Comparative analysis of mononuclear cells isolated from mucosal lymphoid follicles of the human ileum and colon.

Studies of human mucosal lymphoid follicles are rare and have been limited to children's Peyer's patches, which are visible at endoscopy. We investigated lymphoid follicles in ileum biopsies of 87 patients and surgical colon specimens from 66 cancer patients, and examined phenotype and function of isolated follicular immune cells. Two (0-10) and 12 (0-117) follicles per patient were found in ileum and colon samples respectively (P < 0.001). The number of lymphoid follicles mononuclear cells (LFMC) that could be isolated per patient was higher from colon compared with ileum specimens [725 000 (0-23 Mio) versus 100 000 (0-1.3 Mio), P < 0.001]. T cells were predominant in both LFMC and lamina propria mononuclear cells (LPMC), but B cells were more and plasma cells less frequent in LFMC. T cells from mucosal follicles were more frequently CD4-positive and CD62L-positive, but less frequently CD8-positive, CD103-positive and CD69-positive than lamina propria T cells. LFMC from ileum compared with colon showed no differences in mononuclear cell composition. Anti-CD3/CD28 stimulation induced similar proliferation of LFMC and LPMC from ileum and colon, as well as secretion of high levels of interferon-gamma, tumour necrosis factor-alpha and interleukin (IL)-2, but lower levels of IL-4, IL-6 and IL-10. LFMC from colon secreted more IL-2 than those from ileum. Our study shows that mucosal lymphoid follicles can be identified clearly in adult human colon and yield viable immune cells sufficient for phenotypical and functional analysis. The cellular composition of LFMC from ileum and colon is similar, and both secrete predominantly T helper type 1 cytokines.

Capsule endoscopy in refractory celiac disease.

BACKGROUND AND STUDY AIMS: Patients with refractory celiac disease (RCD) are at risk of intestinal T-cell lymphoma, which is difficult to diagnose because it often develops in the small bowel. We therefore studied whether wireless capsule endoscopy was able to detect ulcerative jejunitis or intestinal T-cell lymphomas that were missed by standard endoscopic and imaging procedures in patients with RCD. PATIENTS AND METHODS: Detection of ulcerative jejunitis and overt T-cell lymphoma by capsule endoscopy or by upper and lower endoscopy, abdominal computed tomography (CT) or abdominal magnetic resonance tomography (MRT) was compared in 14 consecutive patients with RCD: in seven patients who showed loss of T-cell antigens on intraepithelial lymphocytes and/or clonality of the T-cell receptor gene (i. e. type II RCD) and in seven patients who did not have these features (i. e. type I RCD). RESULTS: Complete evaluation of the small bowel by capsule endoscopy was achieved in 9/14 patients. Signs of ulcerative jejunitis or intestinal T-cell lymphoma, affecting further clinical management, were found in two patients with type II RCD: in one patient these signs were found only by capsule endoscopy (ulcerations and stenosis) and in another patient the abnormalities were identified by CT/MRT (mesenteric lymph nodes harboring lymphoma). No clinically relevant abnormalities were found in patients with type I RCD by lower endoscopy or by small-bowel imaging (capsule endoscopy, CT, or MRT). CONCLUSIONS: In patients with type II RCD, capsule endoscopy can detect additional cases with ulcerative jejunitis and could be included in the diagnostic armamentarium, subject to confirmation by larger series. In patients with type I RCD, our study confirmed the low diagnostic yield of imaging procedures, including wireless capsule endoscopy.

CD44v7 ligation downregulates the inflammatory immune response in Crohn's disease patients by apoptosis induction in mononuclear cells from the lamina propria.

