In previous histochemical studies the distribution of the two Ca²⁺-binding proteins MRP8 and MRP 14 as well as their heterocomplex MRP8/14 has been demonstrated in different inflammatory diseases. Monoclonal antibodies against MRP8 and MRP14 and their heterodimer MRP8/14 (27E10 epitope) were used to investigate immunohistochemically the distribution of these proteins in routinely processed small and large bowel tissues from patients with Crohn’s disease (CD). Furthermore, we used a sandwich immunoassay to measure serum concentrations of MRPs in 62 patients with CD and in 20 healthy controls. Disease activities of CD patients were simultaneously assessed by the Crohn’s disease activity index (CDAI) and the severity activity index of Goebell (SAI). In our immunohistochemical study, MRP8, MRP 14 and the heterocomplex MRP8/14 were demonstrated in the majority of granulocytes and macrophages in active CD. Additionally, a strong complex MRP8/14 immunoreactivity was present in epithelial cells adjacent to ulcer-ative and fissuring lesions in the bowel. Serum MRP 8/14 concentrations were significantly (p < 0.0001) increased in patients with active CD (CDAI > 150, SAI > 120). No correlations were found for level of MRP 14 and MRP8 alone, respectively. The follow-up of individual patients with initially active CD showed a further increase in MRP8/14 levels during acute attacks of the inflammatory process. We suggest that our assay for MRP8/14 discriminates well between active and inactive CD and may have considerable potential in the analysis of clinical disease activity in CD patients. Our morphological results confirm the finding of increased MRP8/14 serum levels in patients with active CD.

SUMMARY IBD is characterized by increased serum concentrations of different cytokines. IL‐10 inhibits the production of proinflammatory cytokines such as IL‐1, tumour necrosis factor‐alpha (TNF‐a), interferon‐gamma (IFN‐γ) and IL‐6 through inhibitory action on Th1 cells and macrophages, and it is thought to be a suppressor type cytokine. In the present study we determined serum concentrations of IL‐10 in patients with ulcerative colitis (UC) and Crohn's disease (CD). We measured human IL‐10 by our own newly established ELISA system using PharMingen antibodies. Serum antibodies were assessed in 44 patients with UC, 40 patients with CD, and in 30 healthy controls. Human IL‐10 serum levels were significantly increased in patients with active UC (144 ± 34 pg/ml (mean ± s.e.m.), P <0.001) and in active CD (132 ± 32 pg/ml, P <0.001) compared with healthy controls (44.9.5pg/ml). Only patients with active CD and active UC presented with significantly increased IL‐10 serum levels, while patients with inactive disease did not show any significant increase. There was no statistically significant difference between IL‐10 serum levels in patients with CD or UC. Compared with clinical disease activity indices there was a significant correlation between IL‐10 serum concentration and CDAI in patients with CD (r= 0.45, P <0.01) and CAI in VC patients (r= 0.39, P <0.05). Comparing IL‐10 serum levels with serum concentrations of other proinflammatory cytokines there was a significant correlation to scrum levels of sIL‐2R (r= 0.417, P <0.05) and IL‐6 (r= 0.387, P <0.05) in patients with CD. Serum cytokine levels in patients with UC did not show any significant correlation to IL‐10 serum concentration. IL‐10 is elevated in serum of patients with active CD and UC. suggesting that IL‐10 acts as a naturally occurring damper in the acute inflammatory process of IBD.

