Expression of the 60 kDa heat shock protein in normal and inflamed liver.

The 60 kDa heat shock proteins (HSP 60) have been well conserved throughout evolution and are highly immunogenic. Cross-reactivity between bacterial and mammalian HSP 60 is considered a likely mechanism in the pathogenesis of autoimmune diseases. T cell and B cell reactivity to HSP 60 is found in patients with rheumatoid or juvenile arthritis, and the expression of HSP 60 in the inflamed joint is found to be increased. In this study the presence of HSP 60 was demonstrated in normal and inflamed lives. HSP 60 was found to be predominantly expressed in hepatocytes and Kupffer cells, and mainly localized in mitochondria. Heat stress in the form of a 1 h incubation at 42 degrees C increased HSP…

Hepatocellular expression of lymphocyte function—associated antigen 3 in chronic hepatitis

T lymphocyte-mediated cytolytic immune reactions are considered a major cause of hepatocyte injury in chronic viral and autoimmune hepatitis. To further investigate local immune responses, we studied the expression of lymphocyte antigens and cell-cell interaction molecules known to be involved in effector-target cell interactions by light and electron microscopy in liver biopsy specimens from patients with chronic viral and autoimmune hepatitis. CD8+ lymphocytes were found to be the predominant population of cells in the inflammatory infiltrate in chronic hepatitis B and non-A, non-B hepatitis. In contrast, CD4+ cells constituted a comparably higher proportion of cells and were more numerou…

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

Abstract 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, γ-glutamyltranspeptidase and IgG. Anti-n…

Cultured Ito cells of rat liver express the alpha 2-macroglobulin gene.

Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize a2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized a2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis and fluorography. a2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5 - 11 days after the isolation of the cells, increasing amounts of a2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled a2-macroglob…

Development of cytochrome P450 2D6-specific LKM-autoantibodies following liver transplantation for Wilson's disease -- possible association with a steroid-resistant transplant rejection episode.

Abstract Background/Aims: Antibodies to cytochrome P450 2D6, also knownas LKM1-autoantibodies, are characteristic for a subgroup of patients with autoimmune hepatitis, but can also occasionally be found in hepatitis C. We observed the occurrence of LKM1-autoantibodies 4 months after liver transplantation for Wilson's disease, in close association with a steroid-resistant rejection episode, in the absence of evidence for autoimmune hepatitis or hepatitis C. Methods: Sera from several time points prior to and following transplantation were tested for LKM-reactivity by immunofluorescence, ELISA and Western blotting. Antigen specificity was confirmed by Western blotting analysis on different cy…

Identification of cyclin A as a molecular target of antinuclear antibodies (ANA) in hepatic and non-hepatic autoimmune diseases.

Abstract Background/Aims: Antinuclear antibodies (ANA) are a diagnostic of various autoimmune diseases and also of autoimmune hepatitis type 1. The designation ANA describes a heterogeneous group of autoantibodies. In liver diseases, only a few nuclear target antigens have been molecularly identified and characterized. Cyclins play a central role in a cell cycle regulation, DNA transcription, and cell proliferation. Cyclin A was also identified as an integration site of the hepatitis B virus in a patient with hepatocellular carcinoma. In this study we identify cyclin A as a novel nuclear target protein of ANA. Methods: Sera of patients with autoimmune hepatitis (AIH) type 1 ( n =61), type 2…

Actin is a target antigen of anti-neutrophil cytoplasmic antibodies (ANCA) in autoimmune hepatitis type-1.

Abstract Background/Aim: Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies first associated with Wegener's granulomatosis and microscopic polyangiitis. The signifiance of ANCA in autoimmune hepatitis remains uncertain; the nature of the antigen or antigens has not been defined yet. The purpose of this study was to identify the target antigen of ANCA in patients with autoimmune hepatitis. Method/Results: Sera from 32 type-1 autoimmune hepatitis patients were used in the present study. ANCA were detected in 24 of 32 sera (75%). A diffuse cytoplasmic staining pattern (C-ANCA) was detected in 14 patients; the P-ANCA pattern was observed in 10 patients. An extract of hu…

Isolation of naturally processed peptides recognized by cytolytic T lymphocytes (CTL) on human melanoma cells in association with HLA-A2.1.

Cytolytic T lymphocyte (CTL) clones have previously been derived from peripheral blood of melanoma patient SK29(AV). They lyse autologous melanoma cells but not autologous Epstein-Barr virus (EBV)-transformed B lymphocytes. Immunoselection experiments indicate that these CTL clones recognize 4 different antigens (Aa, Ab, B, C) in association with a single HLA restriction element, HLA-A2.1. While the expression of antigens B and C appears to be confined to SK29-melanoma cells, antigens Aa and Ab are shared by a high proportion of allogeneic HLA-A2-positive melanoma lines. HLA-A2.1 and total HLA class I molecules have now been purified from SK29-melanoma cells using affinity chromatography an…

Cytokine Gene Transcription By NF-kappaB Family Members in Patients with Inflammatory Bowel Disease

We examined the expression of the transcription factor NF-kappa B, a nuclear trans-acting factor known to play a key role in cytokine gene regulation, in patients with inflammatory bowel disease (IBD). It was found that LP macrophages in Crohn's disease (CD) and ulcerative colitis (UC) display high levels of NF-kappa B DNA-binding activity accompanied by an increased production of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF) alpha. Western blot studies showed an increased expression of the p50 and c-rel subunits of NF-kappa B; however, the most striking finding was an increased expression level of NF-kappa B p65 in patients with CD and UC. Selective downregulation of p65 in IBD…

Quantification of CD8+ T lymphocytes responsive to human immunodeficiency virus (HIV) peptide antigens in HIV-infected patients and seronegative persons at high risk for recent HIV exposure.

/ T cells responding to HIV-1 peptides were observed in none of 11 HIV- seronegative donors without a history of HIV exposure. ELISPOT assays are relatively fast and easy to perform and appear to reliably detect T cell reactivity due to previous exposure to HIV. These findings support the use of the ELISPOT assay for monitoring T cell responsiveness to HIV peptides. In acute infection with the human immunodeficiency virus We described recently an enzyme-linked immunospot (ELISPOT) assay to detect and quantitate single blood-de- type 1 (HIV-1), initial reduction in virus load is associated with the appearance of a high frequency of antiviral cytotoxic T rived CD8 / T lymphocytes forming tumo…

Regulation of Protein-DNA Interactions at the Interferon-gamma Gene Promoter by Corticosteroids: Implications for Inflammatory Bowel Diseases

Liver Membrane Autoantibodies

Publisher Summary This chapter includes the intensive efforts to standardize the assay, define associations with disease or clinical presentation, and characterize antigenic and biological properties of anti-liver membrane antibodies. The characterization of different target antigens on the hepatic plasma membrane shows a polyvalent and heterogenous humoral immune response against the liver surface. Liver membrane antibodies can induce liver cell damage in vitro, including dysfunction of the target antigen. Among the established liver membrane-related autoantigens, the asialoglycoprotein receptor (ASGPR) represents the only component of the hepatic plasma membrane that is fully characterize…

Proliferative response of CD4+ T cells and hepatitis B virus clearance in chronic hepatitis with or without hepatitis B e-minus hepatitis B virus mutants.

To assess the significance of cell-mediated immunity, T cells were derived from the peripheral blood and liver tissue of hepatitis B virus (HBV)-infected patients and controls. The analysis of the 3H-thymidine-uptake in response to a panel of recombinant HBV antigens revealed that peripheral blood mononuclear cells (PBMC) of the 25 viremic patients with inflammatory active, chronic hepatitis B, 16 with wild-type and nine with HBe-minus HBV mutant infection, showed stronger proliferative responses to HBc and HBe antigens than 16 asymptomatic nonviremic HBsAg carriers with normal aminotransferase levels (HBc: SI 19.3 +/- 3.9 vs. 13.0 +/- 3.2 vs. 8.0 +/- 1.2; P.01 and HBe: SI 16.6 +/- 4.0 vs. …

Yeast expression of the cytokine receptor domain of the soluble interleukin-6 receptor

Abstract The complex of the soluble interleukin-6 receptor (sIL-6R) and IL-6 (IL-6) is a potent agonist on cells expressing the signal transducing protein gp130. In contrast, IL-6 alone only stimulates cells which express a membrane bound form of the IL-6R and gp130. The natural occurring sIL-6R is generated by shedding of the membrane receptor and to a lesser extend by alternative splicing. We have inserted the coding sequence of the 323 amino acid residues of the human sIL-6R into an expression/secretion vector suitable for the methylotrophic yeast Pichia pastoris . We obtained, however, no detectable expression and secretion of the recombinant protein. When we used only the coding sequen…

Stimulation of pancreas and gastric carcinoma cell growth by interleukin 3 and granulocyte-macrophage colony-stimulating factor.

