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.

Identification of the predominant antigenic epitopes in intestinal flora in IBD.

The normal intestinal flora is required for the development of intestinal inflammation in animal models of inflammatory bowel disease (IBD). In humans, several studies indicated a potential association of Escherichia coli (E. coli) with IBD. In addition, we have shown that T-cell clones of IBD patients cross react toward different enteric bacterial species and thus likely respond to conserved bacterial antigens. Therefore, we hypothesized that highly conserved E. coli proteins might be a reasonable candidate to screen for abnormal T-cell responses in IBD. We used high-throughput techniques for cloning, expression, and purification under native conditions of a set of 271 conserved proteins of E. coli, of which 196 were used for whole blood stimulations to assess peripheral T helper (T(H))-cell responses. In addition, because of the association of an adherent-invasive E. coli with Crohn's disease (CD), we included 13 pathogenicity factors of E. coli in the study. We observed that pools of these conserved E. coli proteins less frequently induced interferon-gamma (IFNgamma) production in peripheral T(H) cells in patients with CD and ankylosing spondylitis (AS) compared with healthy controls. In addition, lower percentage of patients with CD and AS responded toward single proteins. The reason for the decreased frequency of an in vitro T(H)-cell IFNgamma response toward E. coli proteins in peripheral blood of CD and AS patients, e.g., increased suppression needs to be clarified.

Autochthonous hepatitis E virus infection in Germany with sequence similarities to other European isolates.

Hepatitis E virus (HEV) is a common cause of acute hepatitis in endemic areas. Yet reports on autochthonous cases in other areas such as middle Europe are increasing. Here we report on a patient, who obviously acquired his HEV infection in Germany. Sequence analysis of the virus gained from his serum revealed homologies to other European isolates and swine isolates.

Safety and efficacy of intravenous pulse cyclophosphamide in acute steroid refractory inflammatory bowel disease.

BACKGROUND AND AIMS: One major problem in the management of steroid refractory attacks of patients with inflammatory bowel disease (IBD) is the establishment of a rapidly acting immunosuppressive regimen. Based on its well known efficacy in systemic vasculitis, intravenous cyclophosphamide pulse therapy was used in refractory IBD patients to evaluate both its efficacy and safety. METHODS: Between December 1998 and May 2001, seven patients (Crohn's disease, n=5; indeterminate colitis, n=1) with severe steroid refractory IBD (Crohn's disease activity index (CDAI) 264-479 points) received 4-6 cycles of monthly treatment with intravenous cyclophosphamide (750 mg) in a prospective uncontrolled pilot study. RESULTS: All patients improved after two intravenous pulses of cyclophosphamide and six of seven patients achieved complete remission (CDAI <150 points). One patient with Crohn's disease of the small and large bowel showed an impressive clinical response but did not enter into remission. Tapering to low dose steroids was possible in all responders. Remission was maintained in all patients for 18 months (median) but required a second course of intravenous pulse cyclophosphamide in one patient. The drug was well tolerated except for two episodes of candida oesophagitis. CONCLUSIONS: Intravenous pulse cyclophosphamide may be a safe and effective treatment in patients with severe IBD unresponsive to steroid treatment and merits evaluation in a controlled trial.

Conserved TCR beta chain usage in reactive arthritis; evidence for selection by a putative HLA-B27-associated autoantigen.

Previous work suggested that expanded CD8+ T-cell clones in the synovial fluid (SF) of HLA-B27+ patients with reactive arthritis (ReA) preferentially use the T-cell receptor variable region (TCRBV) 1, similar CDR3 sequences, and joining region (BJ) 2S3. To determine the range of conservation and disease-specificity of CDR3-sequences, we analyzed the TCRBV1-J2S3 repertoire from 33 healthy HLA-B27+ individuals, patients with various types of spondyloarthropathies (SpA), and with rheumatoid arthritis (RA) by CDR3-spectratyping. After collection and database submission of all available TCRB-CDR3 from HLA-B27-restricted or SpA-derived T cells, we systematically screened the entire human sequence database for sequences similar to the B27/SpA-related CDR3. Spectratyping revealed expanded T cell clones using conserved TCRBV1J2S3 in the SF from 5/6 of the patients with acute ReA but not among the controls. In database searches, 50 HLA-B27 or SpA-related CDR3-sequences generated similar clusters of matched sequences, and matched reciprocally. Identical or closely related sequences were identified in 15 different individuals and a canonical ReA-associated TCRB was defined [BV1-CASSVG(V/I/L)(Y/F)STDTQYF-J2S3]. All but one patient-derived conserved sequences originated from acute stage ReA-patients, and were not present among approximately 3800 other human TCRB sequences in the database. Five of the conserved sequences originated from T cell clones that recognized uninfected cells in an HLA-B27-restricted fashion, implying a role of HLA-B27-restricted CD8+ T cells specific for a ubiquitous self- or cross-reactive microbial determinant in the early phase of ReA. Related sequences were independently identified in four different laboratories. The consensus TCRB motif could be a helpful diagnostic marker in HLA-B27-associated 'undifferentiated arthritis'.

