be ethically acceptable, even if proven therapy is available…where for compelling and scientifically sound methodological reasons its use is necessary to determine the efficacy or safety of a prophylactic, diagnostic or therapeutic method” [ 1 ]. Our viewpoint was essentially an evidence-based discussion of this clarification and its ethical implications. Assuming that medical research successfully rids itself of publication bias [ 13 , 14 ], it should become increasingly obvious that, for many drug classes ( Box 1 ), the emperor of “proven therapy” is wearing no clothes [ 15 ]. But this debate is not only about efficacy; it is also about harm. In the absence of a placebo group, it may be impossible to interpret a drug's potential for harm. Let us look at Stang and colleagues' example of analgesics. The Vioxx Gastrointestinal Outcomes Research (VIGOR) trial showed a five-fold difference in the incidence of myocardial infarction in the rofecoxib (Vioxx) group compared with the naproxen group [ 16 ]. Nonsteroidal anti-inflammatory drugs such as naproxen, however, inhibit platelet function and therefore might have a myocardial protective effect [ 17 ]. Since the VIGOR trial did not include a placebo group, it remained unclear whether there was an increased risk of myocardial infarction with rofecoxib or a decreased risk with naproxen. Four years later, and after tens of millions of patients had received rofecoxib [ 18 ], Merck announced they were withdrawing the drug because of an increased cardiovascular risk [ 19 ]. The decision was based on the unpublished Adenomatous Polyp Prevention on Vioxx (APPROVe) study, a placebo-controlled three-year trial of rofecoxib. In his November 2004 testimony before the United States Senate, David Graham of the FDA provided an estimate of the rate of excess cases of Vioxx-related myocardial infarction and sudden cardiac death. He testified that it was as if, for the five years that Vioxx was on the United States market, “2 to 4 jumbo jetliners were dropping from the sky every week” [ 20 ]. Of those cases, he added, 30% to 40% probably died. If those who believe that “proven therapy” trials are ethically preferable to placebo-controlled trials had had their way, the APPROVe study would have been blocked, and Vioxx would still be on the market today. It seems ironic that such a stance could be taken in the name of ethics. (Illustration: Margaret Shear, Public Library of Science) (Illustration: Margaret Shear, Public Library of Science)

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1069667

Serum Uric Acid and Coronary Heart Disease in 9,458 Incident Cases and 155,084 Controls: Prospective Study and Meta-Analysis

Background It has been suggested throughout the past fifty years that serum uric acid concentrations can help predict the future risk of coronary heart disease (CHD), but the epidemiological evidence is uncertain. Methods and Findings We report a “nested” case-control comparison within a prospective study in Reykjavik, Iceland, using baseline values of serum uric acid in 2,456 incident CHD cases and in 3,962 age- and sex-matched controls, plus paired serum uric acid measurements taken at baseline and, on average, 12 y later in 379 participants. In addition, we conducted a meta-analysis of 15 other prospective studies in eight countries conducted in essentially general populations. Compared with individuals in the bottom third of baseline measurements of serum uric acid in the Reykjavik study, those in the top third had an age- and sex-adjusted odds ratio for CHD of 1.39 (95% confidence interval [CI], 1.23–1.58) which fell to 1.12 (CI, 0.97–1.30) after adjustment for smoking and other established risk factors. Overall, in a combined analysis of 9,458 cases and 155,084 controls in all 16 relevant prospective studies, the odds ratio was 1.13 (CI, 1.07–1.20), but it was only 1.02 (CI, 0.91–1.14) in the eight studies with more complete adjustment for possible confounders. Conclusions Measurement of serum uric acid levels is unlikely to enhance usefully the prediction of CHD, and this factor is unlikely to be a major determinant of the disease in general populations.