Hematopoietic growth factors have recently been well characterized by complementary DNA scloning. For human epidermal growth factor, granulocyte-macrophage colony-stimulating factor recombinant proteins have been expressed in Escherichia coli . To reduce the toxic side effects of chemotherapy on the bone marrow, recombinant human granulocyte-macrophage colony—stimulating factor and recombinant human interleukin 3 were applied to patients suffering of gastrointestinal cancers. To determine the influence of recombinant human granulocyte-macrophage colony—stimulating factor and recombinant human interleukin 3 on human pancreas and gastric cancer cell cells in vitro, a sensitive microculture te…

T cells involved in psoriasis vulgaris belong to the Th1 subset

Although the pathogenesis of psoriasis vulgaris is still unknown, several characteristics point to an immunologically mediated process. Epidermal psoriatic lesions are characterized by a hyperproliferation of keratinocytes and an infiltration of T lymphocytes and granulocytes. Because the former may be mediated in part by lymphokines secreted by T cells, we have focused our interest on the in vivo and in vitro cytokine secretion patterns of T lymphocytes from psoriatic lesions. In five patients T lymphocytes were obtained from epidermal specimens. The cells were propagated with lectin and irradiated feeder cells and subsequently cloned by limiting dilution. The resulting T-cell clones were …

Enterobacteria-infected T cells as antigen-presenting cells for cytotoxic CD8 T cells: a contribution to the self-limitation of cellular immune reactions in reactive arthritis?

In enterobacteria-induced reactive arthritis (ReA), different T cell subsets play a role in the induction and maintenance of the synovitic process. Synovial fluid-derived alphabeta CD4, alphabeta CD8, and gammadelta T lymphocyte clones (TLC) that recognize Yersinia or Salmonella antigens on professional antigen-presenting cells (APC) have been characterized, and T cells themselves can function as nonprofessional APC. T cells were infected with the facultatively intracellular, arthritogenic enterobacterium Yersinia enterocolitica O:3. A CD8 TLC isolated from a patient with Yersinia-induced ReA recognized and efficiently lysed autologous and allogeneic Yersinia-infected T cells. Infected cyto…

Ultrastructural localization of interferon-producing cells in the livers of patients with chronic hepatitis B

Cells expressing alpha- and gamma-interferon were localized in the liver tissue of patients with chronic hepatitis B by means of light and electron microscopy using monoclonal antibodies. Interferon-positive cells were regularly seen in the infiltrating mononuclear cells, and the number showed a good correlation with the degree of the necroinflammatory activity of the disease. In chronic persistent hepatitis and in normal livers, they were infrequent or virtually absent. alpha-Interferon was shown to be positive in lymphocytes, polymorphonuclear leukocytes and fibroblasts, Kupffer cells and, weakly, in the cytoplasm of a few hepatocytes in cases of active hepatitis, whereas gamma-interferon…

Hepatitis B virus with antigenically altered hepatitis B surface antigen is selected by high-dose hepatitis B immune globulin after liver transplantation

“Escape” variants of hepatitis B virus (HBV) can cause infection despite previous immunization. These viruses show alterations of the immunogenic major hydrophilic loop of the HBV small surface protein (s-protein). We studied whether HBV “escape” variants were selected in patients with graft infection after liver transplantation for HBV-related diseases who received passive immunoprophylaxis with high-dose polyclonal hepatitis B hyperimmune globulin (HBIG). For that, pre- and posttransplantation sera of 34 patients were analyzed for the occurence of HBV S-gene variants. In addition, binding of in vitro–translated variant s-proteins to HBIG was studied. Variants with exchanges of amino acid …

Mixed cryoglobulinemia type II in chronic hepatitis B associated with HBe-minus HBV mutant: Cellular immune reactions and response to interferon treatment

The case of a young female patient with chronic active hepatitis B, vasculitic purpura, edema, and circulating immune complexes due to mixed Cryoglobulinemia is described. Serum transami-nases were elevated. Serological assays showed hepatitis B surface antigen (HBsAg), antibody to hepatitis B e antigen (anti-HBe), and antibody to hepatitis B core antigen (anti-HBc) antibodies but no antibody to hepatitis C virus (anti-HCV) or antibody to hepatitis delta virus (anti-HDV) antibodies. Using hepatitis B virus-polymerase chain reaction (HBV-PCR) and direct sequencing a precore/core (preC/C) mutant unable to synthesize HBeAg was detected in serum. HBV antigens were demonstrated in the circulatin…

HLA-B27-derived peptides as autoantigens for T lymphocytes in ankylosing spondylitis

Objective.To study whether peptides derived from the HLA-B27 molecule sequence can stimulate peripheral blood T lymphocytes (PBL) from patients with HLA-B27-associated spondylarthropathies. Methods.PBL from 55 HLA-B27+ patients with ankylosing spondylitis (AS), 28 HLA-B27+ patients with other spondylarthropathies, 7 rheumatoid arthritis patients, and 30 HLA-B27+ and 22 HLA-B27- healthy controls were tested in lymphocyte proliferation assays with 4 synthetic peptides derived from the HLA-B*2705 molecule. Results. A 13-mer peptide (B27PA) induced significant proliferative responses in 17 of the 55 AS patients (stimulation index [SI] 2.5–17.5), as well as in 3 of the HLA-B27+ healthy controls …

Significance of IgG and IgM HCV antibody secretion in vitro in patients with chronic hepatitis C: correlation with disease activity and response to interferon-alpha.

Hepatitis C virus antibodies are found in the serum of most patients with chronic hepatitis C. However, the significance of the humoral response is still uncertain. In this study, in vitro IgG and IgM anti-hepatitis C virus secretion by peripheral blood mononuclear cells of patients with chronic hepatitis C was analyzed. Peripheral-blood mononuclear cells from 21 of 36 patients (58.3%) secreted IgG anti-hepatitis C virus in vitro, as demonstrated with anti-hepatitis C virus—specific enzyme immunoassays and recombinant immunoblot assays. Ten of the 36 patients (27.8%) showed both IgG and IgM anti-hepatitis C virus core in vitro. In 9 of these 10 patients, IgM anti-hepatitis C virus was also …

Hepatic expression patterns of the large and middle hepatitis B virus surface proteins in viremic and nonviremic chronic hepatitis B.

The envelope of hepatitis B virus consists of large, middle, and small hepatitis B surface proteins. Recent data from in vitro studies suggest that intracellular expression and distribution of the three polypeptides may be variable. These observations in artificial expression systems prompted this analysis of the occurrence and distribution of the three hepatitis B surface proteins in the liver tissue of substantial viremic (hepatitis B virus DNA- and hepatitis B e antigen-positive) and low-viremic or nonviremic (hepatitis B virus DNA-negative, anti-hepatitis B e antigen-positive) carriers by specific monoclonal antibodies against large, middle, and small proteins. Patients with an active f…

Biosynthesis of the subcomponents Clq, Clr and Cls of the first component of complement (Cl) by guinea pig hepatocyte primary cultures

Thus far, the synthesis of C1q by liver cells has not been demonstrated. To investigate this possibility, viable hepatocytes were isolated from the liver of guinea pigs and primary cultures were established. The cells (10(6) cells/ml) were cultured under serum-free conditions for 8 days and the culture medium was changed every 24 h. The few contaminating Kupffer cells were lysed by preincubating the cell cultures with a monoclonal (22C4-8) antibody directed against a nonpolymorphic Ia determinant and preabsorbed rabbit serum. The hemolytic activity of C1 and its subcomponents C1q and C1r/C1s was tested in the supernatants. Guinea pig hepatocyte primary cultures synthesize and secrete up to …

Limited T cell receptor Vbeta-chain repertoire of liver-infiltrating T cells in autoimmune hepatitis.

Abstract Background/Aims: To characterize the cellular immune reactions in autoimmune hepatitis, the T cell receptor repertoire of livre-infiltrating and circulating T cells was studied. Methods: Nucleic acids of liver-tissue and peripheral blood-derived T cells from 12 patients with untreated autoimmune hepatitis, four patients with chronic hepatitis C and three patients with toxic liver injury were extracted and analysed using a semiquantitative RT-PCR with a panel of T cell receptor Vβ family specific primers. After agarose gel electrophoresis, the distribution of T cell receptor (TCR) Vβ molecules was assessed by densitometry. Furthermore, results were compared to the TCR Vβ distributio…

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

Abstract 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-α 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 st…

Fat-storing cells of the rat liver synthesize and secrete C1-esterase inhibitor; modulation by cytokines.

During liver fibrogenesis, fat-storing cells transform into myofibroblast-like cells and produce increasing amounts of extracellular matrix proteins. Because fat-storing cells produce α2-macroglobulin, an important serine protease inhibitor (serpin), we investigated whether fat-storing cells also synthesize C1-esterase inhibitor, another important serpin. C1-esterase inhibitor synthesis was studied in rat fatstoring cells at day 0, 3 and 7 after isolation by biosynthetic labeling, immunoprecipitation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Messenger RNA was examined by Northern-blot analysis. C1-esterase inhibitor gene expression and synthesis were detectable in fresh…

Pre-core mutants of hepatitis B virus in patients receiving immunosuppressive treatment after orthotopic liver transplantation.

