Minilaparoscopy in the diagnosis of cirrhosis: superiority in patients with Child-Pugh A and macronodular disease.

BACKGROUND AND STUDY AIMS: The diagnosis of cirrhosis has prognostic and therapeutic implications, but early forms are difficult to diagnose. Laparoscopy with histology has been reported to be superior to histology alone, but is often considered to be too invasive. This study aimed to assess whether minilaparoscopy offers similarly high sensitivity coupled with only minor invasiveness. PATIENTS AND METHODS: Minilaparoscopy with biopsy was performed in 226 consecutive patients with chronic liver disease. Cirrhosis was diagnosed macroscopically primarily on the basis of nodularity in a nontumorous liver. A histological diagnosis using the modified Knodell score was made without knowledge of the macroscopic assessment. RESULTS: Biopsies from 22 patients were inadequate for histological assessment, and 16 of these were considered to be cirrhotic from macroscopic observation. Out of 204 liver biopsies, 94 (46 %) were macroscopically identified as cirrhotic; 68/204 (33 %) showed stage 5 or 6 fibrosis (incomplete or complete cirrhosis). Histological understaging occurred mainly in patients who were otherwise diagnosed as having early Child-Pugh A cirrhosis, macroscopically incomplete cirrhosis and macronodular cirrhosis; 4/204 (2 %) of patients with cirrhosis histologically were understaged macroscopically. CONCLUSIONS: Macroscopic evaluation during minilaparoscopy increases the sensitivity of detection of liver cirrhosis, compared with biopsy alone, by more than 30 %. Because of its minimal invasiveness, minilaparoscopy combined with biopsy is recommended as a superior method for the staging of chronic liver disease.

Antisense phosphorothioate oligonucleotides to the p65 subunit of NF-kappaB abrogate fulminant septic shock induced by S. typhimurium in mice.

The aim of this study was to characterize the functional relevance of the transcription factor NF-kappaB in the pathogenesis of septic shock. BALB/c mice were infected with two wild-type (WT 1, WT 2) strains of S. typhimurium that induce NF-kappaB or an escape variant that lacks this ability (P21) at a dose of 1 x 109/animal, respectively. Furthermore, wild-type infected mice were treated with antisense oligonucleotides directed against NF-kappaB 24 h before and 3 or 6 h after infection, while mismatched oligonucleotides were used as controls. Subsequently, the clinical course, histological and immunological alterations were monitored. Infection with WT 1 and WT 2 strains led to lethal septic shock within 24-36 h. In contrast, infection with the P21 variant was not followed by fulminant septic shock. Treatment with specific antisense oligonucleotides against the p65 subunit of NF-kappaB 24 h before infection prevented the development of fulminant, lethal septic shock and was associated with a significant increase of survival. After 20 h, markedly depressed serum levels of interferon (IFN)-gamma and interleukin (IL)-6 but not IL-10 and tumour necrosis factor (TNF)-alpha were observed in p65 antisense-treated compared to mismatched-treated animals. These data show that the ability of S. typhimurium to induce lethal septic shock is critically dependent on their capacity to induce NF-kappaB.

Cobalt-specific T lymphocytes in synovial tissue after an allergic reaction to a cobalt alloy joint prosthesis.

Metals such as cobalt and nickel are common contact allergens. We studied the mechanisms underlying an allergic reaction with marked synovial inflammation in a patient with a cobalt alloy arthroplasty. After removing the joint prosthesis the adjacent synovial tissue was examined for cobalt-specific T lymphocytes. Synovial membrane mononuclear cells were expanded in interleukin 2 and cloned using a representative cloning protocol. T cell clones were tested for their proliferative response to cobalt and further characterized with regard to cytokine secretion, phenotype, and HLA restriction. Additionally, synovial fibroblasts were tested for their function as antigen presenting cells (APC). Almost 30% of the T cell clones reacted to cobalt, but not to the control nickel. All these T cell clones were CD4 positive. The cobalt induced proliferative response could be blocked by anticlass II antibodies. Also, synovial fibroblasts expressing class II molecules induced by interferon-gamma were able to serve as APC. However, when testing a panel of APC of HLA class II mismatched donors, no requirement for a certain HLA class II molecule could be defined. Further studies are necessary to determine mechanisms of presentation and recognition of cobalt by T lymphocytes, a prerequisite for improved prevention and treatment of metal induced allergic reactions.