Introduction Numerous genetic and environmental factors have been associated with uric acid [ 1 ], and serum uric acid values are markedly elevated in patients with gout ( Table 1 ). Since at least fifty years ago, modestly higher serum uric acid concentrations have been reported in patients with coronary heart disease (CHD) than in controls [ 2 ], and there have been suggestions that measurement of serum uric acid can enhance the prediction of CHD [ 3 ]. Prospective epidemiological studies have, however, reported apparently conflicting findings, with several studies reporting positive associations only among women [ 4 , 5 ], The interpretation of the data has been further complicated by the correlation of serum uric acid concentrations with several established coronary risk factors (such as blood pressure), with the use of cardiovascular medications (such as diuretics), and with clinical conditions associated with CHD (such as chronic renal disease [ 6 ]). It has been difficult, therefore, to determine whether serum uric acid values are predictive of CHD, and, if so, whether any such associations are independent from established risk factors or from the effects of disease or both. Table 1 Characteristics of Uric Acid a Values are mean (SD) b Conversion to SI units: 1 μmol/l = 59.48 mg/dl c Approximate correlation between two measurements taken some years apart in the same individuals To help address these uncertainties, we report a prospective study with more CHD cases than any previous report on serum uric acid, involving 2,459 incident cases of nonfatal myocardial infarction (MI) and CHD death, and 3,969 controls from within a prospective observational study of about 19,000 middle-aged Icelanders without a previous history of MI. To help put these results in context, we also report a meta-analysis of 15 previously published prospective studies of serum uric acid, involving a total of an additional 7,002 incident CHD cases and an additional 151,122 controls, including supplementary information obtained by correspondence from investigators to help assess in more detail the impact of possible confounders. The present analyses have been restricted to prospective cohorts sampled from essentially general populations (i.e., excluding cohorts selected on the basis of existing vascular or other diseases, or on the basis of having risk factors for vascular disease, such as high blood pressure) to reduce any distorting effects of preexisting disease on serum uric acid levels. Methods The Reykjavik Study The Reykjavik Study, initiated in 1967 as a prospective study of cardiovascular disease, has been described in detail previously [ 7 ]. All men born between 1907 and 1934 and all women born between 1908 and 1935 who were resident in Reykjavik, Iceland, and its adjacent communities on 01 December 1966 were identified in the national population register and then invited to participate in the Reykjavik Study during five stages of recruitment between 1967 and 1991, yielding 8,888 male and 9,681 female participants without a history of MI (72% response rate). Nurses administered questionnaires, made physical measurements, recorded an electrocardiogram, performed spirometry, and collected fasting venous blood samples, which were stored at −20 °C for subsequent analysis. All participants have been monitored subsequently for all-cause mortality and for cardiovascular morbidity, with a loss to follow-up of less than 1% to date. A total of 2,459 men and women with available serum samples had major coronary events between the beginning of follow-up and 31 December 1995, yielding mean durations of follow-up among CHD cases of 17.5 (standard deviation [SD] 8.7) years and, among controls, of 20.6 (SD, 8.2) years. In total, 1,073 CHD deaths and 701 nonfatal MIs were recorded among men (including 564 confirmed MIs and 137 possible MIs), and 385 CHD deaths and 300 nonfatal MIs among women (including 237 confirmed MIs and 63 possible MIs). Deaths from coronary heart disease were ascertained from central registers on the basis of a death certificate with International Classification of Diseases codes 410–414, and the diagnosis of nonfatal MI was based on MONICA criteria. We selected 3,969 controls that were “frequency-matched” to cases on calendar year of recruitment, sex, and age in 5-y bands from among participants who had survived to the end of the study period without a MI. The National Bioethics Committee and the Data Protection Authority of Iceland approved the study protocol, and participants provided informed consent. Laboratory Methods Serum uric acid levels were measured with a Technicon autoanalyzer [ 8 ]. The measurement of other biochemical analytes has been described previously [ 7 ]. Baseline measurements of serum uric acid were available on 2,456 out of 2,459 CHD cases and 3,962 out of 3,969 controls. To assess the within-person consistency of serum uric acid levels over time, measurements were made in pairs of samples collected at an interval of about 12 y apart in 379 individuals in the present study. Statistical Methods and Meta-Analysis Case-control comparisons were made by unmatched stratified logistic regression fitted by unconditional maximum likelihood. Analysis of serum uric acid values was previously specified to be by sex-specific thirds of values in the controls (with subsidiary analyses involving other cut-off values). Adjustment was made for age, sex, smoking status (never, former, current), daily cigarette consumption, blood pressure, body mass index, fasting concentrations of total cholesterol and triglycerides, and various markers of socioeconomic status related to occupation, education, home ownership, and type of accommodation. We assessed variation in the strength of association according to pre-specified sub-groups, using likelihood ratio tests for interaction after adjusting for these factors, with 99% confidence intervals (CIs) used in these exploratory analyses. For the meta-analysis, studies of serum uric acid and CHD published before May 2003 with greater than a year's follow-up conducted in essentially general populations (i.e., excluding cohorts defined on the basis of preexisting cardiovascular or other diseases) were sought by computer-based searches, scanning the reference lists of all relevant studies and review articles, hand-searching of relevant journals, and correspondence with authors of studies. Computer searches using Medline, PubMed, Web of Science, and Embase databases used keywords relating to uric acid in combination with CHD (e.g., coronary heart disease, ischemic heart disease, vascular disease, MI, and atherosclerosis). Relevant endpoints included nonfatal MI (generally using World Health Organization criteria) and CHD death (generally using International Classification of Disease criteria). The following factors were abstracted from each study: numbers of cases and controls, mean age of cases and percentage of males, mean duration of follow-up, assay type, and those used for adjustment in multivariable assessments. Five studies were excluded because they reported insufficient data or only unadjusted risk ratios [ 9 , 10 , 11 , 12 , 13 ], but these involved only a total of about 590 CHD cases (or < 6% of the total number of cases in the present report). Of 16 studies (including four studies that had not previously reported in relation to CHD [ 14 , 15 , 16 , 17 ]), 11 provided supplementary tabular data on sex-specific “relative risks” (i.e., incidence rate ratios according to sex-specific thirds of serum uric acid distribution in controls) and details of factors adjusted for in multivariable analyses. We excluded female-specific estimates based on fewer than 30 CHD cases, owing to very small sample sizes from two studies [ 16 , 18 ]. Where data were not available by thirds of serum uric acid levels, the log-relative risk (and its standard error) was estimated from the reported relative risks using log-linear scaling and assuming normality of the uric acid distribution, as described previously [ 19 ]. Where data were available only in separate age strata, a single pooled estimate was used. Fixed-effect summary measures were calculated as the inverse-variance weighted average of the log-relative risks. Heterogeneity was assessed by the heterogeneity Q statistic and by random effect regression models with restricted maximum likelihood estimation. Subsidiary analyses (conducted only on studies known to exclude individuals with existing CHD) grouped studies by sex, study size, geographical location, sampling framework (population- or workforce-based), degree of adjustment for other cardiovascular risk factors, type of assay, and duration of follow-up. Statistical analyses were conducted using Stata version 7.0. To make some allowance for multiple comparisons, 99% CI were used for individual studies, and 95% CI were reserved for the combined estimates. Results The Reykjavik Study The mean age at CHD event among cases was 70.2 (SD, 9.7) y. There were highly significant differences between cases and controls with respect to established vascular risk factors such as smoking, body mass index, blood pressure, and serum lipid concentrations ( Table 2 ). Serum uric acid values were highly significantly associated with male sex, nonmanual occupation, body mass index, diastolic blood pressure, triglycerides, and serum creatinine ( p < 0.0001 for each), although most of these associations weakened after adjustment for other vascular risk factors ( Table S1 ). In 379 participants who provided paired blood samples, on average about 12 y apart, the within-individual correlation coefficient among serum uric acid values was 0.60 (CI, 0.54–0.66), similar to the decade-to-decade consistency observed in values of systolic blood pressure [0.66 (CI, 0.60–0.72)] and total serum cholesterol [0.60 (CI, 0.54–0.66)] in these participants. Table 2 Baseline Characteristics of Cases and Controls in the Reykjavik Study Values are mean (SD) unless indicated otherwise a Information on occupation was available for only 1,742 cases and 2,888 controls, respectively b Information on education was available for only 1,292 cases and 2,157 controls, respectively c Information on home ownership was available for 2,323 cases and 3,754 controls, respectively d Information on type of residence was available for 2,258 cases and 3,646 controls, respectively. Other categories included “duplex” and “villa.” e Information on serum uric acid was available for 2,456 cases and 3,962 controls, respectively f Value log transformed for analysis and presented as geometric mean (SD) The odds ratio for CHD was 1.39 (CI, 1.20–1.61; Wald test statistic, χ 2 1 = 18.4) in males in the top third compared with those in the bottom third of baseline serum uric acid levels (tertile cut-offs, > 339 versus < 286 μmol/l [ Table 3 ]), and this fell to 1.12 (CI, 0.94–1.33; χ 2 1 = 1.5) after adjustment for smoking, other established risk factors, and indicators of socioeconomic status ( Table 3 ). The odds ratio for CHD was 1.42 (CI, 1.13–1.79; χ 2 1 = 9.1) in females in the top third compared with those in the bottom third of baseline serum uric acid levels (tertile cut-offs, > 280 