Orthotopic liver transplantation (OLT) is a possible treatment for acute or chronic liver failure due to hepatitis B virus (HBV) infection, but reinfection of the graft can be a serious complication. The aim of this study was to monitor HBV markers, to analyse pre-core-/core-mutations as well as to identify the viral population causing reinfection after OLT, and to investigate the emergence or disappearance of these mutants in patients receiving immunosuppressive treatment. Fifty-four pre-and posttransplant serum samples of 17 patients were analysed. All patients underwent OLT for HBV-related liver disease and had HBV-DNA before and after OLT. Total DNA was extracted from all sera and a 240…

Normal interleukin-12 production in individuals with antibodies toHelicobacter pylori

It is increasingly recognized that the inability of the immune system to clear H. pylori infection is caused by an inadequate immune response and is associated with chronic gastric inflammation. To further investigate the cellular immune response to H. pylori, we studied PBMC from 31 H. pylori antibody-negative and 16 H. pylori antibody-positive individuals for H. pylori-induced DNA synthesis, secretion of the Th1-type cytokine IFN-gamma and secretion of IL-12, a cytokine produced by bacteria-stimulated monocyte/macrophages and a potent inducer of antibacterial immune responses and Th1-type T cells. All experiments were performed using Y. enterocolitica 03 as control. Our results demonstrat…

Clonal analysis of human T lymphocytes infiltrating the liver in chronic active hepatitis B and primary biliary cirrhosis

Human T lymphocytes infiltrating the liver in chronic active hepatitis B (CAH-B) and primary biliary cirrhosis were isolated from liver biopsy cores, cloned by limiting dilution technique and expanded in vitro. Phenotypic and functional analysis demonstrates that this tissue infiltrate represents a heterogeneous cell population. However, when compared to peripheral blood lymphocytes of the same patients, a marked enrichment for T8+ cytotoxic T cells was found to exist at a local site in both types of chronic liver disease. These data provide support for the notion that liver cell injury in CAH-B and PBC may be mediated by a common immunologic mechanism likely executed by cells of the T line…

Hepatocellular carcinoma after thorotrast exposure: establishment of a new cell line (Mz-Hep-1).

A human hepatoma cell line, associated with thorotrast exposure, from an hepatitis B marker-negative patient was established as a permanent cell line (Mz-Hep-1) in tissue culture. Histology of the primary tumor, as well as phase contrast, transmission and scanning electron microscopy of the cultured cells showed typical characteristics of liver cells. Mz-Hep-1 cells secreted complement components (C2, C3, C4), carcinoembryonic antigen, lactate dehydrogenase, chymotrypsin, haptoglobin and retinol-binding protein and expressed HLA-, transferrin-, blood group B-related determinants and complement component C5 and carcinoembryonic antigen on their cell surface. Mz-Hep-1 cells represent the firs…

Induction of tumor-cell lysis by bi-specific antibody recognizing ganglioside GD2 and T-cell antigen CD3

Human tumor cells expressing ganglioside GD2 were lysed by various effector populations targeted with an anti-CD3-anti-GD2 bi-specific antibody (BAb CD3 x GD2). This antibody-heteroconjugate was prepared by chemically cross-linking the OKT-3 monoclonal antibody (MAb) reactive with CD3 antigen on T lymphocytes with the ganglioside MAb ME 361, which binds preferentially to the tumor-associated ganglioside GD2. The specificity of target-cell lysis by the cytotoxic T cells (CTL) was mediated by the specificity of the targeting antibody: GD2-negative cells were not lysed in the presence of the CD3 x GD2 BAb. A dose-dependent response was observed in a range of 10 to 10,000 ng/ml. In contrast, 2 …

klebsiella pneumoniae-reactive t cells in blood and synovial fluid of patients with ankylosing spondylitis comparison with hla–b27 + healthy control subjects in a limiting dilution study and determination of the specificity of synovial fluid t cell clones

Objective To study the frequency of Klebsiella pneumoniae-responsive T cells in the peripheral blood (PB) of ankylosing spondylitis (AS) patients compared with that in healthy HLA-B27+ donors, and to examine T lymphocyte clones (TLC) derived from AS patient synovial fluid (SF) for the presence of Klebsiella reactivity. Methods Limiting dilution analysis of PB T cells in 8 patients with active AS and in 8 HLA-B27+ healthy subjects was used to determine the frequency of PB T cells responsive to K pneumoniae and Escherichia coli GroEL. SF T cells from a patient with active AS were cloned, and 125 TLC were characterized in proliferation assays. Results There were fewer T cells in the PB of AS p…

Kinetics of hepatitis B surface antigen-specific immune responses in acute and chronic hepatitis B or After HBs vaccination: Stimulation of thein vitroantibody response by interferon gamma

Because cellular and humoral immune responses against the hepatitis B virus (HBV) surface antigen (HBs) might be crucial to overcome HBV infection, HBs-specific B- and T-cell responses of HBV patients and HBs vaccine recipients were analyzed quantitatively and functionally. In patients with acute hepatitis B (AHB), transient high anti-HBs-secreting B-cell frequencies were observed early after clinical onset, whereas 1 patient who probably developed chronic infection and chronic HBV carriers had absent or weak B- and T-cell responses. In HBs vaccine recipients, maximal HBs-specific B- and T-cell responses were detected after the first injection that decreased gradually before anti-HBs antibo…

Autoimmunity and liver disease

Experimental Hepatitis

Publisher Summary This chapter presents the animal models of experimental hepatitis (EAH). EAH was developed as a model of human autoimmune hepatitis (AIH). AIH in humans is a heterogeneous disease, which most often occurs in young females and generally responds very well to immunosuppressive therapy. AIH is often not recognized or is misdiagnosed as chronic non-A, non-B (non-C) viral hepatitis. Even when left untreated for considerable time periods, the disease in some patients is moderate to mild, and transient spontaneous remissions can be observed. EAH in many ways reflects this disease process. EAH also is often mild to moderate, and spontaneous remission occurs. Autoantibodies are cri…

Pyruvate dehydrogenase specific T cells in primary biliary cirrhosis show restricted antigen recognition sites

:  Background/Aims: The aim was to characterise the antigen recognition sites of the variable T cell receptor α-chain (TCRAV) and β-chain (TCRBV) of T cells specific to the pyruvate dehydrogenase (PDC) in primary biliary cirrhosis. Methods: In 21 PDC-specific T cell clones isolated from five patients we analysed TCRAV and TCRBV usage by RT-PCR and sequenced the CDR3 regions. Results: Preferential expression of the TCR elements BV6 (6 clones), BV12 (4 clones) and BV1 (3 clones), and frequent usage of the joining elements JB2.3 and JB2.1 were seen. Analysis of the α chain revealed rearrangement of AV2 in 7 clones (35%) and AV7 in 3 clones, however, distribution of the joining elements was het…

Two different subtypes of antimitochondrial antibodies are associated with primary biliary cirrhosis: identification and characterization by radioimmunoassay and immunoblotting.

Antimitochondrial antibodies from patients with primary biliary cirrhosis react with different mitochondrial polypeptides as demonstrated by Western blots. The IgG fractions of a patient with primary biliary cirrhosis Stage I reacting exclusively with a pair of polypeptides at 48,000 daltons (p 48) on Western blot and from a patient with Stage III primary biliary cirrhosis reacting exclusively with a single 62,000 dalton polypeptide (p 62) were labeled with 125I; two radio-immunoassays were established detecting antimitochondrial antibodies against p 62 and p 48, respectively. Autologous sera blocked the assay, but the two reference sera did not block each other. Fourteen of 40 patients wit…

HLA DRw8 and primary biliary cirrhosis

Dominant TCRB-V-J chain usage and clonal expansion of sarcoma-reactive CD4+ HLA-DR-restricted T cells suggest a limited set of immunodominant sarcoma antigens

Tumor-reactive CD4+ T cells have been isolated from tumor patients, and their specifity but not T-cell receptor (TCR) repertoire has been analyzed. Since we have described CD4+ sarcoma-reactive T-cell clones, we now sought to determine whether the TCR repertoire of these clones provides information on the spectrum of recognized sarcoma antigens. We analyzed the TCR beta (TCRB) chain repertoire of 19 CD4+ HLA-DR-restricted T-cell clones reactive with the autologous sarcoma cell line MZ-MES-1, with HLA-DR-matched tumor cell lines of different tissue origins and B-cell blasts. We identified 7 different clonotypes, which used a limited set of TCRBV and TCRBJ segments. Although the CDR3 of the d…

The presence of high amounts of HBV-DNA in serum is associated with suppressed costimulatory effects of interleukin 12 on HBV-induced immune response

