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Biomedical subjects

M Neurath

Publications and source records attributed to M Neurath.

At least 19 recordsLinked to original sources

[Massive hemorrhage in a 28-year-old male patient with intestinal Behçet's disease].

A 28-year-old man was admitted to the emergency room of our hospital after syncope and acute gastrointestinal bleeding. On the basis of numerous oral and genital ulcerations as well as uveitis anterior and erythema nodosum, the diagnosis of Behçet's disease had been confirmed previously. The bleeding focus could not be detected by endoscopy. During the next days recurrent massive hemorrhages occurred in spite of immunosuppressive therapy with steroids. Angiography revealed a circumscribed bleeding source in the colon transversum near the left colonic flexure, which was treated by superselective coil embolization. A massive hemorrhage reoccurred and required a surgical approach with a Hartmann procedure. Histology depicted bleeding into the submucosa and mucosa and an ulcer in this area as well as lymphatic infiltration of the mucosa. Immunosuppressive therapy was intensified with azathioprine and resulted in a dramatic improvement of the oral and genital ulcers. In addition, no further gastrointestinal hemorrhage occurred. Due to its higher prevalence in patients from eastern Asia, Behçet's disease poses an important differential diagnosis of intestinal hemorrhage in patients of this descent.

Adult↗

An interleukin 12 p40-IgG2b fusion protein abrogates T cell mediated inflammation: anti-inflammatory activity in Crohn's disease and experimental colitis in vivo.

BACKGROUND AND AIMS: Interleukin-12 (IL-12), a p35/p40 heterodimer, plays a pivotal role in the immune response in Crohn's disease (CD). Since IL-12 p40 dimers act as IL-12 antagonists, we assayed p40 dimer proteins to modulate chronic intestinal inflammation. METHODS: We generated a fusion protein consisting of the IL-12(p40) subunit fused to the constant region of IgG2b. IL-12(p40)-IgG2b was tested in a murine 2,4,6,-trinitrobenzene sulphonic acid (TNBS) colitis model and in lamina propria mononuclear cells (LPMNC) from patients with CD in vitro. RESULTS: Dimeric IL-12(p40)-IgG2b fusion protein bound specifically to the IL-12 receptor. In concentrations <10(-7) M, it acted as an IL-12 antagonist as it inhibited interferon gamma (IFN-gamma) secretion, suppressed proliferation, and increased apoptosis of LPMNC from patients with CD. However, in concentrations >10(-6) M, IL-12(p40)-IgG2b increased IFN-gamma secretion and lymphocyte proliferation thereby acting as an IL-12 agonist. In TNBS colitic mice, IL-12(p40)-IgG2b decreased mortality (10% v 68%), prevented body weight loss, reduced tumour necrosis factor alpha, and increased IL-10 secretion. CONCLUSIONS: The IL-12(p40)-IgG2b fusion protein has dichotomic properties as a specific IL-12 antagonist and selective repressor of mucosal inflammation at low concentration and as an IL-12 agonist at high concentration.

Adult↗

[Anti-TNF antibodies in the treatment of inflammatory intestinal stenoses in Crohn's disease].

Stenoses are a frequent complication in patients with Crohn's disease and represent a major diagnostic and therapeutic challenge. The proper assessment of the nature of a stenosis as inflammatory or fibrotic is critical for appropriate treatment, since symptomatic fibrotic stenoses require surgical resection. Standard diagnostic procedures to assess the nature of a stenosis include endoscopy, conventional contrast radiography and magnetic resonance tomography. Recent data suggest, that the positron-emission-tomography possesses a high sensitivity and specificity to confirm inflammatory activity in the bowel. The recombinant monoclonal anti-TNF-antibody Infliximab (Remicade) has been approved for the treatment of steroid refractory and steroid dependent Crohn's disease in Germany since 9/2000 and the efficacy of Infliximab is well documented. However, few data exist about the treatment of inflammatory stenoses with Infliximab. We performed a retrospective analysis of our experience with Infliximab in patients with Crohn's disease with special reference to patients with inflammatory stenoses. Among a total of 21 patients treated with Infliximab 11 patients had an inflammatory stenosis. 9 of these patients responded well to Inflimab and became completely asymptomatic for a considerable period of time. Infliximab was tolerated well except for one patient who developed an intrabdominal abscess. The notable clinical response of patients with inflammatory stenoses to Infliximab suggests that treatment with Infliximab might be helpful to postpone or avoid surgical intervention. This finding should be further investigated in a prospective randomized study.