v < 232 μmol/l), and this fell to 1.12 (CI, 0.85–1.46; χ 2 1 = 0.6) after adjustment for smoking, other established risk factors, and indicators of socioeconomic status. In a combined analysis of males and females, the odds ratio for CHD was 1.39 (CI, 1.23–1.58; χ 2 1 = 27.2) and this fell to 1.12 (CI, 0.97–1.30; χ 2 1 = 2.4) after adjustment. In analyses restricted to the 2,083 cases without evidence of CHD at baseline, the adjusted odds ratios fell further to 1.08 (CI, 0.90–1.31) in males and 1.00 (CI, 0.75–1.33) in females ( Table 3 ), but the findings were materially unchanged in analyses excluding the 200 CHD cases with “possible” MI or in analyses varying cut-off levels (e.g., by quarters, fifths, or increases of 1 SD; see Table 3 legend). Figure 1 indicates that there was no substantial variation in the strength of association between serum uric acid and CHD at different levels of established risk factors, and, in particular, there was no good evidence of interactions with sex or systolic blood pressure (sex, χ 2 1 = 0.03, p = 0.86; smoking, χ 2 1 = 0.28, p = 0.60; body mass index, χ 2 2 = 1.13, p = 0.57; total cholesterol, χ 2 2 = 2.42, p = 0.30; systolic blood pressure, χ 2 2 = 4.63, p = 0.10). Figure 1 Associations between Serum Uric Acid and CHD in 2,456 cases and 3,962 Controls in the Reykjavik Study at Different Levels of Established Risk Factors Squares indicate odds ratios, with the size of the square proportional to the effective sample size. Table 3 Relative Odds of Coronary Heart Disease in Individuals Who Had Serum Uric Acid in the Top Third of the Sex-Specific Distribution of Controls Relative to Those Who Had Values in the Bottom Third of This Distribution in the Reykjavik Study a Systolic blood pressure, total cholesterol, triglycerides, body mass index, smoking (former or current, including number cigs per day), FEV1, history of diabetes b Odds ratios (males and females combined) using alternative comparisons were: 1.24 (0.99–1.55) top fifth vs. bottom fifth; 1.22 (1.03–1.45) top quarter vs. bottom quarter; 1.08 (1.02–1.15) per standard deviation increase. Sex-specific odds ratios using thirds of the overall (not sex-specific) distribution of serum uric acid were: in males, 1.19 (0.98–1.43); in females 1.34 (0.98–1.82) Meta-Analysis In aggregate, 16 prospective reports [ 6 , 14 , 15 , 16 , 17 , 18 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ] on serum uric acid (including the present study) have involved a total of 9,458 CHD cases and 155,084 controls, with a weighted mean age at entry of 50 y and weighted mean follow-up of 10.5 y ( Table 4 ). Studies were conducted in the USA [ 22 , 23 , 24 , 25 , 28 ], Western Europe [ 6 , 14 , 15 , 17 , 18 , 20 , 27 ], Israel [ 21 ], and Japan [ 16 , 26 ], and all reported adjustment for at least smoking and some other established risk factors. Overall, in a comparison of individuals with serum uric acid values in the top third with those in the bottom third of the population, the relative risk for CHD was 1.13 (CI, 1.07–1.20: Figure 2 ), with statistically compatible results in male and females (χ 2 1 = 1.1; p = 0.3). In a subsidiary analysis of seven studies [ 20 , 22 , 23 , 24 , 25 , 27 ], involving 6,357 CHD cases and 65,978 controls, all of which excluded individuals with known cardiovascular disease at the baseline examination, the relative risk for CHD was 1.10 (CI, 1.03–1.18). There was significant heterogeneity among the 23 sex-specific study estimates (χ 2 2 = 38.1, p = 0.02), but only some of this was explained by study characteristics such as sample size (χ 2 2 = 11.1), geographical location (χ 2 2 = 1.0), sampling framework (χ 2 1 = 0.5), degree of adjustment for possible confounders (χ 2 2 = 10.0), duration of follow-up (χ 2 1 = 0.1), and assay type (χ 2 3 = 4.2) ( Figure 3 ). In a random-effects model, that takes additional account of study variation and the joint impact of these characteristics, only degree of adjustment for possible confounders remained a significant source of heterogeneity at the 1% level of significance (sex, p = 0.36; sample size, p = 0.41; geographical location, p = 0.71; sampling framework, p = 0.02; degree of adjustment for possible confounders, p = 0.001; duration of follow-up, p = 0.67; and assay type, p = 0.06). Figure 2 Meta-Analysis of Prospective Observational Studies of Serum Uric Acid and CHD in Essentially General Populations, Subdivided by Sex Conventions are the same as in Figure 1 . Combined odds ratios and their CIs are indicated by unshaded diamonds for subtotals and shaded diamonds for grand totals. +, adjustment reported only for age and sex; ++, adjustment for these plus smoking; +++, adjustment for these plus some additional established risk factors; ++++, adjustment for these plus existing cardiovascular disease. Study abbreviations: ARIC, Atherosclerosis Risk in Communities; BIRNH, Belgium Interuniversity Research on Nutrition and Health; BRHS, British Regional Heart Study; CHA, Chicago Heart Association Detection Project in Industry; GRIPS, Göttingen Risk Incidence and Prevalence Study; IIHDS, Israeli Ischemic Heart Disease Study; MONICA, World Health Organization Monitoring Trends and Determinants in Cardiovascular Disease; NHANES, National Health and Nutrition Examination Survey; NHEFS, NHANES I Epidemiologic Follow-Up Study; PROCAM, Prospective Cardiovascular Munster Study. Figure 3 Prospective Studies of the Association of Serum Uric Acid and CHD, Grouped by Various Characteristics Conventions are the same as in Figure 1 . *, each sex-specific estimate was treated as a “study”; †, two studies (6 and 13) were drawn from general practice registers; §, risk factors adjusted for included: smoking, blood pressure, total cholesterol, triglycerides, alcohol consumption, obesity, use of cardiovascular medication, history of hypertension, and history of diabetes. PTA, phosphotungstic acid. Table 4 Prospective Studies of Serum Uric Acid and Coronary Heart Disease in Essentially General Populations: Study Characteristics a Sampling method: Random, a randomly selected subset of eligible persons was invited to participate; complete, all eligible persons in the study population were invited to participate b Only men were included in analyses, due to a small numbers of female cases: BIRNH, 26 women; Osaka, 4 women; MONICA Augsberg, number of female cases not stated c Duration of follow-up was “at least 10 years.” Table abbreviations: NS, not specified; Mn, mean; GP, general practice; SD, standard deviation. Study abbreviations: IIHDS, Israeli Ischemic Heart Disease Study; BRHS, British Regional Heart Study; NHANES, National Health and Nutrition Examination Survey; NHEFS, NHANES I Epidemiological Follow-up Study; PROCAM, Prospective Cardiovascular Munster Study; ARIC, Atherosclerosis Risk in Communities; GRIPS, Göttingen Risk Incidence and Prevalence Study; CHA, Chicago Heart Association Detection Project in Industry; BIRNH, Belgium Interuniversity Research on Nutrition and Health; MONICA, World Health Organization Monitoring Trends and Determinants in Cardiovascular Disease Discussion The present report provides prospective evidence from the largest study so far of serum uric acid and CHD—plus a meta-analysis of 15 previous relevant studies—involving a total of more than 9,000 incident cases and more than 150,000 controls. The overall findings suggest that individuals with baseline serum uric acid values in the top third of the population have about a 10% greater risk of CHD over the subsequent decade than those in the bottom third (with the likelihood that this association would be about twice as strong if based on long-term usual levels of serum uric acid). It is likely, however, that this modest association has been exaggerated by the preferential publication of striking findings in smaller studies (“publication bias”), or by residual confounding by established risk factors, or both. For example, the observation of weaker associations in studies with more comprehensive adjustment for possible confounders lessens the likelihood that any association between serum uric acid and CHD is independent from possible confounders; the odds ratio was only 1.02 (CI, 0.91–1.14), which is not significant, in the eight studies with the most complete reported adjustment for possible confounders ( Figure 3 ). The present data also provide no good evidence to support previous claims that the association between serum uric acid and CHD is stronger in females than in males [ 5 ], or stronger at higher levels of established risk factors, such as in individuals with higher blood pressure recordings [ 29 ]. The main implication of these data is to refute suggestions made throughout the past half-century that measurement of serum uric acid can importantly enhance the prediction of CHD in general populations. These data do not directly address the question of whether or not serum uric acid may be involved in the causation of CHD through a number of potentially relevant vascular effects (such as through the formation of free radicals or through the oxidation of low-density-lipoprotein cholesterol [ 1 , 30 ]), but they suggest that serum uric acid levels are unlikely to be a major determinant of CHD. Supporting Information Table S1 Comparison of Baseline Values of Risk Factors and Other Characteristics in Controls in the Reykjavik Study by Thirds of Serum Uric Acid Concentration (67 KB DOC). Click here for additional data file. Patient Summary Background Defining which risk factors are important for disease is useful for clinicians and patients not only because the presence of risk factors allows the prediction of who is more likely to get a disease, but also because they provide some insight into the underlying causes of disease. One such suspected risk factor for coronary heart disease is the level of uric acid in the blood. The debate over whether uric acid is useful for predicting heart disease has been going on for over fifty years. Most evidence for risk factors comes from studies of populations, in which it can be hard to tease out the effects of many different factors; often studies come to different conclusions. One way of finding out which results are reliable is to pool the results of many studies. What Did the Researchers Find? They looked at the uric acid levels of around 2,500 people with coronary heart disease and almost 4,000 controls measured at the start of a large study in Iceland, and then investigated whether there was a relation between levels of uric acid and development of heart disease. After adjusting for all the other factors that could affect the chance of heart disease, they found that uric acid did not predict heart disease. They then combined these results with those from 15 other studies, and confirmed the findings. What Do These Findings Mean? After fifty years, it now seems clear that measurement of uric acid does not help to predict heart disease. It may still be involved in triggering heart disease, but any effect must be subtle. Where Can I Get More Information? The National Heart Lung and Blood Institute has many pages of information on heart disease: http://www.nhlbi.nih.gov/health/public/heart/index.htm#ami

