Odette Viñas

Human hepatic stellate cells show features of antigen‐presenting cells and stimulate lymphocyte proliferation

Following cell activation, hepatic stellate cells (HSCs) acquire proinflammatory and profibrogenic properties. We investigated whether activated HSCs also display immune properties. Here we show that cultured human HSCs express membrane proteins involved in antigen presentation, including members of the HLA family (HLA-I and HLA-II), lipid-presenting molecules (CD1b and CD1c), and factors involved in T-cell activation (CD40 and CD80). Exposure of HSCs to proinflammatory cytokines markedly up-regulates these molecules. Importantly, cells freshly isolated from human cirrhotic livers (in vivo activated HSCs) highly express HLA-II and CD40, suggesting that HSCs can act as antigen-presenting cells (APCs) in human fibrogenesis. We also explored whether human HSCs can efficiently process exogenous antigens. Activated HSCs internalize low- and high-molecular-weight dextran and transferrin, indicating that they can perform fluid-phase and receptor-mediated endocytosis. Moreover, HSCs can perform phagocytosis of macromolecules because they internalize latex particles as well as bacteria. Interestingly, both culture-activated and in vivo activated HSCs express high levels of CD68, a protein involved in antigen trafficking. Finally, we studied whether HSCs modulate T-lymphocyte proliferation. In basal conditions, coculture of irradiated HSCs barely induces allogeneic T-lymphocyte proliferation. However, cytokine-stimulated HSCs stimulate the allogeneic T-lymphocyte response in an HLA-II-dependent manner. In conclusion, human activated HSCs express molecules for antigen presentation, internalize macromolecules, and modulate T-lymphocyte proliferation. These results suggest that HSCs may play a role in the immune function of the liver.

Cross-reactivity of anti-Mycobacterium gordonae antibodies with the major mitochondrial autoantigens in primary biliary cirrhosis

Primary biliary cirrhosis is a chronic cholestatic liver disease associated with autoimmune disorders. Antimitochondrial autoantibodies and granulomatous portal lesions are characteristic in primary biliary cirrhosis. Since granuloma may be induced by Mycobacteria, and there is evidence implicating Mycobacteria as infectious agents capable of initiating autoimmunity, a study was performed to determine the presence of antibodies against 10 atypical Mycobacteria in 19 patients with primary biliary cirrhosis, and in 35 controls (25 patients with other chronic liver diseases and 10 healthy subjects). All primary biliary cirrhosis sera and none of the controls reacted with the extract from Mycobacterium gordonae, showing identical recognition profiles with two polypeptides of 70-65 and 55 kDa. No other reaction was found in primary biliary cirrhosis patients and in controls with the extracts from the other nine atypical Mycobacteria tested. Eluted immunoglobulins which reacted with the 70-65 and 55 kDa polypeptides from M. gordonae, bound to the mitochondrial antigens PDH-E2 and BCKDH-E2. Furthermore, when the extract from M. gordonae was tested with eluted immunoglobulins from recognized PDH-E2 and BCKDH-E2 by primary biliary cirrhosis patients, we observed both 70-65 and 55 kDa polypeptides. These data indicate that antibodies to M. gordonae, found in all primary biliary cirrhosis patients, cross-react with the major mitochondrial targets of the disease. We suggest that M. gordonae may play a potential pathogenic role in primary biliary cirrhosis.

Antibodies to mycobacterial 65-kD heat shock protein cross-react with the main mitochondrial antigens in patients with primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease associated with autoimmune disorders. The aetiology is unknown, although it has been suggested that the disease may be related to infectious agents. Previous studies revealed that sera from patients with PBC react against Mycobacterium gordonae. This specific reactivity, characterized by a recognition of two membrane polypeptides of 70–65 and 55 kD, cross‐react with the two major mitochondrial autoantigens of PBC. As the most immunogenic components of mycobacteria are the heat shock proteins (hsp), which have been associated with autoimmunity, this study has been undertaken to characterize whether the reacting polypeptides in PBC are hsp from M. gordonae. Cultures of M. gordonae were incubated at 37 °C and 46 °C before sonication, protein extraction and separation by SDS‐PAGE. Exposure of M. gordonae to heat shock treatment resulted in membrane protein overexpression, similar to the 70–65‐kD polypeptide recognized by the sera from patients with PBC. Immunoprecipitation assays with a monoclonal antibody directed against the Hsp65 kD of mycobacteria and with sera from patients with PBC revealed similar reacting profiles characterized by the precipitation of the overexpressed 65‐kD polypeptide from M. gordonae. Competitive immunoblotting showed that binding of the monoclonal antibody to the Hsp65 kD protein was prevented by preincubation with sera from patients with PBC, but not with sera from healthy subjects. Furthermore, monoclonal antibody to the Hsp65 kD protein recognized the main mitochondrial autoantigens of PBC (PDH‐E2 and BCKDH‐E2). These data indicate the existence of cross‐reacting epitopes contained on M. gordonae Hsp65 kD and the main mitochondrial antigens in patients with PBC.

