[Detection by flow cytometry of T cell subsets secreting IL-2 and UFNy(Gamma): optimalisation for the technic and the establishment of reference values]. / Détection par cytométrie en flux des sous-populations lymphocytaires T sécrétrices d'IL-2 et IFN-gamma : optimisation de la technique et établissement des valeurs de référence.

A dysregulation in Th1/Th2 balance has been described for different pathological situations. Knowing the cytokine profile in a given pathology could assist in understanding the disease mechanism and in choosing an immune intervention most effective for the management of this condition. In this work, the production of two Th1 cytokines, IL-2 and IFN- gamma, was analyzed for different T-cell subsets from 20 normal subjects (mean age 33.5 years) and reference values were defined using the flow cytometric analyses. The optimum operating conditions were set as following: mononuclear cells were stimulated with PMA (20 ng/ml) and ionomycin (1 uM) for 6 h in the presence of brefeldin A (10 ug/ml). Cells were fixed with 4% paraformaldehyde and then dually stained, with anti-CD3 or anti-CD4 or anti-CD8 for the membrane and with anticytokine antibody for the intracytoplasma after being permeabilized with 0.5% saponine solution. The frequency determination of cells that produce IL-2 or IFN-gamma revealed large 95% confidence intervals: (CD3-IL-2: 4.60-10.67%, CD8-IL-2: 1.47-23%, CD3-IFN-gamma: 2,97-32,49%, CD4-IFN-gamma: 2.83-21%, CD8-IFN-gamma: 4.60-35.28%). CD4+ lymphocytes produce the majority of IL-2 (85 vs 13% for CD8+). For IFN-gamma, the situation is more balanced, but the CD4+ lymphocytes remain the predominant producer cells (63 vs. 41%).

Association between anticardiolipin antibodies and mortality in patients with peripheral arterial disease.

PURPOSE: Anticardiolipin antibodies may be associated with recurrent thromboembolic events in patients with myocardial infarction or stroke. We sought to determine the prevalence of anticardiolipin antibodies in patients with peripheral arterial disease and their association with subsequent thromboembolic events and mortality. METHODS: We ascertained anticardiolipin antibodies using a standardized enzyme-linked immunosorbent assay (immunoglobulin G [IgG] anticardiolipin > or =15 GPL units or IgM anticardiolipin > or =15 MPL units) in 232 patients with peripheral arterial disease and 100 control subjects. Patients were observed to determine overall and cardiovascular mortality, and incident thromboembolic events. RESULTS: IgG anticardiolipin antibodies were significantly more common in the patients with peripheral arterial disease (36 of 232 [16%]) than in the controls (7 of 100 [7%], P = 0.03). During a median follow-up of 3.5 years, 3 of the 232 patients were lost to follow-up and 56 (24%) died. Overall mortality was significantly greater in the IgG anticardiolipin-positive patients (16 of 35 [46%]) compared with those who were IgG anticardiolipin-negative (40 of 194 [21%], P = 0.0003), largely due to an increase in cardiovascular mortality among the IgG anticardiolipin-positive patients. In a multivariate proportional hazards analysis, IgG anticardiolipin antibodies were an independent risk factor for overall mortality (hazard ratio [HR] = 2.1, 95% confidence interval [CI]: 1.2 to 4.0) and cardiovascular mortality (HR = 4.4, 95% CI: 1.6 to 12). CONCLUSIONS: IgG anticardiolipin antibodies are common in patients with peripheral arterial disease and are associated with an increased risk of overall and cardiovascular mortality.

Enzyme-linked immunosorbent assays using the recombinant dense granule antigens GRA6 and GRA1 of Toxoplasma gondii for detection of immunoglobulin G antibodies.

