bluuebunny/crossref_metadata_2025 huggingface dataset:
Epitope mapping of anti-PR3 antibodies using chimeric human/mouse PR3 recombinant proteins
Authors: SELGA D , SEGELMARK M , WIESLANDER J , GUNNARSSON L , HELLMARK T
Published: December 2003

Autoantibodies against proteinase 3 (PR3) and myeloperoxidase (MPO) (ANCA = anti-neutrophil cytoplasmic antibodies) are used as diagnostic tools for patients with small vessel vasculitis. ANCA are detected by different assays, but the correlation between the results of these assays is generally poor. The overall aim of the study was to provide a framework for the future development of new assays with an increased diagnostic yield. In order to express discrete epitopes of human PR3 (hPR3), the nonantigenic molecules murine PR3 (mPR3) and human leucocyte elastase (HLE) were used as a framework. We constructed recombinant chimeric vectors and were able to produce 6 hPR3/mPR3 proteins and 3 hPR3/HLE proteins. Anti-PR3 monoclonal antibodies differed in their binding pattern to the chimeras, but no distinct binding region could be identified for any monoclonal antibody. The recombinant hPR3/mPR3 were also tested in ELISA with sera from patients with Wegener's granulomatosis with renal involvement. The results show that patients have antibodies to different constructs, indicating that the patients vary in their antibody repertoire from the beginning of the disease, and that patients may have antibodies from a broad range of clones early in the course of the disease. Recombinant hPR3/mPR3 chimeric proteins have a potential to be used as antigens in future ANCA assays.
Nicotinamide does not influence cytokines or exhaled NO in human experimental endotoxaemia
Authors: SOOP A , ALBERT J , WEITZBERG E , BENGTSSON A , NILSSON C-G , SOLLEVI A
Published: December 2003

This study examined the hypothesis that nicotinamide could attenuate endotoxin-induced inflammatory responses in humans as indicated by levels of cytokines and nitric oxide. Ten healthy male volunteers participated in a randomised, double-blind, cross-over design with regard to the effects of nicotinamide. The volunteers received orally 4 g nicotinamide or placebo at 14 h and at 2 h preceding the experiment (total dose of 8 g). Endotoxin (E. coli, 2 ng/kg), was administered intravenously. Blood samples and haemodynamic data were collected prior to and up to 6 h after the endotoxin infusion. Orally exhaled NO was measured hourly. Following endotoxin, body temperature increased from baseline 36·3 ± 0·09°C to a maximum of 38·0 ± 0·1°C for all (mean ± SEM, P <0·001) and heart rate increased from 59 ± 1·9 to 87·0 ± 2·6 beats/min after 3 h (mean ± SEM, P <0·001). Endotoxin challenge also markedly elevated the TNF-α, IL-6, IL-8 and IL-10 concentrations (P < 0·001 versus baseline for all) during the study period. Orally exhaled NO also increased (P < 0·01) compared to baseline. Nicotinamide treatment did not influence the patterns of cytokine and NO response to endotoxin.

In conclusion, there was no effect on the inflammatory parameters by oral nicotinamide at a dose of 8 g, limiting the potential use of this agent for anti-inflammatory purpose in man.
Antibodies to inner ear antigens in Meniere's disease
Authors: RIENTE L , BONGIORNI F , NACCI A , MIGLIORINI P , SEGNINI G , DELLE SEDIE A , URSINO F , TOMMASI S , FATTORI B
Published: December 2003

Meniere's disease (MD) is an idiopathic inner ear disorder characterized by fluctuating hearing loss, episodic vertigo and tinnitus. Its aetiology is unknown, although there is growing evidence that autoimmunity may be involved in its development. Using the Western blot immunoassay, we examined the reactivity to bovine inner ear antigens of sera from a series of MD patients who had previously been extensively studied for the presence of antibodies to collagens and membrane proteins. Reactivity to inner ear antigens of molecular weight 44 and 53 kD was found in 11/25 (44%) and 10/25 (40%) of the patients, respectively; both antigens were absent in the sera of healthy donors. It is still unclear whether the antibodies to 44 and 53 kD proteins play a role in the pathogenesis of MD or if they instead represent the result of inflammation and tissue destruction. Even if the latter is true, they may contribute to the perpetuation of the disease or play a role as a cofactor in association with other mechanisms.
Lung cell responses toM. tuberculosisin genetically susceptible and resistant mice following intratracheal challenge
Authors: ERUSLANOV E B , MAJOROV K B , ORLOVA M O , MISCHENKO V V , KONDRATIEVA T K , APT A S , LYADOVA I V
Published: December 2003

One approach to study the role of distinct cellular mechanisms in susceptibility/resistance to tuberculosis (TB) is to compare parameters of response to infection in the lungs of mouse strains exhibiting genetically determined differences in TB susceptibility/severity. Interstrain differences in antimycobacterial macrophage reactions, T cell responses & inflammation in the lungs of TB-susceptible I/St, TB-resistant A/Sn and (I/St × A/Sn)F1 mice were analysed following intratracheal inoculation of 103 CFUs of M. tuberculosis H37Rv. The antimycobacterial responses in the lungs of susceptible I/St mice were characterized by: (i) increased inflammatory infiltration by all major immune cell subsets; (ii) decreased type 1 cytokine production; (iii) impaired antimycobacterial activity of lung macrophages; (iv) unusually high proliferation of lung T lymphocytes. Differences in several parameters of anti-TB immunity between susceptible and resistant mice corresponded well to the polygenic pattern of TB control previously established in this mouse model. Importantly, lung macrophages isolated from noninfected mice were unable to respond to IFN-γ by increasing their mycobactericidal function, but between weeks 3 and 5 of the infection this capacity developed in all mice. However, by this time point susceptible but not resistant mice demonstrated a pronounced decrease in IFN-γ production by lung cells. This chain of events may explain the inability of I/St mice to control both early and chronic TB infection.
Graves’ hyperthyroidism and thyroiditis in HLA-DRB1*0301 (DR3) transgenic mice after immunization with thyrotropin receptor DNA
Authors: FLYNN J C , RAO P V , GORA M , ALSHARABI G , WEI W , GIRALDO A A , DAVID C S , BANGA J P , KONG Y M
Published: December 2003