Diagnostic and prognostic value of antibodies against chimeric fibrin/filaggrin citrullinated synthetic peptides in rheumatoid arthritis

IntroductionEvidence suggests that citrullinated fibrin(ogen) may be a potential in vivo target of anticitrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis (RA). We compared the diagnostic yield of three enzyme-linked immunosorbent assay (ELISA) tests by using chimeric fibrin/filaggrin citrullinated synthetic peptides (CFFCP1, CFFCP2, CFFCP3) with a commercial CCP2-based test in RA and analyzed their prognostic values in early RA.MethodsSamples from 307 blood donors and patients with RA (322), psoriatic arthritis (133), systemic lupus erythematosus (119), and hepatitis C infection (84) were assayed by using CFFCP- and CCP2-based tests. Autoantibodies also were analyzed at baseline and during a 2-year follow-up in 98 early RA patients to determine their prognostic value.ResultsWith cutoffs giving 98% specificity for RA versus blood donors, the sensitivity was 72.1% for CFFCP1, 78.0% for CFFCP2, 71.4% for CFFCP3, and 73.9% for CCP2, with positive predictive values greater than 97% in all cases. CFFCP sensitivity in RA increased to 80.4% without losing specificity when positivity was considered as any positive anti-CFFCP status. Specificity of the three CFFCP tests versus other rheumatic populations was high (> 90%) and similar to those for the CCP2. In early RA, CFFCP1 best identified patients with a poor radiographic outcome. Radiographic progression was faster in the small subgroup of CCP2-negative and CFFCP1-positive patients than in those negative for both autoantibodies. CFFCP antibodies decreased after 1 year, but without any correlation with changes in disease activity.ConclusionsCFFCP-based assays are highly sensitive and specific for RA. Early RA patients with anti-CFFCP1 antibodies, including CCP2-negative patients, show greater radiographic progression.

Antiphospholipase A2 Receptor Antibody Levels Predict the Risk of Posttransplantation Recurrence of Membranous Nephropathy.

Background Secretory phospholipase A2 receptor (PLA2R) is the target antigen of the auto-antibodies produced in most (∼70%) patients with primary membranous nephropathy (pMN). The applicability of anti-PLA2R1 antibody monitoring for the prediction of MN recurrence in kidney transplant recipients still is a matter of debate. Methods We sought to characterize the presence and concentration of anti-PLA2R antibodies by enzyme-linked immunosorbent assay (ELISA) in a cohort of 21 patients with pMN before and after transplantation to evaluate whether anti-PLA2R concentrations could predict pMN recurrence. Results The presence of pMN recurrence was significantly correlated with the existence of a positive ELISA assay at graft biopsy or with high level of anti-PLA2R1 activity before transplantation (P = 0.03). In the receiver operating characteristic analysis, anti-PLA2R levels (cut-off of 45 U/mL) during the pretransplantation period accurately predicted pMN recurrence, with a sensitivity of 85.3%, specificity of 85.1%, negative predictive value of 92%, and an area under the curve of 90.8%. This finding supports the hypothesis that anti-PLA2R cause pMN recurrence in humans and indicates the need to prove in an experimental model. Furthermore, 6 of 7 patients with recurrence were carriers of HLA DQA1* 05:01/05 and DQB1* 02:01, confirming these DQ alleles as those associated with higher anti-PLA2R levels. Conclusions This study is the first to demonstrate pretransplantation circulating anti-PLA2R antibodies in a cohort of renal transplant recipients who prospectively developed recurrent disease. Currently, anti-PLA2R levels measured by ELISA may be a rational tool to establish the risk of MN recurrence in renal allograft recipients.

Effects of smoking on disease activity and radiographic progression in early rheumatoid arthritis.

Objective. To analyze the effects of cigarette smoking on disease activity and radiographic damage in patients with early rheumatoid arthritis (RA). Methods. Study subjects were 156 patients with early RA (< 2 yrs). Disease activity, therapeutic response, and radiographic progression were compared in smokers and nonsmokers at 24 months. Results. At baseline, ever-smokers had earlier disease onset and a closer association with the shared epitope (SE), but not more seropositive disease. No significant differences were observed in disease activity and European League Against Rheumatism therapeutic responses between smokers and nonsmokers. Multivariate analysis showed that baseline Larsen score, the HLA-DRB*04 genotype, being female, and current smoking were associated with radiographic progression. Conclusion. In patients with early RA, smoking was associated with earlier disease onset and the SE. Smoking was an independent factor of radiographic progression.

Increased CD5-positive B lymphocytes in type I diabetes.

