Guido Schürmann

Cytokine Gene Transcription By NF-κB Family Members in Patients with Inflammatory Bowel Disease

ABSTRACT: We examined the expression of the transcription factor NF‐κB, a nuclear trans‐acting factor known to play a key role in cytokine gene regulation, in patients with inflammatory bowel disease (IBD). It was found that LP macrophages in Crohns disease (CD) and ulcerative colitis (UC) display high levels of NF‐κB DNA‐binding activity accompanied by an increased production of interleukin (IL)‐1, IL‐6, and tumor necrosis factor (TNF)α. Western blot studies showed an increased expression of the p50 and c‐rel subunits of NF‐κB; however, the most striking finding was an increased expression level of NF‐κB p65 in patients with CD and UC. Selective downregulation of p65 in IBD macrophages by a specific antisense phosphorothioate oligonucleotide was sufficient to considerably reduce production of proinflammatory cytokines. These results demonstrate a characteristic increase of NF‐κB binding levels in patients with IBD. The data suggest that antisense DNA targeting NF‐κB p65 can be used as a novel molecular approach for the treatment of patients with IBD.

Activation and Signaling Status of Human Lamina Propria T Lymphocytes

In this study, proliferative responses of human lamina propria T lymphocytes were examined in vitro. The response of lamina propria T lymphocytes to Sepharose-bound anti-CD3 antibody plus interleukin 2 was significantly lower than the response of autologous peripheral blood T lymphocytes, whereas the responses of lamina propria T lymphocytes to anti-T11(2/3) antibodies plus sheep erythrocytes or anti-CD28 antibody plus interleukin 2 were largely preserved. After coculture with mucosa supernatant, peripheral blood T lymphocytes showed a similar pattern of reactivity as lamina propria T lymphocytes. This reduced reactivity to T-cell antigen receptor stimulation appears to exist at the level of signal transduction, because triggering of CD3 induces low amounts of intracellular inositol 1,4,5-triphosphate and no free calcium increase in lamina propria T lymphocytes when compared with peripheral blood T lymphocytes. This study indicates that the antigen receptor-dependent activation pathway of lamina propria T lymphocytes for proliferation is down-regulated by intestinal mucosa derived factor(s) and that the alternative pathways mediated by CD2 or CD28 are largely preserved. Based on previous data that lamina propria T lymphocytes can provide help to B cells, it is possible that these alternative activation pathways play an important role in T-B cell interaction in the gut.

Human intestinal mucosa alters T-cell reactivities

BACKGROUND Compared with T cells in peripheral blood, lamina propria T lymphocytes have a low proliferative response to stimulation via the T-cell antigen receptor/CD3 complex or by protein kinase C activators yet largely preserve or even show an enhanced reactivity to CD2 and CD28 triggering. Coculture of peripheral blood T lymphocytes with intestinal mucosa supernatant leads to a similar functional behavior as found in freshly recovered lamina propria T lymphocytes. The aim of this study is to characterize the nature of substances in the mucosal supernatant responsible for downregulation of T-cell receptor-dependent signals. METHODS Mucosal supernatant was produced, dialyzed, digested with proteinase K, reduced by 2-mercaptoethanol or dithiothreitol, and tested for its activity on peripheral blood T lymphocytes. RESULTS Supernatant lost its activity after dialysis through a membrane (pore size 12,000-14,000). Digestion with proteinase K does not abolish its activity suggesting that the substances are neither proteins nor peptides. However, its effects on T lymphocyte proliferation can be reversed by reducing agents like 2-mercaptoethanol or dithiothreitol, suggesting that oxidative substances are contained in mucosal supernatants. CONCLUSIONS Our data support the view that mucosal substances that down-regulate antigen receptor-induced T lymphocyte proliferation are small, nonprotein, nonpeptide molecules with oxidative properties.

Cytokine messenger RNA expression and proliferation status of intestinal mononuclear cells in noninflamed gut and Crohn's disease

T-cell activation and local cytokine production probably contribute to the pathogenesis of Crohns disease. This study investigates the proliferative status of intestinal mononuclear cells (MNC) and cytokine messenger RNA (mRNA) production in gut tissue sections from patients with Crohns disease and noninflamed controls. mRNA in situ hybridization was performed using 33P-labelled riboprobes for human interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, tumour necrosis factor-α and interferon-γ. The expression of the proliferation-associated antigen Ki-67 was analysed by immunohistochemical single and double staining. Compared with controls, where proliferation of MNC and cytokine expression was restricted to mucosal lymphoid follicles, inflamed gut tissue contained increased numbers of cells expressing cytokine mRNA, most prominently IL-1β and IL-6, but also interferon-γ and tumour necrosis factor-α. Proliferating T-cells were increased in number, and small amounts of IL-2-expressing cells were detected. IL-4 was expressed by a few cells exclusively in follicular germinal centres. IL-5 was negative. Proinflammatory cytokines are strongly expressed in situ in Crohns disease and largely predominate over lymphokine mRNA. Our results provide in situ evidence of a local lymphocyte response in Crohns disease with characteristics of a delayed-type hypersensitivity reaction.