Abstract Background/Aims: The aim of this study was to examine the influence of the viral load on costimulatory effects of rhIL-12 on the hepatitis B virus (HBV)-induced immune response. Methods: Peripheral blood mononuclear cells of HBsAg positive patients without cirrhosis were stimulated with HBsAg, HBcAg, preSlAg and tetanus toxoid in the absence or presence of IL-12 (0.01, 0.1 and 1 ng/ml). Stimulation by α-CD3+α-CD28, pokeweed mitogen (PWM) and lipopolysaccharide (LPS) were used as controls. Then, proliferation and cytokine production were determined by 3 H-thymidine uptake and ELISA after 72 h. The patients were divided into group 1 ( n =21): HBV-DNA: not detectable, group 2 ( n =13)…

A new hepatocyte stimulating factor: cardiotrophin-1 (CT-1)

Abstract Recently, a novel cytokine, cardiotrophin-1 (CT-1), was cloned and found to induce cardiac myocyte hypertrophy in vitro. Amino acid sequence similarity showed CT-1 to be a member of the IL-6/LIF/CNTF/OSM/IL-11 cytokine family. Since all known members of the IL-6 cytokine family induce an hepatic acute phase protein (APP) gene expression, we investigated the ability of CT-1 to induce a liver acute phase response. Upon stimulation of rat hepatoma cells, CT-1 and LIF induced the strongest rat fibrinogen mRNA expression, OSM and IL-6 induced a less pronounced response. When human hepatoma cells and primary rat hepatocytes were stimulated with CT-1, the expression of human haptoglobin a…

Transforming growth factor-β1 in autoimmune hepatitis: correlation of liver tissue expression and serum levels with disease activity

Abstract Background/Aims: Transforming growth factor-β 1 (TGF-β 1 ) is considered the most important mediator of hepatic fibrogenesis. At the same time, TGF-β 1 is an immunosuppressive cytokine. Development of fibrosis, often rapid, is a characteristic of autoimmune hepatitis, as is spontaneous systemic immunosuppression. The aim of our study was therefore to define the role of TGF-β 1 in autoimmune hepatitis. Methods/Results: Using the MV 1Lu bioassay, we found markedly elevated serum levels of TGF-β 1 (median 109 ng/ml) in active autoimmune hepatitis, which normalised when patients reached biochemical remission following immunosuppressive therapy (median 34 ng/ml; p =0.0001 compared to ac…

Mycophenolate mofetil for treatment of active inflammatory bowel disease. Clinical and immunological studies.

Overlapping peptides of melanocyte differentiation antigen melan-A/MART-1 recognized by autologous cytolytic T lymphocytes in association with HLA-B45.1 and HLA-A2.1

From the peripheral blood lymphocytes (PBLs) of melanoma patient SK29(AV) we have previously isolated 2 independent cytolytic T lymphocyte (CTL) clones (CTL7/147 and CTL13/211), which lysed autologous tumor cells in association with HLA-B45.1. As demonstrated here, both CTL clones were directed against melanocyte differentiation antigen Melan-A/MART-1, which also was recognized by HLA-A2.1-restricted CTLs from the same patient. By generating and transfecting 3'-deletion mutants of Melan-A/MART-1 cDNA, we localized its peptide-coding regions. The HLA-B45.1-presented peptides were derived from a hydrophobic region of the protein and largely overlapped the peptides recognized by CTLs from the …

Cellular and humoral immune responses against cancer: implications for cancer vaccines.

The key issue in tumor immunology is to identify antigens as target structures for a cancer-selective immunological attack in the tumor-bearing host, resulting in tumor rejection. There is a growing detailed understanding of structural and regulatory gene alterations giving rise to candidate rejection antigens and peptides in tumor cells. As well as reviewing the development of new adjuvant and recombinant vector systems, new approaches are suggested for the construction of cancer vaccines.

MHC-unrestricted recognition of bacteria-infected target cells by human CD8+ cytotoxic T lymphocytes.

Abstract A CD8 + αβTCR + T cell clone (A35) was isolated from the synovial fluid of a patient with postenteric reactive arthritis caused by Yersinia enterocolitica . This clone efficiently killed autologous and allogeneic target cells that had been preincubated with live but not with heat-killed bacteria. There was no restriction by polymorphic parts of HLA-A, -B. or -C molecules and a HLA class II-deficient mutant cell line was lysed as efficiently as its normal counterpart, whereas infected HLA class I-deficient cells (Daudi cells) were not. The clone showed crossreaction between Yersinia enterocolitica , Escherichia coli , Pseudomonas aeruginosa , and Streptococcus pyogenes , but did not…

Receptor Recognition Sites of Cytokines Are Organized as Exchangeable Modules

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-bindin…

Suppression of murine experimental autoimmune hepatitis by T-cell vaccination or immunosuppression

Patients with autoimmune hepatitis (AIH) usually require immunosuppressive therapy for many years, if not for a lifetime. Experimental immunotherapy such as T-cell vaccination aims at manipulating the immune system in such a way that autoimmunity is specifically regulated to enable long-lasting correction of the disease process. We aimed to test the feasibility of T-cell vaccination as well as conventional immunosuppression in the murine model of experimental autoimmune hepatitis (EAH). EAH was induced in 5- to 7-week-old BALB/c mice by immunization with syngeneic liver homogenate in complete Freund's adjuvant. For T-cell vaccination, splenocytes were removed from animals 14 days after indu…

Intercellular adhesion molecule-1 concentration in sera of patients with acute and chronic liver disease: relationship to disease activity and cirrhosis.

To study the influence of chronic hepatitis on intercellular adhesion molecule-1 serum concentration, we measured intercellular adhesion molecular-1 in the serum of 84 patients with chronic liver disease (17 chronic persistent hepatitis, 42 chronic active hepatitis and 25 active cirrhosis) caused by hepatitis B virus (n = 46), hepatitis C virus (n = 10) and autoimmunity (n = 28). Furthermore, 20 patients with acute viral hepatitis (16 hepatitis B virus and 4 hepatitis A virus) and 6 patients with acute drug-induced hepatitis were included. Sera from 20 healthy persons were used as control. Follow-up examinations were performed during immunosuppressive therapy in 20 patients with autoimmune …

Autoimmune hepatitis following hepatitis A virus infection.

A 7-year-old patient is reported who suffered from fatigue and jaundice due to chronic hepatitis. He had acquired hepatitis A virus infection in his community and communicated the disease to his German family 4 weeks later. While the other family members recovered from acute viral hepatitis A, the patient presented 10 weeks after the onset of hyperbilirubinemia (12 mg/dl) with the histology of chronic hepatitis, absence of markers for viral persistence, presence of autoantibodies against smooth muscle (1:320) and the asialoglycoprotein receptor (1:600), and marked hypergammaglobulinemia (3700 mg/dl), leading to the diagnosis of autoimmune hepatitis. The patient received immunosuppressive th…

Assay of hepatitis B virus DNA by polymerase chain reaction and its relationship to Pre-S- and S-encoded viral surface antigens

The polymerase chain reaction was evaluated as a diagnostic tool in 72 chronic hepatitis B virus carriers. Hepatitis B virus DNA was detectable in the serum of HBsAg—positive virus carriers using aliquots as small as 100 al. The detection limit for cloned hepatitis B virus DNA was 100 ag. Primer pairs for different regions of the HBV genome resulted in different sensitivity. Detection of the amplified hepatitis B virus DNA by Southern blotting and subsequent scintillation counting or densitometry allowed a semiquantitative assay. Using several primer pairs in parallel for optimal detection, all HBeAg-positive HBsAg carriers, 80% of HBe antibody—positive symptomatic HBsAg carriers and 57% of…

LOW-DOSE INTERLEUKIN-2 INDUCES SYSTEMIC IMMUNE RESPONSES AGAINST HBsAg IN IMMUNODEFICIENT NON-RESPONDERS TO HEPATITIS B VACCINATION

Abstract A metabolic monocyte defect appears to correlate with non-responsiveness to hepatitis B vaccine in many patients on haemodialysis. This defect prevents production of interleukin-2 during T-cell activation after antigen contact. Receptors for interleukin-2 are, however, expressed in greater numbers than in healthy subjects or uraemic responders to hepatitis B vaccination. In this study, ten uraemic patients, previous non-responders to vaccination against hepatitis B, were revaccinated with the same vaccine combined with one intramuscular injection (2·5 × 10 5 U) of natural human interleukin-2. Systemic production of antibodies against hepatitis B surface antigen was initiated in tho…

Lack of requirement for CD8+ cells in recovery from and resistance to experimental autoimmune encephalomyelitis.

