Correlation of peptide specificity and IgG subclass with pathogenic and nonpathogenic autoantibodies in pemphigus vulgaris: a model for autoimmunity.

Pemphigus vulgaris (PV) is a rare, potentially fatal, autoimmune disease that affects the skin and mucous membranes. The PV antigen (PVA) has been characterized as desmoglein 3. PV patients carry HLA-DR4- or HLA-DR6-bearing extended haplotypes. We recently demonstrated that patients with active disease have high titers of PV autoantibodies of the IgG1 and IgG4 subclasses. Patients in remission, healthy unaffected relatives, and some MHC-matched normal individuals have low levels of PV autoantibodies, which are IgG1 only. Furthermore, intraperitoneal injection of IgG from patients with active disease caused clinical disease in mice, but IgG from patients in remission, healthy relatives, or MHC-matched normal individuals did not. We prepared 12 peptides of 30 amino acids each (peptides Bos 1-12) spanning the extracellular domain of PVA. Patients with active disease recognize peptides Bos 1 and Bos 6 with high titers of IgG1 and IgG4 autoantibodies. Patients in remission have IgG1 autoantibodies to peptide Bos 1 only, in statistically significantly lower titers (P < 0.01). They no longer have IgG4 subclass autoantibodies to peptide Bos 6. Healthy relatives and normal unrelated individuals have low levels of only IgG1 autoantibodies that recognize only Bos 1. In vitro studies indicate that Bos 6-specific IgG and, to a lesser extent, Bos 1-specific IgG can cause acantholysis. Our data suggest that Bos 6-specific IgG4 is probably the main acantholytic autoantibody, while Bos 1-specific IgG4 may act as a facilitator or enhancer of the process. In this study we illustrate some of the paradigms that demonstrate the interactions between the MHC, subclass of autoantibodies, and peptide specificities of the autoantibodies in the autoimmune process. Thus, PV provides an important model to study the pathogenesis of autoimmunity.

Autoimmunity in Chagas disease cardiopathy: biological relevance of a cardiac myosin-specific epitope crossreactive to an immunodominant Trypanosoma cruzi antigen.

Heart tissue destruction in chronic Chagas disease cardiopathy (CCC) may be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after Trypanosoma cruzi infection. Indirect evidence suggests that there is antigenic crossreactivity between T. cruzi and heart tissue. As there is evidence for immune recognition of cardiac myosin in CCC, we searched for a putative myosin-crossreactive T. cruzi antigen. T. cruzi lysate immunoblots were probed with anti-cardiac myosin heavy chain IgG antibodies (AMA) affinity-purified from CCC or asymptomatic Chagas disease patient-seropositive sera. A 140/116-kDa doublet was predominantly recognized by AMA from CCC sera. Further, recombinant T. cruzi protein B13--whose native protein is also a 140- and 116-kDa double band--was identified by crossreactive AMA. Among 28 sera tested in a dot-blot assay, AMA from 100% of CCC sera but only 14% of the asymptomatic Chagas disease sera recognized B13 protein (P = 2.3 x 10(-6)). Sequence homology to B13 protein was found at positions 8-13 and 1442-1447 of human cardiac myosin heavy chain. Competitive ELISA assays that used the correspondent myosin synthetic peptides to inhibit serum antibody binding to B13 protein identified the heart-specific AAALDK (1442-1447) sequence of human cardiac myosin heavy chain and the homologous AAAGDK B13 sequence as the respective crossreactive epitopes. The recognition of a heart-specific T. cruzi crossreactive epitope, in strong association with the presence of chronic heart lesions, suggests the involvement of crossreactivity between cardiac myosin and B13 in the pathogenesis of CCC.

Leptin directly promotes T-cell glycolytic metabolism to drive effector T-cell differentiation in a mouse model of autoimmunity.

