Anna Carla Goldberg

Genetic heterogeneity in susceptibility to autoimmune hepatitis types 1 and 2

OBJECTIVES:Susceptibility to autoimmune hepatitis (AIH) type 1 has been associated with DRB1*03, DRB1*04, and DRB3 alleles in European and North-American whites, with DRB1*04 in Japan, and with DRB1*04 and DRB1*13 in Latin America. Very few studies have been performed on AIH type 2. The aim of the present study was to evaluate the association of AIH types 1 and 2 with HLA-DR and DQ loci.METHODS:We performed HLA-DRB and -DQB1 typing by polymerase chain reaction amplification with sequence-specific primers (PCR-SSP) in 139 AIH patients. Most had AIH type 1 associated with circulating anti-smooth muscle antibody with F-actin specificity or antinuclear antibody. Twenty-eight patients presented AIH type 2 with anti-liver/kidney microsome type 1 or anti-liver cytosol type 1 antibodies.RESULTS:We observed a significant increase of DRB1*13 (70%vs 26% of controls, p < 0.00001) and DRB3 (93%vs 69% of controls, p < 0.00001) in AIH type 1 patients. Analysis of patients without DRB1*13 disclosed a secondary association with DRB1*03 (70%vs 30% of controls, p= 0.0001) and either the DRB1*13 or the DRB1*03 alleles were present in the majority of these patients (91%vs 48% of controls, p= 0.001). Comparison of DRB1*13- and DRB1*03-positive subjects revealed that the former alleles conferred susceptibility to younger patients with AIH type 1. DQB1 typing showed a significant increase in DQB1*06 (68%vs 41% of controls, p= 0.00007) in strong linkage disequilibrium with DRB1*13, and a decrease in DQB1*0301 (8%vs 47% of controls, pc= 0.0003). On the other hand, HLA typing of patients with AIH type 2 disclosed a significant increase in the DRB1*07 (68%vs 20% of controls, pc < 0.00014), DRB4 (79%vs 43% of controls, pc= 0.004), and DQB1*02 (86%vs 42%, p= 0.00002) alleles. After exclusion of DRB1*07, a secondary association with HLA-DRB1*03 was further observed in these patients (78%vs 30%, p= 0.007) and most of them had either DRB1*07 or DRB1*03 (93%vs 44% of controls, pc < 0.0001).CONCLUSIONS:Our data indicate that predisposition to AIH types 1 and 2 is associated, respectively, with the DRB1*13 or DRB1*03 and DRB1*07 or DRB1*03 alleles, and suggest that protection against type 1 disease may be conferred by DQB1*0301. In addition, the cluster of DRB1*13 in children with AIH type 1 also supports the concept that different HLA alleles might influence the onset of the disease.

T-Cell Reactivity against Streptococcal Antigens in the Periphery Mirrors Reactivity of Heart-Infiltrating T Lymphocytes in Rheumatic Heart Disease Patients

ABSTRACT T-cell molecular mimicry between streptococcal and heart proteins has been proposed as the triggering factor leading to autoimmunity in rheumatic heart disease (RHD). We searched for immunodominant T-cell M5 epitopes among RHD patients with defined clinical outcomes and compared the T-cell reactivities of peripheral blood and intralesional T cells from patients with severe RHD. The role of HLA class II molecules in the presentation of M5 peptides was also evaluated. We studied the T-cell reactivity against M5 peptides and heart proteins on peripheral blood mononuclear cells (PBMC) from 74 RHD patients grouped according to the severity of disease, along with intralesional and peripheral T-cell clones from RHD patients. Peptides encompassing residues 1 to 25, 81 to 103, 125 to 139, and 163 to 177 were more frequently recognized by PBMC from RHD patients than by those from controls. The M5 peptide encompassing residues 81 to 96 [M5(81–96) peptide] was most frequently recognized by PBMC from HLA-DR7+DR53+ patients with severe RHD, and 46.9% (15 of 32) and 43% (3 of 7) of heart-infiltrating and PBMC-derived peptide-reactive T-cell clones, respectively, recognized the M5(81–103) region. Heart proteins were recognized more frequently by PBMC from patients with severe RHD than by those from patients with mild RHD. The similar pattern of T-cell reactivity found with both peripheral blood and heart-infiltrating T cells is consistent with the migration of M-protein-sensitized T cells to the heart tissue. Conversely, the presence of heart-reactive T cells in the PBMC of patients with severe RHD also suggests a spillover of sensitized T cells from the heart lesion.