The potential of the dense granule antigens GRA1 and GRA6 of Toxoplasma gondii to be used as diagnosis reagents in a recombinant form was evaluated. Both proteins were expressed in Escherichia coli as glutathione-S-transferase (GST) fusions. The GST-GRA1 fusion comprises the entire GRA1 sequence devoid of its N-terminal signal peptide. Separate expression of the two N- and C-terminal hydrophilic regions of GRA6 showed that only the N-terminal hydrophilic part of the protein was recognized by a pool of positive human sera in an immunoblot. One hundred T. gondii-positive and 98 negative human sera were tested in two separate immunoglobulin G (IgG)-direct enzyme-linked immunosorbent assays (ELISAs) using either GST-GRA1 or GST-GRA6-Nt recombinant protein. Whereas the sensitivity of the GST-GRA1 IgG ELISA was low (68%), the GST-GRA6-Nt IgG ELISA reached a sensitivity of 96%. The reactivity to GRA6-Nt was shown to be high even with human sera of low IgG titers. In addition, comparison of the optical density values for each serum revealed that GRA1 may complement GRA6-Nt to reach an overall sensitivity of 98%. Therefore, the GST-GRA6-Nt ELISA could be used together with another antigen like GRA1 for the development of a recombinant antigen-based test for serodiagnosis of toxoplasmosis.

Anti-neutrophil cytoplasmic auto-antibodies (ANCA) in ulcerative colitis (UC): no relationship with disease activity.

The relationship between anti-neutrophil cytoplasmic auto-antibodies (ANCA) and disease activity in inflammatory bowel diseases remains controversial. The aim of this study was to highlight the relationship between ANCA presence or titers and disease activity in ulcerative colitis (UC). Three groups of patients with UC were studied: 1) group A included 39 patients who had not undergone colectomy, 2) group B, 43 patients with subtotal colectomy and ileo-rectal anastomosis, 3) group C, 98 patients with proctocolectomy and ileo-anal anastomosis, including 88% with pouchitis and 12% without pouchitis at the time of the study. Determination of ANCA was performed using the standardized indirect immunofluorescence assay. ANCA were positive in 59%, 65%, and 54% of patients from groups A, B, and C, respectively (NS). No relationship between ANCA presence or titers and UC activity could be detected within groups A and B. In group C, 45 of 86 patients (52%) without pouchitis and 8 of 12 patients (67%) with pouchitis, were ANCA positive (NS). These results do not support a relationship between ANCA and UC activity in this cohort of 180 patients.

[Prevalence of antiphospholipid antibodies in Horton's disease and in polymyalgia rheumatica]. / Prévalence des anticorps antiphospholipides dans la maladie de Horton et dans la pseudo-polyarthrite rhizomélique.

PURPOSE: Antiphospholipid antibodies (APL) are frequently observed in the course of giant cell arteritis and polymyalgia rheumatica. However, their role and relationships with potential ischemic events are still debated. METHODS: To determine the prevalence of APL in relation with ischemic events, 62 patients with giant cell arteritis and/or polymyalgia rheumatica were retrospectively studied. RESULTS: Before corticosteroid treatment 41% of the 51 patients with giant cell arteritis and 64% of the 11 patients with isolated polymyalgic rheumatica had high IgG ACL levels, with a frequency significantly higher than that (15.6%) of the control group which was composed of healthy elderly. IgM ACL were found in only two patients with giant cell arteritis. There was no correlation between the occurrence of an ischemic event (22 ischemic events in the 51 patients with giant cell arteritis) and the presence of ACL, even though the latter were more frequently observed in the giant cell arteritis group (52% versus 41% in non ischemic patients). The prevalence of ACL increased, reaching 59% if lupus anticoagulant was associated, but the difference was not significant. ACL disappeared soon after corticosteroid therapy had been initiated. CONCLUSION: Though ACL are frequently seen in giant cell arteritis and/or polymyalgia rheumatica, they are not related to ischemic events and disappear rapidly after corticosteroid treatment.

[Multiple sclerosis and antiphospholipid antibodies: study of 62 consecutive patients]. / Sclérose en plaques et anticorps antiphospholipides: étude consécutive de 62 patients.