Familial and twin studies in Caucasians have established that the MHC class II allele HLA-DRB1*0301 (DR3) is a strong susceptibility gene in Graves’ hyperthyroid disease (GD). To determine if a DR3 transgene could help establish an animal model for GD, we expressed DR3 molecules in class II-knockout NOD mice (H2Ag7–). DR3+g7– mice were given cardiotoxin prior to immunization on weeks 0, 3 and 6 with plasmid DNA encoding human thyrotropin receptor (TSHR). Two groups of mice were also coimmunized with plasmid DNA for IL-4 or GM-CSF. Serial bleeds on weeks 8, 11 and 14 showed that approximately 20% of mice produced thyroid-stimulating antibodies (Abs), and approximately 25% had elevated T4 levels. In particular, a subset displayed both signs of hyperthyroidism, resulting in approximately 30% with some aspect of GD syndrome. Additional mice had thyroid-stimulating blocking Abs and/or TSH-binding inhibitory immunoglobulins, while most mice showed strong labelling of TSHR+ cells by flow cytometry. Interestingly, lymphocytic infiltration with thyroid damage and Abs to mouse thyroglobulin were also noted. Vector controls were uniformly negative. Thus, DR3 transgenic mice can serve as a model for GD, similar to our earlier reports that this allele is permissive for the Hashimoto's thyroiditis model induced with human thyroglobulin.
NK cells modulate the cytotoxic activity generated byMycobacterium leprae-hsp65 in leprosy patients: role of IL-18 and IL-13
Authors: DE LA BARRERA S , FINIASZ M , FINK S , ILARREGUI J , ALEMÁN M , OLIVARES L , FRANCO M C , PIZZARIELLO G , DEL CARMEN SASIAIN M
Published: December 2003

Protection against intracellular pathogens such as Mycobacterium leprae is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages. In the present report we evaluated the role of IL-18 and IL-13, two cytokines that can influence NK cell activity, in the generation of M. leprae-derived hsp65-cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) of leprosy patients. We demonstrated that IL-18 modulates hsp65-induced CTL generation and collaborates with IL-12 for this effect. In paucibacillary (PB) patients and normal controls (N) depletion of NK cells reduces the cytolytic activity. Under these conditions, IL-12 cannot up-regulate this CTL generation, while, in contrast, IL-18 increases the cytotoxic activity both in the presence or absence of NK cells. IL-13 down-regulates the hsp65-induced CTL generation and counteracts the positive effect of IL-18. The negative effect of IL-13 is observed in the early stages of the response, suggesting that this cytokine affects IFNγ production by NK cells. mRNA coding for IFNγ is induced by IL-18 and reduced in the presence of IL-13, when PBMC from N or PB patients are stimulated with hsp65. Neutralization of IL-13 in PBMC from multibacillary (MB) leprosy patients induces the production of IFNγ protein by lymphocytes. A modulatory role on the generation of hsp65 induced CTL is demonstrated for IL-18 and IL-13 and this effect takes place through the production of IFNγ.
High-level endothelial E-selectin (CD62E) cell adhesion molecule expression by a lipopolysaccharide-deficient strain ofNeisseria meningitidisdespite poor activation of NF-κB transcription factor
Authors: DIXON G L J , HEYDERMAN R S , VAN DER LEY P , KLEIN N J
Published: December 2003

Binding of host inflammatory cells to the endothelium is a critical contributor to the vascular damage characteristic of severe meningococcal disease and is regulated by endothelial cell adhesion molecules such as ICAM-1, VCAM-1 and CD62E. Intact meningococci induce far higher levels of CD62E than lipopolysaccharide (LPS) alone, whereas LPS is at least as potent as meningococci at inducing both VCAM-1 and ICAM-1 expression. This suggests that meningococci possess additional factors other than LPS present in whole bacteria that result in differential adhesion molecule expression. To investigate this possibility, we studied the capacity of an LPS-deficient isogenic strain of serogroup B Neisseria meningitidis H44/76 (lpxA-) to induce endothelial cell adhesion molecule expression and translocation of the transcription factor NF-κB, and compared it to both parent and unencapsulated strains of both B1940 and H44/76 and purified LPS. Although the LPS-deficient isogenic mutant of strain H44/76 was found to be a poor inducer of NF-κB, it induced higher levels of CD62E expression than LPS alone. These data provide evidence that intact meningococci induce a range of signals in the endothelium that are distinct from those seen with purified LPS alone and that they occur in a LPS-dependent and LPS-independent manner. These signals may explain the potent effects of N. meningitidis on CD62E expression on vascular endothelium and provide a basis for the complex endothelial dysregulation seen in meningococcal sepsis.
Mushroom plant workers experience a shift towards a T helper type 2 dominant state: contribution of innate immunity to spore antigen
Authors: SAIKAI T , TANAKA H , SATO N , ABE S , MATSUURA A
Published: December 2003