The popliteal artery entrapment syndrome: Presentation, morphology and surgical treatment of 13 cases

The morphology, clinical parameters and treatment of the popliteal artery entrapment syndrome (PAES) are presented on the basis of 13 of our own cases and from the literature. PAES is based on a segmental vascular compression due to an anatomical anomaly of the popliteal region and a new classification is presented distinguishing three variants according to the different anatomical conditions. PAES is mostly found in young sportsmen with well-developed muscles. Clinical symptoms are acute or chronic, and the diagnosis is made by physical examination, angiography and Doppler ultrasound, both in neutral position and in plantar flexion. Although thromboendarterectomy in some cases leads to good results, the preferred surgical therapy is decompression of the entrapped artery and reconstruction of the arterial pathway by vein graft interposition. The results were excellent in nine of 13 cases. In two patients, a recurrent thrombosis necessitated a femoro-crural bypass and in two others the entrapment was only diagnosed during reoperation for aneurysms of the venous graft.

Stereology and flow-cytometry of well-differentiated follicular neoplasms of the thyroid gland

A retrospective analysis of surgically resected thyroid nodules by stereology and DNA flow cytometry was performed in 15 follicular adenomas and 15 well-differentiated follicular carcinomas. The criteria for diagnosis were based on the WHO classification of thyroid tumours. By area-weighted random sampling of the visual fields for light-microscopic stereology, any subjective selection bias was precluded, and each point within the embedded neoplastic tissue was given equal probability of being analyzed. 150–250 tumour cell nucleus (TCN) profiles were studied per case by a semiautomatic image analyzing system. Flow cytometric analyses included measurement of the DNA index, and the percentages of cells in S-phase and in G2/M-phase. Adenomas and carcinomas did not differ in stereological estimates related to TCN size. As examination of the stereological techniques by nested analysis of variance showed that this result cannot be ascribed to inaccurate methods, it follows that determination of TCN size is not a useful tool for the diagnosis of malignancy in well-differentiated thyroid tumours. Both groups included similar proportions of diploid and aneuploid neoplasms. In the carcinoma group the percentage of tumour cells in the G2/M-phase was more than twice as high than in the adenoma group (P<0.01). The ratio of short to long TCN profile axis was significantly smaller, and the coefficient of variation of TCN profile area was significantly higher in carcinomas than in adenomas. These findings are consistent with more unequiaxed TCN and higher anisokaryosis in the malignant tumours. Despite the significant differences, however, overlap of data from individual cases precludes the use of these estimates as diagnostic criteria. Pooling of the follicular tumours and dichotomizing the sample by the DNA-index showed that mean TCN profile area is increased and surface-to-volume ratio of TCN is decreased in aneuploid as compared to diploid tumours. This finding indicates that aneuploidy is associated with an increase of TCN size.

T cell receptor repertoire and mitotic responses of lamina propria T lymphocytes in inflammatory bowel disease.

Human intestinal lamina propria T lymphocytes (LPL‐T) physiologically exhibit minimal proliferation in response to antigen receptor stimulation in vitro. This is thought to occur as a consequence of regulatory influences which are exerted by the mucosal microenvironment. The present study is aimed at investigating whether proliferative responses of intestinal LPL‐T to antigen receptor stimulation are altered in patients with inflammatory bowel disease. Accordingly, proliferative responses of LPL‐T in patients with Crohns disease and ulcerative colitis to stimulation with CD3 MoAb plus IL‐2 were examined and compared with controls. In addition, T cell receptor (TCR) repertoires of LPL‐T and peripheral blood T lymphocytes were determined by indirect immunofluorescenee using a panel of 11 TCR Vβ specific antibodies. In most patients with inflammatory bowel disease, LPL‐T showed enhanced proliferation to antigen receptor stimulation compared with controls. Moreover, perhaps as a consequence, an enhanced frequency of in vivo preactivated T cells was seen as judged from an increased spontaneous proliferative response to low concentrations of exogenous IL‐2. LPL‐T and peripheral blood T lymphocytes exhibited similar percentages of TCR Vβ gene usage both in controls and in patients. In summary, polyclonal activation of LPL‐T due to impairment of local adjustment, i.e. insufficient down‐regulation of TCR/CD3‐dependent signalling processes, may contribute to the pathogenesis of inflammatory bowel disease.