Adult↗

A novel rat model of chronic fibrosing cholangitis induced by local administration of a hapten reagent into the dilated bile duct is associated with increased TNF-alpha production and autoantibodies.

BACKGROUND/AIM: The cholangiopathies represent hepatobiliary diseases in which bile-duct epithelial cells are targets for destructive processes, including immune-mediated damage. We describe a novel rat model of chronic fibrosing cholangitis induced by administration of the hapten reagent 2,4,6-trinitrobenzenesulfonic acid (TNBS) into the dilated bile duct. METHODS: The common bile duct was dilated due to a mild stenosis in 8-week-old female Lewis rats. TNBS (50 mg/kg) was injected during a second laparotomy. RESULTS: TNBS-treatment reproducibly resulted in chronic fibrosing cholangitis. In retrograde cholangiography the bile ducts showed irregularities, beading and strictures. Alkaline phosphatase levels remained abnormal throughout the study period. Immunohistochemical staining showed an increased number of macrophages, CD3+ T-lympbocytes and MHC class II antigen upregulation. The spontaneous interferon-gamma, tumor necrosis factor-alpha and interleukin-10 production of liver-derived mononuclear cells was increased. Anti-neutrophil cytoplasmic antibodies with specificity against myeloperoxidase, catalase and actin were found between 1 and 12 weeks after TNBS injection. CONCLUSIONS: We established a novel rat model of chronic fibrosing cholangitis with histologic, cholangiographic, serologic and immunologic similarities to human primary sclerosing cholangitis. This model may be used to study pathomechanisms of chronic cholangitis without concomitant inflammatory bowel disease.

Animals↗

TNBS-colitis.

Disease states, such as the occurrence of gastrointestinal inflammation (Crohn's disease and ulcerative colitis), can be secondary to a host of determinants that act in conjunction to bring about pathologic change. The underlying factors that mediate the development of such mucosal inflammation has recently been brought to the forefront with the advent of animal models. The examination of these animal models have given researchers a better understanding of the mechanisms involved in the pathogenesis of inflammatory bowel disease. This review discusses one such model, TNBS-colitis, and the insights that it provides into the occurrence of IBD and its future treatment.

Animals↗

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.

Animals↗

Anti-PR-3 antibodies induce endothelial IL-8 release.

BACKGROUND: It has been shown that interaction of anti-PR-3 antibodies with human endothelial cells (EC) leads to an activation of EC in vitro, i.e. induction of adhesion molecules like E-selectin, VCAM-1 and tissue factor. The aim of this study was to investigate the effect of anti-PR-3 antibodies on endothelial IL-8 expression. MATERIALS AND METHODS: EC were cultured in 96-well plates and stimulated with TNF-alpha and IL-1beta for 1 h to induce membrane expression of endothelial PR-3. Anti-PR-3 antibodies were purified from sera from patients with clinically active Wegener's granulomatosis. Purified anti-Ro, anti-centromere, anti-dsDNA antibodies and a monoclonal anti-PR-3 antibody (WGM2) served as controls. Induction of IL-8 mRNA was detected by RT-PCR. IL-8 was measured in the supernatant of EC by ELISA. In addition, induction of NFkappaB was investigated by PAGE of nuclear extracts of EC and Western blot with ab against p65. RESULTS: In contrast to controls, interaction of anti-PR-3 antibodies (patients and WGM2) with cytokine activated EC led to the highest amount of IL-8 synthesis. Priming of EC with cytokines alone induced a markedly lower IL-8 level. The lowest levels of IL-8 could be measured after incubation of unprimed EC with anti-PR-3 antibodies. Anti-PR-3 antibody induced endothelial IL-8 expression could be inhibited by cycloheximide. In addition, we established that the activation of NF-kappaB is critically involved in anti-PR-3 antibody induced endothelial IL-8 production. CONCLUSION: In summary, we were able to show that anti-PR-3 antibodies induce endothelial IL-8 synthesis by activating NF-kappaB. As IL-8 represents a powerful neutrophil chemoattractant, our data provide further evidence for a direct pathogenic effect of anti-PR-3 antibodies in ANCA related diseases.

Antibodies, Antineutrophil Cytoplasmic↗

T cell specificity and cross reactivity towards enterobacteria, bacteroides, bifidobacterium, and antigens from resident intestinal flora in humans.

