Idriss Djilali-Saiah

A murine model of type 2 autoimmune hepatitis: Xenoimmunization with human antigens

Autoimmune hepatitis (AIH) is characterized by an immune‐mediated injury of the hepatic parenchyma of unknown pathogenesis. Type 2 AIH is identified by the presence of anti‐liver‐kidney microsomes type 1 (anti‐LKM1) and anti‐liver cytosol type 1 (anti‐LC1) autoantibodies. The current study shows that a murine model of AIH can be generated by DNA immunization against type 2 AIH self‐antigens (P450 2D6 and formiminotransferase‐cyclodeaminase). A pCMV plasmid containing the N‐terminal region of mouse CTLA‐4 and the antigenic region of human CYP2D6 (672‐1,377 bp) and human formiminotransferase cyclodeaminase (FTCD; 1,232‐1,668 bp) was used for DNA immunization of C57BL/6 female mice. Immunized mice showed elevated levels of alanine aminotransferase (ALT), with peaks at 4 and 7 months postinjection. Periportal, portal, and intralobular liver inflammatory infiltrates were observed at histology. Mainly CD4+ lymphocytes, but also CD8+ and B lymphocytes, were found in the liver. Cytotoxic‐specific T cells were found in both the liver and spleen of these animals. Mice developed anti‐LKM1 and anti‐LC1 antibodies of immunoglobulin G2 (IgG2) subclass, against specific mouse autoantigens. The ALT levels correlated with both the presence of anti‐LKM1/anti‐LC1 antibodies and the presence of liver necroinflammation. In conclusion, in mice, DNA immunization against human autoantigens breaks tolerance and induces an autoimmune liver disease. Molecular mimicry between foreign and self‐antigens explains the liver injury. This model of AIH resembles human type 2 AIH and will be helpful for the study of its pathogenesis. (HEPATOLOGY 2004;39:1066–1074.)

DNA Vaccination Breaks Tolerance for a Neo-Self Antigen in Liver: A Transgenic Murine Model of Autoimmune Hepatitis

Understanding the pathogenesis of autoimmune hepatitis requires an animal model in which chronic progressive immune injury develops spontaneously or with minimal manipulations. The new transgenic mouse model proposed in this study is based on the hypothesis that infectious agents have the potential to initiate autoreactivity through molecular mimicry. A transgenic mouse expressing lymphocytic choriomeningitis virus nucleoprotein (NP) in a H-2b background developed liver injury when vaccinated with plasmids expressing NP as an intracellular or a secretory protein. Coinjection of plasmids coding for NP and IL-12 facilitated the induction of a Th1 phenotype as detected by a specific B lymphocyte response characterized by a predominance of IgG2 subclass anti-NP Abs. CTLs activated in peripheral lymphoid organs by DNA vaccination migrated to the periportal and lobular areas of the liver. Their presence was associated with a significant degree of cytolysis, as evidenced by elevated transaminases several weeks after immunization. As activated specific T lymphocytes proliferated in the periphery and caused cytolysis of target cells, this study suggests that autoimmune hepatitis can be triggered by molecular mimicry, and that local injury may not be essential to initiate autoreactivity in the liver.

Polymorphism of antigen processing (TAP, LMP) and HLA class II genes in celiac disease

Susceptibility to CD is strongly associated with particular HLA class II molecules. However, additive genetic factors are likely to be required for the development of the disease. The polymorphic TAP and LMP genes, located within the HLA class II region, are involved in the antigen presentation pathway and thus represent candidate susceptibility genes. HLA class II DRB1, DRB3, DQA1, DQB1, and DPB1 as well as TAP1, TAP2, and LMP2 polymorphism was studied in 80 Caucasian CD patients and 213 normal controls by DNA oligotyping. The DQB1*0201 allele was found in 96.3% of CD patients and provided the highest risk (RR = 50), whereas only 89% of CD patients carried the DQ alpha 501/beta 201 heterodimer (RR = 30). The participation of the DRB3 and DPB1 locus was ruled out as it was attributed to a linkage disequilibrium on the DR3 haplotype. TAP1 and LMP2 allelic distribution was not significantly different among CD patients and controls. The TAP2-C allele was completely absent from the CD population, while it was found in 22.5% of controls. Although linkage disequilibrium between TAP2 and class II loci clearly exists in some haplotypes, TAP could act as additional susceptibility genes.