INTRODUCTION: Although multiple sclerosis (MS) and antiphospholipid syndrome (AS) are usually defined by specific criteria that make them distinguishable, in some cases, transition between the two diseases based on clinical and brain imaging findings is not clear. METHODS: Our study included 62 patients (sex ratio F/M = 1.48; mean age 43.4 +/- 23.6 years) with diagnosis of MS according to Poser criteria and 31 control subjects (sex ratio F/M = 9.3, mean age 37 +/- 17 years). We examined the level of antibodies against phospholipids (anticardiolipid, anti beta 2-glycoprotein 1 and antiphosphatidylethanolamine antibodies), antinuclear, anti native DNA, antiprothrombinase antibodies and rheumatoid factor. RESULTS: Antiphospholipid antibodies were found with a significant level (anticardiolipid > 30 UI, anti beta 2-glycoprotein 1 positive) in only five patients (8%) with MS; two others showed an increase in antinuclear antibodies (1/320 degrees and 1/1280 degrees). CONCLUSION: In contrast with data recently reported, this study failed to find a significant level of antiphospholipid antibodies in MS. This result argues for the existence of different pathogenic mechanisms in MS and AS.

Enhancing the effect of secreted cyclophilin B on immunosuppressive activity of cyclosporine.

BACKGROUND: Cyclophilin B (CyPB) is a cyclosporine (CsA)-binding protein, located within intracellular vesicles and secreted in biological fluids. In previous works, we reported that CyPB specifically interacts with the T-cell membrane and potentiates the ability of CsA to inhibit CD3-induced proliferation of T lymphocytes. METHODS: CyPB levels were measured in plasma from healthy donors and transplant patients. The role of extracellular CyPB on the distribution and activity of CsA was investigated first by studies on the uptake of free and CyPB-complexed drug by blood cells, and second by studies on the inhibitory effects of these two compounds on the CD3-induced proliferation of peripheral blood mononuclear cells. RESULTS: A significant increase in plasma CyPB level was observed for CsA-treated patients (13+/-6.4 nM, n=42) in comparison with untreated donors (4.3+/-2.1 nM, n=34). In vitro, extracellular CyPB dose dependently modified CsA distribution between plasma, erythrocyte, and lymphocyte contents, by both retaining the complexed drug extracellularly and promoting its specific accumulation within peripheral blood mononuclear cells. Moreover, the enhanced ability of CyPB-complexed CsA to suppress CD3-induced T-cell proliferation was preserved in the presence of other blood cells, implying specific targeting of the drug to sensitive cells. Furthermore, although a large interindividual variability of sensitivity to the drug was confirmed for 18 individuals, we found that CyPB potentiated the activity of CsA in restoring a high sensitivity to the immunosuppressant. CONCLUSION: These results suggest that plasma CyPB may contribute to the acceptance and the good maintenance of organ transplantation by enhancing the immunosuppressive activity of CsA through a receptor-mediated incorporation of CyPB-complexed CsA within peripheral blood lymphocytes.

Cerebral magnetic resonance imaging in patients with or without antiphospholipid antibodies.

OBJECTIVE: To determine in patients with systemic lupus erythematosus (SLE) or with primary antiphospholipid syndrome (PAPS) the prevalence of cerebral magnetic resonance imaging changes (MRI) and the relationship with antiphospholipid antibodies. METHODS: Twenty-nine consecutive SLE patients, 24 PAPS patients and 31 healthy controls were prospectively included in the study and underwent MRI Scan over a 1-year period. MRI scans were analyzed separately by a neuroradiologist for white matter changes [periventricular hyperintensity (PVH) (0-6 scale), deep white matter hyperintensity (WMH) (0-24 scale)], and one neurologist for cerebral atrophy (0-39 scale) and stroke subtypes. Statistical assessment consisted of a discriminant analysis performed with SAS-package with MRI data as dependent variables and, as independent variables, age, sex, arterial hypertension, diabetes mellitus, cardiopathy, migraine, neurological symptoms, antiphospholipid antibodies, SLE, steroid treatment. RESULTS: The prevalence of cerebral atrophy was increased in both SLE and PAPS groups relative to controls. PVH and WMH scores were significantly higher in SLE and PAPS than in controls. Focal infarct did not differ in the SLE group when compared with PAPS. PVH and WMH scores were significantly higher in patients with neurological symptoms. Using a correlation test we found a weak significant correlation between cerebral atrophy and lupus anticoagulant. The multivariate analysis found only three independent variables related to PVH and WMH: age, the diagnosis of SLE and cerebral atrophy. CONCLUSIONS: Age, presence of SLE and presence of neurological symptoms were independently related with WMH and PVH, but not antiphospholipid antibodies.