Contemporary mushroom factories are places where there is a substantial risk of the occurrence of respiratory allergy. The aims of this investigation were to estimate its causative agents and to evaluate the contribution of innate immune response in mushroom workers who cultivate Hypsizigus marmoreus (Bunashimeji). Cross-sectional and follow-up studies were performed in the factory. We investigated CD1b, CD3, CD4, CD8, CD14, CD45RO, CD62L and CD161 expression in peripheral blood mononuclear cells (PBMC) by flow cytometry, and serum levels of interleukin (IL-2), IL-4, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-13 and interferon (IFN)-γ by enzyme-linked immunosorbent assay (ELISA). Co-culture experiments of PBMC with spore extracts were also performed. Percentages of CD1b+ monocytes, natural killer (NK), NK T and CD4+ T cells were increased in the workers compared with controls. Increases in Th2 type cells, Th2/Th1 ratio and serum IL-13 and decreased IFN-γ were detected, indicating a Th2-biased status of the workers. The follow-up study showed that monocytes and NK cells increased soon after employment while CD4+ T, Th2 and NK T cells increased gradually as employment time lengthened. Serum precipitating antibody to the mushroom antigen could be detected at a later stage. Co-cultivation of PBMC with the spore extracts induced much higher CD1b expression, and suppressed secretion of Th1 cytokine in culture supernatants. These results indicate that the mushroom antigen contains highly immunogenic substances which stimulate PBMC into a Th2-biased in vivo status, and innate immune cells might also play a critical role in developing respiratory allergy in mushroom workers.
CTLA-4 blockade inhibits induction ofHelicobacter pylori-associated gastritis in mice
Authors: WATANABE K , MURAKAMI K , SATO R , OKIMOTO T , MAEDA K , NASU M , NISHIZONO A , FUJIOKA T
Published: December 2003

The balance between Th1 and Th2 response determines the outcome of Helicobacter pylori infection. Interferon (IFN)-γ plays an inductive role in gastric inflammation, whereas interleukin (IL)-4 counterbalances Th1 response and suppresses the development of gastritis. Th cell response is regulated by co-stimulatory factors. A co-stimulatory molecule, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), plays an inhibitory role in IL-2-dependent cell growth and mediates an optimal inhibitory signal to Th1 and Th2 cells. We administered anti-CTLA-4 monoclonal antibody (MoAb), which blocks CTLA-4 signalling, to examine the relative role for this signalling during maturation of Th1 and Th2 cells in H. pylori infection in mice. Mice treated by anti-CTLA-4 MoAb within the first week of infection showed an inhibition of gastric inflammation, accompanied by an increasing ratio of H. pylori-specific IgG1/IgG2a in serum following infection. Furthermore, the treatment resulted in the higher ratio of IL-4/IFN-γ by splenocytes in response to H. pylori antigen at 6 weeks after infection, compared with untreated mice. These results suggest that the predominance of Th2 response by CTLA-4 blockade leads to an inhibition of the development of gastric inflammation. CTLA-4 signalling could contribute to the regulation of Th subsets and the development of gastric inflammation in H. pylori infection.
Evidence for autoantibody-induced CD4 depletion mediated by apoptotic and non-apoptotic mechanisms in HIV-positive long-term surviving haemophilia patients
Authors: DANIEL V , SADEGHI M , NAUJOKAT C , WEIMER R , HUTH-KÜHNE A , ZIMMERMANN R , OPELZ G
Published: December 2003

It is believed that autoimmune phenomena and apoptosis contribute to CD4 depletion. We investigated 11 long-term (>20 years) HIV-infected haemophilia patients and 10 healthy controls. Using four-colour-fluorescence flow cytometry, we studied the proportions of CD3+CD4+ and CD3+CD4– blood lymphocytes that were CD95+, CD95L+, immune complex+ (IC+, consisting of IgM, IgG, C3d and/or gp120), and were viable or non-viable (propidium iodide+ = PI+). In addition, we studied viability of CD4+IgG+ patient lymphocytes using the apoptosis marker annexin and the permeability indicator 7-amino actinomycin D (7-AAD). HIV+ patients had a higher proportion of CD3+CD4+IgG+PI+ lymphocytes than healthy controls (median: 3·7%versus 0·3%; P = 0·00001). These non-viable IgG-coated lymphocytes might have been killed in vivo by ADCC or complement lysis; 9·1% of the circulating CD3+CD4+ blood lymphocytes were IgG+PI– (controls: 2·5%; P = 0·001). These viable IgG-coated lymphocytes might be targets for phagocytosis or anti-CD95 autoantibody-mediated apoptosis. Because HIV+ patients and healthy controls had similar proportions of PI+ or PI– CD3+CD4+ lymphocytes that carried CD95L on the surface, and because CD3+CD4+CD95L+ cells that were IgG+, C3d+ and/or gp120– were increased in HIV+ patients, the role of CD95L-induced apoptosis in long-term HIV-infected haemophilia patients remains unclear. The findings that HIV+ patients had higher proportions of CD3+CD4+CD95+ (PI+: 6·5%versus 1·4%; P = 0·00002; PI–: 55·8%versus 44·4%; P = 0·04) blood lymphocytes and that the proportion of CD4+IgG+Annexin+7-AAD– blood lymphocytes was associated inversely with peripheral CD4 counts (r = −0·636; P < 0·05) suggest that attachment of IgG to CD4+ blood lymphocytes (anti-CD95?) induces in some lymphocytes apoptosis with subsequent depletion of these IgG-coated apoptotic CD4+ lymphocytes from the circulation. We found supporting evidence for the contention that autoantibody-induced apoptotic and non-apoptotic mechanisms contribute to CD4 depletion in long-term HIV-infected haemophilia patients.
Age-related bias in function of natural killer T cells and granulocytes after stress: reciprocal association of steroid hormones and sympathetic nerves
Authors: SAGIYAMA K , TSUCHIDA M , KAWAMURA H , WANG S , LI C , BAI X , NAGURA T , NOZOE S , ABO T
Published: December 2003