No Major Role for the CTLA-4 Gene in the Association of Autoimmune Thyroid Disease With IDDM

IDDM is a T-cell-mediated autoimmune disease depending on both genetic and environmental susceptibility factors. The HLA class II region (IDDM1) and the insulin promoter region (IDDM2) account for -50 and 10%, respectively, of IDDM genetic risk. At least 15 other IDDM susceptibility markers have been identified by genomewide scanning studies (1). One must consider that IDDM is a heterogeneous disorder that can vary in terms of age at clinical onset, duration of hyperglycemia before strict insulin dependency, existence of familial aggregation, occurrence of complications, and presence of extrapancreatic autoimmune diseases, so that different genes might influence the course or the presentation of the disease. The CTLA-4 gene, which has been mapped to the IDDM12 locus (2q33), is a good candidate gene in IDDM (2). Apart from recognition of major histocompatibility complex (MHC)/peptide complex by the T-cell receptor (TCR), T-cell activation requires a co-stimulatory signal mediated by CD28/B7 interaction. The CTLA-4 gene encodes a T-cell surface molecule whose binding to the B7 molecule on antigen-presenting cell delivers a negative signal to the T-cell and can mediate its apoptosis (3). Thus, CTLA-4 expression on T-cells might well influence the course of an ongoing immune process. CTLA4-deficient mice develop a severe lymphoproliferative disease with multiorgan lymphocytic infiltration and tissue destruction, including the pancreas (4,5). In the same respect, the blockade of the CD28/B7 co-stimulatory signal prevents the occurrence of diabetes but not of insulitis in the NOD mouse model, suggesting that it either promotes a shift of the immune response or makes an additional signal unavailable for the final destructive stage (6). Recently, linkage to IDDM of a point mutation in exon 1 of CTLA-4 (position 49 A/G) leading to a Thr/Ala substitution in the leader peptide has been

CTLA-4/CD 28 region polymorphisms in children from families with autoimmune hepatitis

Susceptibility to autoimmune hepatitis is associated with particular human leucocyte antigen class II alleles. However, non-HLA genetic factors are likely to be required for development of the disease. Among the candidate genes, the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD28 genes, located on chromosome 2q33 in humans, encode a cell surface molecule playing a dominant role in the regulation of T-cell activation. The CTLA-4 and CD28 polymorphisms were investigated in children from 32 families with autoimmune hepatitis (AIH). The transmission/disequilibrium test revealed increased transmission of the (AT)8 (dinucleotide repeat) and A (exon 1) alleles of CTLA-4 gene from heterozygous parents to affected offspring (87.5% and 83.5%) with type 1 AIH, compared with unaffected offspring (50.0% for both, p = 0.009 and 0.02, respectively). In contrast, no deviation in transmission for CTLA-4 polymorphisms was found between type 2 AIH patients and unaffected offspring. No evidence for association was found between CD28 gene polymorphism or D2S72 genetic marker and both types of AIH. This study identified the CTLA-4 gene polymorphism as a non-HLA determinant that predisposes to AIH type 1 in children. The genetic heterogeneity seen in the present study provides a new argument in favor of pathogenic differences between type 1 and type 2 AIH.

Anti-soluble liver antigen/liver-pancreas (SLA/LP) antibodies in pediatric patients with autoimmune hepatitis.