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1069668

Tumor Cell Recognition Efficiency by T Cells

Stuge et al. report a detailed analysis of the fine specificity of CD8+ T cells against tumor-associated antigen in melanoma patients [ 1 ]. They compared peptide-vaccination-driven with naturally arising T cell responses against the HLA-A*0201 restricted melanoma peptide antigens M26 (derived from Melan-A/MART-1) and G209-2M (derived from gp100 protein). A major endpoint of this study was in vitro tumor cell recognition by T cells. Fortunately, this is increasingly used as a “golden” standard in the assessment of tumor-specific T cells. The authors suggest that spontaneously arising antigen-specific T cell populations are qualitatively different from those induced by vaccination with heteroclitic peptides (which are altered for increased HLA binding): tumor cell recognition was found in nearly all T cells from the former, but only in a minority from the latter. As reported previously, these results correlated with recognition efficiency of antigenic peptides. We agree that this has considerable implications for immunotherapy and congratulate the authors for analyzing T cell recognition in great detail. However, in one point our own studies lead to different results: we repetitively found that the majority of T cells generated with the heteroclitic Melan-A M26 peptide were tumor reactive. This was the case for Melan-A-specific T cell populations generated in HLA-A*0201 transgenic mice [ 2 ], in vitro [ 3 ], and in melanoma patients [ 4 ]. The latter studies also assessed T cells from vaccination-site sentinel lymph nodes, containing T cells that are very likely selected and activated by vaccination and not by the tumor. The authors point out correctly that tetramer+ T cells comprise many cells unable to recognize and kill tumor cells in an antigen-specific manner, presumably owing to low T cell receptor avidity to cognate antigen. An extreme case is naïve T cell populations, of which the majority are unable to recognize tumor cells, despite their specific binding to MHC/ peptide tetramers [ 5 ]. Therefore, it is crucial to exclude naïve T cells from studies analyzing tumor recognition. HLA-A*0201+ humans (healthy individuals and melanoma patients) have 0.07% ± 0.05% naïve Melan-A tetramer+ cells within peripheral blood CD8+ T cells [ 5 , 6 ]. The three patients studied by Stuge et al. had 0.23%, 0.12%, and 0.50% Melan-A tetramer+ cells. Thus, one can estimate that the studied populations from the three patients contained approximately 30%, 60%, and 15% naïve Melan-A-specific T cells, respectively. This is only a rough estimate—tetramer analysis before vaccination and assessment of CD45RA/ CCR7 expression would give more insight. Nevertheless, it remains likely that the first two patients had considerably more naïve cells than the third patient (i.e., the one without immunotherapy). In addition, naïve-derived CD8+ T cells have a higher clonogenic potential than activated Melan-A-specific T cells from melanoma patients (unpublished data). This means that overrepresentation of clones derived from naïve CD8+ T cells is likely to occur when both naïve and activated antigen-specific CD8+ T cells co-exist in a given lymphocyte population. As mentioned, Stuge et al. found unexpected high frequencies of T cell clones not recognizing tumor cells in the two vaccinated patients. It is conceivable that this was due to the presumably high percentages of naïve Melan-A-specific cells present in the populations used for generating the clones, which would provide an explanation for the discrepancy with the results of our studies [ 2 , 3 , 4 ]. Ethical considerations limit vaccination studies in healthy humans. In patients, candidate antigens should therefore be tested with strong adjuvants [ 7 ], to increase the likelihood that the studied responses are predominantly vaccination-driven, with only minor contribution of spontaneous T cell activation [ 8 ]. It would be desirable to directly compare vaccination with heteroclitic peptide versus vaccination with natural peptide. However, this is hampered by the lack of ex vivo detectable responses to native peptides owing to their low immunogenicity. Another option is to analyze clonal distributions (T cell receptors) of responding T cells extensively: Further support for the notion that spontaneous (tumor driven) responses have increased potential for tumor recognition would be obtained if mono/oligoclonal T cell repertoires are indeed significantly more often found in spontaneous than vaccination-induced responses. We certainly agree that vaccines must be optimized. Thus, more such studies are desirable, since they have high potential to lead to better understanding of the differences between clinically irrelevant and relevant T cell responses, and to rapidly identify the most promising vaccine formulations that can subsequently be tested in large-scale clinical trials.

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1069669

T Cell Epitope Immunotherapy Induces a CD4+ T Cell Population with Regulatory Activity

Background Synthetic peptides, representing CD4 + T cell epitopes, derived from the primary sequence of allergen molecules have been used to down-regulate allergic inflammation in sensitised individuals. Treatment of allergic diseases with peptides may offer substantial advantages over treatment with native allergen molecules because of the reduced potential for cross-linking IgE bound to the surface of mast cells and basophils. Methods and Findings In this study we address the mechanism of action of peptide immunotherapy (PIT) in cat-allergic, asthmatic patients. Cell-division-tracking dyes, cell-mixing experiments, surface phenotyping, and cytokine measurements were used to investigate immunomodulation in peripheral blood mononuclear cells (PBMCs) after therapy. Proliferative responses of PBMCs to allergen extract were significantly reduced after PIT. This was associated with modified cytokine profiles generally characterised by an increase in interleukin-10 and a decrease in interleukin-5 production. CD4 + cells isolated after PIT were able to actively suppress allergen-specific proliferative responses of pretreatment CD4 neg PBMCs in co-culture experiments. PIT was associated with a significant increase in surface expression of CD5 on both CD4 + and CD8 + PBMCs. Conclusion This study provides evidence for the induction of a population of CD4 + T cells with suppressor/regulatory activity following PIT. Furthermore, up-regulation of cell surface levels of CD5 may contribute to reduced reactivity to allergen.