Stress-associated immune responses were compared between young (8 weeks of age) and old (56 weeks) mice. Since stress suppresses the conventional immune system (i.e. T and B cells) but inversely activates the primordial immune system (i.e. extrathymic T cells, NKT cells, and granulocytes), these parameters were analysed after restraint stress for 24 h. The thymus became atrophic as a function of age, and an age-related increase in the number of lymphocytes was seen in the liver. Although the number of lymphocytes in both the thymus and liver decreased as the result of stress, the magnitude was much more prominent in the thymus. To determine stress-resistant lymphocyte subsets, two-colour immunofluorescence tests were conducted in the liver and spleen. NKT cells were found to be such cells in the liver of young mice. On the other hand, an infiltration of granulocytes due to stress was more prominent in the liver of old mice than in young mice. Liver injury as a result of stress was prominent in young mice. This age-related bias in the function of NKT cells and granulocytes seemed to be associated with a difference in the responses of catecholamines (high in old mice) and corticosterone (high in young mice) after stress. Indeed, an injection of adrenaline mainly induced the infiltration of granulocytes while that of cortisol activated NKT cells. The present results suggest the existence of age-related bias in the function of NKT cells and granulocytes after stress and that such bias might be produced by different responses of sympathetic nerves and steroid hormones between young and old mice.
Autoantibodies recognizing citrullinated rat filaggrin in an ELISA using citrullinated and non-citrullinated recombinant proteins as antigens are highly diagnostic for rheumatoid arthritis
Authors: VITTECOQ O , INÇAURGARAT B , JOUEN-BEADES F , LEGOEDEC J , LETOURNEUR O , ROLLAND D , GERVASI G , MÉNARD J F , GAYET A , FARDELLONE P , DARAGON A , JOLIVET M , LE LOËT X , TRON F
Published: December 2003

The objective of the study was to determine the diagnostic value for rheumatoid arthritis (RA) of anti-filaggrin autoantibodies (autoAb) recognizing citrullinated recombinant rat filaggrin (ACRF) in community cases of very early arthritis. To evaluate the diagnostic value of ACRF, were studied sera from patients with different classified rheumatic diseases and healthy subjects (group 1, n= 422) and 314 community cases of very early arthritis (group 2) that were classified as RA (n = 176), non-RA (n = 63) and undifferentiated (n = 75) arthritides after 1 years of follow-up. ACRF were measured using a new ELISA, with results expressed as the difference between the OD value obtained on citrullinated minus that on noncitrullinated rat filaggrin (differential ACRF; dACRF). For both groups, rheumatoid factors (RF), anti-keratin autoAb (AKA) and anti-perinuclear factor (APF) were tested; for group 2, anti-CCP autoAb were also tested. Different reactivity patterns against citrullinated and noncitrullinated filaggrin were observed. Almost all sera reacting with citrullinated but not noncitrullinated filaggrin were from RA patients. Among RA and non-RA sera that recognized both forms of filaggrin, a positive result was obtained only with RA sera. For groups 1 and 2, dACRF sensitivity was 58·4% and 30·7%, and specificity for RA was 99·5% and 98·4%, respectively. In group 2, dACRF specificity for RA was better than that of RF (92·1%), APF (95·2%), AKA (96·8%) and anti-CCP (95·2%). dACRF positive predictive value was high (98·2) and close to that given by the concomitant positivity of RF and anti-CCP autoAb. Despite a high positive correlation between AKA, APF, anti-CCP and dACRF test results, they were complementary since some sera were positive for only one test. Thus, in a community setting, anti-citrullinated rat filaggrin reactivity detected by a new ELISA, whose originality is based on the difference between serum's reactivities on the citrullinated and native forms of filaggrin, had a higher diagnostic value for RA than other autoAb.
Molecular mechanisms of interleukin-10-mediated inhibition of NF-κB activity: a role for p50
Authors: DRIESSLER F , VENSTROM K , SABAT R , ASADULLAH K , SCHOTTELIUS A J
Published: December 2003

Nuclear factor kappa B (NF-κB) is a transcription factor pivotal for the development of inflammation. A dysregulation of NF-κB has been shown to play an important role in many chronic inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease and psoriasis. Although classical NF-κB, a heterodimer composed of the p50 and p65 subunits, has been well studied, little is known about gene regulation by other hetero- and homodimeric forms of NF-κB. While p65 possesses a transactivation domain, p50 does not. Indeed, p50/p50 homodimers have been shown to inhibit transcriptional activity. We have recently shown that Interleukin-10 exerts its anti-inflammatory activity in part through the inhibition of NF-κB by blocking IκB kinase activity and by inhibiting NF-κB already found in the nucleus. Since the inhibition of nuclear NF-κB could not be explained by an increase of nuclear IκB, we sought to further investigate the mechanisms involved in the inhibition of NF-κB by IL-10. We show here that IL-10 selectively induced nuclear translocation and DNA-binding of p50/p50 homodimers in human monocytic cells. TNF-α treatment led to a strong translocation of p65 and p50, whereas pretreatment with IL-10 followed by TNF-α blocked p65 translocation but did not alter the strong translocation of p50. Furthermore, macrophages of p105/p50-deficient mice exhibited a significantly decreased constitutive production of MIP-2α and IL-6 in comparison to wild type controls. Surprisingly, IL-10 inhibited high constitutive levels of these cytokines in wt macrophages but not in p105/p50 deficient cells. Our findings suggest that the selective induction of nuclear translocation and DNA-binding of the repressive p50/p50 homodimer is an important anti-inflammatory mechanism utilized by IL-10 to repress inflammatory gene transcription.
Modulation of antigen presentation by autoreactive B cell clones specific for GAD65 from a type I diabetic patient
Authors: BANGA J P , MOORE J K , DUHINDAN N , MADEC A M , VAN ENDERT P M , ORGIAZZI J , ENDL J
Published: December 2003