Antibodies against soluble liver antigen/liver-pancreas (SLA/LP) have been associated with severe autoimmune hepatitis (AIH) and poor outcome, but most of these reports have focused on adult patients. The aim of this study was to assess the prevalence and clinical significance of anti-SLA/LP antibodies in a pediatric population with AIH. We developed a quantitative enzyme-linked immuno-assay (ELISA), a Western blot (WB) and an immunoprecipitation assay (IPA) based on recombinant cDNA from activated Jurkat cells. The specificity of these tests was validated by testing 200 serum samples from healthy subjects, and from patients with liver and non-liver diseases. Anti-SLA/LP antibodies were found in patients with type 1 and type 2 AIH. The prevalence of these antibodies in patients with type 1 AIH was: 42% when tested by ELISA, 15% by WB and 50% by IPA. In patients with type 2 AIH, the prevalence rates were 42% by ELISA, 18% by WB and 44% by IPA. The mean titer values for anti-SLA/LP antibodies was significantly higher in type 2 AIH (1:1,300 - 339) than in type 1 AIH (1:600 - 71; p < 0.0001) and closely associated with higher titers of anti-liver kidney microsome type 1 (LKM1) and anti-liver cytosol type 1 (LC1) antibodies in sera. The presence of anti-SLA/LP showed a significant female preponderance in type 1 and 2 AIH patients (p = 0.0003 and p = 0.003, respectively), and was significantly correlated with a lower age at diagnosis (p = 0.05) in type 1 AIH patients. In conclusion, anti-SLA/LP antibodies in pediatric patients are associated with both type 1 and 2 AIH.

Family study of linkage disequilibrium between TAP2 transporter and HLA class II genes. Absence of TAP2 contribution to association with insulin-dependent diabetes mellitus.

The polymorphic TAP1 and TAP2 genes encode a transporter protein required for delivery of cytosolic peptides to class I molecules in the endoplasmic reticulum. Associations have been observed between TAP2 alleles and predisposition to autoimmune diseases such as IDDM but their interpretation has been complicated by the existence of LD between TAP2 and HLA class II loci, and conclusions are still contradictory. In order to precisely define LD on class II haplotypes, we performed an extensive familial analysis. A total of 466 individuals from 55 normal families and 49 IDDM multiplex families was studied, providing information on 420 independent haplotypes. The IDDM-predisposing DRB1*03 and DRB1*04 alleles were in strong negative LD with TAP2-B (delta = -0.035 and -0.034, respectively), and positive LD with TAP2-A (delta = + 0.055 and + 0.012). Positive LD was also found between TAP2-B and DRB1*01 and TAP2-C and DRB1*11 alleles. We then addressed the question of whether TAP2 is an independent additional IDDM-protective or predisposing genetic factor. No TAP2 effect was evidenced when considering DRB1*03 and/or 04 patients. A decreased TAP2-B phenotype frequency was observed in DRB1*03- and DRB1*04-negative IDDM patients compared with DRB1*03- and DRB1*04-negative normal controls (38.6% vs 63%, pc < 0.05), but was probably related to a combination of different weak LD between DRB1 and TAP2 alleles. It thus appears that there is no primary association between TAP2 alleles and IDDM. However, TAP polymorphism may allow us to define particular extended HLA haplotypes involved in susceptibility to autoimmune diseases.

Anti-soluble liver antigen (SLA) antibodies in chronic HCV infection.