Deletion of exon CD44v7 abrogates experimental colitis by apoptosis induction in intestinal mononuclear cells. Here we show that CD44v7 expression was upregulated upon CD40 ligation in human mononuclear cells, and examined whether ligation of CD44v7 also affects activation and apoptosis in lamina propria mononuclear cells (LPMC) from Crohn's disease (CD) patients. Thirty five patients with chronic inflammatory bowel disease (IBD), fourteen controls and four patients with diverticulitis were evaluated. CD44v7 was upregulated predominantly in the inflamed mucosa of CD patients. Furthermore, incubation with an anti-CD44v7 antibody induced apoptosis in LPMC isolated from inflamed mucosa of CD patients, but not from non-inflamed mucosa, from patients with ulcerative colitis (UC) or from normal controls. CD40 ligation and simultaneous incubation with anti-CD44v7 significantly downregulated CD80 in dendritic cells, thus inhibiting a critical second signal for naive T-cell activation. The apoptotic signal was mediated via the intrinsic mitochondrial pathway with decreased Bcl-2 and increased 7A6 (a mitochondrial membrane protein) expression. It was Fas independent and required caspases-3 and -9 activation. The process is highly specific for macrophage activation via CD40. These findings point to a novel mechanism of apoptosis induction in CD patients mediated by CD44v7 ligation.

Changes in expression and distribution of claudin 2, 5 and 8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease.

BACKGROUND: Epithelial barrier function is impaired in Crohn's disease. AIM: To define the underlying cellular mechanisms with special attention to tight junctions. METHODS: Biopsy specimens from the sigmoid colon of patients with mild to moderately active or inactive Crohn's disease were studied in Ussing chambers, and barrier function was determined by impedance analysis and conductance scanning. Tight junction structure was analysed by freeze fracture electron microscopy, and tight junction proteins were investigated immunohistochemically by confocal laser scanning microscopy and quantified in immunoblots. Epithelial apoptosis was analysed in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and 4',6-diamidino-2-phenylindole staining. RESULTS: Patients with active Crohn's disease showed an impaired intestinal barrier function as indicated by a distinct reduction in epithelial resistance. As distribution of conductivity was even, focal epithelial lesions (eg, microerosions) did not contribute to barrier dysfunction. Instead, freeze fracture electron microscopy analysis showed reduced and discontinuous tight junction strands. Occludin and the sealing tight junction proteins claudin 5 and claudin 8 were downregulated and redistributed off the tight junction, whereas the pore-forming tight junctions protein claudin 2 was strongly upregulated, which constitute the molecular basis of tight junction changes. Other claudins were unchanged (claudins 1, 4 and 7) or not detectable in sigmoid colon (claudins 11, 12, 14, 15 and 16). Claudin 2 upregulation was less pronounced in active Crohn's disease compared with active ulcerative colitis and was inducible by tumour necrosis factor alpha. As a second source of impaired barrier function, epithelial apoptosis was distinctly increased in active Crohn's disease (mean (SD) 5.2 (0.5)% v 1.9 (0.2)% in control). By contrast, barrier function, tight junction proteins and apoptosis were unaffected in Crohn's disease in remission. CONCLUSION: Upregulation of pore-forming claudin 2 and downregulation and redistribution of sealing claudins 5 and 8 lead to altered tight junction structure and pronounced barrier dysfunction already in mild to moderately active Crohn's disease.

REPDOSE: A database on repeated dose toxicity studies of commercial chemicals--A multifunctional tool.

A database for repeated dose toxicity data has been developed. Studies were selected by data quality. Review documents or risk assessments were used to get a pre-screened selection of available valid data. The structure of the chemicals should be rather simple for well defined chemical categories. The database consists of three core data sets for each chemical: (1) structural features and physico-chemical data, (2) data on study design, (3) study results. To allow consistent queries, a high degree of standardization categories and glossaries were developed for relevant parameters. At present, the database consists of 364 chemicals investigated in 1018 studies which resulted in a total of 6002 specific effects. Standard queries have been developed, which allow analyzing the influence of structural features or PC data on LOELs, target organs and effects. Furthermore, it can be used as an expert system. First queries have shown that the database is a very valuable tool.

Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test.

As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays (lacI model; commercially available as the Big Blue(R) mouse, and the lacZ model; commercially available as the Mutatrade markMouse), were compared with the results on the same substances in the more traditional mouse bone marrow micronucleus test. 39 substances were found which had been tested in the micronucleus assay and in the above transgenic mouse systems. Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.