We used a GAD65-specific human B–T cell line cognate system in vitro to investigate the modulation of GAD65 presentation by autoantibody, assessed in a proliferation assay. Generally, if the T cell determinant overlaps or resides within the antibody epitope, effects of presentation are blunted while if they are distant can lead to potent presentation. For three different autoreactive B–T cell line cognate pairs, the modulation of GAD65 presentation followed the mode of overlapping or distant epitopes with resultant potent or undetectable presentation. However, other cognate pairs elicited variability in this pattern of presentation. Notably, one B cell line, DPC, whose antibody epitope did not overlap with the T cell determinants, was consistently poor in presenting GAD65. Using the fluorescent dye Alexa Fluor 647 conjugated to GAD65 to study receptor-mediated antigen endocytosis showed that all the antigen-specific B cell clones were efficient in intracellular accumulation of the antigen. Additionally, multicolour immunofluorescence microscopy showed that the internalized GAD65/surface IgG complexes were rapidly targeted to a perinuclear compartment in all GAD-specific B cell clones. This analysis also demonstrated that HLA-DM expression was reduced strongly in DPC compared to the stimulatory B cell clones. Thus the capability of antigen-specific B cells to capture and present antigen to human T cell lines is dependent on the spatial relationship of B and T cell epitopes as well other factors which contribute to the efficiency of presentation.
The cardioprotector dexrazoxane augments therapeutic efficacy of mitoxantrone in experimental autoimmune encephalomyelitis
Authors: WEILBACH F X , CHAN A , TOYKA K V , GOLD R
Published: December 2003

The present study investigates the immunological effects of a combination treatment of mitoxantrone and the cardioprotector dexrazoxane in experimental autoimmune encephalomyelitis (EAE). Mitoxantrone, an anthracycline-derived immunosuppressive drug has been approved recently for treatment of very active multiple sclerosis (MS). Its prolonged use is limited due to its cardiotoxic properties. Dexrazoxane (DZR (S)-(+)-1,2-bis (3,5.dioxopiperazinyl)propane, ICRF-187) is an iron III chelator which in animal models and in cancer patients reduces anthracycline and mitoxantrone induced cardiotoxicity when given immediately before these agents. We examined the immunological effects of dexrazoxane in combination with mitoxantrone in experimental autoimmune encephalomyelitis (EAE) in Lewis rats. EAE was induced by active immunization with myelin basic protein (MBP) or by adoptive transfer of MBP specific T cells (AT-EAE). The clinical course, spinal cord pathology, activity of metalloproteinases (MMP-2 and MMP-9) and T cell apoptosis were assessed. Monotherapy with DZR ameliorated slightly the course of actively induced EAE and AT-EAE. The combination of DZR and mitoxantrone was superior to mitoxantrone given alone. Clinical amelioration ran in parallel with the marked reduction of inflammatory infiltration which was nearly abolished by the combination treatment. DZR did not affect the activity of metalloproteinase 9 and did not increase the proportion of apoptotic lymph node cells ex vivo or T cells in situ. We conclude that in addition to its cardioprotective role, DZR augments mitoxantrone-mediated immunosuppressive effects in animal models of human central nervous system (CNS) autoimmune disease. Clinical trials in MS patients are warranted to evaluate the unexpected immunosuppressive efficacy of DZR as add-on treatment.
Anti-C1q autoantibodies in murine lupus nephritis
Authors: TROUW L A , SEELEN M A , VISSEREN R , DUIJS J M G J , BENEDIKTSSON H , DE HEER E , ROOS A , VAN KOOTEN C , DAHA M R
Published: December 2003

Autoantibodies against C1q can be found in the circulation of patients with several autoimmune diseases including systemic lupus erythematosus (SLE). In SLE there is an association between the occurrence of these antibodies and renal involvement. How anti-C1q autoantibodies contribute to renal disease is currently unknown. Cohorts of MRL-lpr mice, which are known to develop age-dependent SLE-like disease, were used to study the relationship between levels of anti-C1q autoantibodies and renal disease. We collected serum, urine and renal tissue and analysed autoantibodies, complement levels and renal deposition as well as renal function. At 2 months of age all mice already had elevated levels of anti-C1q autoantibodies, and elution of kidneys revealed the presence of these antibodies in renal immune deposits in MRL-lpr mice and not in control MRL+/+ mice. In conclusion, anti-C1q antibodies are already present in serum and immune deposits of the kidney early in life and therefore can play a role in nephritis during experimental SLE-like disease in mice.
Multiple leucocyte activation markers to detect neonatal infection
Authors: HODGE G , HODGE S , HAN P , HASLAM R
Published: December 2003

