H Löhr

Evidence for an overlap syndrome of autoimmune hepatitis and primary sclerosing cholangitis

BACKGROUND/AIMS Autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis are chronic liver diseases with probable autoimmune background. Overlapping features have been described for primary biliary cirrhosis and autoimmune hepatitis. In contrast, there have been only a few case reports on an overlap of autoimmune hepatitis and primary sclerosing cholangitis. METHODS We describe three male patients with clinical and histological overlapping features of primary sclerosing cholangitis and autoimmune hepatitis. RESULTS All initially asymptomatic patients had elevated levels of aminotransferases, alkaline phosphatase, gamma-glutamyltranspeptidase and IgG. Anti-nuclear antibodies and/or smooth muscle antibodies were positive and anti-neutrophil cytoplasmic antibodies were detected in all patients. Retrograde endoscopic cholangiography showed bile-duct strictures characteristic for primary sclerosing cholangitis. Histopathology showed necro-inflammatory activity of portal tracts with bridging necrosis in all patients at the time of first diagnosis. Aminotransferase levels and the necro-inflammatory activity responded well to immunosuppressive treatment. Predominant periductular fibrosis as a typical histopathological feature of primary sclerosing cirrhosis was seen to develop in all patients. Cholestatic serum parameters remained elevated and periductular fibrosis as endoscopic bile duct changes progressed despite immunosuppression. CONCLUSIONS We suggest that these patients present an overlap syndrome of autoimmune hepatitis and primary sclerosing cholangitis as they fulfill the diagnostic criteria for both conditions.

Clinical significance of autoantibodies to soluble liver antigen in autoimmune hepatitis

BACKGROUND/AIMS Classification of autoimmune hepatitis (AIH) into different subgroups according to autoantibody status has been proposed: type I (ANA/SMA), type II (LKM-1) and type III (anti-SLA). However, whether type III AIH forms a clinically distinct disease entity remains controversial. The aim of this study was to evaluate the subclassification of AIH into ANA/SMA and anti-SLA positive patients with regard to clinical, biochemical and histologic differences. METHODS Ninety-seven consecutive patients with a well-documented long-term course of AIH with ANA/SMA and/or anti-SLA autoantibodies were studied. Clinical, biochemical and histological features of patients with ANA/SMA and/or anti-SLA autoantibodies were compared in a secondary analysis of data acquired prospectively. RESULTS Anti-SLA autoantibodies were found in 21.6% of patients. Anti-SLA-positive patients tended to have lower transaminases (mean: 153 vs. 247 IU/l), gamma-globulins (25 vs. 31%) and bilirubin (1.8 vs. 3.3 mg/dl) in comparison to ANA/SMA positive patients, but there was a large overlap. HLA-type A1 B8 was more frequent in anti-SLA positive patients, while there was no difference in HLA DR3 and DR4 allotype. Response to immunosuppressive therapy was excellent, but relapse occurred frequently. Diagnosis of anti-SLA positive AIH was often delayed (mean: 68 months from first elevation of transaminases) since testing for anti-SLA autoantibodies is currently not generally available. CONCLUSIONS ANA/SMA and anti-SLA positive patients share most clinical, biochemical, histologic and prognostic features. Distinction between type I and type III AIH is therefore clinically not helpful. However, testing for anti-SLA autoantibodies helps in the diagnosis of AIH in many patients who may otherwise be misdiagnosed.

Clonal analysis of liver-infiltrating T cells in patients with LKM-1 antibody-positive autoimmune chronic active hepatitis