BACKGROUND: T cell responses to normal intestinal bacteria or their products may be important in the immunopathogenesis of chronic enterocolitis. AIMS: To investigate the T cell specificity and cross reactivity towards intestinal bacteria. PATIENTS/METHODS: T cell clones were isolated with phytohaemagglutinin from peripheral blood and biopsy specimens of inflamed and non-inflamed colon from five patients with inflammatory bowel disease (IBD) and two controls. T cell clones were restimulated with anaerobic Bacteroides and Bifidobacteria species, enterobacteria, and direct isolates of aerobic intestinal flora. T cell phenotype was analysed by single-cell immunocyte assay. RESULTS: Analysis of 96 T cell clones isolated from peripheral blood and biopsy specimens from two patients with IBD showed that both Bifidobacterium and Bacteroides species specifically stimulate proliferation of CD4+TCRalphabeta+ T cell clones from both sites and that cross reactivity exists between these anaerobic bacteria and different enterobacteria. Analysis of 210 T cell clones isolated from three patients with IBD and two controls showed that indigenous aerobic flora specifically stimulate T cell clones from peripheral blood and biopsy specimens from a foreign subject. Some of these flora specific T cell clones were cross reactive with defined enterobacteria. In addition, T cell clones stimulated by their own indigenous aerobic flora were identified in patients with IBD. CONCLUSION: Immune responses to antigens from the intestinal microflora involve a complex network of T cell specificities.

Adult↗

CD44 variant isoforms on blood leukocytes in chronic inflammatory bowel disease and other systemic autoimmune diseases.

We have described recently that TNBS-induced colitis, an animal model of chronic inflammatory bowel disease (IBD), can be cured by treatment with anti-CD44v7. This finding led us to evaluate whether CD44v7 may be of functional importance in patients with IBD. Expression of CD44 variant isoforms (CD44v) has been evaluated on PBMC of 46 patients with IBD, 43 patients with autoimmune diseases not affecting the gastrointestinal tract, 26 patients with nonautoimmune disease, and 24 healthy donors. In all groups, expression of CD44v on freshly harvested PBMC was not above or was borderline above background levels. After in vitro stimulation, expression of CD44 standard (CD44s) and CD44v6 was strongly up-regulated. Exclusively on PBMC of patients with autoimmune disease, high expression of CD44v3 and CD44v7 was observed. CD44v3 and CD44v7 were mainly expressed on subsets of CD4+ lymphocytes, B cells, and monocytes; CD44v6 was predominantly detected on CD4+ and CD8+ cells. Considering functional activity, CD44v7 apparently exerted a dual effect. After culturing PBMC in the presence of anti-CD44v7, a higher percentage of cells produced IL-10. This was irrespective of whether the PBMC were derived from healthy donors or from patients with autoimmune disease or IBD. On the other hand, PBMC of all donors proliferated upon cross-linking of CD3 and CD44s or CD3 and CD44v6. Instead, costimulatory activity of CD44v7 was seen only in PBMC of patients with autoimmune disease and IBD. Because expression and function of CD44v7 in patients with systemic autoimmune disease and IBD have been much like the ones in mice suffering of TNBS-induced colitis, it is tempting to speculate that blockade of CD44v7 could also be of therapeutic relevance in the human diseases.

Adult↗

Identification and characterization of a novel Ets-2-related nuclear complex implicated in the activation of the human interleukin-12 p40 gene promoter.

Interleukin-12 (IL-12) is a proinflammatory cytokine produced by antigen-presenting cells in response to many microbial infections. IL-12 plays an important role in the generation of T helper type-1 cells, which favor cell-mediated immune response. IL-12 is composed of two different subunits, p40 and p35, whose expression can be regulated concomitantly or differentially. Monocytic cells, the major producers of IL-12, can be primed by interferon-gamma (IFN-gamma) to produce optimal amounts of IL-12 in response to LPS stimulation as a consequence of bacterial infection. The priming effect is exerted primarily at the transcriptional level on the p40 promoter in conjunction with the effects of LPS, possibly by inducing specific transcription factors, which individually have no direct effect but which cooperatively can activate the promoter. We examined in detail one of these DNA-protein interactions observed around an Ets-2 element situated at -211/-207 of the p40 promoter, which is known to be a functionally critical site. This region interacts with a nuclear complex termed F1 that appears to be highly inducible by either IFN-gamma treatment for 16 h or lipopolysaccharide stimulation for 8 h. F1 binding to the Ets-2 site requires a considerable amount of spacing around the Ets-2 site, as revealed by gel mobility shift and in vitro methylation assays. Supershift experiments and DNA affinity purification indicated that both Ets-2 and a novel, antigenically related protein with an approximate molecular mass of 109 kDa are part of the F1 complex, together with additional components including IRF-1 and c-Rel. This novel protein is designated GLp109 for its inducibility by IFN-gamma or lipopolysaccharide. Its possible role in the activation of the IL-12 p40 promoter is discussed.