Diagnosis of congenital or neonatal infection is often based on clinical signs. However, clinical symptoms of infections may not be specific, and for this reason early diagnosis is often determined on results of laboratory tests, which may not currently be adequate. A more reliable method of detection of infection may be the demonstration of activated lymphocytes, which can be conducted rapidly and before the isolation of the infected organism. We have shown that detection of up-regulation of CD45RO, an activated/memory isoform of CD45 present on T cells, provides a reasonably sensitive screening test for neonatal infection. We also showed that dual expression of CD45RA/CD45RO was up-regulated early during the infective process in neonates with documented infection. However, other leucocytes are also activated during the infective process. To improve the sensitivity of the neonatal infection screening test and to identify the types of leucocytes involved in the immune response to the infective organism, we studied further the up-regulation of a comprehensive range of surface activation markers on T cells, monocytes and natural killer (NK) cells from a group of 17 newborn patients with positive culture, a group of 40 possibly infected patients based on clinical signs and a control group. ‘Normal’ ranges were established for each activation marker for each leucocyte subset from 1 to 7 and 7-14-day-old newborns <35 weeks’ gestation and 35-40 weeks’ gestation. There was a significant increase in the percentage of T cells expressing CD25 in the peripheral blood from infants at 2 weeks of age. Expression of HLA-DR on T cells, CD25 and CD69 on monocytes and HLA-DR on NK cells was also increased significantly in the peripheral blood from infants at 2 weeks of age and may reflect a maturation of these functional surface molecules. Up-regulation of CD69 on NK cells was the most sensitive marker for neonatal sepsis (positive in 13/16 patients). CD69 and CD25 expression was increased significantly on T cells in 11/17 and 10/17 patients, respectively. A combination of CD45RA/CD45RO and CD45RO identified 11/16 infected patients. Measurement of CD69 expression on NK cells with CD45RA, CD45RO, CD25 and CD69 expression on T cells resulted in a significant increase in at least two leucocyte activation markers from infected patients. In conclusion, this is the first report of the up-regulation of CD69 on NK cells as a sensitive marker of neonatal infection. A combination of this marker with CD45RA, CD45RO, CD25 and CD69 expression on peripheral blood derived T cells is the most sensitive and specific for neonatal infection.
Application of differential display, with in situ hybridization verification, to microscopic samples of breast cancer tissue
Authors: Kao Ruey Ho , Francia Giulio , Poulsom Richard , Hanby Andrew M. , Hart Ian R.
Published: December 2003

Summary.  The technique of differential display (DD) has been used widely to identify potentially interesting overexpressed or repressed genes in a variety of compared samples. When used in studying tissue samples, it inevitably confronts problems of limited amount of input material and cell‐type heterogeneity. We report here the application of in situ hybridization as a method of confirmatory test for DD as well as definition of cell type expressing differential cDNA. This procedure employed material derived from a single case of human mammary, grade III, infiltrating ductal carcinoma, using free‐hand microdissection, where we have compared gene expression profiles in invasive tumour with those in adjacent normal tissue. A total of 21 cDNAs were found to be differentially expressed between the two tissue types; 11 upregulated in the tumour sample and 10 upregulated in the normal sample. Six cDNAs were utilized as probes for in situ hybridization analysis of a further five cases of comparably staged breast cancer. One of these clones, 11AT1, which was found to be homologous to Hsc70, was shown to be overexpressed in tumour cells relative to adjacent normal stroma and to benign glandular epithelium in all five cases; an increase in expression was further confirmed at protein level by immunohistochemistry. The study demonstrated the applicability of in situ hybridization as a screening test in DD strategy for studying tissue material and a reasonable technique combination of identifying changes in gene expression associated with tumour development.
Roles of inducible nitric oxide synthase in the development and healing of experimentally induced gastric ulcers
Authors: Tatemichi Masayuki , Ogura Tsutomu , Sakurazawa Nobuyuki , Nagata Hiroshi , Sugita Minoru , Esumi Hiroyasu
Published: December 2003

Summary.  The roles of inducible nitric oxide synthase (iNOS) in the development and healing of gastric ulcers have not been fully characterized. We characterized iNOS expression in experimentally induced ulcers in rat and mouse stomachs and investigated the roles of iNOS using iNOS gene‐deficient (iNOS–/–) mice and wildtype mice. Gastric ulcers were induced in rats and mice by the application of acetic acid and cryoinjury, respectively. iNOS expression was detected on days 1–7 and peaked 3 days after ulcer induction in the rat. iNOS‐positive cells were distributed mainly among the infiltrating cells and fibroblasts in the ulcer bed. The almost similar courses of healing and iNOS expression were observed in the ulcers of mice. During the course of healing, the iNOS gene status did not affect cell proliferation in the healing zone or vessel formation in the ulcer bed. iNOS deficiency, however, caused larger ulcers and severer inflammation during ulcer healing; the clearance of inflammatory cells in the ulcer bed by apoptosis was also delayed when the ulcer was re‐epithelialized in the iNOS‐deficient mice. These results indicate that iNOS is expressed in the ulcer bed and that iNOS activity may play beneficial roles in the ulcer repair process, possibly by regulating inflammation.
The endothelin‐1 receptor antagonists ameliorate histology and ultrastructural alterations in the pancreas and decrease trypsinogen activation in severe taurocholate pancreatitis in rats
Authors: Andrzejewska Anna , Dlugosz Jan W.
Published: December 2003