Abstract Experimental autoimmune encephalomyelitis (EAE) is a model of T-cell mediated autoimmune disease. Active disease is mediated by myelin basic protein specific CD4+T-cells, whose adoptive transfer can also induce passive disease. In the Lewis rat EAE is a transient disease inducing lasting resistance to rechallenge. The mechanisms of recovery and resistance are poorly understood. CD8+suppressor T-cells have mostly been thought to be central, especially in resistance to reinduction of the disease. In this study we showed by complete depletion of CD8+cells that this subset does not influence either recovery or resistance to EAE in the Lewis rat. This was further confirmed by depleting …

Nature of autoantigens and autoantibodies in autoimmune hepatitis

Autoimmune chronic active hepatitis (AI-CAH) is characterized by young age at onset, predominance of females, hypergammaglobulinemia, response to immunosuppressive treatment and characteristic circulating autoantibodies. This clinical syndrome was first described by Waldenstr6m in 1950 [47]. Later the association of autoimmune hepatitis with antinuclear antibodies (ANA) lead to the term "lupoid hepatitis" [19]. Additional autoantibodies have been described [21]. At least three subgroups of AI-CAH can be distinguished serologically and clinically [28]. As diagnostic tools, autoantibodies help to further differentiate the heterogeneous group of hepatitis B virus (HBV) surface antigen (HBsAg)-…

Proliferative response of synovial fluid and peripheral blood mononuclear cells to arthritogenic and non-arthritogenic microbial antigens and to the 65-kDa mycobacterial heat-shock protein

Cellular immune responses to microbial antigens have been implicated in the pathogenesis of some forms of arthritis including reactive arthritis, Reiter's syndrome, ankylosing spondylitis and rheumatoid arthritis. We investigated the proliferative T cell responses of paired peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MC) to so-called arthritogenic bacteria (Yersinia enterocolitica and Salmonella typhimurium), to control antigens, such as Candida albicans, mumps virus and purified protein derivative, to the recombinant mycobacterial 65-kDa heat-shock protein (hsp 65) and the mitogen phytohemagglutinin (PHA) in 16 patients with different inflammatory rheumatic diseases. T…

Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice

Antibodies to tumor necrosis factor (TNF)-alpha have been recently proposed as effective treatment for patients with Crohn's disease. Here, we analyze the functional role of TNF-alpha in a mouse model of chronic intestinal inflammation induced by the hapten reagent 2,4,6,-trinitrobenzene sulfonic acid (TNBS) that mimics some characteristics of Crohn's disease in humans. Macrophage-enriched lamina propria (LP) mononuclear cells from mice with TNBS-induced colitis produced 10-30-fold higher levels of TNF-alpha mRNA and protein than cells from control mice. When mice with chronic colitis were treated by intraperitoneal injection of antibodies to TNF-alpha, an improvement of both the clinical a…

Ganglioside GD3 shedding by human malignant melanoma cells

Gangliosides appear to be important target molecules for immunological effector mechanisms on neuro-ectodermal tumors. Therefore in vitro studies were performed to examine whether ganglioside GD3, which is highly expressed on the cell surface of cultured human melanoma cells, is being shed into the culture medium. Measurable quantities of gangliosides GM3 and in particular GD3 were shed by the melanoma cells we have tested as detected on thin-layer chromatograms (TLC) stained with orcinol. Ganglioside GD3 was also evidenced by immunostaining with anti-GD3 MAb and by ELISA. The concentration of GD3 in the supernatant of human melanoma cells depended on the ganglioside pattern of the cell lin…

Duration of immunosuppressive therapy in autoimmune hepatitis

Immune blot analysis of viral surface proteins in serum and liver of patients with chronic hepatitis B virus infection

The small and the middle surface proteins of hepatitis virus form either the virion or the 22 nm particle both of which are secreted. The large surface protein by itself remains cell bound in artificially transfected cell culture unless it is accompanied by an excess of the smaller protens. Its behavior in vivo is not yet well studied. Using specific monoclonal antibodies for immunoblotting, we found an abundance of small surface protein in the serum of chronic virus carriers and moderate amounts in the liver irrespective of viremia. The large surface protein was present in the serum and the liver of viremic carriers. In nonviremic carriers, the large protein was absent from serum, but in t…

De novo expression of intercellular adhesion molecule 1 (ICAM-1, CD54) in pancreas cancer.

We examined the expression of intercellular--adhesion molecule-I (ICAM-I, CD54) in 6 surgically removed pancreatic tumors and 8 pancreatic tumor cell lines. Immunohistochemistry revealed a varying percentage of ICAM-I-positive pancreas tumor cells, while normal pancreatic tissue (except for slight reactivity of endothelial cells) was not stained. The presence of the ICAM-I molecule on the cell surface and the expression of ICAM-I mRNA were investigated for 8 different pancreatic tumor cell lines. Three of these (Capan-I, Capan-2, QGP-I) expressed ICAM-I constitutively. In 4 of the ICAM-I-negative pancreas cancer cell lines, it was possible to induce a remarkable expression of ICAM-I by incu…

Interleukin-10 expression is autoregulated at the transcriptional level in human and murine kupffer cells

Interleukin 10 (IL-10) is known to downregulate immune responses. The regulation of IL-10 gene expression therefore determines the outcome of local immune reactions. We investigated time course and downregulation of IL-10 production in primary Kupffer's cells (KC), which are known to secrete IL-10 in response to endotoxin challenge. Human and murine KC were isolated by centrifugal elutriation and investigated for IL-10 gene expression by a two-step amplification procedure (reverse transcriptase-polymerase chain reaction [PCR] followed by T7-polymerase chain reaction). We show that IL-10 messenger ribonucleic acid (mRNA) showed a >450 fold increase in KC 2 hours after endotoxin challenge. IL…

Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis.

Summary Background Autoantibodies are a hallmark of autoimmune hepatitis, but most are not disease specific. Autoantibodies to soluble liver antigen (SLA) and to liver and pancreas antigen (LP) have been described as disease specific, occurring in about 30% of all patients with autoimmune hepatitis, but no standardised assays are available. Methods We tested 2000 serum samples from patients with various liver diseases and controls for SLA autoantibodies by inhibition ELISA. Serum samples positive for SLA antibodies were used for immunoscreening of cDNA expression libraries. Identified clones were tested against a panel of serum samples positive for SLA and LP autoantibodies and control seru…

Corynebacterium parvum (Propionibacterium acnes): an inducer of tumor necrosis factor-alpha in human peripheral blood mononuclear cells and monocytes in vitro.

The present study investigates the potential capacity of the immunostimulant Corynebacterium parvum (C.p.) to induce tumor necrosis factor-alpha (TNF-alpha) in human peripheral blood mononuclear cells (PBMC) and blood monocytes (BMo) in vitro. Both at the mRNA and protein level, stimulation of PBMC and BMo upon C.p. induces TNF-alpha. Compared to the hitherto used TNF-alpha inducers in vitro such as Sendai virus, phytohemagglutinin or lipopolysaccharide the C.p. stimulus displayed a threefold stronger induction of TNF-alpha production (p less than 0.001). Using C.p. as an inducer it was possible to demonstrate that TNF-alpha production is regulated by prostaglandin E2; preincubation of the …

Active immunization of homosexual men using a recombinant hepatitis B vaccine

Twenty homosexual men [13 anti-human immunodeficiency virus (HIV)-positive, seven anti-HIV negative] without HBsAg, anti-HBs, and anti-HBc were vaccinated with three 20 micrograms doses of a recombinant hepatitis B vaccine. All anti-HIV-positive homosexuals were nonresponders independent of the initial number of CD4-positive cells. Among seven anti-HIV-negative individuals, five responded. After three doses of the vaccine, CD4-positive cells fell in anti-HIV positive individuals by 22.4%. A similar fall in CD4-positive cells of an average 24.9% was noted in 17 matching, but nonvaccinated, anti-HIV-positive homosexuals. The study indicates that the efficacy of vaccination in anti-HIV-positiv…

Autoimmune hepatitis in the elderly.