T cell receptor Vbeta 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 beta-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 BV9+ T cell clones rearranged the joining element JB1S3 within their CDR3 regions. JB2S3 was detected in another four clones together with BV5S2+S3 or BV13S1. A conserved sequence motif, Q(N)G(X)N, was seen in the diversity regions of five clones (36%). In order to identify T cells expressing the preferred TCRBV molecules in situ, immunohistologic examination of liver biopsies was performed. In AIH patients an accumulation of T cells expressing TCRBV 13S1, BV8 and BV5S3 was observed. CONCLUSIONS: Our data define TCRBV restriction and preferred CDR3 features of LKM-1 specific T cells. The in situ localization of T cells expressing these restricted TCR molecules may suggest a pathogenic relevance of LKM-1 specific cellular immune responses.

Combined interleukin 6 and soluble interleukin 6 receptor accelerates murine liver regeneration.

BACKGROUND & AIMS: Liver regeneration after loss of hepatic tissue leads to hepatocyte and nonparenchymal cell proliferation and rapid restoration of liver parenchyma. Interleukin (IL)-6 is a key inducer of transcription factors involved in liver regeneration. Whenever IL-6 activates target cells, it binds to a specific IL-6 receptor (IL-6R). The IL-6/IL-6R complex then associates with the signal transducer gp130, leading to activation of intracellular signaling. METHODS: We have recently constructed the designer cytokine Hyper-IL-6 consisting of soluble IL-6R covalently linked to IL-6, which directly stimulates gp130 even in the absence of membrane-bound IL-6R. We compared the influence of IL-6 and Hyper-IL-6 on liver regeneration after partial hepatectomy in mice. RESULTS: The IL-6/soluble IL-6 fusion protein Hyper-IL-6, but not IL-6 alone, led to an earlier onset of hepatocellular proliferation resulting in an acceleration of liver weight restoration. Also, during liver regeneration, soluble IL-6R levels were increased. CONCLUSIONS: These results emphasize a central role for IL-6 and soluble IL-6R in liver regeneration and indicate a possible therapeutic potential for the designer cytokine Hyper-IL-6 in clinical situations associated with liver regeneration such as acute hepatic failure or resection of chronically damaged liver tissue.

Identical T-cell expansions in the colon mucosa and the synovium of a patient with enterogenic spondyloarthropathy.

Intestinal T lymphocytes activated by antigen are suspected to play a key role in enterogenic spondyloarthropathies (SpA). Therefore, we aimed to identify and functionally characterize T-cell clones that are coexpanded in the intestinal mucosa and the synovium. Colon, peripheral blood, and synovium of a patient with enterogenic SpA were screened for clonal T-cell expansions by TCRB-CDR3 length analysis and sequencing. T-cell clones expanded in vivo were isolated from archived synovial cells by targeted T-cell cloning and characterized for phenotype, cytokine production, and antigen specificity. The synovial TCRBV18(+) T-cell repertoire of the patient was dominated by 2 CD8(+) T-cell clones using related CDR3. Both clones were expanded throughout the colon and were present in the peripheral blood. Upon in vitro stimulation with PDB/ionomycin, they showed predominantly interferon gamma and interleukin (IL)-4 but also tumor necrosis factor alpha and IL-10 production and did not specifically lyse autologous T-cell blasts, B-cell lines, or other autologous or allogeneic target or CD1d-transfected cells. These findings strongly suggest that T lymphocytes activated by antigen in the intestinal mucosa contribute to joint inflammation in enterogenic SpA by recognition of antigens specific for the inflamed synovium.

Selection of hepatitis B virus variants with aminoacid substitutions inside the core antigen during interferon-alpha therapy.