CD5+ B lymphocytes have been implicated in the production of polyspecific and monospecific antibodies that bind self‐antigens, and increased proportions of this B cell subset occur in patients with some autoimmune diseases. We investigated the proportion of peripheral blood CD5+ B lymphocytes in type I diabetic patients. Compared with 18 age‐matched healthy subjects. 11 out of 28 (39.2%) type I diabetic patients had increased proportions of circulating CD5+ B lymphocytes with no alterations in the numbers of circulating B and T lymphocytes. Although all patients with increased CD5 B lymphocytes also had serum islet cell antibodies and/or insulin autoantibodies. the occurrence of increased proportions of CD5+ B lymphocytes and scrum autoantibodies was not significantly correlated. Increased proportions of CD5+ B lymphocytes was not related to the time elapsed since the clinical onset of diabetes. In addition, regardless of being increased or normal, the proportion of CD5+ B lymphocytes appeared as a relatively constant phenotype after 1 year of follow‐up studies at 3‐month intervals in eight patients. Although the significance of these findings remains to be established, the possibility exists that CD5+ B cells play a role in the pathogenesis of type I diabetes.

Follow-up study of the revascularization process of purified rat islet beta-cell grafts

The revascularization of islets of Langerhans transplanted in heterotopic sites like the liver by portal vein embolization or the renal subcapsular space is a major process necessary for the viability of grafted cells. This process has been extensively studied by different techniques and the results have shown that islet revascularization is an early phenomenon that takes place soon after transplantation. In this report we have analyzed by a double indirect immunofluorescence technique, the revascularization process of purified endocrine islet beta-cells transplanted in the renal subcapsular space of syngeneic rats. Lewis rats were grafted with islets cultured for 24 h, with a suspension of purified beta-cells cultured for 24 h, and with a suspension of purified beta plus nonbeta-cells cultured for 24 h. Rats were killed at different days after implantation and the kidney bearing the grafts were snap frozen and immunohistochemically stained with a rabbit anti factor VIII antiserum (which labels endothelial cells). Immunocytochemical analysis revealed that cultured islets completed revascularization by days 3-5 after transplantation, as shown by the detection of capillary endothelial cells within and surrounding the islets. Within purified endocrine beta-cell grafts, the presence of numerous endothelial cells was not observed until days 10-14, indicating that revascularization of beta-cells with host vessels is not such an early phenomenon as it takes place in whole isolated islets. Conversely, the addition of a population of endocrine nonbeta-cells to the purified islet cell grafts, partially accelerated the revascularization of pure beta-cell grafts, which showed the presence of abundant capillary endothelial cells already at day 7 after transplantation, indicating that some other unidentified factors besides the absence of endothelial cells may explain the retardation of beta-cell grafts revascularization.

Palindromic Rheumatism with Positive Anticitrullinated Peptide/Protein Antibodies Is Not Synonymous with Rheumatoid Arthritis. A Longterm Followup Study

Objective. To analyze longterm progression to rheumatoid arthritis (RA) and the predictive value of anticitrullinated peptide/protein antibodies (ACPA) in palindromic rheumatism (PR). Methods. We selected all patients in our clinic with PR who had at least 1 ACPA measurement. We included only patients with pure PR, defined as no evidence of associated rheumatic disease at the first serum ACPA measurement. Clinical characteristics, serum ACPA levels, duration of PR until serum ACPA measurement, and total followup time were recorded. The outcome variable was the definitive diagnosis of RA. The prognostic value of ACPA status in pure PR for a definite diagnosis of RA was analyzed by different statistical methods. Results. Seventy-one patients (54 women/17 men) with a PR diagnosis were included. Serum ACPA were positive in 52.1%. After a mean followup of 7.6 ± 4.7 years since the first ACPA measurement, 24 patients (33.8%) progressed to chronic disease: 22% RA, 5.6% systemic lupus erythematosus, and 5.6% other diseases. The positive likelihood ratio of ACPA status for RA was 1.45, and the area under the receiver-operating characteristic curve of ACPA titers was 0.60 (95% CI 0.45−0.75). Progression to RA was more frequently seen in ACPA-positive than in ACPA-negative patients (29.7% vs 14.7%), but the difference was not significant (hazard ratio 2.46, 95% CI 0.77−7.86). Mean ACPA levels of patients with pure PR did not differ significantly from those of patients who progressed to RA. Conclusion. ACPA are frequently found in the sera of patients with PR, and a significant proportion of these patients do not progress to RA in the long term.

Synthesis of Overlapping Fibrin Citrullinated Peptides and their use for Diagnosing Rheumatoid Arthritis

With the aim of developing a new enzyme‐linked immunosorbent assay test to detect autoantibodies in the sera of rheumatoid arthritis patients with a high sensitivity and specificity using synthetic citrullinated peptides of fibrin (which is abundant in rheumatoid synovium) as antigenic substract, peptides belonging to α‐ and β‐fibrin chains were selected by computer‐aided prediction of antigenicity and epitope mapping and synthesized in solid phase. We analysed by enzyme‐linked immunosorbent assay 133 sera from patients with well‐characterized rheumatic diseases, including 67 patients with rheumatoid arthritis. The results of the immunoassays reported highlight the usefulness of fibrin‐related peptides in rheumatoid arthritis diagnosis and, especially, the ability and specificity of the [Cit621,627,630]α‐fibrin(617–631) (αfib617) peptide sequence to recognize the autoantibodies that are present in rheumatoid arthritis patients.