Celiac disease-like abnormalities in a subgroup of patients with irritable bowel syndrome.

BACKGROUND & AIMS: Abdominal symptoms in the absence of mucosal abnormalities are features of both the irritable bowel syndrome (IBS) and latent/potential celiac disease (cd). To identify a possible subgroup of IBS patients with latent/potential cd, surrogate markers of cd were investigated in IBS patients. METHODS: IBS patients suffering from diarrhea (n = 102), and patients with active cd (n = 10), treated cd (n = 26), and latent cd (n = 5) were included in the study. We measured serum immunoglobulin (Ig) A against gliadin and tissue-transglutaminase, and IgA and IgM against gliadin, tissue-transglutaminase (intestinal cd-associated antibodies), and the dietary proteins beta-lactoglobulin and ovalbumin in duodenal aspirate by enzyme-linked immunosorbent assay. Intraepithelial lymphocytes (IELs) were counted in histology sections, and the expression of HLA-DQ2 (A1*0501/B1*0201) was investigated by polymerase chain reaction. In 26 IBS patients, the effect of 6 months of gluten withdrawal was examined. RESULTS: Most cd patients expressed HLA-DQ2 and had increased intestinal cd-associated antibodies, whereas cd-associated serum IgA and IEL counts were increased in active cd in contrast to treated or latent cd. In IBS patients, 35% were HLA-DQ2-positive, 23% had increased IEL counts, and 0% and 30% had increased cd-associated antibodies in serum and duodenal aspirate, respectively. Furthermore, stool frequency and intestinal IgA decreased significantly under a gluten-free diet in the subgroups of HLA-DQ2-positive and intestinal antibody-positive IBS patients when compared with IBS patients without these markers. CONCLUSIONS: HLA-DQ2 expression and increased intestinal cd-associated antibodies are markers that can identify latent/potential cd in a subgroup of IBS patients who consequently appear to profit from a gluten-free diet.

Rapid increase of mucosal CD4 T cells followed by clearance of intestinal cryptosporidiosis in an AIDS patient receiving highly active antiretroviral therapy.

Highly active antiretroviral therapy (HAART) suppresses the replication of human immunodeficiency virus (HIV) and leads to an increase in circulating CD4 T lymphocytes, but its effects on other immune compartments such as the intestinal mucosa are not well understood. We describe a severely immunodeficient HIV-infected patient with intractable watery diarrhea and weight loss caused by infection with Cryptosporidium parvum in whom we studied virologic and immunologic changes in both peripheral blood and the intestinal mucosa after initiating HAART. Mucosal biopsies were performed by rectoscopy before and at several time points after HAART was begun. Nucleic acids were extracted from rectal biopsy specimens and blood samples, and HIV RNA was measured by reverse-transcription polymerase chain reaction. Lymphocytes were isolated from rectal biopsy specimens after mechanical disaggregation, and circulating and mucosal CD4 T cells were determined by flow cytometry. HAART led to clinical recovery and eradication of cryptosporidiosis. In both blood and mucosa, HIV RNA decreased below the limit of detection and CD4 T cells increased. Mucosal CD4 T cells increased much faster and to much higher levels than circulating CD4 T cells. Our findings show a rapid repopulation of the intestinal mucosa with CD4 T cells after initiation of HAART that can effectively restore mucosal immunity, leading to eradication of opportunistic pathogens.

Vinyl chloride: still a cause for concern.