Animals↗

Two different negative regulatory elements control the transcription of T-cell activation gene 3 in activated mast cells.

T-cell activation gene 3 (TCA3) encodes a beta-chemokine that is transcriptionally regulated in mast cells; the gene has a functional NF-kappaB element at positions -194 to -185. The 5'-flanking region of this gene is also known to have a negative regulatory region between -2057 and -1342. To characterize the negative regulatory elements (NREs), this region was sequenced and then digested by HindIII enzyme into two fragments, NRE-1 (-2057 to -1493) and NRE-2 (-1492 to -1342). Both NRE-1 and NRE-2 in the 5'-3' orientation inhibited chloramphenicol acetyltransferase (CAT)-protein synthesis by a TCA3-CAT construct transfected into mast cells that were then activated. Only NRE-1 inhibited CAT-protein synthesis in the 3'-5' orientation. Further deletion of the 5' region of NRE-1 partially abolished the inhibitory activity. Both NRE-1 and NRE-2 inhibited the activity of a CD20-CAT construct independent of cell activation. Electrophoretic mobility shift assays showed DNA-protein complex formation with subsequences (CCCCCATTCT) of NRE-1 (NRE-1a) and (CCATGA) of NRE-2 (NRE-2b). NRE-1a appears to be novel. NRE-2b is identical with a putative silencer motif in the alphaIIb integrin gene. Site-directed mutagenesis demonstrated that both NRE-1a and NRE-2b are important in the negative regulation of TCA3 promoter activity. In vivo ligation-mediated PCR footprinting of the NRE-2 region revealed protection between -1372 and -1354, which contains NRE-2b. The data thus demonstrate identity of a silencer motif, here termed NRE-2b, in both the alphaIIb integrin gene and the TCA3, and that this silencer region in mast cells is functional both in vivo and in vitro. Further, evidence is presented that the promoter for TCA3 contains a novel silencer motif, termed NRE-1a, characterized by a CT-rich sequence.

Animals↗

Induction and prevention of colonic inflammation in IL-2-deficient mice.

Gene-targeted mice deficient for IL-2 (IL-2 -/- mice) are free of apparent disease when maintained under germfree conditions but develop colitis and autoimmunity in a conventional environment. Here we show that colitis can be reproducibly induced in IL-2 -/- mice, but not in IL-2 +/+ mice, by i.p. immunization with Ag in CFA; thus enabling the systematic study of the immunopathogenesis of the colitis. We found that TNP-KLH or TNP-OVA had the most significant effect in inducing colitis, and while TNP-KLH immunization leads to the early appearance of activated T cells in the colons of both IL-2 -/- and IL-2 +/+ mice, only lamina propria cells of IL-2 -/- mice produced high amounts of INF-gamma. Moreover, both infiltrating colon CD4+ (69%) and CD8+ (6%) T cells secrete large amounts of IFN-gamma; however, only the depletion of CD4+ T cells leads to abrogation of the inflammation. In further analysis, we showed that the high IFN-gamma production is IL-12 driven, since colonic tissues of IL-2 -/- mice but not IL-2 +/+ mice show the presence of heterodimeric IL-12 and co-administration of anti-IL-12 with TNP-KLH completely prevented colitis and significantly reduced IFN-gamma production. Finally, we demonstrate that IL-2 -/- mice are deficient in their ability to induce Th2 responses after TNP-KLH challenge and that such immunization also leads to autoimmune-like phenomena in other organs of IL-2 -/- mice. These findings suggest that in the absence of IL-2 systemic administration of Ag induces primarily Th1 cells driven by overexpression of heterodimeric IL-12.

Animals↗

Reciprocal IFN-gamma and TGF-beta responses regulate the occurrence of mucosal inflammation.