Summary.  The role of potent vasoconstrictor endothelin‐1 (ET‐1) in acute pancreatitis (AP) remains controversial. The aim was to compare the effect of nonselective ET RA/B (LU‐302872) and selective ET RA (LU‐302146) antagonists on pancreatic histology, ultrastructure and trypsinogen activation in severe taurocholate AP in rats. Male Wistar rats with AP were treated with an intraperitoneous injection of 1, 5 and 10 mg/kg of body weight of each antagonist at 0, 6, 12 and 18 h after taurocholate administration. After 24 h, the samples of pancreases were taken for histological and ultrastructural examinations and for assessment of trypsinogen activation. Both antagonists, at all investigated doses, decreased the damage to the acinar cells detected in the light microscope and ultrastructurally. Trypsinogen activation increased to 29.7 ± 3.9% in the AP untreated in comparison to the control group [12.7 ± 1.4% (P < 0.001)]. This increase was attenuated to 13.8 ± 2.2% in AP treated with a high dose of the nonselective antagonist and to 8.4 ± 1.7% with low dose of selective antagonist. The obtained results indicate that ET‐1 could participate in the damage to the pancreas during AP. Both antagonists of ET‐1 receptors exerted a similar beneficial effect on the morphological changes of the pancreas in AP. One of the probable mechanism could be the attenuation of trypsinogen activation.
The effects of lectins on indomethacin‐induced small intestinal ulceration
Authors: Yasuoka Takashi , Sasaki Masaya , Fukunaga Tetsuya , Tsujikawa Tomoyuki , Fujiyama Yoshihide , Kushima Ryouji , Goodlad Robert A.
Published: December 2003

Summary.  Growth factors, such as epidermal growth factor and keratinocyte growth factor, have considerable therapeutic potential for repairing mucosal injury in the intestine when given systemically. Recently, several lectins have been shown to have trophic effects on the intestine when given orally. We examined the effects of phytohaemagglutinin (PHA) and concanavalin A (Con‐A) on indomethacin‐induced intestinal injury in rat. Five‐week‐old rats were randomized to four groups (n = 5), and intestinal injury was induced by indomethacin injection in three of these groups. Elemental diet (ED) feeding was then commenced. The groups were thus ED feeding/indomethacin untreated (control group), ED feeding/indomethacin treated (ED group), 0.1% PHA‐supplemented ED feeding/indomethacin treated (PHA group) and 0.1% Con‐A‐supplemented ED feeding/indomethacin treated (Con‐A group). After 7 days of feeding, macroscopic inflammatory scores, mucosal permeability, myeloperoxidase (MPO) activities and cell proliferation were determined. Macroscopic inflammatory scores, mucosal permeability and MPO activities were significantly lower in both lectin groups than that in control group. Twenty‐four hour excretion rate of phenolsulphonphthalein was significantly lower in both lectin groups than that in ED group. Cell proliferation of the small intestine was significantly increased by both lectins. Lectin supplementation can induce ulcer healing following indomethacin‐induced damage.
Oriented immobilization of Pseudomonas putida putidaredoxin at a gold (111)–buffer interface: a real time scanning tunnelling microscopy study
Authors: Mukhopadhyay R. , Lo K. K. , Wong L. L. , Hill H. A. O.
Published: December 2003

A scanning tunnelling microscopy study of adsorption of wild‐type Pseudomonas putida putidaredoxin at a gold (111)–buffer interface has been made in real time. Reversible adsorption has been observed reflecting weak interaction of the wild‐type protein with a gold (111) electrode. A genetically engineered mutant, C73S‐D58C, which contains a surface thiol, has been used for ‘immobilization’ and ‘orientated adsorption’ on the gold surface. The implication of such orientated immobilization in development of a bio‐electrode surface has been predicted.
φFLIM: a new method to avoid aliasing in frequency‐domain fluorescence lifetime imaging microscopy
Authors: Van Munster E. B. , Gadella T. W. J.
Published: December 2003

In conventional wide‐field frequency‐domain fluorescence lifetime imaging microscopy (FLIM), excitation light is intensity‐modulated at megahertz frequencies. Emitted fluorescence is recorded by a CCD camera through an image intensifier, which is modulated at the same frequency. From images recorded at various phase differences between excitation and intensifier gain modulation, the phase and modulation depth of the emitted light is obtained. The fluorescence lifetime is determined from the delay and the decrease in modulation depth of the emission relative to the excitation. A minimum of three images is required, but in this case measurements become susceptible to aliasing caused by the presence of higher harmonics. Taking more images to avoid this is not always possible owing to phototoxicity or movement. A method is introduced, φFLIM, requiring only three recordings that is not susceptible to aliasing. The phase difference between the excitation and the intensifier is scanned over the entire 360° range following a predefined phase profile, during which the image produced by the intensifier is integrated onto the CCD camera, yielding a single image. Three different images are produced following this procedure, each with a different phase profile. Measurements were performed with a conventional wide‐field frequency‐domain FLIM system based on an acousto‐optic modulator for modulation of the excitation and a microchannel‐plate image intensifier coupled to a CCD camera for the detection. By analysis of the harmonic content of measured signals it was found that the third harmonic was effectively the highest present. Using the conventional method with three recordings, phase errors due to aliasing of up to ± 29° and modulation depth errors of up to 30% were found. Errors in lifetimes of YFP‐transfected HeLa cells were as high as 100%. With φFLIM, using the same specimen and settings, systematic errors due to aliasing did not occur.
Analysis of boron‐10 in soft tissue by dynamic secondary ion mass spectrometry
Authors: Oyedepo A. C. , Brooke S. L. , Heard P. J. , Day J. C. C. , Allen G. C. , Patel H.
Published: December 2003

