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Background: Recently, we demonstrated in vivo effects of acutely induced hyperglycemia, diabetes and mannitol infusions on rat mesenteric microcirculation concerning leukocyte-endothelial-cell interaction (Schäffler et al. EJCI 28: 886-893, 1998)., Design: In this study we have investigated the possible involvement of the protein kinase C (PKC) and p38 MAP kinase cascade as signal transducing elements during hyperglycemic and osmotic stress in an in vivo rat model., Results: Acutely induced hyperglycemia resulted in a significant increase in leukocyte adhesion. This effect could be mimicked by mannitol. Both PKC and p38 MAP kinase were involved in the effects mediated by glucose and mannitol. Acutely induced hyperglycemia resulted in a significant increase in leukocyte emigration. This effect could be imitated by mannitol. However, PKC and p38 MAP kinase were only involved under osmotic stimulation. The hyperglycemia-induced reduction in leukocyte rolling velocity seemed to be a glucose-specific effect, since mannitol did not influence leukocyte rolling velocity. This glucose effect on leukocyte rolling velocity was mediated by an activation of the PKC/p38 MAP kinase cascade. Both hyperglycemia and osmotic stimuli alone were able to reduce venular shear rate without recruitment of the p38 MAP kinase cascade. The observed reduction of shear rate seems to be mediated only by the osmotic effects of glucose via activation of the PKC system., Conclusion: The observed effects of glucose on adhesion, emigration and shear rate are due to osmotic effects. The PKC/MAP kinase cascade is involved as a signal transducing component. The reduction of leukocyte rolling velocity is a glucose-specific effect, mediated by the activation of both the PKC and the p38 MAP kinase cascade. Venular shear rate and leukocyte emigration can be influenced by glucose and mannitol due to different regulation mechanisms. It is concluded, that isoform-specific inhibitors of PKC and specific MAP kinase inhibitors represent a potential drug target for preventing microvascular dysfunction in diabetes.

Background: The recently described A-->G (-3826) point mutation within the distal region of the UCP-1 promoter is possibly involved in the development of obesity, diabetes and related metabolic disorders. It was the aim of this study to examine the allelic frequency and the prevalence of the three UCP-1 genotypes in a broad caucasian cohort and to investigate the significance of this polymorphism for obesity and diabetes., Methods: 1020 subjects were randomly chosen from 6450 participants in the Diabetomobile Study. The UCP-1 genotype was determined by genomic PCR and Bcl-I-RFLP analysis in 1020 subjects and tested for association with a variety of metabolic parameters. In addition, the influence of this mutation on adipocyte nuclear factor binding was investigated by electrophoretic mobility shift assays (EMSA)., Results: The genotype frequencies in 1020 subjects were: AA genotype, 57.0%; AG genotype, 35.4%; GG genotype, 7.6%; with allelic frequencies of 0.75 for allele A and 0.25 for allele G. No significant differences between the genotypes and age, gender, BMI, leptin, glucose, fasting insulin, C-peptide, HbA1c, diabetes manifestation, total cholesterol, and HDL cholesterol were found. Analysis of the Trp64Arg polymorphism of the beta3-adrenergic receptor in a subgroup of 343 subjects revealed no additive effect to the UCP-1 polymorphism. An yet unknown adipocyte-specific factor of nuclear extracts from 3T3-L1 adipocytes during differentiation is able to bind specifically to the distal UCP-1 promoter region and this binding ability can not be abolished by the mutation., Conclusions: We determined the genotype and allelic frequency of the UCP-1 promoter polymorphism in the largest known population-based study. The results from genotyping demonstrate clearly that this polymorphism does not play a major role in the pathogenesis obesity and diabetes. A yet unknown adipocyte derived and differentiation-dependent regulated transcription factor is able to bind to the distal UCP-1 promoter surrounding -3826 bp. This binding is not affected by presence of the mutation.