Upon activation, T cells require energy for growth, proliferation, and function. Effector T (Teff) cells, such as Th1 and Th17 cells, utilize high levels of glycolytic metabolism to fuel proliferation and function. In contrast, Treg cells require oxidative metabolism to fuel suppressive function. It remains unknown how Teff/Treg-cell metabolism is altered when nutrients are limited and leptin levels are low. We therefore examined the role of malnutrition and associated hypoleptinemia on Teff versus Treg cells. We found that both malnutrition-associated hypoleptinemia and T cell-specific leptin receptor knockout suppressed Teff-cell number, function, and glucose metabolism, but did not alter Treg-cell metabolism or suppressive function. Using the autoimmune mouse model EAE, we confirmed that fasting-induced hypoleptinemia altered Teff-cell, but not Treg-cell, glucose metabolism, and function in vivo, leading to decreased disease severity. To explore potential mechanisms, we examined HIF-1α, a key regulator of Th17 differentiation and Teff-cell glucose metabolism, and found HIF-1α expression was decreased in T cell-specific leptin receptor knockout Th17 cells, and in Teff cells from fasted EAE mice, but was unchanged in Treg cells. Altogether, these data demonstrate a selective, cell-intrinsic requirement for leptin to upregulate glucose metabolism and maintain function in Teff, but not Treg cells.

Non-Celiac Gluten Sensitivity and Autoimmunity. A Case Report

Introduction, objective: To present a case report in which the finding of non-coeliac gluten sensitivity was decisive for the treatment of a complex autoimmune disease. Materials and methods: A 43-year-old woman with polyarthritis, psoriatic features, anti-SSA/Ro and anti-cyclic citrullinated peptide antibodies, with refractory course, was evaluated for gluten sensitivity despite negative serology for coeliac disease. Results: The patient carried the HLA DQ2 haplotype and duodenal biopsy showed lymphocytic enteritis. A gluten-free diet resolved the clinical picture and permitted tapering of immunosuppressive therapy. Conclusion: Non-coeliac gluten sensitivity can be associated with autoimmunity despite the absence of the specific autoantibodies of coeliac disease.

Cervical Spine Involvement In Rheumatoid Arthritis--a Systematic Review.

Rheumatoid arthritis (RA) is a systemic chronic inflammatory disorder that can compromise the cervical spine in up to 80% of the cases. The most common radiological presentations of cervical involvement are atlantoaxial subluxation (AAS), cranial settling and subaxial subluxation (SAS). We performed a systematic review in the PubMed Database of articles published later 2005 to evaluate the prevalence, progression and risk factors for cervical spine involvement in RA patients. Articles were classified according to their level of evidence. Our literature review reported a wide range in the prevalence of cervical spine disease, probably explained by the different studied populations and disease characteristics. Uncontrolled RA is probably the main risk factor for developing a spinal instability. Adequate treatment with DMARD and BA can prevent development of cervical instabilities but did not avoid progression of a pre-existing injury. MRI is the best radiological method for diagnosis cervical spine involvement. AAS is the most common form of RA. Long term radiological follow-up is necessary to diagnosis patients with late instabilities and monitoring progression of diagnosed injuries.

Lymphocyte Activation In Silica-exposed Workers.

Exposure to silica dust has been examined as a possible risk factor for autoimmune diseases, including systemic sclerosis, rheumatoid arthritis, systemic lupus erythematosus and ANCA-associated vasculitis. However, the underlying cellular and molecular mechanisms resulting in the increased prevalence of autoimmunity remain elusive. To clarify these mechanisms, we studied various markers of immune activation in individuals occupationally exposed to silica dust, i.e., serum levels of soluble IL-2 receptor (sIL-2R), levels of IL-2, other pro- and anti-inflammatory cytokines and lymphoproliferation. Our results demonstrate that silica-exposed individuals present important alterations in their immune response when compared to controls, as shown by increased serum sIL-2R levels, decreased production of IL-2 and increased levels of the pro-inflammatory (IFN-γ, IL-1α, TNF-α, IL-6) as well as anti-inflammatory (IL-10 and TGF-β) cytokines. Furthermore, silica-exposed individuals presented enhanced lymphoproliferative responses. Our findings provide evidence that the maintenance of immune homeostasis may be disturbed in silica-exposed individuals, possibly resulting in autoimmune disorders.