We report here a preliminary study in which dynamic secondary ion mass spectrometry (SIMS) has provided images of boron‐10 (10B) in biological tissue as used in research into boron neutron capture therapy. Cultured tumour cells incubated in media containing known concentrations of a 10B‐containing compound, p‐boronophenylalanine (BPA), and intracranial tumour tissue from animals previously injected with BPA were analysed by an in‐house constructed SIMS. Investigations were conducted in positive secondary ion detection mode using a 25‐keV, 5‐nA gallium primary ion source. For calibration purposes, tissue standards were also analysed and their boron‐to‐carbon signal ratios correlated to bulk boron concentrations measured by inductively coupled plasma atomic emission spectroscopy (ICP‐AES). Ion maps of 10B, 12C, 23Na and 39K showing gross tissue and cell features were acquired. SIMS and ICP‐AES standard measurements were in good agreement. Tissue regions with high or low 10B concentrations were identified along with 10B hotspots in normal brain areas. Cultured cells revealed the intracellular localization of 10B. SIMS is capable of producing images showing the distribution of 10B at p.p.m. levels in cells and in normal and tumour‐bearing brain tissue.
Optimizing imaging parameters for the separation of multiple labels in a fluorescence image
Authors: Neher R. , Neher E.
Published: December 2003

A theoretical analysis is presented on how to separate the contributions from individual, simultaneously present fluorophores in a spectrally resolved image. Equations are derived that allow the calculation of the signal‐to‐noise ratio of the estimates for such contributions, given the spectral information on the individual fluorophores, the excitation wavelengths and intensities, and the number and widths of the spectral detection channels. We then ask how such imaging parameters have to be chosen for optimal fluorophore separation. We optimize the signal‐to‐noise ratio or optimize a newly defined ‘figure of merit’, which is a measure of efficiency in the use of emitted photons. The influence of photobleaching on the resolution and on the choice of imaging parameters is discussed, as well as the additional resolution gained by including fluorescence lifetime information. A surprisingly small number of spectral channels are required for an almost optimal resolution, if the borders of these channels are optimally selected. The detailed consideration of photobleaching is found to be essential, whenever there is significant bleaching. Consideration of fluorescence lifetime information (in addition to spectral information) improves results, particularly when lifetimes differ by more than a factor of two.
Freeze‐substitution and low‐temperature embedding of dairy products for transmission electron microscopy
Authors: Smith A. K. , Goff H. D. , Sun B. D.
Published: December 2003

Dairy products are comprised largely of fat, air and water, which makes it difficult to preserve their ultrastructure for electron microscopy. Keeping the samples frozen throughout fixation and embedding protects the structure and distribution of the components of emulsions and foams. Therefore, dairy products were freeze‐substituted and embedded at low temperature (−20 °C) to prepare them for transmission electron microscopy. Whipped cream, ice cream mix and dairy/non‐dairy mixed systems were frozen by plunging in propane, at its boiling point (−187 °C). Ice cream, because it is already frozen, was fractured into 1‐mm3 pieces in liquid nitrogen and then added to frozen fixative (−196 °C). Fixative solution consisted of glutaraldehyde, osmium tetroxide and uranyl acetate dissolved in either methanol or acetone. When material was to be stained after sectioning the fixative was limited to glutaraldehyde in methanol. The temperature was increased step‐wise from −80 to −20 °C. Solvent was replaced with resin; the polar resin Lowicryl HM4, the non‐polar resin Lowicryl HM20, LR White and LR Gold were tested. Samples were embedded and polymerized at −20 °C using ultraviolet light to cross‐link the resin. Methanol proved to be the most effective solvent for substituting the ice; the hydrophobic resin Lowicryl HM20 was the most effective resin for retaining fat structure following osmium fixation.
Graphical representation and multicomponent analysis of single‐frequency fluorescence lifetime imaging microscopy data
Authors: Clayton A. H. A. , Hanley Q. S. , Verveer P. J.
Published: December 2003

Graphical representation of fluorescence lifetime imaging microscopy data demonstrates that a mixture of two components with single exponential decays can be resolved by single frequency measurements. We derive a method based on linear fitting that allows the calculation of the fluorescence lifetimes of the two components. We show that introduction of proper error‐weighting results in a non‐linear method that is mathematically identical to a global analysis algorithm that was recently derived. The graphical approach was applied to cellular data obtained from a lifetime‐based phosphorylation assay for the epidermal growth factor receptor and yielded results similar to those obtained by a global analysis algorithm.
Measurement of specimen‐induced aberrations of biological samples using phase stepping interferometry
Authors: Schwertner M. , Booth M. J. , Neil M. A. A. , Wilson T.
Published: December 2003

Confocal or multiphoton microscopes, which deliver optical sections and three‐dimensional (3D) images of thick specimens, are widely used in biology. These techniques, however, are sensitive to aberrations that may originate from the refractive index structure of the specimen itself. The aberrations cause reduced signal intensity and the 3D resolution of the instrument is compromised. It has been suggested to correct for aberrations in confocal microscopes using adaptive optics. In order to define the design specifications for such adaptive optics systems, one has to know the amount of aberrations present for typical applications such as with biological samples. We have built a phase stepping interferometer microscope that directly measures the aberration of the wavefront. The modal content of the wavefront is extracted by employing Zernike mode decomposition. Results for typical biological specimens are presented. It was found for all samples investigated that higher order Zernike modes give only a small contribution to the overall aberration. Therefore, these higher order modes can be neglected in future adaptive optics sensing and correction schemes implemented into confocal or multiphoton microscopes, leading to more efficient designs.