Introduction The central role of T cells in the pathogenesis of allergic disease is well established [ 1 ]. Through production of interleukin (IL)-4, IL-5, and IL-13, allergen-specific T helper (Th) 2 cells direct IgE synthesis, eosinophil growth/differentiation, and induction of airway hyperreactivity [ 2 , 3 ].Until recently, it was assumed that the basis for allergic disease was an imbalanced Th cell response to certain allergens, manifest as a predominance of Th2 cytokines over Th1 cytokines. However, immune suppression may also be a normal consequence of a protective immune response, serving to limit excessive responses that lead to immunopathology [ 4 ]. The role of regulatory T cell (T reg ) populations in maintaining homeostasis is increasingly well understood. The term T reg is used to describe a variety of T cell functional phenotypes that display common features. Several studies have described the dependence of T reg function on cell–cell contact. In certain cases regulation was demonstrated to be dependent on IL-10 and/or transforming growth factor β secretion [ 5 , 6 , 7 , 8 , 9 ]. Regulation of immune responses may be attributable to both naturally occurring (thymus-derived, or “natural”) regulatory cells and also naïve or effector T cells that have acquired suppressive activity (adaptive regulatory cells) [ 10 , 11 ]. Therapeutic administration of short, soluble peptide sequences, in the absence of inflammatory signals, may result in presentation by immature or quiescent antigen-presenting cells (APCs). Immature allogeneic human dendritic cells (DCs) induced non-proliferating, IL-10-producing CD4 + T cells with regulatory properties [ 12 ], while peptide-specific human T reg were induced following administration of antigen-pulsed immature DCs in vivo [ 13 , 14 ]. DCs producing IL-10 were able to suppress airway inflammation in a murine model of asthma [ 15 ]. Thus, partially immature or “steady state” DCs, circulating in the lymphatics, may interact with T cells in a tolerogenic milieu, in the absence of concomitant pro-inflammatory stimuli such as pattern recognition receptor triggering [ 16 ]. An additional mechanism for limiting immune responses may be reducing sensitivity to cognate signals. Up-regulation of CD5, a suppressor of T cell signalling [ 17 ], has been associated with regulatory cells arising as a consequence of competition for space and resources [ 18 ]. Under such conditions, suppression was shown to lack antigen specificity and to be mediated by cells that did not exhibit any of the hallmarks of “professional” T reg . Recently, Hawiger and colleagues delivered antigen to steady-state DCs via the DEC-205 molecule. Following cognate interaction with these cells, antigen-specific T cells were unresponsive and expressed enhanced levels of CD5 [ 19 ]. Chronic low-level antigen exposure in the periphery has also been shown to result in anergy in CD8 + cells that was associated with increased expression of CD5, further illustrating a role for CD5 in regulation of T cell function [ 20 ]. In animal models, the administration of low-dose peptide is a well-established mechanism for the induction of T reg that may arise as a result of presentation by steady-state DCs and “non-professional” APCs [ 21 , 22 , 23 , 24 ]. Similarly, administration of soluble peptides to allergic asthmatic individuals has been shown to result in markedly reduced cutaneous reactions to allergen injection [ 25 , 26 , 27 ], reduced airway hyperreactivity [ 27 ], and improvements in symptom scores after nasal allergen challenge [ 28 ]. Changes in clinical reactivity were associated with decreased Th1 and Th2 cytokines and increased IL-10 production [ 25 , 26 ]. In the current study, we address the hypothesis that low-dose peptide therapy in allergic individuals results in antigen-specific hyporesponsiveness associated with the induction of a suppressive population of CD4 + T cells, together with up-regulation of surface CD5 levels on antigen-specific T cells. Methods Patients and Study Design Individuals who were cat-allergic and asthmatic were recruited, diagnosed, and assessed as described in detail elsewhere [ 29 ]. The study received prior approval from the Ethics Committee of the Royal Brompton and Harefield Hospitals National Health Service Trust (London, United Kingdom). Written, witnessed informed consent was obtained from all patients. Peripheral blood mononuclear cells (PBMCs) were obtained from patients enrolled in two consecutive studies (open study design) of immunotherapy employing short synthetic peptides derived from the sequence of the major cat allergen Felis domesticus allergen 1 (Fel d 1). The studies employed different dosing regimes in order to evaluate dose effects on clinical and mechanistic outcomes. The first study included eight patients (referred to hereafter as Group 1) who received incremental doses of Fel d 1 peptides (0.1, 1, 1, 5, 10, and 25 μg) totalling 42.1 μg of each peptide, while the second study comprised 12 patients (referred to hereafter as Group 2) who received a total of 291 μg (1, 5, 10, 25, 50, 100, and 100 μg) of each peptide. Peptides were synthesised by Fmoc chemistry, purified by HPLC, and presented as lyophilised solids (Advanced Biotechnology Centre, Imperial College London, United Kingdom). Peptides were reconstituted with sterile physiological saline and dispensed into sterile vials for single patient use (Nova Laboratories, Leicestershire, United Kingdom). Peptide sequences were as follows: EICPAVKRDVDLFLTGT, LFLTGTPDEYVEQVAQY, EQVAQYKALPVVLENA, KALPVVLENARILKNCV, RILKNCVDAKMTEEDKE, KMTEEDKENALSLLDK, KENALSLLDKIYTSPL, LTKVNATEPERTAMKK, TAMKKIQDCYVENGLI, SRVLDGLVMTTISSSK, ISSSKDCMGEAVQNTV, and AVQNTVEDLKLNTLGR. Clinical parameters and outcome measures associated with peptide intervention in donors from whom PBMC samples were obtained are described in detail elsewhere [ 27 , 28 ]. Briefly, in the first study peptide immunotherapy (PIT) resulted in improved non-specific bronchial hyperreactivity, since a significantly ( p = 0.02) greater concentration of histamine was required to induce a 20% reduction in forced expiratory volume measured in 1 s. Additionally, a significant reduction ( p = 0.03) in the magnitude (area in square millimeters) of the late-phase skin reaction was observed post-treatment. In the second study, treatment was associated with a reduction in the magnitude of the late asthmatic reaction induced by inhaled allergen challenge, together with a significant decrease in nasal outcome measurements (number of sneezes, nasal blockage, and weight of nasal secretion; all measurements at 15 min post-challenge, p = 0.02). PBMC Cultures PBMCs were isolated from venous blood by density gradient centrifugation (Histopaque-1077; Sigma Chemicals, Poole, United Kingdom) and cryopreserved. All experiments were performed on pre- and post-PIT PBMCs of the same patient in single experiments, to reduce inter-experiment variation within single patients. Prior to in vitro culture, PBMCs were thawed, washed, and labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, Oregon, United States), as follows: 2.5 × 10 6 each of pre-PIT and post-PIT PBMCs were resuspended in 0.5 ml of RPMI-1640 (Invitrogen, Paisley, United Kingdom), and 0.5 ml of 1 μM CFSE added under constant, gentle agitation, to achieve a final CFSE concentration of 0.5 μM. After 10 min, 1 ml of human AB serum (Sigma, Poole, United Kingdom) was added to terminate labelling, and cells were washed twice. Cells were resuspended at 2.5 × 10 6 cells/ml of complete medium (RPMI-1640 supplemented with L -glutamine and 5% human AB serum) and plated at 5 × 10 5 cells/well in 96-well flat-bottom culture plates (Nunc, Merck Eurolab, Lutterworth, United Kingdom) in 200 μl of final volume, under the following culture conditions: unstimulated, stimulated with 30 μg/ml whole cat allergen (generous gift of Leti Laboratories, Madrid, Spain) and stimulated with plate-bound α-CD3/α-CD28 (10/1 μg/ml; BD Pharmingen, Cowley, United Kingdom). For suppression experiments, PBMCs were separated into CD4 + and CD4 neg populations. Limited quantities of peripheral blood were available from study patients. Therefore, for reasons of economy, CD4-depleted PBMCs (CD4 neg ) remaining after selection of CD4 + cells were used as target cells in all suppression assays. For each patient, 20 × 10 6 each of pre- and post-PIT PBMCs were labelled with αCD4 magnetic beads (MACS; Miltenyi, Bisley, United Kingdom) and positively sorted to a mean purity of 94%. CD4 neg pre- and post-PIT cells were labelled with CFSE as described above, while CD4 + pre- and post-PIT T cells were labelled with PKH-26 (Sigma) as follows: cells were resuspended in diluent C (Sigma) at no more than 10 7 cells/ml, and an equal volume of a PKH-26 dilution (1 μM) was added to reach a final concentration of 0.5 μM. After 2 min, the reaction was stopped with the addition of 1 ml of human serum, and cells were washed twice. Cells were cultured in the following combinations: pre- and post-PIT CD4 neg cells alone, pre-PIT CD4 neg plus pre- or post-PIT CD4 + , and post-PIT CD4 neg plus pre- or post-PIT CD4 + (CD4 neg cells at 0.5 × 10 6 cells/well and CD4 + cells at 0.125 × 10 6 cells/well in 96-well flat-bottom tissue culture plates, to achieve a ratio of 4:1). All were cultured in the absence or presence of cat allergen (30 μg/ml) for 1 wk in a humidified incubator at 37 °C gassed with 5% CO 2 in air. Flow Cytometry To determine changes in the proliferation of T cell subpopulations associated with PIT, cells were recovered after 1 wk of culture, washed twice, and stained for 30 min at 4 °C with a combination of αCD4-PE + αCD8-Cy, or αCD45-PE. Isotype controls used were mouse IgG 2a -PE, mouse IgG 1 -PE, and mouse IgG 1 -Cy. Mean fluorescence intensity (MFI) was determined by FACS (FACScan, BD Pharmingen) of at least 2 × 10 4 events within the lymphocyte gate. In suppression experiments, the extent of proliferation was measured as above on the CFSE-labelled read-out population without additional antibody staining. Percentages of CD4 + CD25 + T cells, CD4 + CD5 + cells, or CD8 + CD5 + cells were measured for unstimulated cells by double staining with αCD4-Cy + αCD25-FITC, αCD4-Cy + αCD5-PE, or αCD8-Cy + αCD5-PE. Isotype controls used were mouse IgG 1 -Cy and mouse IgG 1 -FITC (all antibodies were from BD Pharmingen). Cytokine Measurements Culture supernatants of 100 μl were collected from wells 48 h after the start of culture. Cytokines were measured by cytometric bead array Th1/Th2 kit (BD Pharmingen) according to the manufacturer's instructions. A total of six cytokines were measured simultaneously. Data for IL-5, IL-10, and interferon (IFN)-γ are shown. Cytokine concentrations were determined using cytometric bead array analysis software (BD Pharmingen). The sensitivity of the assays was 2.4 pg/ml for IL-5, 2.8 pg/ml for IL-10, and 7.1 pg/ml for IFN-γ. Data Analysis FACS cell surface data and CFSE–PKH-26 mixing experiment proliferation data were acquired with Cellquest (BD Pharmingen), and events within the live lymphocyte gate were interpreted using Winmdi 2.8 software (Scripps Research Institute, http://facs.scripps.edu/software.html ). CFSE proliferation data of T cell subsets were acquired with Cellquest, and events within the CD4 + , CD8 + , or CD45RO + gate analysed with the Proliferation Wizard module in ModFit LT software (Verity Software House, Topsham, Massachusetts, United States). Percentage proliferation is defined as the fraction of the starting population that has proliferated during the course of the experiment. Statistical Analysis For statistical analysis data were analysed for normality using the Shapiro-Wilks test. Normally distributed data were analysed using the paired t-test (parametric). Non-normal data were analysed using the Wilcoxon signed rank test (non-parametric). Analysis was performed by an independent