Background: Growing evidence indicates that the immune system and the hypothalamic-pituitary-adrenal system are linked by several mechanisms, for example intracellular glucocorticoid receptors (hGR). Glucocorticoids are the standard treatment of acute attacks of inflammatory bowel disease (IBD). Binding of glucocorticoids to hGR down-regulates the transcription of inflammatory genes that can propagate IBD., Patients and Methods: IBD patients were either treated with 5-60 mg of prednisolone for more than 1 week or were without glucocorticoid treatment for more than 4 weeks. hGR levels were determined from isolated cytosol of peripheral blood mononuclear cells (PBMCs) or mucosal biopsies using a radioassay with [3H]-dexamethasone. Interleukin (IL) 6 levels were determined by enzyme-linked immunosorbent assay (ELISA)., Results: The systemic (PBMC) hGR levels of corticosteroid-treated IBD patients were significantly lower than those of control subjects (59.6 +/- 57.1 dpm mg-1 cytosol protein vs. 227.0 +/- 90.8 dpm mg-1 cytosol protein, P = 0.007) and IBD patients not receiving glucocorticoid treatment (179.7 +/- 171.3 dpm mg-1 cytosol protein, P = 0.002). Systemic hGR levels in untreated IBD patients did not differ significantly from those in control subjects. In patients with connective tissue diseases, systemic hGR levels were also found to be decreased in the absence of glucocorticoid treatment. Systemic hGR levels in patients with Crohn's disease (CD) treated with steroids (66.6 +/- 61.0 dpm mg-1 cytosol protein) were not different from those in patients with ulcerative colitis (UC) (56.1 +/- 51.6 dpm mg-1 cytosol protein). In contrast to these findings, mucosal hGR levels were significantly decreased in both steroid-treated (18.0 +/- 15.5) and not steroid-treated (37.8 +/- 30.5) patients compared with control subjects (125.6 +/- 97.1; P = 0.00009 and P = 0.0008 respectively). IL-6 levels in all IBD groups with and without steroids were significantly different from those in control subjects., Conclusion: In IBD there is no difference in systemic hGR levels between not steroid-treated patients and control subjects, in spite of inflammatory activity (IL-6). Mucosal hGR levels were decreased independently of treatment, probably leading to a decreased protection against NF-kappaB action in the intestinal mucosa.

Background: Glutamine (Gln) is a major energy source for the intestinal mucosa. Its depletion results in epithelial atrophy and in bacterial translocation. Clinical substitution of this nonessential amino acid in critically ill persons results in a reduction of epithelial atrophy and in an accelerated recovery. The objective of this study was to assess the effect of Gln on leukocyte-endothelial cell interaction in an indomethacin (Indo)-induced long-lasting ileitis in Sprague-Dawley rats., Methods: Indo (7.5 mg/kg subcutaneously) was injected at time 0 and 24 hours later. Animals were fed with standard rat chow (ST) for 10 days until 12 hours before intravital microscopy analysis. Gln (3 g/kg body wt) was gavaged twice a day in the morning 4 hours apart (1) for 10 days between Indo administration and the experiment (ST/Gln, therapy), (2) for 14 days before Indo (Gln/ST, prophylaxis), or (3) from 14 days before Indo until the experiment (Gln/Gln, prophylaxis and therapy). Ten mesenteric venules (30 microm diameter) per animal (n = 5 per group) were observed using intravital microscopy, and the following parameters were monitored: number of adherent and emigrated leukocytes, leukocyte rolling velocity, erythrocyte velocity, venular blood flow, and shear rate. Macroscopically visible injury was scored 0 to 5., Results: Ten days after Indo treatment the macroscopic score was 3.5+/-0.4 vs. 0.6+/-0.2 of controls, and leukocyte adherence and emigration were increased (2.2-fold and 3.3-fold vs. control, respectively), whereas leukocyte rolling velocity and venular wall shear rate were reduced (both parameters to 81% of control). Glutamine prophylaxis, therapy, and the combination of both significantly attenuated macroscopic damage and prevented the microcirculatory disturbances to a similar extent. The beneficial effects of glutamine were accompanied by a normalization of fecal pH to control level, which had been lowered by Indo treatment., Conclusions: The long-lasting Indo-induced ileitis was accompanied by macroscopic ulceration and microcirculatory disturbances. Oral therapy and prophylaxis with glutamine reduced macroscopic and microcirculatory inflammatory activity, indicating a special demand for glutamine in this type of inflammation.

Background: Microsatellite instability (MSI) is a new mechanism described in carcinogenesis of several tumours and seems to predispose for cancer in some chronic inflammatory diseases. As thyroiditis is thought to be both the cause and the consequence of thyroid carcinoma, it was the aim of this retrospective study to investigate the epidemiology and the influence of a coexistent thyroiditis on prognosis and clinicopathological parameters in an iodine-deficient area., Methods: The grade of lymphocytic infiltration (LI) of 153 thyroid carcinomas was determined in paraffin-embedded tissues: G0 = no LI, G1 = peritumoral LI, G2 = peritumoral LI with follicle, G3 = diffuse LI. A non-radioactive PCR-based screening method was used for detection of MSI., Results: Twenty-seven (17.7%) out of 153 carcinomas were accompanied by thyroiditis (G1, 16; G2, 5; G3, 6). Ten cases fulfilled the criteria necessary for diagnosing Hashimoto's thyroiditis. Nine out of 10 (90%) Hashimoto's cases and 16 out of 17 (94%) other thyroiditis cases were associated with a significantly (chi2 < 0.01) lower pT stage (pT1, pT2) than cases without thyroiditis. No statistical association was found by comparing multifocality or sclerosing variants with the grade of lymphocytic infiltration. MSI was detected neither in patients with severe inflammation nor in the absence of thyroiditis., Conclusions: MSI does not seem to play a role in the pathogenesis of thyroid carcinoma. Coexistent thyroiditis is associated with a lower pT stage and thus could be an indicator of a better prognosis.