The above new findings concerning the immunological mechanisms governing mucosa, immune responses and oral tolerance in TCR-transgenic mice, as well as those operative in mice with experimental colitis, greatly expand our understanding of the processes that normally control mucosal inflammation and possibly other types of inflammation as well (Fig. 1). They indicate that, in the nondiseased mouse, ingested proteins evoke a Th1-cell (IFN-gamma) response in the mucosal follicles that is quickly counter-regulated by induction of T-cell anergy/deletion, if this Th1-cell response is inhibited (experimentally by anti-IL-12), TGF beta-producing cells appear, and these are capable of active immune suppression. This reciprocal relationship between IFN-gamma production and TGF-beta production is further supported in mouse models of mucosal inflammation. Thus, in the TNBS-colitis model, there is direct stimulation of the immune cells in the lamina propria as a result of diffuse haptenization of mucosal proteins, which leads to a massive Th1-cell response capable of overwhelming any suppressive counter-regulatory mechanisms normally generated in the PPs. This highly polarized Th1-cell response is controlled only by direct abrogation of IL-12 production with exogenous administration of anti-IL-12, or with indirect inhibition of this response via induction of oral tolerance and accompanying production of TGF-beta (Refs 6-8). The data obtained from this model are consistent with those obtained with another model of intestinal inflammation--inflammation in severe combined immunodeficiency (SCID) mice following lymphoid repletion with CD45Rbhi (naive) T cells. In this model, inflammation is again mediated by Th1 cells and is prevented by co-repletion with CD45Rbhi (memory) T cells, which appear to work by secreting TGF-beta (Refs 9, 10). Thus, a common feature of the various experimental models of intestinal inflammation studied to date is the Yin-Yang relationship of IFN-gamma and TGF-beta, with the former being proinflammatory and the latter anti-inflammatory. Is the IFN-gamma TGF-beta dichotomy that is evident both in the normal state and in models of inflammation simply a reflection of an underlying Th1 Th2 dichotomy? The answer to this important question is not yet known. Thus, while it is clear from the in vitro studies already discussed that IL-12 and/or IFN-gamma inhibit TGF-beta production, the role of IL-4 in this process is more elusive. These in vitro studies indicate that IL-4 is not required for TGF-beta production, a finding that is consistent with studies in which the transfer of CD45Rbhi, T cells from IL-4-/- mice protected SCID mice from colitis induced by CD45Rbhi T cells. However, the addition of IL-4 to in vitro cultures containing anti-IL-12 augmented TGF-beta production, most probably by IL-4 acting as a growth factor for TGF-beta-producing cells rather than as an inducing factor (T. Marth et al., unpublished). Obviously, more work will be necessary to resolve this issue. Finally, it should be noted that the above considerations apply to human inflammatory diseases of the gastrointestinal tract, such as Crohn's disease. Recently, it has been shown that T cells extracted from Crohn's disease tissues manifest skewed Th1-cell responses. The hypothesis can therefore be put forward that this disease results from a dysregulated Th1-cell response to ubiquitous mucosal antigens that is not appropriately controlled by normal counter-regulatory mechanisms. Interventions that artificially bring the excessive Th1-cell response back into balance, such as administration of IL-12 antagonists, should therefore find a central place in the treatment of the disease.

Animals↗

Temporal control of IgH gene expression in developing B cells by the 3' locus control region.

The suggested roles of the downstream 3' regions acting as a Locus Control Region (LCR), have allowed comparisons to be made between the regulation of the IgH locus with other model systems whose gene expression is governed by LCR activity. Here we summarize the importance of the IgH 3'LCR and its putative functional role in IgH gene expression and compare it with the 5'LCR regulatory region of the human beta-globin locus.

Animals↗

Defects of monocyte interleukin 12 production and humoral immunity in Whipple's disease.

BACKGROUND & AIMS: Whipple's disease (WD) is a systemic infection in which the causative bacteria typically accumulate within macrophages. The aim of this study was to test whether this macrophage dysfunction is the cause or result of previously shown T-cell defects. METHODS: In vitro production of interleukin (IL)-12, IL-10, tumor necrosis factor alpha, interferon gamma (IFN-gamma), and transforming growth factor beta (TGF-beta) from purified monocytes and peripheral blood mononuclear cells, cytokine expression on duodenal biopsy specimens, and serum cytokine and immunoglobulin (Ig) levels were tested in 9 patients with WD. RESULTS: Reduced monocyte IL-12 production and decreased IFN-gamma secretion by peripheral blood mononuclear cells in vitro were found, as well as reduced immunohistological staining for IL-12 and IFN-gamma, but no decrease in other cytokines in patients with WD. A similar but less severe defect in 2 relatives with WD argued for a genetic basis of this abnormality. Serum IgG2, an IFN-gamma-dependent Ig subclass, and serum TGF-beta levels were reduced in patients with WD. CONCLUSIONS: The described monocyte defects in WD may result in a secondary reduction of IFN-gamma production and IgG2 serum levels. This provides a rationale for additive immunotherapy in patients with antibiotic-refractory WD.

Antibody Formation↗