Clinical distinctions and pathogenic implications of type 1 autoimmune hepatitis in Brazil and the United States

BACKGROUND/AIMS Type 1 autoimmune hepatitis has a strong genetic predisposition that varies among different ethnic groups. Our aims were to determine if the clinical manifestations differed between patients with type 1 autoimmune hepatitis from Brazil and the United States and if classical disease could be associated with region-specific susceptibility markers. METHODS The clinical manifestations and genetic risk factors of 161 patients from the United States were compared to those of 115 patients from Brazil. RESULTS The patients from Brazil had earlier disease onset, lower frequency of concurrent immune diseases, higher serum levels of aspartate aminotransferase and gamma-globulin, greater occurrence of smooth muscle antibodies, and lower frequency of antinuclear antibodies than the patients from the United States. Human leukocyte antigen (HLA) DR13 and DRB1*1301 occurred more commonly in the Brazilian patients and HLA DR4 less often. Normal subjects from each country had similar frequencies of HLA DR13 and DR3. CONCLUSIONS Type 1 autoimmune hepatitis in Brazil has different features at presentation than the disease in Caucasoid patients from the United States, and it is associated with HLA DR13. Background populations in each country have similar frequencies of HLA DR13 and DR3, and region-specific etiologic factors may determine the HLA association.

Immunological and non-immunological effects of cytokines and chemokines in the pathogenesis of chronic Chagas disease cardiomyopathy

The pathogenesis of Chagas disease cardiomyopathy (CCC) is not well understood. Since studies show that myocarditis is more frequent during the advanced stages of the disease, and the prognosis of CCC is worse than that of other dilated cardiomyopathies of non-inflammatory aetiology, which suggest that the inflammatory infiltrate plays a major role in myocardial damage. In the last decade, increasing evidence has shown that inflammatory cytokines and chemokines play a role in the generation of the inflammatory infiltrate and tissue damage. CCC patients have an increased peripheral production of the inflammatory Th1 cytokines IFN-gamma and TNF-alpha when compared to patients with the asymptomatic/indeterminate form. Moreover, Th1-T cells are the main producers of IFN-gamma and TNF-alpha and are frequently found in CCC myocardial inflammatory infiltrate. Over the past several years, our group has collected evidence that shows several cytokines and chemokines produced in the CCC myocardium may also have a non-immunological pathogenic effect via modulation of gene and protein expression in cardiomyocytes and other myocardial cell types. Furthermore, genetic polymorphisms of cytokine, chemokine and innate immune response genes have been associated with disease progression. We will review the molecular and immunological mechanisms of myocardial damage in human CCC in light of recent findings.

HLA-DRB1∗0405 is the Predominant Allele in Brazilian Patients With Vogt-Koyanagi-Harada Disease

Vogt-Koyanagi-Harada (VKH) disease is a rare disorder affecting pigmented structures especially the eye and is the main cause of autoimmune non-infectious uveitis in the Brazilian population. The autoimmune target is believed to be the melanocyte. A strong association of VKH disease with HLA-DR4 in the Japanese population is well known. The same association, albeit with lower relative risks has been found in other populations. A secondary association to HLA-DR1 involving a sequence linked with susceptibility to Rheumatoid Arthritis has also been described. VKH disease is more common in non-Caucasian populations. Brazilian patients of varying ethnic origins have been typed for HLA class II antigens. Several of the features found in other population samples are present. Over half of the patients typed HLA-DR4 (20/37) and typing with sequence-specific oligonucleotides disclosed predominance of the DRB1*0405 allele with a relative risk of 11.76 over the general population. In addition, HLA-DR1 and DQ4 were also present, in patients both positive and negative for HLA-DR4. These results suggest that, as in other autoimmune diseases, multiple overlapping susceptibility factors encoded by the MHC complex contribute to the overall susceptibility for the disease, the major factor however, being the presence of the DRB1*0405 allele.