The hepatitis B virus (HBV) core antigen carries many epitopes relevant for B and T cell response that show aminoacid variation during viral infection. In a longitudinal analysis, sequential serum samples of 15 patients that suffered from chronic HBV infection were collected before, during, and after high-dose IFN-alpha treatment. The HBV preCore/Core (preC/C) sequence of the selected samples in each patient was determined and analysed for sequence variations compared to the pretreatment sample. The positions of HBV core aminoacid substitutions were assigned to immunodominant B, CD4(+) and CD8(+) cell epitopes. Seventy-five percent of all aminoacid substitutions were found within immunodominant T and B cell epitopes (12.5% were inside known HBV core mutation cluster regions) that show an increased number of clustered aminoacid substitutions during chronic HBV infection and overlap partially with the immunodominant epitopes. Only 12.5% of the detected core antigen aminoacid substitutions could not be assigned to any epitope or mutation cluster region. Stable HBV core antigen aminoacid substitutions, which were found between pretreatment sequence and the last sequence analysed during therapy, were found most frequently inside T helper cell epitopes. This longitudinal analysis of aminoacid substitutions inside the HBV core antigen in patients with chronic HBV infection shows that core aminoacid variations occur most frequently inside immunodominant HBV core epitopes, possibly due to an immuneselective pressure of the host against the virus. The data also suggest that stable HBV variants with aminoacid substitutions in immunodominant core epitopes can be selected during high-dose IFN-alpha therapy and persist after the end of treatment.

There is no disease-specific role for streptococci-responsive synovial T lymphocytes in the pathogenesis of psoriatic arthritis.

The initiation or exacerbation of psoriasis vulgaris is associated with infections by group A streptococci. T lymphocytes specific for streptococcal antigens or expressing a restricted, for streptococcal superantigens typical T cell receptor Vbeta chain repertoire have been described in psoriatic skin lesions. The aim of our study was, therefore, to clarify whether streptococci-reactive T lymphocytes played a role in the pathogenesis of psoriatic arthritis (PsA), and by which antigens they might be stimulated. Synovial membrane mononuclear cells from patients with PsA and other arthropathies, separated by collagenase digestion, were expanded in interleukin-2-supplemented medium and subsequently cloned in a representative cloning procedure. The T cell lines and about 30% of the T cell clones proliferated in response to preparations of group A streptococci but not to other bacteria as tested by [3H]thymidine incorporation assays. Interestingly, they did not proliferate in response to exotoxin-negative streptococci, but did so in response to the streptococcal pyrogenic exotoxins A and C, which are known to be superantigens. Accordingly, no HLA-DR restriction was seen for the proliferative response. The remaining 70% of the established T cell clones did not react to an antigen of group A streptococci. Our results show that in patients with PsA, osteoarthritis or rheumatoid arthritis a significant number of synovial T lymphocytes were responsive to streptococcal superantigens, but not to conventional streptococcal antigens. A disease-specific role of streptococci-reactive T lymphocytes in the pathogenesis of PsA is, therefore, unlikely.

Dendritic cells lose ability to present protein antigen after stimulating antigen-specific T cell responses, despite upregulation of MHC class II expression.

Immature dendritic cells (DC) take up, process and present protein antigens; mature DC are specialized for stimulating primary T cell responses with increased expression of MHC class II and co-stimulatory molecules, but are incapable of processing and presenting soluble protein. The current study examined whether maturation of DC is triggered by T cell recognition of antigens presented by immature DC. Human DC derived from CD34+ progenitor cells by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) in serum-free medium could prime naive CD4+ T cells to keyhole limpet hemocyanin (KLH) and ovalbumin (OVA). The cultured DC retained the ability to prime T cells to native protein for at least 15 days. To test for changes in DC function after participation in an immune response, DC were co-cultured with either allogeneic or autologous CD4+ T cells. DC co-cultured with autologous T cells retained the ability to prime T cells to intact protein antigens. By contrast, DC which had previously stimulated an allogeneic T cell response lost ability to prime T cells to soluble proteins. However, such <> induced a MLR and stimulated peptide-specific primary CD4+ T cell responses. This indicated that <> did not die or lose the ability to prime, but lost the ability to process and present subsequent antigens. Following participation in T cell activation, DC increased surface expression of MHC class II, co-stimulatory molecules CD40 and B7.2, and the intercellular adhesion molecule-1 (ICAM-1). In addition, our data suggest that interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) are involved in this T cell-mediated DC maturation.