Immune response to autologous and heterologous Helicobacter pylori antigens in humans.

Infection of humans with Helicobacter pylori results in the development of chronic gastritis and plays an important role in gastric ulcer pathogenesis. Despite the infiltration of the mucosa with specific immunocompetent cells and production of specific antibodies, the infection usually persists for life. This study was performed to investigate if immunologic mechanisms exist which could contribute to the inability of the host to terminate the infection. Therefore, we compared the in vitro immunoreactivity of peripheral blood mononuclear cells (PBMC) from H. pylori-infected patients after stimulation with sonicated H. pylori bacteria from the stomach of the patient (autologous bacterial strain) with stimulation by bacteria from other patients (heterologous bacteria). We measured cell proliferation, expression of T cell activation markers CD25, HLA-DR, and CD71, as well as production ofinterleukin-10 (IL-10), an inhibitory cytokine. We found that the proliferative response of PBMC was significantly lower after autologous than after heterologous stimulation. Furthermore, secretion of IL-10 in the culture supernatants was significantly higher when PBMC were incubated with autologous than with heterologous H. pylori antigens. No significant differences between autologous or heterologous stimulation were observed in the increased expression of T cell activation markers. These data indicate that systemic immunologic response to H. pylori are strain-dependent. For further studies of the immune responses towards H. pylori, the use of an autologous stimulatory system seems necessary.

[Abdominal tuberculosis: a rare differential diagnosis of pancreatic carcinoma]. / Abdominale Tuberkulose. Eine seltene Differenzialdiagnose zum Pankreaskarzinom.

HISTORY AND ADMISSION FINDINGS: A 79-year-old local resident, presenting with abdominal pain, sweating and weight loss and suspected of having cancer of the pancreas was referred for diagnosis and treatment. Physical examination was negative except for pain on pressure over the right upper abdomen and the epigastrium. INVESTIGATIONS: Erythrocyte sedimentation rate was increased; as were the transaminases and cholestasis parameters. Ultrasonography and computed tomography of the abdomen revealed an echo-poor mass with cystic areas in the region of the head of the pancreas, as well as extra- and intrahepatic dilatation of the biliary tract. Endoscopic retrograde cholangiopancreatography failed to demonstrate a ductal pancreatic carcinoma. Biopsies of a macroscopically peculiar-looking duodenal ulcer demonstrated a noncaseous epithelioid granuloma. A fine-needle biopsy was performed for further diagnosis. DIAGNOSIS, TREATMENT AND COURSE: Histological examination of the needle biopsy revealed a caseous granuloma and acid-fast bacteria. The tuberculin test (GTI) was strongly positive (14-15 mm), indicating tuberculosis of the pancreas and duodenum. Multiple tuberculostatics rapidly improved the patient's symptoms, and the further course was without complications. CONCLUSION: Tuberculosis should be included in the differential diagnosis of consumptive disease with an atypical presentation, especially because treatment could well be curative.

CD4+ and CD8+ clonal T cell expansions indicate a role of antigens in ankylosing spondylitis; a study in HLA-B27+ monozygotic twins.