Hepatitis C infection is associated with autoimmune disorders, such as the production of autoantibodies. Anti-LKM1 and anti-LC1, immunomarkers of type 2 autoimmune hepatitis, have been previously associated with a HCV infection. Anti-Soluble-Liver-Antigen autoantibodies (SLA) are specifically associated with type 1 and type 2 autoimmune hepatitis and more closely related to patients who relapse after steroid therapy. The recent molecular cloning of the soluble liver antigen provides the opportunity to develop more specific tests for the detection of antibodies against it. The aim of this work is to characterize anti-soluble-liver autoantibodies in sera from patients chronically infected by HCV. A recombinant cDNA from activated Jurkat cells coding for the full length tRNP(Ser)Sec/SLA antigen was obtained. ELISA, Western Blot and immunoprecipitation tests were developed and used to search for linear and conformational epitopes recognized by anti-SLA antibodies in sera from patients chronically infected by HCV. Anti-soluble liver antigen antibodies were found in sera from 10.4% of HCV-infected patients. The prevalence was significantly increased to 27% when anti-LKM1 was also present. Most anti-SLA reactivity was directed against conformational epitopes on the antigen. The means titers by ELISA were lower than those obtained in type 2 AIH. The result of autoantibody isotyping showed a subclass restriction to IgG1 and also IgG4. This study shows the presence of anti-SLA antibodies in approximately 10% of HCV infected patients. The prevalence of SLA autoantibodies in HCV infected patients increases when LKM1 autoantibodies are also present. The relationship between the prevalence of this characteristic autoimmune hepatitis autoantibody and the implication of an autoimmune phenomenon in the liver injury of patients chronically infected by HCV needs further investigation.

Autoantibody detection in type 2 autoimmune hepatitis using a chimera recombinant protein.

Autoantibodies against cytochrome P450 2D6 (CYP2D6), known as anti-liver/kidney microsome type 1 (LKM1) and/or anti-human formiminotransferase cyclodeaminase, formally known as anti-liver cytosol type 1 (LC1) define type 2 autoimmune hepatitis (AIH). The aims of this work are to develop a sensitive and specific test to detect anti-LKM1 and/or anti-LC1 autoantibodies and to establish the prevalence of anti-LC1. Sera from children with type 2 AIH (n=48) and those from a control group (n=100) were evaluated for anti-LKM1 and anti-LC1 by Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. Each serum sample was assayed for reactivity against formiminotransferase cyclodeaminase and CYP2D6 alone or as part of a recombinant chimera protein. By ELISA with recombinant chimera protein, 50 serum samples were positive, 48 from patients with type 2 AIH and 2 from patients with chronic hepatitis C. Twenty-five of 48 (52%) patients studied were positive for both CYP2D6 and LC1 autoantibodies. Anti-LC1, either as the only marker or associated with anti-LKM1, was positive in 34/48 (71%). By Western blotting, anti-LC1 was found in 27/48 (56%) patients. This ELISA technique has proven to be antigen-specific and more sensitive than Western blot for the detection of anti-LC1 and anti-LKM1 autoantibodies. The prevalence of anti-LC1 (71%) confirms it as an important immunomarker in type 2 AIH.

Absence of primary association between DM gene polymorphism and insulin-dependent diabetes mellitus or celiac disease

The DMA and DMB genes encode class II-like heterodimetric molecules located in a specialized endocytic compartment, where they facilitate efficient loading of antigenic peptides on HLA class II molecules. Both genes are located within the MHC class II region and present a limited allelic polymorphism. Here we report the distribution of DM alleles in a group of 75 IDDM patients, 72 CD patients, and 162 random controls. We found a pronounced decreased frequency of DMA*0102 in both patient groups relative to controls. This difference was, however, mainly secondary to a strong negative linkage disequilibrium (LD) between this allele and the IDDM and CD-associated DRB1*03 allele. The DMB phenotype frequencies were similar in CD patients and controls. By contrast, we observed a decreased frequency of DMB*0101 and an increased frequency of DMB*0102 and DMB*0104 in IDDM patients. These differences disappeared when matching individuals for DRB1*03 or DRB1*04 alleles, which was in accordance with strong negative LD between DMB*0101 and DRB1*04 or DQB1*0302 alleles, and positive LD between DMB*0104 and DQB1*0201. Our data suggest that the apparent associations of IDDM or CD with given DM alleles are mostly secondary to primary associations with alleles at the DRB and DQB loci.