Abstract OBJECTIVES: Autoimmune hepatitis (AIH) is widely believed to be a disease of young women and menopause. Little is known about the frequency and clinical characteristics in patients aged ≥65 yr. METHODS: We reviewed charts of 120 consecutive outpatients with known AIH to identify patients who were diagnosed at the age of 65 or older. These 20 patients (median age, 69 yr) were compared to the same number of younger patients (median age, 24 yr) with well-documented AIH from the same cohort. RESULTS: Seventeen percent (20/120) of our patients were ≥65 yr at the time of diagnosis. In the older patients median time to diagnosis was significantly longer than in the younger patients (8.5 >…

Failure of acyclovir to enhance the antiviral effect of α lymphoblastoid interferon on HBe-seroconversion in chronic hepatitis B

Serum HBeAg levels and HBe-seroconversion were investigated in patients with chronic HBeAg-positive hepatitis who were randomized to receive either alpha lymphoblastoid interferon (5 megaunits subcutaneously daily for 16 weeks) plus acyclovir (2 g intravenously daily during weeks 1 and 2 and weeks 9 and 10) (n = 49) or no treatment (n = 48). HBeAg levels in serial dilutions of patient serum were assessed quantitatively by radioimmunoassay and compared with the values found for negative control serum. One year after the start of therapy 44 treated patients and 43 control patients were available for follow-up. A complete response (HBe-seroconversion) occurred in 11 treated patients (25%) and …

Experimental autoimmune hepatitis: Disease induction, time course and t-cell reactivity

This study describes a murine model of autoimmune hepatitis: experimental autoimmune hepatitis. Experimental autoimmune hepatitis could be induced most effectively in male C57BL/6 mice by intraperitoneal immunization with the 100,000 g supernatant of syngeneic liver homogenate (S-100) in complete Freund's adjuvant. BALB/C and C3H mice were less susceptible than C57BL/6 mice. Experimental autoimmune hepatitis could not be induced in Lewis rats. Intraperitoneal immunization was more effective than intramuscular or subcutaneous injections, and the amount of protein administered above a threshold was of little influence. A single intraperitoneal injection of S-100 in complete Freund's adjuvant …

Treatment of hepatitis B surface antigen (HBsAg)-positive chronic hepatitis with recombinant leucocyte α-A interferon

A total of 32 individuals with HBsAg-positive and anti-delta-negative chronic hepatitis were treated with recombinant alpha-A interferon in phase I and phase II studies. In 5/32 patients HBsAg could be eliminated and in 19/32 individuals HBeAg became negative including all those who also eliminated HBsAg. Side-effects were tolerable in most patients and were readily reversible upon discontinuation of interferon therapy. In conclusion, treatment of HBsAg-positive chronic hepatitis with interferon seems to be a promising therapeutic approach. Future studies will have to establish the optimal dose, duration of treatment and factors predicting a favourable outcome of the treatment.

Immune Response to Tumor Stress Proteins—Implications for Vaccine Development Against Cancer

Publisher Summary Stress proteins or heat shock proteins (HSP) belong to the most conserved proteins. The conservation of stress proteins stems from their basic and vital role in cells: Prevention of protein aggregation under stress and physiological conditions. Stress proteins are important target antigens in autoimmune diseases and during certain bacterial infections. This chapter reviews the immunogenicity of stress proteins of tumor cells, stimulation of T cell response by tumor stress proteins and implications this Tcell response have for immunity against the tumor or autoimmunity. The expression of stress proteins in cancer is altered. An overexpression of constitutively expressed or …

Persistent expression of hepatitis C virus genome in primary tumor and adrenal metastasis of a hepatocellular carcinoma developed in a non-cirrhotic liver

To the Editor: There is increasing evidence that chronic infection with hepatitis C virus (HCV) is a risk factor for hepatocellular carcinoma (HCC) in patients seronegative for hepatitis B virus (HBV) surface antigen (HBsAg). In western countries, HCCs occur in anti-HCV positive patients mostly in association with cirrhosis, which can be considered as a precancerous condition (1). However, there are rare cases of HCC that were found in anti-HCV positive patients without pre-existing liver cirrhosis (2). We report here the detection of HCV RNA in a primary HCC derived from an HCV-infected patient with a non-cirrhotic liver and its persistent expression in an adrenal metastasis that developed…

The quantitative humoral immune response to the hepatitis C virus is correlated with disease activity and response to interferon-alpha.

Virus-host interactions may have pathogenetic significance in chronic hepatitis. Thus the humoral immune response was evaluated during the clinical course of HCV-infected patients.Eighteen selected chronic HCV patients received three doses of 3 or 6 MU interferon-alpha 2a weekly for 6 to 12 months and were followed up for 6 to 60 months. Anti-HCV antibody levels were serially measured either in end-point diluted sera with the Matrix-Assay or with quantitative anti-HC34-IgG and -IgM ELISA. Circulating immune complexes were assessed by flow cytometry and the results were correlated with histology, quantitative HCV-RNA levels and genotypes.Nine complete responders (CR; genotypes 1a n = 4; 1b n…

Expression of differentiation antigens and growth-related genes in normal kidney, autosomal dominant polycystic kidney disease, and renal cell carcinoma.

Cellular differentiation and mRNA levels of genes involved in kidney growth were investigated in normal kidney cells, cyst-lining epithelial cells of polycystic kidney disease, and renal carcinoma cells (RCC). All cells comparatively studied exhibited an antigenic phenotype of proximal tubular cells as shown by the expression of a panel of brush border membrane enzymes and kidney-associated cell surface antigens. The epithelial developmental antigen Exo-1 was expressed in 50% to 80% of cyst-lining epithelia in polycystic kidney tissue and in 20% to 30% of polycystic kidney cells cultured in vitro. Normal kidney cells and RCC were negative under identical culture conditions. The expression o…

Enteropathische Spondarthritiden bei chronisch entzündlichen Darmerkrankungen: Prävalenz, Befallsmuster und HLA-Assoziation

Enteropathische Spondarthritiden stellen die haufigste extraintestinale Manifestation der chronisch entzundlichen Darmerkrankungen (CED), Morbus Crohn (MC) und Colitis ulcerosa (CU), dar. Ziel der vorliegenden Studie war es daher, ein groses Patientengut mit chronisch entzundlichen Darmerkrankungen auf die Pravalenz und das Befallsmuster von Spondarthritiden (SpA) sowie auf eine Assoziation der Spondarthritiden mit der Ausdehnung der Darmerkrankung und dem HLA-Typ des Patienten hin zu untersuchen. 521 Patienten (409 mit Morbus Crohn, 112 mit Colitis, ulcerosa) wurden prospektiv uber den Zeitraum eines Jahres untersucht. Bei der Diagnose einer Spondarthritis wurden anamnestische, klinische, …

Active hepatitis B vaccination of dialysis patients and medical staff

AbstractActive hepatitis B vaccination of dialysis patients and medical staff. One hundred six patients with terminal renal insufficiency and 29 medical personnel were given three doses of hepatitis B vaccine at an interval of 0, 1, and 6 months (Merck, Sharp and Dohme, West Point, Pennsylvania, part of a joint study no. 649). Chronic hemodialysis patients (N = 99) received 40 µg vaccine (V) i.m. Uremic patients, who were just about to start chronic dialysis treatment (N = 7), were given 40 µg V, and at the first vaccination 3ml hyperimmune globulin (HBIG) in addition. The medical personnel was alternately vaccinated with 20 µg V (N = 8), 40 µg (N = 11), 40 µg V, and 3ml HBIG at the first v…

T cell receptor Vβ chain restriction and preferred CDR3 motifs of liver-kidney microsomal antigen (LKM-1)-reactive T cells from autoimmune hepatitis patients

Aims/Background : The liver-kidney-microsomal antigen (LKM-1) has been recognized as a major CD4 + T cell antigen in autoimmune hepatitis (AIH). The aim of this study was to characterize the antigen recognition sites of the variable T cell receptor β-chain (TCRBV) of T cells specific to LKM-1. Methods: By repeated stimulation of T cells with a recombinant LKM-1 antigen or an LKM-derived peptide followed by limited dilution, we generated T cell clones. Usage of TCRBV was analyzed by RT-PCR and CDR3 antigen recognition sites were sequenced. Results: The 18 LKM-1 specific T cell clones isolated from six AIH patients preferentially expressed the TCR elements BV9, BV5S2+S3, BV6, and BV13S1. Four…

Activation and methotrexate-mediated suppression of the TNF alpha promoter in T cells and macrophages.

Relationship between the target antigen of liver-kidney microsomal (LKM) autoantibodies and rat isoenzymes of cytochrome P-450

Chronic active hepatitis (CAH) is a clinical syndrome of different etiologies. Liver-kidney microsomal (LKM) autoantibodies characterize a subgroup of HBsAg negative CAH, which is considered to be an autoimmune liver disease. By immunoblotting analysis (IB) LKM positive sera have been shown to react strongly with a poly-peptide band at 50 kD. Therefore we investigated various rat microsomal enzymes with a molecular weight around 50 kD as potential candidate target antigens. These included epoxide hydrolase, cytochrome P-450 reductase, and phenobarbital-inducible isoenzymes of cytochrome P-450 (PB1, PB2, PB3a, PB3b). By radioimmunoassay (RIA) and IB LKM positive sera were shown to react with…

Effects of cytokines on synthesis and function of the hepatic asialoglycoprotein receptor

In this study we have investigated whether cytokines, critical mediators of the immune response, might have a direct effect on the expression and/or function of the human hepatic asialoglycoprotein receptor (ASGPR). Binding and uptake of asialoglycoproteins by the human hepatoma cell line, HepG2, and by freshly isolated rat hepatocytes were inhibited by 50% after 3-6 hours and completely abolished following a 24 hour exposure to tumor necrosis factor (TNF) alpha, interferon (INF) alpha or gamma, or interleukin-2 (IL-2). The loss of ASGPR binding activity mediated by IL-2 was reversible up to 4 hours of exposure and accompanied by the selective phosphorylation of the cell-surface receptor. S…

Cellular immune response to human renal-cell carcinomas: Definition of a common antigen recognized by HLA-A2-restricted cytotoxic T-Lymphocyte (CTL) clones

Cytotoxic T lymphocyte (CTL) clones directed against autologous renal-cell carcinoma (RCC) cell lines were generated by mixed lymphocyte/tumor-cell culture (MLTC) using peripheral blood lymphocytes (PBL). A CD8+, CD4- CTL clone MZ1257-CTL 5/30 with high cytolytic activity for the autologous tumor cell line MZ1257-RCC was established. No lysis of the autologous EBV-transformed B lymphocytes (EBV-B) or K562 cells was observed. A panel of HLA-A2-matched allogeneic RCC lines was recognized by CTL 5/30. Further specificity analysis showed a cross-reactivity with HLA-A2-matched allogeneic tumor cells of various origins, especially melanoma. CTL 5/30 was also cross-reactive with several HLA-A2-pos…

The therapeutic potential of interleukin-6 hyperagonists and antagonists.