Autoantibodies against microsomal antigen of liver and kidney (LKM‐1) are diagnostic markers for a subgroup of autoimmune chronic active hepatitis (AI‐CAH). Cytochrome P4S0dbl, now classified as cytochrome P450 IID6, is the major antigen of LKM‐1 antibodies. Immunohistological studies suggest that hepatic injury in AI‐CAH is mediated by liver‐infiltrating T cells. In the present study the specificity and function of liver‐infiltrating T cells was analysed at the clonal level. Phenotypical characterization of 189 T cell clones isolated from four liver biopsies of LKM‐1 antibody‐positive patients showed an enrichment of CD4+CD8‐ T cells. Five CD4+CD8‐ T cell clones proliferated specifically in the presence of recombinant human LKM‐1 antigen (rLKM). This reaction was restricted to autologous antigen‐presenting cells and to HLA class II molecules. In order to see whether rLKM was also recognized by peripheral blood T lymphocytes (PBL) we tested the proliferative response of PBL from several individuals. PBL from three of the four patients with LKM‐1 antibody‐positive AI‐CAH proliferated to rLKM, whereas no response was seen with PBL from patients with LKM‐1 antibody‐negative chronic liver diseases and from healthy blood donors. These data demonstrate that the LKM‐1 antigen is recognized by liver‐infiltrating T cells in LKM‐1 antibody‐positive AI‐CAH. For further functional characterization, liver‐derived T cell clones were tested for their cytotoxic activity. In the presence of phytohacmagglutinin 24 out of 26 CD4‐CD8+ but also 20 out of 63 CD4+CD8‐ T cell clones lysed autologous as well as allogenic EBV‐transformed B cell lines or K562 cells. Five CD4‐CD8+ T cell clones lysed autologous but not allogenic B cell lines spontaneously in a HLA class I‐restricted manner. Although the antigen specificity of these clones is still unknown the data show the presence of autoreactive T cells at the site of inflammation that could contribute in the pathogenesis of AI‐CAH.

Bile duct epithelia as target cells in primary biliary cirrhosis and primary sclerosing cholangitis

Abstract Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are chronic autoimmune-mediated diseases of the biliary tree, resulting in a loss of bile ducts. There are morphological features that clearly distinguish them from each other: in PBC, there is overt destruction of the bile ducts with disruption of the basement membrane; in PSC there is abundant periductular fibrosis with shrinkage and subsequent loss of the bile ducts. In order to see if the disparate histopathology is paralleled by different immunohistology we looked at a panel of epitopes on bile duct epithelia especially to see if biliary epithelial cells may present as targets for cell mediated immune respone. In PBC bile duct epithelial cells mostly expressed CD58 (lymphocyte function-associated antigen 3), CD80 (B7 BB1), and CD95 (Fas). In PSC, however, these epitopes were only expressed in a few examples to a lower degree. The respective effector T lymphocytes were positive for CD2 and CD28. Subtyping of the lymphocytes in the liver tissue further showed a predominance of CD4 positive T cells over CD8 cells up to 2-to-1 in both diseases. Determination of lymphocytes by cytokines to Th1 or Th2 subtype showed a majority of Th1 lymphocytes in PBC and PSC. We conclude that in PBC bile duct epithelial cells may display features of target cells of a T cell-mediated immune reaction with the Th1 cells predominating. In PSC other mechanisms of bile duct loss may play a role, since in this disease the majority of cells lack essential epitopes that constitute targets of cell mediated immunity.

The viral clearance in interferon-treated chronic hepatitis C is associated with increased cytotoxic T cell frequencies

BACKGROUND/AIMS Cytotoxic T lymphocytes have been demonstrated in peripheral blood and liver tissue of patients with chronic hepatitis C virus infection, but their significance for viral clearance is unknown. Therefore, we analyzed hepatitis C virus-specific cytotoxic T lymphocyte precursor frequencies in chronic hepatitis C virus carriers during interferon-alpha treatment. METHODS Blood mononuclear cells or CD8+ T cells from HLA-A2 positive and negative patients and controls were analyzed in chromium-release assays using a panel of 18 synthetic peptides from the HCV core, E1 and NS4 antigens bearing HLA-A2 binding motifs. Specific cytotoxic T lymphocyte precursor frequencies were studied within CD8+ T cells derived from interferon-alpha-treated patients using a TNF-alpha-based ELISPOT assay and compared to viremia levels. RESULTS T cells from 16 of 24 HLA-A2+ but none of the six HLA-A2- patients with chronic hepatitis C and six HLA-A2+ healthy controls lysed targets pulsed with peptide cocktails. Fine specificity revealed four very immunogenic epitopes in the core (C36-44, C132-140) and the envelope regions (E332-340, E363-372). Cytotoxic T lymphocyte precursor frequencies were prospectively analyzed in 11 interferon-alpha-treated HLA-A2+ hepatitis C virus patients. Four sustained and two transient therapy responders showed lower pretreatment viremia levels and significantly higher specific cytotoxic T lymphocyte precursor frequencies during viral clearance compared to five therapy non-responders and untreated controls. CONCLUSIONS The quantitative induction of HLA-class I restricted responses by interferon-alpha could contribute to a beneficial outcome of hepatitis C virus infections. Furthermore, it appears that the balance between viral load and specific cellular immune responses is critical for successful viral clearance.