Vinyl chloride (VC) is both a known carcinogen and a regulated chemical, and its production capacity has almost doubled over the last 20 years, currently 27 million tons/year worldwide. According to recent reports it is still a cause for concern. VC has been found as a degradation product of chloroethylene solvents (perchloroethylene and trichloroethylene) and in landfill gas and groundwater at concentrations up to 200 mg/m(3) and 10 mg/L, respectively. Worldwide occupational exposure to VC still seems to be high in some countries (e.g., averages of approximately 1,300 mg/m(3) until 1987 in one factory), and exposure may also be high in others where VC is not regulated. By combining the most relevant epidemiologic studies from several countries, we observed a 5-fold excess of liver cancer, primarily because of a 45-fold excess risk from angiosarcoma of the liver (ASL). The number of ASL cases reported up to the end of 1998 was 197 worldwide. The average latency for ASL is 22 years. Some studies show a small excess risk for hepatocellular carcinoma, and others suggest a possible risk of brain tumors among highly exposed workers. Lung cancer, lymphomas, or leukemia do not seem to be related to VC exposure according to recent results. The mutation spectra observed in rat and human liver tumors (ASL and/or hepatocellular carcinoma) that are associated with exposure to VC are clearly distinct from those observed in sporadic liver tumors or hepatic tumors that are associated with other exposures. In rats, the substitution mutations found at A:T base pairs in the ras and p53 genes are consistent with the promutagenic properties of the DNA adduct 1,N(6)-ethenoadenine formed from VC metabolites. Risk assessments derived from animal studies seem to overestimate the actual risk of cancer when comparing estimated and reported cases of ASL.

Increased immunoglobulin G production by short term cultured duodenal biopsy samples from HIV infected patients.

BACKGROUND: Secretory immunity is a major defence mechanism against infections at mucosal surfaces which are common in HIV infected patients. AIMS: To analyse intestinal immunoglobulin production in HIV infection in comparison with that in saliva and serum. PATIENTS AND METHODS: Immunoglobulin G (IgG), A (IgA), and M (IgM) concentrations were determined in supernatants of short term cultured duodenal biopsy samples, serum, and saliva from HIV infected patients (n = 28) and controls (n = 14) by radial immunodiffusion. RESULTS: IgG was increased in the supernatants of short term cultured biopsy samples and saliva from HIV infected patients compared with controls (p < 0.01), but IgA and IgM levels were normal. In contrast, both IgG and IgA concentrations in serum were higher in HIV infected patients than in controls (p < 0.002). No correlation was found between IgA produced by duodenal biopsy specimens and serum IgA. CONCLUSION: Abnormalities in mucosal immunoglobulin production in HIV infection were surprisingly small, indicating that specific secretory immunity rather than quantitative immunoglobulin production may be impaired. However, increased production of IgG could contribute to mucosal inflammation by complement activation. Our findings of normal mucosal IgA production and the lack of correlation between serum and mucosal IgA argues against an intestinal origin for the increased serum IgA levels in HIV infected patients.

Ela 1.0--a framework for life-cycle impact assessment developed by the Fraunhofer-Gesellschaft. Part A: The conceptual framework.

The Fraunhofer-Gesellschaft has sponsored the development of a conceptual and flexible, computer aided tool to perform the impact assessment within LCA (life cycle assessment) for technical products and processes. The developed general framework "Ela 1.0" (environmental loads analysis) consists of four elements: the selection of appropriate impact categories, the categorization of emissions and wastes leaving the systems as well as of resource and energy consumption, the characterization and an analysis of the results of the impact assessment. The latter compares the product-based emissions with the total of emissions of a region such as Germany, the EU or OECD countries. The framework Ela 1.0 considers the environmental categories: global warming, ozone depletion, resource and energy consumption, wastes, eutrophication (including COD and BOD as measured parameters), acidification, ecotoxicity, ozone formation and human toxicity. The latter categories are handled by listing of precursors for ozone formation, and by listing of emissions scored according to their human hazard potential. The options, possibilities and limitations of the conceptual framework are presented in part A of a series of publications.

Abnormal predominance of IgG in HIV-specific antibodies produced by short-term cultured duodenal biopsy specimens from HIV-infected patients.