statistician (Turnstat, Reading, United Kingdom). Results PIT Results in the Inhibition of Cat-Allergen-Induced Proliferation of CD45RO + , CD4 + , and CD8 + T cell Subsets The effect of PIT on cellular proliferation of T cell subsets was evaluated by combining CFSE labelling with cell surface staining. As shown in Figure 1 A, the majority of cat-allergen-specific T cells resided within the CD45RO + (memory) T cell population. The proliferation of this population was markedly inhibited following PIT ( Figure 1 A– 1 D). Limited allergen-specific proliferation was detected in the CD45RO − (naïve) population, but this appeared less sensitive to the effects of PIT. Data from all nine individuals tested showed a similar reduction in the post-PIT proliferative response ( Figure 1 E; mean proliferation pre-PIT [20.3%] was decreased post-PIT [5.8%], p = 0.004; pre-PIT range 7.9%–41.8%; post-PIT range 0%–16.7%). Proliferation in the absence of a stimulus was less than 2% in all cases and was subtracted. Proliferation to plate-bound α-CD3/α-CD28 (10 μg/ml and 1 μg/ml, respectively, as a mixture) resulted in mean pre-PIT proliferation of CD45RO + T cells of 64.2% and post-PIT proliferation of 60.1% (data not shown). Figure 1 PIT Reduces Antigen-Specific Proliferation of Memory T Cells (A–D) PBMCs taken before and after PIT were labelled with CFSE to track cell division after antigen stimulaton. Proliferation of cat-allergen-specific CD45RO + lymphocytes was reduced following PIT (A) and (B). (C) and (D) represent CD45RO + T cells as shown in panels (A) and (B), respectively, analysed with ModFit software. The right-hand peaks represent the parental population, and generations of dividing cells are depicted leftwards along the x-axis. (E) Summary of the percentage of proliferating CD45RO + T cells pre- and post-PIT (percent proliferating cells is defined as the fraction of the starting population that has proliferated during the course of the experiment, determined with Modfit) for all nine patients tested. Open symbols represent patients enrolled in treatment Group 1, while solid symbols depict patients from treatment Group 2. Horizontal solid bars show mean levels of proliferation. Background proliferation (in the absence of a stimulus) was less than 2% and was subtracted. The Wilcoxon signed rank test was used for statistical analysis. The effect of PIT on CD4 + and CD8 + populations was also addressed. Both CD4 + and CD8 + subsets proliferated to whole cat allergen. CD4 + post-PIT T cell proliferation was significantly reduced ( p = 0.016; Figure 2 A), despite a slight increase in proliferation for one patient (mean pre-PIT to post-PIT CD4 proliferation was reduced from 5.4% to 2.1% [pre-PIT range 0%–12.7%; post-PIT range 0%–7.3%]). A similar reduction was observed for CD8 + T cells ( p = 0.031; Figure 2 B data from seven patients available for analysis). Post-PIT CD8 + proliferation showed a greater reduction (mean pre-PIT to post-PIT CD8 + proliferation was reduced from 8.0% to 2.5% [pre-PIT range 1.8%–20.8%; post-PIT range 0.3%–4.7%]). Figure 2 PIT Reduces Antigen-Specific Proliferation of CD4 + and CD8 + T Cells CD4 + and CD8 + proliferation data were obtained and interpreted as for Figure 1 . (A) and (B) represent percentage proliferation of PBMCs to cat allergen for each patient as determined with ModFit. Open symbols represent patients from treatment Group 1, while solid symbols depict patients from treatment Group 2. Horizontal solid bars indicate means. Background proliferation has been subtracted. The Wilcoxon signed rank test was used for statistical analysis. Modulation of Cytokine Secretion following Peptide Immunotherapy In order to characterise modulation of cytokine responses following PIT, culture supernatants were collected after 48 h. Cytokines were measured simultaneously by flow cytometry. The majority of patients displayed increased IL-10 secretion although this change did not achieve statistical significance. IL-5 secretion was significantly reduced post-PIT ( p = 0.02; Table 1 ). IFN-γ secretion tended to be reduced following PIT, but heterogeneity was observed. Table 1 Modulation of Cytokine Secretion Profiles in Allergen-Stimulated PBMCs following PIT a Cytokine concentration in picograms per millilitre with background (cells cultured in medium alone) subtracted b Patients from Group 1 c ND indicates not detected, and assigned a value of zero for statistical analysis. The sensitivity of the assays was 2.4 pg/ml for IL-5, 2.8 pg/ml for IL-10, and 7.1 pg/ml for IFN-γ d IFN-γ and IL-10 analysed with paired t-test (normal distribution), IL-5 analysed by Wilcoxon (non-normal distribution). Normality determined by Shapiro-Wilks test PIT Leads to the Induction of a CD4 + T Cell Population with Suppressor Activity To identify populations of T cells with suppressive activity and to attempt to distinguish between active suppression and clonal deletion as potential mechanisms following PIT, pre- and post-PIT CD4 + T cells were isolated and their effect on proliferation of the CD4 neg fraction measured ( Figure 3 ). Two distinct cell-cycle tracking dyes, CFSE and PKH-26, were employed to visually separate the target (CD4 neg ; CFSE) from the effector (CD4 + ; PKH-26) populations, by flow cytometry. PIT resulted in a 69% reduction (14.7% proliferation pre-PIT versus 4.6% proliferation post-PIT) in proliferation of the CD4 neg T cell population for the one representative patient shown in detail ( Figure 3 A and 3 B). When pre- or post-PIT CD4 + T cells were added to CD4 neg PBMCs (at a ratio of 1:4), a marked reduction in proliferation of cat-allergen-specific CD4 neg pre-PIT T cells was observed when co-cultured with post-PIT ( Figure 3 E; 7.9% proliferation) but not with pre-PIT CD4 + T cells ( Figure 3 C; 17.7% proliferation), indicating that the post-PIT CD4 + T cells harboured a suppressor population. As post-PIT CD4 neg T cell proliferation was minimal, addition of post-PIT CD4 + T cells did not have a further suppressive effect on this cell population ( Figure 3 F). Additionally, removal of the CD4 + T cells from post-PIT PBMCs did not cause the depleted PBMC population to proliferate ( Figure 3 B), suggesting that antigen-specific cells in the post-treatment population had already been rendered anergic in vivo as a result of PIT, or possessed the ability to actively suppress responses themselves. Similar experiments were performed in a further four patients. A summary of the results for all five patients is shown in Figure 3 G. Inhibition of proliferation by CD4 + post-PIT T cells ranged from 64.0% to 19.6%, with a mean of 47.5%. Figure 3 CD4 + Cells Isolated after PIT Suppress the Proliferative Response of Baseline CD4 neg Cells PBMCs taken before and after PIT were separated into CD4 + and CD4 neg populations by immunomagnetic separation. CD4 neg cells were labelled with CFSE and served as target cells. CD4 + cells were labelled with PKH-26 and were evaluated for suppressor/regulator function by co-culture with CD4 neg cells. (A) and (B) show antigen-stimulated proliferation of CD4 neg target cells before and after PIT. Proliferation of CD4 neg target cells was reduced after PIT (B). In (C) and (E), pre-PIT CD4 neg cells were employed as target cells. The addition of post-PIT (E), but not pre-PIT (C) CD4 + cells inhibited proliferation. In (D) and (F), post-PIT CD4 neg cells were employed as target cells. Addition of either pre-PIT (D) or post-PIT (F) CD4 + cells had no further effect on proliferation. Proliferation in the absence of a stimulus was less than 2% in all experiments. Representative data for one patient are shown. Data for an additional four patients were obtained using the same protocol. A data summary of percentage proliferation of pre-PIT CD4 neg PBMCs in the presence of pre-PIT or post-PIT CD4 + T cells for five patients from treatment Group 2 is shown in (G). The paired t-test was used for statistical analysis. Phenotypic Characterisation of Candidate Regulatory T Cells Induced Post-PIT T cell surface markers known to be associated with tolerance induction, such as CD25 and CD5, were compared on pre- and post-PIT resting PBMC in an attempt to provide further insight into the nature of the suppressor population. No significant variation was found in CD4 + CD25 + cell numbers (mean pre-PIT to post-PIT proliferation 20.5%–17.9%; data not shown). However, when CD5 expression was determined on both CD4 + and CD8 + cells, a significant increase in MFI in both populations was observed ( p = 0.016 and 0.047, respectively). Figure 4 A and 4 B show increases in CD5 expression on CD4 + and CD8 + cells (MFI of CD5 expression on CD4 + cells: pre-PIT mean, 465.5 [range, 290.3–908.5]; post-PIT mean, 559.7 [range, 302.5–1241.8]; range of post-PIT percentage change in MFI, 4%–37%; MFI of CD5 expression on CD8 + cells: pre-PIT mean, 110.9 [range, 55.3–345.8]; post-PIT mean, 149.2 [range, 60.6–352.2]; range of post-PIT percentage change in MFI, 7%–118%). Increased MFI resulted not only from a decrease in the numbers of CD5 low cells and an associated increase in CD5 + cells in both populations, but from an increase in CD5 expression on the CD5 + cells as well, as is shown in Figure 4 C and 4 D for one representative patient. Figure 4 PIT Enhances CD5 Expression on Resting CD4 + and CD8 + PBMCs (A and B) Box-and-whiskers plots representing changes in MFI of CD5 expression levels on unstimulated CD4 + (A) and CD8 + (B) pre- and post-PIT PBMCs from seven patients in treatment Group 2. Isotype control MFI values have been subtracted. (C and D) CD5 levels on pre-PIT (heavy black line) and post-PIT (grey, filled) CD4 + and CD8 + PBMCs of one representative patient. M1 marks the CD5 low population, with a pre- to post-PIT decrease in CD5 low CD4 + PBMCs from 6.6% (in black) to 1.5% (in grey), and a decrease in CD5 low CD8 + PBMCs from 32.3% to 13.9%. M2 indicates the concomitant increases in CD5 + CD4 + and CD5 + CD8 + cells post-PIT. Changes in MFI values for the total pre- and post-PIT CD4 + or CD8 + populations of the single representative patient are shown in the upper right-hand corner of each histogram. The Wilcoxon signed rank test was used for statistical analysis. The effect of PIT on proliferation, cytokine secretion patterns, phenotype of T cell subsets, and suppressive capacity did not appear to be dependent on the total dose of peptide administered in the two treatment groups. Discussion Following PIT, proliferation of CD4 + , CD8 + , and CD45RO + memory T cells was reduced following culture with whole cat dander allergen extract. Non-specific T cell receptor (TCR) ligation with anti-CD3/CD28 was unaffected, implying that only cat-allergen-specific T cells had been targeted by PIT. The reduction in proliferation was primarily observed within the differentiated memory (CD45RO + ) rather than the naïve T cell population, the latter displaying minimal cell division. While the role of CD4 + T cells in the pathogenesis of allergic disease is well established, that of CD8 + T cells is less well defined. A number of reports suggest that CD8 + T cells may be activated in the asthma process. CD8 + cells from both bronchoalveolar lavage fluid and peripheral blood from atopic donors were found to produce IL-4 and IL-5 in lavage samples and bronchial biopsies [ 30 , 31 ]. Moreover, individuals with severe atopic disease have high frequencies of Dermatophagoides pteronyssinus 1–specific CD8 + T cells that secrete significantly more IL-4, IL-5, and IL-13 than non-atopic individuals [ 32 ]. Here we have shown that CD8 + T cells proliferate markedly to cat allergen in vitro even in the absence of CD4 + T cells. In the context of previous studies, it appears likely that these cells may contribute to disease pathogenesis. Thus, induction of non-responsiveness in CD8 + T cells should have a positive therapeutic outcome in allergic disease. Cytokine profiles of