Analysis of HLA haplotypes in autoimmune hepatitis type 1: identifying the major susceptibility locus

Susceptibility to autoimmune hepatitis type I (AIH-1) has been associated with HLA-DR3, DR52, and DR4 antigens in Caucasian and Oriental patients. However, in Brazil, disease susceptibility is primarily linked to DR13 and DR52. In this highly admixed population, we find different DR13-associated haplotypes, presenting a unique opportunity to discriminate relevant genes within a tightly linked genomic region. To identify the primary susceptibility locus, we sequenced DR13 alleles of 39 patients with AIH-1 and 22 controls. Patients were almost exclusively DRB1*1301, but half of controls typed DRB1*1302. HLA-DQ haplotypes were varied. Oligotyping of DRB3 locus of all patients and also within the HLA-DR13 positive group showed an allele distribution comparable to controls, confirming that the stronger association lies in the DRB1 locus. On the other hand, if DRB1*1301 is the major susceptibility factor in our sample, the only amino acid different from DRB1*1302 in position 86, corresponding to pocket 1 in the peptide-presenting groove, may be important. We propose that peptide presentation leading to pathogenesis of AIH-1 may be quite stringent, but will also be affected by other strong genetic or environmental susceptibility factors, which would explain the various HLA molecules associated to the disease in the different populations.

Heterozygosity for the S180L Variant of MAL/TIRAP, a Gene Expressing an Adaptor Protein in the Toll‐Like Receptor Pathway, Is Associated with Lower Risk of Developing Chronic Chagas Cardiomyopathy

BACKGROUND Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. Among T. cruzi-infected individuals, only a subgroup develops severe chronic Chagas cardiomyopathy (CCC); the majority remain asymptomatic. T. cruzi displays numerous ligands for the Toll-like receptors (TLRs), which are an important component of innate immunity that lead to the transcription of proinflammatory cytokines by nuclear factor-kappaB. Because proinflammatory cytokines play an important role in CCC, we hypothesized that single-nucleotide polymorphisms (SNPs) in the genes that encode proteins in the TLR pathway could explain differential susceptibility to CCC among T. cruzi-infected individuals. METHODS For 169 patients with CCC and 76 T. cruzi-infected, asymptomatic individuals, we analyzed SNPs by use of polymerase chain reaction-restriction fragment length polymorphism analysis for the genes TLR1, TLR2, TLR4, TLR5, TLR9, and MAL/TIRAP, which encodes an adaptor protein. RESULTS Heterozygous carriers of the MAL/TIRAP variant S180L were more prevalent in the asymptomatic group (24 [32%] of 76 subjects) than in the CCC group (21 [12%] of 169) (chi2=12.6; P=.0004 [adjusted P (Pc)=.0084]; odds ratio [OR], 0.31 [95% confidence interval {CI}, 0.16-0.60]). Subgroup analysis showed a stronger association when asymptomatic patients were compared with patients who had severe CCC (i.e., patients with left-ventricular ejection fraction<or=40%) (chi2=11.3; P=.0008 [Pc=.017]; OR, 0.22 [95% CI, 0.09-0.56]) than when asymptomatic patients were compared with patients who had mild CCC (i.e., patients with left-ventricular ejection fraction>40%) (chi2=7.7; P=.005 [Pc=.11]; OR, 0.33 [95% CI, 0.15-0.73]). CONCLUSION T. cruzi-infected individuals who are heterozygous for the MAL/TIRAP S180L variant that leads to a decrease in signal transduction upon ligation of TLR2 or TLR4 to their respective ligand may have a lower risk of developing CCC.

Autoimmune hepatitis, HLA and extended haplotypes.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease. Characteristic liver-infiltrating immune cells in portal and periportal areas, hypergammaglobulinemia and typical autoantibodies indicate an ongoing autoimmune reaction against liver self antigens, which lead to irreversible cellular damage and ultimately to severe hepatic failure. A significant part of adult, but not pediatric AIH patients, exhibit concurrent autoimmune diseases, further strengthening the immunological etiology of the disease. Genetic susceptibility to autoimmune hepatitis is strongly associated with HLA-DRB1 alleles. In Caucasian European and North American patients, AIH-1 is associated with the presence of DRB1*0301, DRB3*0101 and DRB1*0401 alleles, while AIH-2 is associated with DRB1*0301 or DRB1*07. In Brazil, the primary susceptibility allele for AIH-1 is DRB1*1301, but a secondary association with DRB1*0301 has also been identified. We looked for additional susceptibility factors in the extended MHC region. We genotyped 107 AIH-1 children and up to 326 healthy subjects for TNFA G-308A, TNFA G-238A, LTA A+252G, LTA A+80C, NFKBIL1 T-63A, BAT1 C-348T, BAT1 G-22C, MICA, and HLA-B polymorphisms. The TNFA-308 A allele was significantly increased in AIH-1 when compared with healthy controls, confirming data from other studies. Linkage disequilibrium analysis was carried out. The ancestral haplotype comprising TNFA-308A, TNFA-238G, LTA+252G, LTA+80C, NFKBIL1-63A, BAT1-348C, BAT1-22C, HLA-B*08, MICA*08 was more common in DRB1*03 positive patients than in controls (40% vs. 14%), showing a seven-fold increased risk for the disease [OR=7.8 (95%CI 2.04-29.9.2, p=0.0021). In contrast, the remaining patients carrying DRB1*03 exhibited varied haplotypes. Finally, a variety of class III haplotypes was also present in HLA-DRB1*13 patients, without a predominant pattern. The most common of the 98 haplotypes present in patients were completely absent in controls. The extended haplotype analysis in this sample of AIH-1 patients highlights not only the genetic diversity present in the Brazilian population, but is also in accordance with the previously documented microdiversity within the MHC region. The present knowledge of AIH suggests that the same or a very similar disease can be induced by yet unknown, but different, triggers followed by presentation on different HLA-DR molecules of the epitopes derived from the corresponding autoantigens, characterizing a much more complex disease than previously thought.