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

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 serum samples, and the epitope mapped by deletion mutants and exonuclease digestion. FINDINGS: SLA and LP autoantibodies were identical. Of 2000 serum samples screened, 35 were positive for SLA autoantibodies. These positive samples came from patients with autoimmune hepatitis; three from patients with an overlap syndrome (primary biliary cirrhosis and secondary autoimmune hepatitis). Expression cloning and absorption experiments identified a 422 aminoacid protein present in two splice variants as the sole target antigen. Aminoacids 371-409 were critical for immune recognition. INTERPRETATION: The identified cDNA encodes the primary target antigen of SLA/LP autoantibodies. The SLA/LP antigen has a previously unknown aminoacid sequence, and presumably codes for an unindentified enzyme, suggested to be UGA-suppressor tRNA-associated protein. SLA/LP autoantibodies are disease specific and recognise a dominant epitope, suggesting a specific antigen-driven immune response. Identification of the SLA/LP target antigen will allow establishment of a reliable, widely available diagnostic assay. Furthermore, its role in the pathogenesis of autoimmune hepatitis can now be studied.

The early immune response in the liver of BALB/c mice infected with S. typhimurium.

Gram-negative bacteria acquired through gastrointestinal infection can be a serious cause for the development of septic shock especially in immunosuppressed patients. Thus, the aim of this study was to examine the early events of the immune reaction against S. typhimurium. Bacteria were injected into mice at different concentrations. Four animals from each group were killed at five different points of time. Liver cytokine mRNA expression was determined by semiquantitative rt-PCR and liver histology was examined. Serum cytokine levels of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-4 and IL-10 were determined. intravenous (i.v.) infection with 109 bacteria led to lethal septic shock within 24 h. A delayed production of IFN-gamma, TNF-alpha, IL-18 and IL-10 and milder histological alterations in the liver were observed in these animals. The highest expression of cytokines in the liver and the strongest histological alterations were seen after infection with 107 bacteria. Here, an increased mRNA expression of all proinflammatory cytokines began 1 h after infection. Animals infected with 1 x 102 bacteria had the highest detectable serum levels of IL-6 and IL-10. These data indicate that the immediate events in the immune reaction within the liver after infection with S. typhimurium are associated with the outcome of the subsequent sepsis.

Synovial fluid lymphocyte proliferation in response to crude microbial antigens is not useful as a diagnostic test to specifically indicate a bacterial cause of arthritis.

OBJECTIVE: To determine the role of lymphocyte proliferation assay of synovial fluid mononuclear cells (SFMC) with whole fraction bacteria in the diagnosis of reactive arthritis (ReA) or arthritis of unknown origin. METHODS: We stimulated SFMC of 52 unselected patients who consecutively presented in our rheumatology outpatient clinic with the following diagnoses: ReA (n = 8), rheumatoid arthritis (RA) (n = 16), ankylosing spondylitis (AS) (n = 6), osteoarthritis (OA) (n = 5), psoriatic arthritis (PsA) (n = 5) and arthritis of varying origin (AVO) (n = 12) and peripheral blood MC (PBMC) of 10 healthy controls with arthritogenic (Y. entero-colitica, S. enteritidis, C. trachomatis) and non-arthritogenic (E. coli, K. pneumoniae, S. pyogenes, C. albicans) bacteria/mitogens and Tetanus toxoid. T cell proliferation was measured in a standard [3H] Thymidine uptake assay. RESULTS: In all groups of patients tested, SFMC could be stimulated both by arthritogenic and non-arthritogenic bacteria. So-called specific responses were observed in patients with ReA, but also in RA and AS. CONCLUSION: Our findings show that a lymphocyte proliferation assay with SFMC with whole fraction bacteria is not an adequate diagnostic tool to confirm bacterial involvement in inflammatory arthritis.