Budesonide 9 mg Is at Least as Effective as Mesalamine 4.5 g in Patients With Mildly to Moderately Active Crohn's Disease

BACKGROUND & AIMS Comparative data on budesonide vs mesalamine for the treatment of mild-to-moderately active Crohns disease (CD) are sparse. We assessed the efficacy and safety of each therapy in patients with mildly to moderately active CD. METHODS We performed a randomized, double-blind, double-dummy, 8-week, multicenter study in which 309 patients with mildly to moderately active CD received pH-modified-release oral budesonide (9 mg/day once daily or 3 mg/day 3 times daily) or Eudragit-L-coated oral mesalamine (4.5 g/day). RESULTS The primary efficacy variable, clinical remission (defined as Crohns Disease Activity Index ≤150), at the final visit occurred in 69.5% (107 of 154) of patients given budesonide vs 62.1% (95 of 153) of patients given mesalamine (difference, 7.4%; 95% repeated confidence interval, -4.6% to 18.0%; P = .001 for noninferiority). Clinical remission rates did not differ significantly between the 2 budesonide groups. Treatment response, defined as Crohns Disease Activity Index of 150 or less and/or a decrease of 70 or more (Δ70) or 100 or more (Δ100) points from baseline to final visit, did not differ significantly between patients given budesonide vs mesalamine (Δ70, P = .11; Δ100, P = .15), or between the 2 budesonide groups (Δ70, P = .38; Δ100, P = .78). No other efficacy end points differed significantly between groups. Discontinuation because of adverse events occurred in 3% and 5% of budesonide- and mesalamine-treated patients, respectively. There were no clinically relevant differences in adverse events between the 2 budesonide groups. CONCLUSIONS Budesonide (9 mg/day) was numerically, but not statistically, more effective than Eudragit-L-coated mesalamine (4.5 g/day) in patients with mildly to moderately active CD. Budesonide (9 mg/day), administered once daily, was as effective as the standard (3 times daily) regimen.

Induction of strong hepatitis B virus (HBV) specific T helper cell and cytotoxic T lymphocyte responses by therapeutic vaccination in the trimera mouse model of chronic HBV infection

Humanized BALB / c mice (termed trimera mice) conditioned by lethal total body irradiation and bone marrow transplantation from SCID mice have been described to support rapid engraftment of human peripheral blood mononuclear cells (PBMC) and the induction of strong B and T cell responses after immunization in vivo. Moreover, these mice can be infected with the hepatitis B and C viruses (HBV, HCV). The current study employed this model to study therapeutic vaccination approaches against the HBV. Thus, strong primary Th cell responses against the HBV core (HBc) and the Borrelia burgdorferi control antigen were induced by transfer of antigen‐loaded dendritic cells together with autologous PBMC from HBV‐naive donors as well as by vaccination with high doses of antigen or a DNA plasmid encoding for HBcAg. Moreover, primary peptide‐specific CTL responses against the immunodominant epitope HBc18 – 27 were induced by HBc particle or DNA vaccination of chimera engrafted with HBV‐naive PBMC. Finally, strong HBc‐specific Th cell and antibody responses were induced by HBc or DNA vaccination of mice reconstituted with PBMC from a chronic HBV patient. Thus, since HBc represents the immunodominant antigen in self‐limited HBV infection, HBc particles or DNA vectors are good candidates for therapeutic vaccination, that will be further studied in ourmodel and clinical studies.

Reduced virus specific T helper cell induction by autologous dendritic cells in patients with chronic hepatitis B – restoration by exogenous interleukin‐12