cat-allergen-stimulated PBMCs were established following peptide therapy. Levels of IL-2 and IL-4 were generally below the limit of detection of the assays employed. PIT had no effect on secretion of tumour necrosis factor α (data not shown). Production of IL-5, a cytokine considered particularly relevant in asthma, was significantly reduced following PIT. In approximately half of the patients there were reductions in both Th1 and Th2 cytokines, as previously described [ 26 ]. We have observed similar results in an unpublished study of PIT for bee venom hypersensitivity. The majority of patients showed increased IL-10 production after PIT, in agreement with our earlier observations. However, in the present study this did not achieve statistical significance, in contrast to a previous report. Enhanced production of IL-10 has been associated with protection from allergic symptoms in both naturally exposed individuals such as beekeepers and in individuals receiving bee venom immunotherapy [ 33 ]. In contrast, IL-10 production in relation to cat allergen exposure and protection is less well established. A protective effect of high-dose natural exposure to cat allergens (resulting in a “modified Th2 response”) has been reported [ 34 , 35 ]. Woodfolk and colleagues demonstrated elevated IL-10 production in individuals displaying a “modified Th2” profile in which cat-allergen-specific IgG4 appeared to protect from disease [ 36 ]. In their study, particular regions of the Fel d 1 molecule (carboxy terminus of chain 2) appeared to be associated with presentation by HLA-DRB1*0701 and were associated with preferential IL-10 induction. For technical reasons, peptides from this region were not included in the preparation used in the present study. Inclusion of such peptides in future studies may enhance vaccine efficacy. In the present study, cytokine production was evaluated in peripheral blood cells. Cytokine production at local tissues targeted by allergens may provide a more accurate picture of the effects of immunotherapy with peptides or native allergens/allergen extracts. For example, in a related study a significant increase in the number of cutaneous CD4 + IFNγ + cells ( p = 0.03), but not in CD4 + IL-10 + cells, was observed in allergen-challenged skin biopsies [ 27 ]. Similarly, a significant increase in the number of IFN-γ mRNA(+) cells ( p = 0.03) was found in nasal biopsies of patients enrolled in a whole-grass-pollen immunotherapy trial, in the absence of significant modifications in IFN-γ secretion by corresponding in vitro stimulated PBMCs [ 37 ]. Thus, in vivo localization of cells by allergen challenge may reveal patterns of immunomodulation that differ from changes in the blood of the same individual. For this reason, caution should be exercised when interpreting alterations in cytokine profiles in different tissues following immunotherapy. We addressed the possibility that changes in T cell proliferation and cytokine secretion may be related to the induction of a population of T reg or suppressor T cells, similar to that observed following peptide intervention in murine models [ 38 ]. PBMCs were separated into CD4 + and CD4 neg populations. CD4 + cells isolated from post-PIT blood were able to actively suppress the proliferation of pre-treatment CD4 neg cells. The selection of CD4 neg cells, rather than CD4 + cells, as targets was due to limitations in the number of cells available. Nevertheless, the results obtained indicate the induction of regulatory and suppressor CD4 + T cells following PIT. Interestingly, removal of CD4 + cells from the post-PIT PBMC population did not lead to a reversal of the allergen-specific hyporesponsiveness in the pre-PIT CD4 neg population. This observation suggests that in addition to active suppression by CD4 + cells, enduring effects of therapy can also be detected. Explanations for such observations may include the following: (i) clonal deletion of some antigen-specific CD4 neg cells, (ii) the induction of anergy in these cells during the treatment phase, or (iii) the presence of a CD4 neg suppressor population. In support of the last possibility, regulatory CD8 + T cells have recently been described [ 39 ]. Studies identifying allergen-specific CD4 and CD8 T cells will be required to address such issues. In future studies it will be of interest to identify the subpopulation or subpopulations of CD4 and CD8 cells responsible for the suppressive effect, by removing candidate T cells from the pre- and post-treatment CD4 + T cell populations prior to co-culture. No increase in numbers of CD4 + CD25 + cells was observed in PBMCs following PIT, in contrast to studies of whole allergen immunotherapy [ 40 , 41 ]. In fact, numbers of CD25 bright cells significantly decreased following peptide therapy (data not shown). Furthermore, CD4 + CD25 + T cells obtained before and after treatment in a related PIT study did not differ in their ability to suppress allergen-specific effector T cell proliferation and IL-13 production, arguing against a major role for this type of regulatory cell in peptide therapy [ 42 ]. We speculate that PIT results in T cell activation in the absence of inflammatory signals, possibly via presentation by immature APCs, or even by neighbouring T cells. Well-characterised in vitro human models have demonstrated that it is indeed possible to induce T cell anergy following incubation with cognate peptide in the absence of professional APCs [ 43 , 44 ]. Recently, Apostolou and von Boehmer reported induction of antigen-specific hyporesponsiveness, mediated by regulatory cells, following continuous, low-dose peptide administration in mice [ 21 ], an observation that supports our current and previous clinical findings. Additionally, Prakken and colleagues have demonstrated induction of IL-10-secreting T reg following oral peptide therapy in patients with rheumatoid arthritis. These cells may similarly represent an induced population of adaptive T reg [ 45 ]. While CD25 expression is considered to be a marker of a functionally distinct population of T reg (provided the cells have not been recently activated), CD5 expression levels on T cells may be an indicator of a regulatory function [ 18 ]. CD5 has been shown to be a negative regulator of TCR signalling, influencing the fate of developing thymocytes [ 17 ]. In the periphery, CD5 neg T cells show enhanced proliferation to TCR triggering [ 46 ]. Conversely, increased membrane levels of CD5 correlate with a lowering of the T cell response to antigen by targeting downstream signalling events [ 47 ]. In the current study, CD5 levels were significantly elevated on directly ex vivo, unstimulated CD4 + and CD8 + T cells, following peptide therapy. The increases were slight, which likely relates to the low precursor frequency of the cells targeted. Interestingly, the increases observed on CD8 + T cells were partly due to a reduction in the size of the CD8 + CD5 neg T cell population. A distinct CD8 + CD5 neg T cell population that accounts for 3%–10% of the total CD8 + T cell population in healthy donors has previously been described [ 48 ] and appears to be the main producer of lymphotactin (XCL-1) [ 49 ]. As the average size of CD8 + CD5 neg T cell populations in the allergic asthmatic patients in our study is substantially larger (23.2% of the total CD8 + T cells), it is tempting to speculate that this is further evidence for a dysregulated immune response associated with allergic disease. This observation is in agreement with data from lymphopenic mice that developed wasting disease with accelerated kinetics following adoptive transfer of T cells expressing low levels of CD5, whilst CD5 hi cells were protective [ 18 ]. Consistent with these findings, surface levels of CD5 on human T cells also appear to correlate with immune function, as the CD5 neg population was increased in bone marrow transplant recipients as well as in patients with advanced AIDS [ 50 , 51 ]. However, as relatively little is known about the role of CD5 in human T cell tolerance, further investigations are required to establish the relevance of our finding in allergic disease. Isolating Fel d 1–specific T cells should yield valuable information on the functional relevance of increased CD5 expression on allergen-specific cells. To our knowledge, this is the first demonstration that PIT induces a CD4 + T cell population that actively suppresses antigen-induced proliferation of effector T cells. The use of dual labelling with distinctly coloured dyes allowed evaluation of the effect of the CD4 + T cell subset on the proliferation of CD4 neg (including CD8 + cells, natural killer cells, B cells, monocytes, and basophils) using flow cytometry. While dual labelling has been widely used to track cell migration in animal models [ 52 ], its application in in vitro human T cell proliferation experiments has, to our knowledge, not previously been reported. Single-colour labelling of distinct human PBMC populations has been used to characterise, isolate, and clone peanut-allergen-specific T cells [ 53 ] and to determine precursor frequencies of recall-antigen-specific T cells [ 54 ]. Measurement of proliferation by means of CFSE has the additional advantages of requiring relatively low numbers of cells and allowing additional phenotypic (cell surface markers) or functional parameters (intracellular cytokine secretion) to be studied in parallel, in distinct subpopulations [ 54 ]. In conclusion, our data indicate that low-dose PIT targets both CD4 + and CD8 + memory T cells and induces a population of active suppressor/regulatory T cells within the CD4 + compartment. Suppressor activity may also reside within the CD4 neg compartment. Peptide therapy resulted in a heterogeneous modulation of allergen-specific PBMC cytokine responses in vitro, generally characterised by IL-10 induction and IL-5 suppression. Finally, modest but consistent increases were observed in surface CD5 expression on both CD4 + and CD8 + T cells, an observation that may be linked to the induction of antigen-specific hyporesponsiveness. The ability to modulate antigen-specific T cell function in vivo has important implications for the treatment and prevention of allergic, autoimmune, and allograft-related diseases. Supporting Information Accession Numbers The SwissProt ( http://www.ebi.ac.uk/swissprot/ ) accession numbers for the gene products discussed in this paper are CD5 (P06127), DEC-205 (Q60767), Fel d 1 chain 1 (P30438), Fel d 1 chain 2 (P30440), and lymphotactin (P47992). Patient Summary Background Increasing numbers of people are developing allergies to pets and becoming asthmatic. It is not clear what combination of events triggers allergy—for example, whether keeping pets as a child is protective—nor what can be done to treat the allergy once it develops. What Did the Authors Do? They looked at a small group of people who were allergic to cats and asthmatic. They measured the levels of different kinds of T cells in their blood—cells that are associated with the allergy. They then treated the people with small proteins that are very similar to the triggers for the allergy and looked to see how the levels of various T cells changed. They found that the protein treatment triggered a particular type of cell, which seemed able to repress the reactive cells that had triggered the immune reaction previously. What Do These Results Mean for Patients? There are many things that interact to produce allergy, and this study does not help in understanding exactly how this happens. It does suggest a way that treatment with specific small proteins might work in reducing the allergy; however, the results will need to be confirmed in much larger studies. Where Can I Get More Information? Both the American Academy of Allergy Asthma and Immunology, and Asthma UK have large sections of patient information: http://www.aaaai.org/patients.stm ; http://www.asthma.org.uk/