Interleukin-6 (IL-6) is a 4-helical protein that binds to a specific IL-6 receptor on target cells and to two molecules of the promiscuous signal transducing protein, glycoprotein 130 (gp130). Structure-function analysis has led to the definition of molecular contacts between IL-6 and its receptor subunits. This knowledge has led to the design of competitive antagonistic proteins that retain their receptor binding capability, but fail to stimulate one or both gp130 proteins; the properties of such recombinant antagonistic proteins are compared with traditional neutralising monoclonal antibodies targeted at IL-6 or receptor subunits. Furthermore, several strategies have been employed to cons…

In vitro secretion of specific antimitochondrial antibodies in primary biliary cirrhosis

Antimitochondrial antibodies are present in the serum of virtually all patients with primary biliary cirrhosis. They have a well-defined antigen reactivity that is diagnostic for the disease. The role of these autoantibodies in the disease process remains to be defined. In this study we show that antimitochondrial antibodies can be produced in vitro by peripheral blood lymphocytes, that the cells producing antimitochondrial antibodies are present in the peripheral blood in a high frequency and seem to be maximally activated. Stimulation with pokeweed mitogen did not augment the in vitro production of antimitochondrial antibodies in patients nor did it induce the production of these antibodi…

The use of computer-assisted video image analysis for the quantification of CD8+ T lymphocytes producing tumor necrosis factor alpha spots in response to peptide antigens.

Enzyme-linked immunospot (ELISPOT) analysis is a sensitive technique for the detection and quantification of single T lymphocytes forming cytokine spots after antigen contact in vitro. Herein computer-assisted video image analysis (CVIA) was applied to automatically determine the number and size of tumor necrosis factor alpha (TNF-alpha) spots formed by single blood-derived CD8+ T cells after contact with peptide-loaded target cells. With CVIA and TNF-alpha ELISPOT analysis we quantified CD8+ T cells responsive to HLA-A2.1-binding tyrosinase and influenza matrix peptides in healthy donors. We followed the course of the virus-specific T cell response in two HLA-A2-positive patients with reac…

T cell activation defect in hemodialysis patients: Evidence for a role of the B7/CD28 pathway

T cell activation defect in hemodialysis patients: Evidence for a role of the B7/CD28 pathway. The immunosuppressive effect of chronic renal failure is correlated with an impaired proliferation of peripheral blood leukocytes in vitro . This is mainly due to an impaired function of the accessory cells rather than the T cells. Here we tried to define a missing accessory signal for T cell activation in hemodialysis patients. We substituted cell surface bound molecules by adding tumor cell lines to the in vitro assays that express different patterns of accessory molecules. Cell lines that express the costimulatory B7 molecule reconstituted the activation of patients' cells whereas B7 negative c…

The gp130-stimulating designer cytokine hyper-IL-6 promotes the expansion of human hematopoietic progenitor cells capable to differentiate into functional dendritic cells.

Abstract Objective . Hyper-IL-6, a fusion protein of interleukin-6 and its specific receptor, together with stem cell factor leads to the proliferation of primitive hematopoietic progenitor cells. Based on these findings, the current study examined whether hyper-IL-6 promotes the growth of precursor cells that can be further differentiated into dendritic cells in the presence of additional cytokines. Methods . Dendritic cell cultures were generated from CD34 + hematopoietic progenitor cells derived either from bone marrow or from peripheral blood. CD34 + cells were cultured in the presence of cytokines for 2 weeks and then used for phenotyping and T-cell stimulation assays. Results . Hyper-…

Humoral Mechanisms in T cell Vaccination: Induction and Functional Characterization of Anti-lymphocytic Autoantibodies

T cell vaccination, the application of syngeneic attenuated T cells, has been shown to prevent effectively and treat experimental autoimmune diseases, but its mechanisms of action are poorly understood. Here we present data on the induction of a humoral anti-T cell response by T cell vaccination, capable of strongly inhibiting T cell proliferation and of ameliorating experimental autoimmune disease. T cell vaccination in the Lewis rat induced autoantibodies reactive with several syngeneic T cell proteins. These autoantibodies were not detectable in normal Lewis sera as assessed by immunoblotting and flow cytometry with intact syngeneic T cells. The autoantibody reactivity was not restricted…

Impaired HLA-class-I stability in a sarcoma cell line which stimulates exclusively HLA-class-II-restricted autologous T cells

Defects in the generation and transport of antigenic peptides within tumor cells will lead to the expression of unstable HLA-class-I molecules on the cell surface. These defects will allow tumor cells to escape an MHC-class-I-restricted T-cell response. Recently, we described an exclusively HLA-class-II-restricted autologous T-cell response against a human sarcoma cell line MZ-MES-1 in vitro. Here, we show that surface HLA-class-I molecules of MZ-MES-1 cells are unstable at physiological temperature. HLA-class-I surface expression of MZ-MES-1 cells could be strongly enhanced by culture at low temperature in contrast to various other cell lines analyzed in parallel. Furthermore, culture at l…

Synovial fluid-derivedYersinia-reactive T cells responding to human 65-kDa heat-shock protein and heat-stressed antigen-presenting cells

Humoral and cellular immune reactions to heat-shock proteins have been implicated in the pathogenesis of arthritis. Heat-shock proteins occur in bacteria as well as all eukaryotes and have been highly conserved during evolution. Cross-reactivity between bacterial and human heat-shock proteins induced at the site of inflammation may underlie the pathogenesis of some forms of arthritis. In order to test this hypothesis, we raised and cloned a Yersinia-specific T cell line from the synovial fluid lymphocytes of a patient with Yersinia-induced reactive arthritis. From this line we obtained a CD4+ T cell clone that proliferated in response to Yersinia antigens and both to the mycobacterial and t…

Sinusoidal endothelial cells from guinea pig liver synthesize and secrete cellular fibronectin in vitro.

Endothelial liver cells were obtained from guinea pig by enzymatic digestion and centrifugal elutriation. Cells were cultured on gelatin and fibronectin pretreated culture vessels. Endothelial cells were characterized by phase-contrast microscopy, electron microscopy and the presence of Factor VIII-related antigen. Fibronectin secretion was determined in cell-free supernatants by a sensitive and specific ELISA and localized on fixed cultured cells by immunofluorescence. [35S]Methionine endogeneously labeled fibronectin was immunoprecipitated from supernatants and cellular lysates and displayed on sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. After attachment to the culture…

Characterization of the overlap syndrome of primary biliary cirrhosis (PBC) and autoimmune hepatitis: Evidence for it being a hepatitic form of PBC in genetically susceptible individuals

Some patients with autoimmune liver disease present with a clinical and/or histological picture showing characteristic findings of both autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC). Various names, mostly overlap syndrome, have been used to describe these cases, which have thus far not been more closely characterized. The aim of this study was the comparison of 20 patients with overlapping features to representative patients considered suffering from typical AIH or typical PBC (20 patients in each group). We found these patients to indeed show a very mixed picture of both conditions biochemically, serologically, and histologically. However, closer analysis suggested that al…

Chronic hepatitis C: the viral load per liver cell before treatment as a new marker to predict long-term response to IFN-α therapy

Abstract Background/Aims: So far, there are no reliable parameters that can predict the long-term sustained response to treatment with interferon-α in patients with chronic hepatitis C. In this study, we have developed a semi-quantitative method to determine the viral load per liver cell and have correlated this factor with the outcome of hepatitis C patients treated with interferon-α. Methods: Hepatitis C virus RNA levels were measured in serum, peripheral blood mononuclear cells and liver cells of randomly chosen hepatitis C patients before treatment with interferon-α ( n =37). The number of cells present in the liver biopsies was determined by a polymerase chain reaction-based quantitati…

Autoantibodies in experimental autoimmune hepatitis

Experimental autoimmune hepatitis (EAH) can be induced in mice by immunization with syngeneic soluble liver antigens in complete Freund's adjuvant. It has previously been shown that autoreactive T cells play an important role in this animal model of autoimmune hepatitis. We have studied the occurrence of liver autoantibodies in EAH. Characteristic autoantibodies appeared several weeks after disease induction and antibody titres continued to rise when histological and biochemical signs of disease activity had already regressed. Autoantibodies in EAH seemed to recognize autoantigens other than those present in autoimmune chronic active hepatitis patients. We conclude that autoantibodies arise…

Contribution of sinusoidal endothelial liver cells to liver fibrosis: expression of transforming growth factor-beta 1 receptors and modulation of plasmin-generating enzymes by transforming growth factor-beta 1.