Insufficient stimulatory capacities of autologous dendritic cells (DC) may contribute in part to impaired T cell stimulation and therefore viral persistence in patients with chronic hepatitis B virus (HBV) infection. In order to characterize the antigen presenting functions of DC from chronic HBV carriers and controls antigen specific T cell responses were analysed. CD34+ peripheral blood progenitor cells were differentiated to immature DC in the presence of GM‐CSF, IL‐6/IL‐6R fusion protein and stem cell factor. Proliferative CD4+ T cell responses and specific cytokine release were analysed in co‐cultures of DC pulsed with HBV surface and core antigens or tetanus toxoid and autologous CD4+ T cells. Cultured under identical conditions DC from chronic HBV carriers, individuals with acute resolved hepatitis B and healthy controls expressed similar phenotypical markers but chronic HBV carriers showed less frequent and weaker HBV antigen specific proliferative T helper cell responses and secreted less interferon‐γ while responses to the tetanus toxoid control antigen was not affected. Preincubation with recombinant IL‐12 enhanced the HBV specific immune reactivities in chronic HBV patients and controls. In conclusion, the weak antiviral immune responses observed in chronic hepatitis B may result in part from insufficient T cell stimulating capacities of DC. Immunostimulation by IL‐12 restored the HBV antigen specific T cell responses and could have some therapeutical benefit to overcome viral persistence.

Regulation of the neutralizing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vaccine recipients and patients with acute or chronic hepatitis B virus (HBV) infection

Antibodies directed to the HBs antigen indicate viral clearance and the development of life‐long immunity in patients that recovered from HBV infection. In HBs antigen vaccine recipients anti‐HBs antibodies provide protective immunity. However, little is known about the regulation of this HBs‐specific antibody response. The existence of anti‐HBs‐secreting B cells was demonstrated using the highly sensitive ELISPOT technique compared with conventional ELISA in serum and cell culture supernatants. In the peripheral blood of patients with acute self‐limited hepatitis B, HBs‐specific B cells were demonstrated with a high frequency despite undetectable anti‐HBs serum antibodies. HBV‐immunized patients that had recovered from infection and vaccine recipients had significantly lower frequencies, whereas chronic HBV carriers and negative controls showed no anti‐HBs‐secreting B cells. Coculture experiments of isolated B and T cells revealed that the anti‐HBs antibody response was restricted to the presence of T helper cells, but not to identical HLA class II molecules. Allogeneic T cells derived from vaccine recipients or chronic HBV carriers stimulated the HBs‐specific B cell response in HBs vaccine recipients. Otherwise, isolated T helper cells could never provide sufficient help to induce the HBs‐specific B cell response in chronic HBV carriers. Furthermore, peripheral blood mononuclear cells (PBMC) of six out of 10 vaccine recipients, one out of five HBV‐immunized patients, but of no chronic HBV carrier showed a proliferative response to different HBs antigen preparations. This study demonstrated a high frequency of circulating anti‐HBs‐producing B cells in the early phase of acute HBV infection, but a lower frequency of HBs‐specific B cells years after resolution of HBV infection. In chronic HBV carriers, however, deficient HBs‐specific T and B cell responses were observed.

Autoreactive liver-infiltrating T cells in primary biliary cirrhosis recognize inner mitochondrial epitopes and the pyruvate dehydrogenase complex

Primary biliary cirrhosis (PBC) is characterized by lymphoid infiltrates in the portal tracts of the liver and the occurrence of antimitochondrial autoantibodies in serum directed against components of the pyruvate dehydrogenase complex and the other alpha-keto acid dehydrogenase complexes. These enzymes are located on the inner mitochondrial membrane. The destruction of the biliary tract in PBC is thought to be mediated by autoreactive liver-infiltrating T cells exerting cytotoxic activity or releasing certain lymphokines. In this study the reactivity of liver infiltrating T cells was shown to a bovine pyruvate dehydrogenase complex (PDH), a purified E2 subunit (PDH-E2) and a crude preparation of human liver mitoplasts (HLM), i.e. mitochondria depleted of their outer membranes. Peripheral blood lymphocytes (PBL) from 11 of 15 patients (73.3%) with PBC showed a HLA class II-restricted proliferative response to the PDH complex whereas PBL from patients with chronic viral hepatitis, autoimmune hepatitis or extrahepatic cholestatic icterus (n = 20) and healthy controls (n = 5) did not. In addition 13 of 15 PBL from patients with PBC (86.6%) and three of nine PBL from patients with autoimmune hepatitis (33.3%) reacted with the crude HLM preparation whereas no reactivity was found with PBL from eight patients with chronic viral hepatitis, three patients with extrahepatic cholestasis or five healthy controls. Clonal analysis of 115 liver-infiltrating T cells derived from two diagnostic liver biopsies of patients with PBC revealed a predominance of activated CD4+CD8- T helper cells.(ABSTRACT TRUNCATED AT 250 WORDS)