Cytotoxic T lymphocyte antigen–4 gene polymorphisms do not confer susceptibility to autoimmune hepatitis types 1 and 2 in Brazil

OBJECTIVES:Susceptibility to autoimmune hepatitis (AIH) has been linked to different HLA-DR antigens in distinct populations. Recently, an A–G polymorphism in exon 1 of the cytotoxic T lymphocyte antigen-4 (CTLA-4) gene was associated with predisposition to AIH type 1 (AIH-1) in white individuals in North America. This polymorphism has been associated with several other autoimmune diseases, presumably because of its effect in the expression of CTLA-4, an adhesion molecule that downregulates peripheral T cell responses. The aims of this study were to assess the frequency of CTLA-4 genotypes in Brazilian patients with AIH-1 and AIH type 2 (AIH-1), as well as to investigate the influence of these genotypes in disease expression.METHODS:Determination of CTLA-4 genotypes was carried out in 106 patients with AIH-1, 26 subjects with AIH-2, and 67 healthy control subjects by polymerase chain reaction (PCR)–based techniques.RESULTS:No difference in the distribution of CTLA-4 genotypes was observed in subjects with AIH-1 and AIH-2 as compared to healthy controls. Patients with AIH-1 and AIH-2 with the GG genotype exhibited lower γ-globulin and ALT levels, respectively.CONCLUSIONS:Susceptibility to AIH-1 and AIH-2 in Brazilian patients is not influenced by exon 1 CTLA-4 gene polymorphisms at position 49.

BAT1, a Putative Anti-Inflammatory Gene, Is Associated with Chronic Chagas Cardiomyopathy

BACKGROUND It is not understood why only a subset of individuals infected with Trypanosoma cruzi develop chronic Chagas cardiomyopathy (CCC). Patients with CCC display high levels of circulating proinflammatory cytokines. Heart-infiltrating lymphocytes from patients with CCC also express proinflammatory cytokines (tumor necrosis factor- alpha and interferon- gamma ) that are detectable in biopsy samples and surgical heart-tissue samples. BAT1, a putative anti-inflammatory gene, presents functional polymorphisms in its promoter region that influence its transcriptional level. METHODS We assessed, by polymerase chain reaction restriction fragment-length polymorphism analysis, BAT1 variants in the promoter region at positions -22C/G and -348C/T in 154 patients with CCC and in 76 T. cruzi-infected but asymptomatic (ASY) patients. RESULTS Of the patients with CCC, 16% were homozygous for the -22C allele, compared with 4% of the ASY patients (P=.004; odds ratio [OR], 4.7 [95% confidence interval {CI}, 1.4-16]). A similar trend was observed for the -348C homozygotes (P=.01; OR, 1.9 [95% CI, 1.0-3.5]). Susceptibility to CCC was conferred by the C variants at nt -22 (P=.003; OR, 1.8 [95% CI, 1.2-2.8]) and at nt -348 (P=.02; OR, 1.7 [95% CI, 1.0-2.8]). CONCLUSIONS BAT1 variants previously associated with reduced expression of HLA-B-associated transcript 1 are predictive of the development of CCC. These variants may be less efficient in down-regulating inflammatory responses and may contribute to the elevated production of proinflammatory cytokines in patients with CCC.