Circulating Levels Of Chemokines In Psoriasis.

Chemokines may contribute to local and systemic inflammation in patients with psoriasis. Previous studies have demonstrated the importance of chemokine ligands and receptors in the recruitment of T cells into psoriatic lesional skin and synovial fluid. The aim of this study was to evaluate the levels of Th1-related chemokines in psoriasis and to investigate any association with disease severity. We quantified serum levels of CXCL9, CXCL10 and CXCL16 and the frequencies of CD4+CXCR3+ T lymphocytes through ELISA and flow cytometry, respectively. A total of 38 patients with psoriasis and 33 controls were included. There were no significant differences in chemokine levels between psoriasis and control groups. Patients with psoriatic arthritis had lower median level of CXCL10 when compared with controls (p=0.03). There were no significant correlations between serum chemokines analyzed and disease severity. Frequencies of CD4+CXCR3+ T cells were lower in patients with psoriasis than in controls (p<0.01). A sensitivity analysis excluding patients on systemic therapy yielded similar results. Serum concentrations of CXCL9, CXCL10 and CXCL16 were not increased in the psoriasis group or correlated with disease severity. Systemic levels of chemokine ligands do not seem to be sensitive biomarkers of disease activity or accurate parameters to predict response to therapy. Frequencies of CD4+CXCR3+ T cells were decreased in the peripheral blood of psoriasis patients, possibly due to recruitment to inflammatory lesions.

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD

Abnormal collagen deposition in synovia after collagen type V immunization in rabbits

Sinovitis in Scleroderma (SSc) is rare, usually aggressive and fully resembles rheumatoid arthritis. Experimental models of SSc have been used in an attempt to understand its pathogenesis. Previous studies done in our laboratory had already revealed the presence of a synovial remodeling process in rabbits immunized with collagen V. To validate the importance of collagen type V and to explore the quantitative relationship between this factor and synovia remodeling as well as the relationship between collagen type V and other collagens, we studied the synovial tissue in immunized rabbits. Rabbits (N= 10) were immunized with collagen V plus Freund's adjuvant and compared with animals inoculated with adjuvant only (N= 10). Synovial tissues were submitted to histological analysis, immunolocalization to collagen I, III and V and biochemical analysis by eletrophoresis, immunoblot and densitometric method. The synovial tissue presented an intense remodeling process with deposits of collagen types I, III and V after 75 and 120 days of immunization, mainly distributed around the vessels and interstitium of synovial extracellular matrix. Densitometric analysis confirmed the increased synthesis of collagen I, III and V chains (407.69 +/- 80.31; 24.46 +/- 2.58; 70.51 +/- 7.66, respectively) in immunized rabbits when compared with animals from control group (164.91 +/- 15.67; 12.89 +/- 1.05; 32 +/- 3.57) (p<0.0001). We conclude that synovial remodeling observed in the experimental model can reflect the articular compromise present in patients with scleroderma. Certainly, this experimental model induced by collagen V immunization will bring new insights in to pathogenic mechanisms and allow the testing of new therapeutic strategies to ameliorate the prognosis for scleroderma patients.

Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

Background: The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGFbeta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods: Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V ( 25 mu g/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05. Results: IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 +/- 0.118 vs. 0.874 +/- 0.282, p < 0.001), bronchioles (0.294 +/- 0.139 vs. 0.646 +/- 0.172, p < 0.001) and in the septal interstitium (0.027 +/- 0.014 vs. 0.067 +/- 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 +/- 0.07 vs. 1.0 +/- 0.528, p = 0.002) and V (1.12 +/- 0.42 vs. 4.74 +/- 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression ( p < 0.0001). Conclusions: Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

Administration of M. leprae Hsp65 Interferes with the Murine Lupus Progression

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K(409)A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F(1) mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F1 serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K(409)A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K(409)A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process.

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10. RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+)IL-17(+), CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+)IFN-gamma(+) and CD4(+)IL-17(+) T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.