The aim of our study was to analyze HIV-specific humoral immunity in the intestinal mucosa at different stages of HIV infection in comparison with serum and saliva. Duodenal biopsy specimens from 30 AIDS patients and 9 HIV-infected patients without AIDS were cultured for 48 hours. Culture supernatants, as well as simultaneously obtained serum and saliva samples, were adjusted to the same immunoglobulin concentrations and tested for HIV-specific IgG and IgA by Western blot. The HIV antigen pattern differed clearly between IgA and IgG but was similar for each isotype independent of its origin (i.e., serum, saliva, or biopsy specimen supernatants). Short-term cultured duodenal biopsy specimens from HIV-infected patients at all stages produced predominantly IgG, which was broadly reactive with HIV antigens. Lower titers of HIV-specific IgA, which recognized few antigens, were found, mostly the glycoprotein gp160. At later stages of the disease compared with earlier stages, the reaction pattern of mucosal IgA from saliva and biopsy supernatants was even more restricted; secretory component was frequently absent. The abnormal predominance of HIV-specific IgG over IgA in mucosal secretions may result from abnormal antibody production in the mucosa rather than from serum leakage. Mucosal inflammation induced by HIV-IgG immune complexes and insufficient immune exclusion by secretory IgA may not only lead to increased mucosal HIV replication but may also contribute to gastrointestinal disease in HIV-infected patients.

Serum antibody reactivity to recombinant mig and whole cell antigens in Mycobacterium avium infection.

Mycobacterium avium is a significant opportunistic pathogen in immunocompromised patients. Moreover, the prevalence of infections in patients without known predisposing conditions has also been increasing in recent years. Patients would greatly benefit from early diagnosis of disseminated infection. Serodiagnostic tests have already been promising in tuberculosis and immunocompetent patients but studies in HIV-infected patients and humoral response to M. avium antigens resulted in conflicting data. We have evaluated the use of the phagocytosis-induced MIG protein of M. avium as a diagnostic antigen. Serum antibody levels of M. avium-infected, HIV-negative patients were significantly elevated for the recombinant MIG (p < 0.001) and also for M. avium whole-cell antigens (p < 0.025) as compared to controls. In contrast, HIV-infected patients with disseminated M. avium infection demonstrated also elevated levels of antibody for the whole-cell antigen (p < 0.00001) but a decreased reactivity for the MIG antigen (p < 0.007). The recombinant antigen proved to have no cross-reactivity with M. tuberculosis antigens as antibody levels were decreased in tuberculosis patients (p < 0.001). Therefore, a simultaneous serological test using recombinant MIG and the whole cell antigens might be helpful in the sometimes problematic diagnosis of M. avium infections in patients without predisposing conditions.

Does prolonged implantation of depth electrodes predispose the brain to kindling?

Chronically implanted depth electrodes are widely used for the study of electrical signals generated in deep cerebral locations and for electrical stimulation of such locations. Although the effects of lesions resulting from electrode implantation are generally considered minimal, some reports have shown lasting neurochemical, histological, and behavioral alterations in response to such implantation. Furthermore, there is some evidence that prolonged electrode implantation may decrease the seizure threshold of the implanted region and increases the rate of kindling from this region. This prompted us to undertake a study on different periods of post-surgical delay to onset of electrical stimulation and subsequent characteristics of kindling development. Rats were implanted with a bipolar electrode in the basolateral amygdala, and the threshold for induction of focal paroxysmal activity (afterdischarge threshold, ADT) was determined after post-surgical recovery periods of either 1, 2, 4, or 8 weeks. The animals were then kindled by daily administration of an electrical stimulus until all rats exhibited fully kindled seizures. In fully kindled rats, the ADT was redetermined. Compared to animals with 1 week of electrode implantation, the pre-kindling ADT was significantly lower in rats with 2 and 4 weeks of electrode implantation, but returned towards the 1 week values at 8 weeks. An enhanced kindling rate was seen when kindling stimulations were started after 4 and 8 weeks of electrode implantation. Despite the marked differences in pre-kindling ADT, the post-kindling ADT was similar in the groups with 1, 2, or 4 weeks but significantly lower in the group with 8 weeks post-surgical delay to onset of testing. The data suggest that prolonged implantation of a bipolar electrode into a sensitive region of the limbic system predisposes the brain to kindling. Based on previous observation of iron deposits induced by electrode implantation and the epileptogenic effect of iron in cortical and limbic regions, we propose that the present observations are due to deposition of iron from hemoglobin destruction in local microhemorrhages caused by the implantation.