Transforming growth factor-beta 1 is an important cytokine in the pathophysiology of liver fibrosis, stimulating the production of extracellular matrix. Whether this cytokine can also control the degradation of matrix proteins in liver cells has not been investigated. Because plasmin is an important protease for the degradation of matrix glycoproteins, we investigated whether sinusoidal endothelial liver cells could contribute to fibrosing liver disease through the modulation of plasmin-generating enzymes in response to transforming growth factor-beta 1. Sinusoidal endothelial cells from guinea pig liver were investigated in pure monolayer culture. Using 125I-labelled transforming growth fa…

Clinical significance of autoantibodies to soluble liver antigen in autoimmune hepatitis.

Abstract 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…

Autoantibodies to human asialoglycoprotein receptor in autoimmune-type chronic hepatitis.

Autoantibodies to the human asialoglycoprotein receptor (anti-h-ASGPR) were studied with a solid-phase ELISA in the sera of 421 patients with inflammatory liver diseases, 288 patients with various other disorders and 31 controls. Anti-h-ASGPR were found predominantly in autoimmune chronic active hepatitis (44 of 88, 50%) and were closely related to inflammatory activity. In a subpopulation of these patients with untreated, biopsy-proven active disease or relapse, 15 of 17 were positive (88%). In contrast, only 11 of 204 patients (5.3%) with viral hepatitis were anti-h-ASGPR receptors-positive (chi 2 analysis; p less than 0.001). We also compared the occurrence of anti-h-ASGPR with antibodie…

Strain differences in experimental autoimmune hepatitis

The human hepatic asialoglycoprotein receptor is a target antigen for liver-infiltrating T cells in autoimmune chronic active hepatitis and primary biliary cirrhosis.

Autoantibodies to the human hepatic asialoglycoprotein receptor have been found in nearly 50% of the sera of patients with autoimmune chronic active hepatitis and in 15% of patients with primary biliary cirrhosis. In this study we demonstrate that the human hepatic asialoglycoprotein receptor is also a target antigen for T cell-mediated immune responses. Peripheral blood lymphocytes of 37% (7 of 19) of patients with autoimmune chronic active hepatitis and 33% (2 of 6) of patients with primary biliary cirrhosis showed a proliferative response to highly purified human hepatic asialoglycoprotein receptor, whereas no proliferation was found with peripheral blood lymphocytes of patients with chr…

Posttransfusional, LKM-1-autoantibody-positive hepatitis C virus infection, cryoglobulinemia, and aplastic anemia.

Aplastic anemia is occasionally caused by viral hepatitis, hepatitis C virus being the most important factor. Pathogenetically, decreased bone marrow function, abnormalities of the bone marrow microenvironment, and immune-mediated suppression of hematopoiesis are important. Hepatitis C virus infection is associated with a variety of extrahepatic manifestations including autoimmune features like cryoglobulinemia, Sjogren's syndrome, and autoimmune hepatitis. Here we report the case of a 42-year-old man with aplastic anemia due to posttransfusional hepatitis C virus infection associated with cryoglobulinemia and LKM-1 autoantibodies. Following a triple immunosuppressive therapy, there was a c…

Production of interleukin-6, tumor necrosis factor α and interleukin-10 in vitro correlates with the clinical immune defect in chronic hemodialysis patients

Production of interleukin-6, tumor necrosis factor α and interleukin-10 in vitro correlates with the clinical immune defect in chronic hemodialysis patients. In patients with chronic renal failure alterations in monokine production are a common feature. Their clinical relevance has not yet been proven. We show here a correlation between an overproduction of interleukin-(IL)-6 and tumor necrosis factor alpha (TNFα) upon stimulation with LPS by mononuclear cells in vitro and the clinical grade of immunodeficiency found in these patients. Higher levels of IL-6 and TNFα were correlated with an immunocompromized state, that is, non-responsiveness to hepatitis B vaccination, whereas patients with…

Predominance of Th1-type T cells in synovial fluid of patients with Yersinia-induced reactive arthritis

The pathogenetic mechanisms underlying the development of reactive arthritis and the functional capacities of synovial T cells specific for Yersinia enterocolitica are still unclear. In this study we have determined the cytokine secretion patterns of 24 CD4+ synovial fluid (SF)-derived T cell clones from 2 patients with Yersinia-induced reactive arthritis, 16 clones specific for different Yersinia antigens and 8 clones as controls. The clones specific for Yersinia antigens predominantly belong to the T helper cell 1 (Th1) subset with production of interferon (IFN)-gamma and interleukin (IL)-2, but no IL-4, whereas SF T cells not reactive with Yersinia antigens produce IL-2, IL-4 and IFN-gam…

Hepatitis B vaccination and interleukin 2 receptor expression in chronic renal failure

Hepatitis B vaccination and interleukin-2 receptor expression in chronic renal failure. Only 50 to 60% of dialysis patients develop anti-HBs antibodies following hepatitis b vaccination. The nonre-sponder state correlates with impaired monocyte function, decreased interleukin-2 (IL-2) production of T cells, and an upregulation of the IL-2 receptor system. In the present study we examined anti-HBs production after hepatitis B vaccination and the in vitro expression of IL-2 receptors in nondialyzed patients with various degrees of chronic renal failure. Forty-four patients with impaired renal function were immunized with 20 µg recombinant hepatitis B vaccine and boostered after one and six mo…

T lymphocyte control of autoreactivity: analysis with human T cell clones and limiting dilution culture

To investigate cellular mechanisms controlling activated autoreactive T lymphocytes, a limiting dilution system was established employing cloned autoreactive major his-tocompatibility complex class II specific lymphocytes (a2/7) as stimulator cells for autologous peripheral blood mononuclear cells. At low responder/stimulator ratios, cytotoxic effector cells were generated capable of lysing clone a2/7. Importantly, within the population of cells mediating autocytotoxic effector function, differential specificities were found to exist. The generation of such autocytotoxic T lymphocytes appears to be inhibited by an additional population of cells circulating at lower frequency suggesting that…

Detection of hepatitis C virus in paraffin-embedded liver biopsies of patients negative for viral RNA in serum

The diagnosis of hepatitis C is based on serological testing for antibodies against various epitopes of the hepatitis C virus (HCV) and detection of HCV RNA in serum, because anti-HCV antibodies alone cannot discriminate patients who are infectious from those who have resolved the infection. If HCV RNA is not detected, which is the case in at least 20% of enzyme immunoassay (EIA)-positive patients, diagnosis remains unclear in a state of disease possibly well suited for therapeutic intervention. Therefore, we investigated if detection of HCV antigens or HCV RNA in routinely processed, formalin-fixed and paraffin-embedded (ffpe) liver biopsy specimens of patients positive for anti-HCV, but n…

Complete congenital heart block in autoimmune hepatitis (SLA-positive).

Complete congenital heart block is a serious complication of neonatal lupus erythematosus which most often occurs in children of mothers suffering from connective tissue disease. We report the occurrence of complete congenital heart block associated with autoimmune hepatitis (SLA-positive). A 32-year-old woman was treated for more than 10 years for autoimmune hepatitis (SLA-/ANA-positive) and remained in clinical remission under immunosuppressive therapy. She showed an MHC-haplotype typical for autoimmune hepatitis (A1, B8, DR3). After a normal first pregnancy, an emergency caesarean section was performed in the 32nd week of her second pregnancy because of fetal bradycardia. The child died …

Interleukin 12 enhances deficient HCV-antigen-induced Th1-type immune response of peripheral blood mononuclear cells

The aim of this study was to examine the possible immunomodulating effects of rhIL-12 on the immune response induced by different hepatitis C virus (HCV) antigens. Freshly isolated peripheral blood mononuclear cells (PBMC) of 33 patients with chronic HCV infection were stimulated with optimal concentrations of antigens from the NS3, NS4, NS5, and core region of HCV in the absence or presence of interleukin12 (IL-12). Stimulation by α-CD3 + α-CD28, lipopolysaccharide (LPS), and pokeweed mitogen (PWM) were used as controls. Proliferation and cytokine production were determined by 3H-thymidine uptake and enzyme-linked immunosorbent assay (ELISA) after 72 hr. After stimulation with antigen or a…