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1069670

Cholesterol, Statins, and Alzheimer Disease

After reading the excellent research article by Pedrini et al. [ 1 ] and the associated synopsis [ 2 ], one may conclude that the only pathway of statins' effect on Alzheimer disease (AD) is the regulation of amyloid precursor protein (APP) processing and amyloid-ß protein (Aß) generation. The moderation is provided in the research article's patient summary, reminding that “statins are likely to influence the risk for Alzheimer disease by several different pathways.” What are these other pathways? It is essential to note that in addition to APP processing and Aß chemistry being modulated by statins, fine tuning of cholesterol homeostasis also affects cholinergic function, ionotropic and metabotropic receptors, tau phosphorylation, neural oxidative stress reactions, and other features of neurodegeneration (reviewed in [ 3 ]). Moreover, precise regulation of neural cholesterol dynamics and supply is itself essential for synapse function, plasticity, and behaviour [ 3 ]. These data suggest that in addition to its role in sporadic AD, cholesterol homeostasis break is the unifying primary cause of neuromuscular diseases, Niemann-Pick type C disease, and Down syndrome, and explains why rare cases of familial AD (associated with mutations in APP and presenilin genes) are translated into Alzheimer's via membrane cholesterol sensitivity of APP processing by secretases and Aß generation. Also important, is the synopsis's [ 2 ] apparently outdated dividing of APP processing into “harmful” (Aß-generating) and “healthy” (non-amyloidogenic). One should be cautious in calling Aß a harmful molecule. This is because several recent studies have illuminated an essential function for amyloidogenic processing of APP and Aß in neurons [ 4 ] and synapses [ 5 ]. In this context, the reciprocal effect of Aß on cholesterol synthesis, cellular uptake, efflux, and esterification, and its relation to the experimental restoration of long-term potentiation (LTP, a synaptic plasticity measure) may represent one of the poorly comprehended physiological functions of Aß [ 6 , 7 ].

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1069671

Accounting for Individual Differences in Risk of Alzheimer Disease

Gatz's statement, “At least half of the explanation for individual differences in susceptibility to Alzheimer disease is genetic” [ 1 ], is, in my opinion, incorrect. As the one who led the team debating Ashford and Mortimer, whose 2002 article [ 2 ] supports this statement, at the 2001 conference on Alzheimer disease (AD) in Cincinnati (“Challenging Views of Alzheimer's Disease”) [ 3 ], I think that the evidence that dietary and lifestyle factors explain the majority of the individual risk for AD in the US is very strong. My original paper in 1997 [ 4 ] found that total dietary fat and energy intake were the most important dietary risk factors, while fish and cereal intake were the most important risk reduction factors. These findings have been generally confirmed by Drs. Luchsinger and Morris and others. The reason I did my study was that the Honolulu Heart Study reported that Japanese American men in Hawaii had 2.5 times the risk of AD of native Japanese. African-Americans have about four times the risk of AD of native Nigerians. If genetics were the primary risk factor, those living in the US would have a risk of developing AD very similar to that of individuals living in their ancestral home. The reason this is not the case is that the American diet provides too much food, which is a particular problem for those genetically predisposed to AD.

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1069672

A Canadian Perspective

Erick H. Turner [ 1 ] notes that “ClinicalTrials.gov, a registry authorized by the Food and Drug Modernization Act of 1997, appears not to be comprehensive.” While we await the creation of clinical trials registry and results databases that are truly comprehensive, innovative efforts to provide convenient access to credible information about known existing clinical trials need to continue. A Canadian example is provided by OntarioCancerTrials.ca, a consumer-oriented site developed by the Ontario Cancer Research Network (OCRN), with funding from the Ontario government.

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