Immunoglobulin G subclass distribution of anti-endothelial cell antibodies (AECA) in patients with ulcerative colitis or Crohn's disease.

Anti-endothelial cell antibodies have been described in sera from patients with inflammatory bowel disease. The aim of this study was to determine, by ELISA, the IgG subclass distribution of anti-endothelial cell antibodies, in patients with ulcerative colitis (N = 28) or Crohn's disease (N = 82) as compared with blood donors (N = 95). Thirty-six percent of ulcerative colitis and 23% of Crohn's disease patients were positive for at least one of the IgG anti-endothelial cell subclasses. Interestingly, the pattern of IgG anti-endothelial cell subclass observed in the two inflammatory bowel diseases differs. In Crohn's disease, the IgG1 anti-endothelial cell antibody level was significantly increased (P < 0.05) while IgG2 and IgG4 anti-endothelial cell antibody levels were decreased (P < 0.0001 and P < 0.01, respectively) as compared to ulcerative colitis patients. The immunoglobulin G3 anti-endothelial cell antibody level was decreased in both ulcerative colitis and Crohn's disease patients as compared to healthy blood donors. No relationship was detected between disease activity of ulcerative colitis or Crohn's disease patients and anti-endothelial cell IgG subclasses. Finally, the disparity of IgG anti-endothelial cell subclass distribution in these two inflammatory bowel diseases suggests that the ability to activate effector mechanisms is not identical, and hence, deals with the concept of distinctive pathogenetic mechanisms in these two diseases.

Elastase derived elastin peptides: putative autoimmune targets in giant cell arteritis.

OBJECTIVE: Histological analysis of giant cell arteritis (GCA) reveals a granulomatous reaction around the internal elastic lamina. Elastolysis by multinucleated giant cells has also been reported. We investigated elastin derived peptides as putative recall antigens for peripheral blood mononuclear cells (PBMC) from patients with GCA. METHODS: PBMC were collected from 17 patients with GCA (Group 1), 17 patients with vascular diseases, connective tissue diseases, or polymyalgia rheumatica without GCA (Group 2), and 17 healthy controls (Group 3). Cultures of PBMC with different elastin derived peptides or elastase were analyzed. RESULTS: A proliferative response was obtained only with elastate derived elastin peptides in 12/13 untreated patients with GCA. Steroid treatment was believed to abolish this proliferative response in 4 patients with GCA. PBMC from only 3/34 non-GCA subjects responded to these antigens. No proliferative response was obtained for other elastin derived peptides or elastase in any subject. CONCLUSION: Degradation of native elastin by leukocyte elastase can provide elastin derived peptides that act as autoimmune targets for T cells in GCA.

[Autoimmune hepatitis treated with cyclosporin revealed by acute hepatocellular failure]. / Traitement par la ciclosporine d'une hépatite auto-immune révélée par une insuffisance hépatocellulaire aiguë.

BACKGROUND: Autoimmune hepatitis (AIH), usually chronic hepatitis, can be revealed by an acute episode of hepatic failure not easily treatable. CASE REPORT: A 4 year-old boy presented with severe acute hepatic failure (AHF) without hypergammaglobulinemia, revealing AIH. A triple immunosuppressive treatment (prednisolone, azathioprine, ciclosporine) was started when clinical and biological signs worsened (prothrombin time 18%, factor V 32%). Liver functions significantly improved within 10 days, although complications due to intensive immunosuppression occurred (Candida septicemia). CONCLUSION: AIH must be evocated in each case of AHF. Some observations of AIH treated with ciclosporine are published but protocols of its administration are variable: ciclosporine is used alone or associated with other drugs, in first intention or secondarily, when the classical treatment is contra-indicated or fails. Controlled studies are needed to precise ciclosporine indications in AIH and to propose the best protocol.