Ankylosing spondylitis (AS) is a complex genetic disease in which both MHC and non-MHC genes determine disease susceptibility. To determine whether the T cell repertoires of individuals with AS show signs of increased stimulation by exogenous antigens, CD4+ and CD8+ T cell subsets of five monozygotic HLA-B27+ twins (two concordant and three discordant for AS) and CD8+ T cell repertoires of three healthy HLA-B27+ individuals were characterized by TCR beta-chain (TCRB) CDR3 size spectratyping. Selected TCRB-CDR3 spectra were further analysed by BJ-segment analysis and TCRB-CDR3 from expanded T cell clones were sequenced. In an analysis of all data (519/598 possible TCRB-CDR3 spectra), AS was associated with increased T cell oligoclonality in both CD8+ (P = 0.0001) and CD4+ (P = 0.033) T cell subsets. This was also evident when data were compared between individual twins. Nucleotide sequence analysis of expanded CD8+ or CD4+ T cell clones did not show selection for particular TCRB-CDR3 amino acid sequence motifs but displayed sequence homologies with published sequences from intra-epithelial lymphocytes or synovial T cells from rheumatoid arthritis patients. Together, these results provide support for the hypothesis that responses to T cell-stimulating exogenous or endogenous antigens are involved in the induction and/or maintenance of AS.

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.

Anti-TNF antibody in Crohn's disease--status of information, comments and recommendations of an international working group.

The chimeric anti-TNF antibody Remicade (Infliximab) has recently been approved for human use by the FDA and is now available on the market. Since there is considerable interest in this kind of treatment among patients with Crohn's disease, an international working group has summarized the presently available information about efficacy, side effects and possible problems of this treatment. Studies show that Remicade is effective in the treatment of active Crohn's disease, maintaining remission and fistulae. The working group does not see Infliximab as a first-line treatment for Crohn's disease. It may be used in active phase recurrent disease, chronic active disease and fistulae if standard treatment was not successful. For the surveillance special attention has to be given to the unknown malignancy rate of Infliximab. Infusion should be performed in an institution, routinely performing intravenous infusions and a two-hour surveillance of the patients should be guaranteed to recognize anaphylactic reactions or acute side effects. There is presently no information indication that the combination with immunosuppressants might increase risks or side effects of this treatment. Due to the limited information available the working group would prefer to use Remicade in studies only and recommends central collection and documentation of all data on efficacy and side effects for the next year.

Induction of cytokine production in naive CD4(+) T cells by antigen-presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells.

BACKGROUND & AIMS: Murine liver sinusoidal endothelial cells (LSECs) constitutively express accessory molecules and can present antigen to memory Th1 CD4(+) T cells. Using a T-cell receptor transgenic mouse line, we addressed the question whether LSECs can prime naive CD4(+) T cells. METHODS: Purified LSECs were investigated for their ability to induce activation and differentiation of naive CD4(+) T cells in comparison with bone marrow-derived antigen-presenting cells and macrovascular endothelial cells. Activation of T cells was determined by cytokine production. LSECs were further studied for expression of interleukin (IL)-12 by reverse-transcription polymerase chain reaction, and the unique phenotype of LSECs was determined by flow cytometry. RESULTS: We provide evidence that antigen-presenting LSECs can activate naive CD62Lhigh CD4(+) T cells. Activation of naive CD4(+) T cells by LSECs occurred in the absence of IL-12. In contrast, macrovascular endothelial cells from aorta could not activate naive CD4(+) T cells. The unique functional characteristics of microvascular LSECs together with a unique phenotype (CD4(+), CD11b+, CD11c+, CD80(+), CD86(+)) make these cells different from macrovascular endothelial cells. Furthermore, LSECs did not require in vitro maturation to activate naive CD4(+) T cells. Most importantly, LSECs failed to induce differentiation toward Th1 cells, whereas conventional antigen-presenting cell populations induced a Th1 phenotype in activated CD4(+) T cells. Upon restimulation, CD4(+) T cells, which were primed by antigen-presenting LSECs, expressed interferon gamma, IL-4, and IL-10, which is consistent with a Th0 phenotype. Exogenous cytokines (IL-1beta, IL-12, or IL-18) present during T-cell priming by antigen-presenting LSECs could not induce a Th1 phenotype, but neutralization of endogenously produced IL-4 during T-cell priming led to a reduced expression of IL-4 and IL-10 by CD4(+) T cells upon restimulation. The addition of spleen cells to cocultures of LSECs and naive CD4(+) T cells during T-cell priming led to differentiation of T cells toward a Th1 phenotype. CONCLUSIONS: The ability of antigen-presenting LSECs to induce cytokine expression in naive CD4(+) T cells and their failure to induce differentiation toward a Th1 phenotype may contribute to the unique hepatic microenvironment that is known to promote tolerance.

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.

