Natural hidden autoantibodies to tissue transglutaminase cross-react with fibrinogen.

INTRODUCTION: Patients with celiac disease display autoantibodies against tissue transglutaminase (TG2), and the high sensitivity and specificity of these autoantibodies render them a reliable tool for diagnosis. However, we found that denatured sera from healthy persons also showed reactivity against TG2. METHODS: To further examine the specificity of this phenomenon, sera of healthy individuals and celiac patients were denatured by heat or pH shift. RESULTS AND DISCUSSION: Denatured sera of all individuals showed autoantibodies against TG2 in ELISA that could be specifically inhibited by TG2, but the biological role of these autoantibodies remains unknown. The alpha fibrinogen precursor could be isolated as serum protein that reacts with TG2 antibodies and treated sera reacted with fibrinogen in Western blotting. Cross-reactivity of TG2 antibodies with fibrinogen and vice versa was observed. CONCLUSION: We hypothesise that denaturation of sera reveals hidden autoantibodies against TG2, which might be normally masked by fibrinogen.

In vitro differentiation of human monocytes into dendritic cells by peptic-tryptic digest of gliadin is independent of genetic predisposition and the presence of celiac disease.

INTRODUCTION: This study was done to further reveal the role of the innate immune system in celiac disease. METHODS: Dendritic cells were matured from venous blood of patients with active or treated celiac disease and DQ2-DQ8-positive or negative controls. Dendritic cells were treated with a peptic-tryptic digest of gliadin (500 microg/ml) and their activation was analyzed by fluorescent-activated cell sorting analysis, cytokine secretion, and their ability to elicit T cell proliferation. RESULTS AND DISCUSSION: Gliadin upregulated interleukin (IL)-6, IL-8, and IL-12 (p40) secretion in dendritic cells and induced strong expression of the maturation markers human leukocyte antigen (HLA)-DR, CD25, CD83, and CD86 of all subjects irrespective of their genotype or the presence of disease, whereas the digest of bovine serum albumin showed no effect. However, gliadin-stimulated dendritic cells from active celiac showed enhanced stimulation of autologous T cells compared to the other groups. CONCLUSION: Further research should be aimed at identifying the mechanisms that control inflammation in healthy individuals.

Pathomechanisms in celiac disease.

Celiac disease is a complex autoimmune disease which is characterized by a strong genetic association (HLA-DQ2 or -DQ8), gluten as nutritional etiological factor, and the enzyme tissue transglutaminase as endomysial autoantigen. Patients develop highly predictive IgA autoantibodies to tTG. Certain gluten peptides are presented by the disease-associated HLA-DQ2/DQ8 molecules leading to stimulation of gluten-specific T cells. This immune response which is driven in the lamina propria causes the mucosal transformation characteristic for celiac disease. Increased intestinal expression of tTG in patients with CD appears to play an important role in the pathogenesis of CD. Thus, modification of gluten peptides by tTG, especially deamidation of certain glutamine residues, can enhance their binding to HLA-DQ2 or -DQ8 and potentiate T cell stimulation. Furthermore, tTG-catalyzed cross-linking and consequent haptenization of gluten with extracellular matrix proteins allows for storage and extended availability of gluten in the mucosa. New therapeutic approaches aim at proteolytic destruction of immunodominant gliadin peptides that are resistant to intestinal enzymes by bacterial prolyl endopeptidases, the inhibition of tTG activity with highly specific enzyme inhibitors or at HLA-DQ2/DQ8 blocking peptide analogues.

Activation of tumor-specific T lymphocytes by radio-frequency ablation of the VX2 hepatoma in rabbits.

Radio-frequency ablation (RFA) is used as a minimally invasive treatment for inoperable hepatic tumors. Immunological reactions secondary to RFA may play a role in the observed tumor control. In our study, the VX2 carcinoma was implanted into the liver of rabbits. After 3 weeks, tumors were treated with RFA or were left untreated. Peripheral blood lymphocytes were harvested before tumor implantation, 2 weeks postoperatively and at 2-week intervals thereafter. T cells were stimulated with lysates of either tumor tissue or nontumorous liver loaded on autologous antigen-presenting cells and their stimulation index was determined by [(3)H]thymidine incorporation. A 3-fold increase over background or controls was considered significant. Stimulation with phytohemagglutinin served as a positive control. The animals were necropsied, and liver and tumor tissue were analyzed immunohistologically for T-cell infiltration. T cells from tumor-bearing (n = 9) and RFA-treated (n = 11) animals were investigated in a follow-up study. The mean postoperative observation was 45 days. All of the 11 RFA-treated animals exhibited circulating T cells activated specifically toward tumor antigens throughout the observation period, which was accompanied by dense T-cell infiltration. In contrast, T cells of untreated tumor-bearing rabbits showed no reaction and only sparse T cell infiltration. We concluded that RFA induces a tumor-specific T-cell reaction in the otherwise unreactive tumor-bearing host, apparently overcoming immune tolerance and leading to the presentation of otherwise cryptic tumor antigens. Therefore, in addition to destroying tumor tissue, RFA induces an immune response against tumor antigens that may be exploited in multimodal antitumor strategies.

Failure of cyclosporin A to induce transforming growth factor beta (TGF-beta) synthesis in activated peripheral blood lymphocytes.

Induction of transforming growth factor beta (TGF-beta) by the immunosuppressive drug cyclosporin A (CsA) in activated lymphocytes has been claimed to add to the renal pro-fibrotic effects of CsA. The aim of this study was to evaluate CsA-mediated TGF-beta induction in a larger number of lymphocyte preparations from different donors. Peripheral blood lymphocytes (PBL) were obtained from healthy blood donors. The cells were stimulated with phytohemagglutinin E (PHA) and phorbol ester (tetradecanoyl phorbol acetate, TPA) in the presence or absence of CsA. TGF-beta, interleukin-2 (IL-2) and cyclooxygenase-2 (Cox-2) mRNA were detected by Northern blot analysis or by real time reverse transcriptase-polymerase chain reaction (RT-PCR). TGF-beta and IL-2 protein were determined in the cellular supernatants by enzyme-linked immunosorbent assay. TGF-beta mRNA and protein were up-regulated when the cells were stimulated with PHA/TPA. Cyclosporin A at high concentrations (500 ng/mL) caused a transient increase in TGF-beta mRNA which was significant after 2 h. CsA did not induce sustained TGF-beta protein expression (24-72 h) in any of the preparations (n = 14), whereas the up-regulation of IL-2 mRNA and protein was prevented by CsA in the same preparations. Furthermore, up-regulation of Cox-2 mRNA was inhibited by CsA. Taken together, there was no evidence for TGF-beta as a clinically relevant mediator being induced by CsA in activated peripheral blood T-lymphocytes.