Implication of cyclosporine in up-regulation of Bcl-2 expression and maintenance of CD8 lymphocytosis in cytomegalovirus-infected allograft recipients.

T cell homeostasis and CD4/CD8 ratios are normally reestablished by apoptotic clearance of activated T cells after immune stimulation. In allograft recipients with cytomegalovirus infection, CD8 lymphocytosis persists after negativation of viral cultures, contrary to immunocompetent hosts. We investigated the expression of Bcl-2 protein, an intracellular suppressor of apoptosis, and of CD95 (APO-1/Fas), a membrane inducer of apoptosis, in peripheral blood lymphocytes from 45 solid organ recipients. During the viremic phase of CMV infection, we found absence or diminished expression of Bcl-2 protein and increased expression of CD95 antigen in activated CD8+ T cells. Opposite evolution of these molecular regulators of apoptosis was reflected by the presence of 10-25% of apoptotic lymphocytes with fragmented DNA, as shown by both in situ nick translation and electrophoresis. Normalization of Bcl-2 expression was progressive over several months but still lower than in uninfected allograft recipients. These results suggest that the initial evolution of CMV infection in allograft recipients resembles acute viral infection in immunocompetent hosts. Conversely, we showed that overexpression of Bcl-2 protein in lymphocytes from uninfected allograft recipients, and culture of unstimulated normal lymphocytes with 0.5 micrograms/ml cyclosporine led to an increase in the expression of intracellular Bcl-2. This up-regulation of Bcl-2 protein by cyclosporine suggests the acquisition of resistance to apoptosis. Thus, the reversion of balance between T cell death and survival after acute CMV infection might be impeded by cyclosporine. Combination of CMV latent infection and cyclosporine therapy appears therefore critical to shift the homeostatic maintenance of the peripheral lymphocyte compartment toward persistingly high numbers of CD8+ T cells.

Characterization, quantification, and localization of passenger T lymphocytes and NK cells in human liver before transplantation.

Quantification and localization of the main lymphocyte populations were studied in the livers of normal (n = 8) and brain dead (n = 8) subjects. Cytometric analysis performed on mononuclear cell suspensions obtained from liver biopsies was compared to an automatic image analysis of immunostained sections. The overall number of liver associated lymphocytes was in the usual range of peripheral blood content (2 to 9x10(9) cells). Phenotypic analysis showed predominant NK and CD8+ cells that highly expressed class II antigen and CD25 and CD69 activation markers. Quantitative mapping of these activated lymphocytes revealed their preferential localization in the portal tract and the perisinusoidal area as compared to the pericentrolobular zone, especially in donor livers. This strategic localization could suggest a possible early cooperation between donor lymphocytes and initial infiltrating cells from the recipient and could explain the special immunological status of allografted livers.

Individual differences in the in vitro response to cyclosporin A (CsA): possible heterogeneity in the involvement of the CD28-B7/BB1 pathway.

Differences in the response of graft recipients to the immunosuppressive effect of cyclosporin A (CsA) represent a major factor of allograft acceptance. Response to CsA was investigated in vitro in 59 healthy subjects by measuring the inhibition of lymphocyte proliferation and interleukin-2 (IL-2) production. Marked differences were found comparing individuals: 61% of the subjects responded with half inhibitory doses ID50 < 200 ng/ml (responder group), 20% with ID50 within 200-400 ng/ml (intermediate responder) and 19% were non responder with ID50 > 400 ng/ml and sometimes over 1,000 ng/ml. To explain these differences, the CD28-B7, co-stimulatory pathway for T-cell activation was explored since it is the only CsA-resistant pathway known so far. Both responder and non responder individuals showed increased proliferative response and IL-2 secretion by co-stimulation with CD3 and CD28, resulting in increased ID50 by a similar factor. The percentage of CD28+ cells within T-lymphocytes varied markedly among subjects (48.5 +/- 28.9% of the CD8+ cells). However we could not correlate the inter-individual variation of sensitivity to CsA to the size of the CD8+CD28+ T cell subset nor to divergent response to CD3 and CD28 co-stimulation.