Stereology, flow cytometry, and immunohistochemistry of follicular neoplasms of the thyroid gland

A retrospective analysis of surgically resected thyroid cold solitary nodules was performed by stereology, DNA flow cytometry, and immunohistochemistry in 15 follicular adenomas and 15 well-differentiated follicular carcinomas to determine if a differential diagnosis of both follicular neoplasms can be done exclusively from cytologic criteria. Between 150 and 200 tumor cell nuclei (TCN) were studied per case for their TCN profile area, perimeter, and density, as well as for stereologic estimates, including the new parameter, volume-weighted mean particle volume (Vv). Flow-cytometric analyses included measurement of the DNA index and the percentage of cells in S phase and G2M phase. The same tumors were examined for the expression of thyroglobulin and the intermediate filaments vimentin and keratin. Follicular adenomas and follicular carcinomas did not show any significant differences in stereologic estimates related to TCN size. Both groups included similar proportions of diploid and aneuploid neoplasms. Aneuploid follicular neoplasms showed a significantly greater area, perimeter, and volume of TCN as compared with diploid tumors, regardless of their histologic diagnosis. Follicular adenomas and follicular carcinomas expressed a similar staining pattern for the tested immunoreagents, with a few cases coexpressing vimentin and keratin. From our results, a differential diagnosis of follicular neoplasms cannot be performed on the basis of cytologic aspirates exclusively. Infiltration of capsula or vessels remains the only safe indicator of malignancy in the absence of metastases. The lack of cytologic differences suggests that some follicular adenomas are preinvasive carcinomas, not yet showing infiltrative growth at the time of resection.

Effect of anti-CD11b (αM-MAC-1) and anti-CD54 (ICAM-1) monoclonal antibodies on indomethacin induced chronic ileitis in rats

Abstract Leukocyte emigration from blood to sites of inflammation involves sequential interaction of specific adhesion molecules expressed by both leukocytes and endothelial cells. The central steps in leukocyte-endothelial adhesive interactions are leukocyte rolling, sticking, and transmigration. This study investigated the effect of monoclonal antibodies against CD54 (ICAM-1) and CD11b (αM-chain of MAC-1) on intestinal inflammation. Anti-CD54 and anti-CD11b were tested in rats with indomethacin-induced chronic ileitis. Macroscopic changes were assessed by a modified version of the Wallace et al. score. Leukocyte rolling and sticking were investigated by intravital microscopy. Results show that indomethacin administration led to a chronic inflammatory response characterized by significant increase (P<0.05) in rolling (from 5.41±2.87 to 32.41±15.03 100 µm–1 s–1) and sticking (from 0.16±0.18 to 9.11±5.3 100 µm–1 s–1) leukocytes. After antibody treatment only the anti-CD11b group showed significant (P<0.05) reduction in rolling (from 32.41±15.03 to 6.6±2.7 100 µm–1 s–1) and sticking (from 9.11±5.3 to 0.07±0.09 100 µm–1 s–1) leukocytes. This was also the case for macroscopic changes. Indomethacin led to a rise in the Wallace score from 0 to 4.29±0.76 points (P<0.05) and anti-CD11b to a reduction from 4.29±0.76 to 1.29±1.11 points (P<0.05). Anti-CD54 and combined anti-CD11b/CD54 administration was not followed by significant changes. Therefore we suggest that leukocyte-based CD11b but not endothelial-based CD54 contributes most to leukocyte adhesion in the setting of indomethacin-induced ileitis in rats.

Regulation of T Cell Reactivities by Intestinal Mucosa

The large surface area of intestinal mucosa is under constant exposure to pathogens and dietary antigens. The intestinal mucosal immune system together with non-specific barriers like digestive proteases, intestinal mobility, the commensal microflora and mucous coat are believed to provide protection for the host. Mucosal plasma cells produce large amounts of IgA which can traverse the mucosal membrane and prevent the entry of foreign antigens. IgA production needs help by T lymphocytes. Therefore, T cell activation is a prerequisite for IgA production.