Anti-neutrophil cytoplasmic antibodies in a rat model of trinitrobenzenesulphonic acid-induced liver injury.

BACKGROUND: In sera from patients with autoimmune liver diseases, e. g. primary sclerosing cholangitis (PSC) and autoimmune hepatitis, anti-neutrophil cytoplasmic antibodies (ANCAs) can be found. Until now, no animal model of ANCA induction in liver disease has been described. In this study, we describe a novel rat model of acute liver injury and subsequent ANCA production. MATERIALS AND METHODS: The hapten reagent 2,4,6-trinitrobenzenesulphonic acid (TNBS) was injected into the portal vein of female Lewis rats. Two experimental groups were studied: group A (TNBS/ethanol) received different TNBS concentrations; control animals of group B (ethanol) were injected with 10% (v/v) ethanol/0.9% (w/v) NaCl. RESULTS: A dose-dependent acute necrotizing liver injury occurred after injection of TNBS. Histopathological examination revealed acute hepatic injury with confluent parenchymal necrosis, mild bile duct proliferation and periportal infiltration. The periportal infiltration consisted mainly of macrophages and T lymphocytes. ANCAs were found in an allogenic test system between 1 and 8 weeks after TNBS injection in 11 out of 28 (39%) TNBS-treated rats (group A) and did not correlate with the extent of liver injury or TNBS dose. Autoantibody specificities of IgG type were directed against catalase (29%), myeloperoxidase (14%) and actin (7%), as detected by enzyme-linked immunosorbent assay and Western blotting. Moreover, autoantibodies against the asialoglycoprotein receptor were observed. Peripheral blood mononuclear cells and spleen lymphocytes from TNBS-treated rats were shown to produce ANCAs. CONCLUSION: In summary, we were able to show that intraportal administration of the hapten reagent TNBS induces an acute necrotizing liver injury with subsequent ANCA production in Lewis rats. ANCA specificities were mainly directed against catalase, an autoantigen that has recently been identified in sera from patients with primary sclerosing cholangitis and autoimmune hepatitis. This animal model offers the opportunity to study the pathomechanisms of ANCA production in necrotizing liver injury.

Constitutive and inducible in vivo protein-DNA interactions at the tumor necrosis factor-alpha promoter in primary human T lymphocytes.

Tumor necrosis factor-alpha (TNF-alpha) is a key cytokine of lymphocytes with major regulatory functions in immunomodulation, chronic inflammation, and septic shock. However, only limited information on TNF promoter regulation in vivo in primary lymphocytes is available. To determine and compare protein-DNA interactions at the native TNF locus in primary lymphocytes, we analyzed the human TNF-alpha promoter by ligation-mediated polymerase chain reaction (LM-PCR) techniques. Accordingly, primary CD4+ T lymphocytes from peripheral blood were cultured in the presence of various stimuli and analyzed by LM-PCR. Inducible in vivo protein-DNA interactions at the TNF promoter were detected between -120 and -70 bp of the human TNF promoter relative to the transcriptional start site. This area includes binding sites for transcription factors such as ETS-1, NFAT, ATF-2/c-jun, SP-1/Egr-1, and NF-kappaB. In contrast, no protein-DNA interactions were observed at various binding sites with reported regulatory function in tumor cell lines such as the k2 element, the NFAT site at -160, the AP1 site at -50, and the SP1 site at -65. Additional mutagenesis and transfection studies demonstrated that NF-kappaB and CREB/AP-1 are important regulators of inducible TNF promoter activity in primary human T lymphocytes. These results provide novel insights into the complex regulation of TNF gene transcription in primary T lymphocytes in vivo by constitutive and inducible protein-DNA interactions that appear to be at least partially different compared to previously characterized tumor cell lines.

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.

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.