IgG, albumin, and sCD44 in whole-gut lavage fluid are useful clinical markers for assessing the presence and activity of pouchitis.

Pouchitis is the most significant long-term complication in patients with ileoanal pouch anastomosis (IAP) and is especially frequent in patients with ulcerative colitis. There is an urgent need for simple and objective parameters to assess the presence and activity of pouchitis. Whole-gut lavage fluid (WGLF) was collected from 34 patients [8 with pouchitis (PDAI > or = 7 points) and 26 without pouchitis (Pouchitis Disease Activity Index, PDAI, < 7)]. Patients with active ulcerative colitis (n = 8) served as controls. Concentrations of IgG and sCD44 in WGLF were measured by enzyme-linked immunosorbent assays and those of albumin by immunoturbidimetry. Similar to the case in active ulcerative colitis, concentrations of IgG, albumin, and sCD44 in WGLF were significantly increased in acute pouchitis and reached high specificity (IgG 96%, albumin 96%, sCD44 100%) and acceptable sensitivity (75%) for the diagnosis of acute pouchitis. These parameters were also closely correlated with disease activity as determined by PDAI and endoscopic scoring indices. Assay of protein concentrations in WGLF is thus a simple and objective means for grading inflammation of the pouch and may be useful as a quantitative index of disease activity in clinical studies.

[Crohn disease, ulcerative colitis. When bacteria attack the intestinal wall....]. / Morbus Crohn, Colitis ulcerosa. Wenn Bakterien die Darmwand attackieren....

Crohn's disease (CD) and ulcerative colitis (UC) are clinical entities characterized by spontaneous relapses and are thought to be caused in large part by a dysregulated immune response to inflammatory stimuli. Specific infectious agents or antigens inducing or perpetuating inflammation, however, are not known. Recent results in contrast support the hypothesis, that the normal intestinal flora plays a central role in the pathogenesis of both diseases. Studies performed with E. coli Nissle 1917 demonstrated that this bacterium can positively affect the course of disease in UC and CD patients. The clinical efficacy of probiotics can yield valuable information about disease pathogenesis and, as a modification of current standard therapy, opens new and interesting therapeutic alternatives.

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-10 protein release from KC strictly depended on de novo protein synthesis. Endotoxin mediated increase in IL-10 gene expression was downregulated by exogenous (>350-fold reduction of IL-10 mRNA level), as well as endogenous IL-10 protein, showing a negative autoregulatory feedback loop. IL-10 receptor expression was found to be constitutive and functional in KC. Early expression of IL-10 in KC may be of functional relevance to the outcome of immune and inflammatory reactions in the liver sinusoid. The negative autoregulation of IL-10 expression may represent a mechanism to regain a state of functional responsiveness in the microenvironment towards new proinflammatory stimuli. In conclusion, autoregulatory downregulation of IL-10 expression in KC may account for important regulatory steps of local immune response in the liver sinusoid.

Normal interleukin-12 production in individuals with antibodies to Helicobacter 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 demonstrate that DNA synthesis, IFN-gamma production and IL-12 production induced by H. pylori or Y. enterocolitica 03 did not differ significantly between H. pylori antibody-positive and H. pylori antibody-negative individuals. However, in the H. pylori-positive group there was a tendency, although not statistically significant, to produce less IFN-gamma in response to H. pylori but not Y. enterocolitica. These results demonstrate that monocyte/macrophages from H. pylori-positive individuals secrete normal amounts of IL-12 upon bacterial challenge and suggest that the decreased production of IFN-gamma in H. pylori-positive individuals observed in previous studies is selective for H. pylori and not caused by decreased IL-12 secretion.

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 different clonotypes was diverse, repeated restimulation with sarcoma cells led to a monoclonal expansion of T cells with particular TCR clonotypes in 5/6 mixed lymphocyte tumor cell cultures (MLTC). One clonotype was found in 2 independent MLTC experiments. Sarcoma-reactive T cells were demonstrated in patient tumor-infiltrating lymphocytes (TIL) and peripheral blood mononuclear cells (PBMC) by clonotypic PCR. Our results indicate a limited number of immunodominant antigens expressed by the sarcoma cells. The junctional diversity of the TCR clonotypes shows that these antigens did not lead to extensive negative thymic selection as classical autoantigens would have. Therefore, the recognized antigens might represent cryptic autoantigens related to cellular transformation or proliferation.