M cells are known as specialized epithelial cells of the follicle‐associated epithelium of the gastrointestinal tract. As M cells have a high capacity for transcytosis of a wide range of microorganisms and macromolecules, they are believed to act as an antigen sampling system. The primary physiological role of M cells seems to be the rapid uptake and presentation of particular antigens and microorganisms to the immune cells of the lymphoid follicle to induce an effective immune response. In contrast to absorptive enterocytes, M cells do not exert direct defense mechanisms to antigens and pathogens in the gut lumen. Therefore, they provide functional openings of the epithelial barrier. Although M cells represent a weak point of the epithelial barrier, even under noninflamed conditions, there seems to be a balance between antigen uptake and immunological response. The low number of M cells in the gastrointestinal tract and the direct contact to immune cells in the lamina propria usually prevent the occurrence of mucosal inflammation. During chronic intestinal inflammation we observe an increase of M cell number and apoptosis selectively in M cells. M cell damage seems to be responsible for the increase of the uptake of microorganisms that is observed during intestinal inflammation. Under inflammatory conditions in the intestine, the maintenance of the epithelial barrier is broken and M cells seem to play a major role during this process.

Several studies have demonstrated that intestinal epithelial cells play a major role in the initiation and perpetuation of intestinal inflammation by secreting proinflammatory cytokines and chemokines. MCP-1 is suggested to be a chemokine that plays a major part during intestinal inflammation in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL-4, IL-10 and IL-13 have been described to exert anti-inflammatory properties on various cell types. The aim of our study was to determine the effect of Th2 cytokines on the production of MCP-1 by activated intestinal epithelial cells. We examined Caco-2 cells as well as intestinal epithelial cells which were isolated from surgical specimens. Production of the chemokine MCP-1 was determined under stimulated and non-stimulated conditions. IL-4, IL-10 and IL-13 were added to stimulated epithelial cells under various culture conditions. Supernatants were analysed for cytokine concentrations using ELISAs. Under stimulation with physiological agents like IL-1beta or tumour necrosis factor-alpha (TNF-alpha), we observed markedly increased concentrations of MCP-1 in supernatants of Caco-2 cells and intestinal epithelial cells. IL-4, IL-10 and IL-13 all had the capacity to down-regulate the production of MCP-1 in Caco-2 cells as well as in freshly isolated epithelial cells. Caco-2 cells which were primed with Th2 cytokines 24 h before stimulation were subsequently decreased in their ability to be stimulated by IL-1beta or TNF-alpha for MCP-1 production. As MCP-1 has been shown to play a major role during intestinal inflammation, the in vitro suppression of MCP-1 in enterocytes suggests the in vivo use of regulatory cytokines in patients with active IBD.

The gastropathy associated with the ingestion of non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin is a common side effect of this class of drugs, but the precise mechanisms by which they cause mucosal damage have not been fully explained. During continued use of an injurious substance, such as aspirin, the extent of gastric mucosal damage decreases and this phenomenon is named gastric adaptation. To assess the extent of mucosal damage by aspirin and subsequent adaptation the effects of 14 days of continuous, oral administration of aspirin (2 g per day) to eight healthy male volunteers was studied. To estimate the rate of mucosal damage, gastroscopy was performed before (day 0) and at days 3, 7, 14 of aspirin treatment. Gastric microbleeding and gastric mucosal blood flow were measured using laser Doppler flowmeter and mucosal biopsy specimens were taken for the estimation of tissue DNA synthesis and RNA and DNA concentration. In addition, the activation of neutrophils in peripheral blood was assessed by measuring their ability to associate with platelets. Aspirin induced acute damage mainly in gastric corpus, reaching at day 3 about 3.5 on the endoscopic Lanza score but lessened to about 15 at day 14 pointing to the occurrence of gastric adaptation. Mucosal blood flow increased at day 3 by about 50% in the gastric corpus and by 88% in the antrum. The in vitro DNA synthesis and RNA concentration, an index of mucosal growth, were reduced at day 3 but then increased to reach about 150% of initial value at the end of aspirin treatment. The gastric microbleeding rate rose from about 0-38 mi/day at day 0 to about 7-7 mi/day at day 3 but then decreased significantly to virtually normal values at the end of the study. The neutrophil/platelet adherence showed significant increase during aspirin treatment. It is concluded that the treatment with aspirin in humans induces gastric adaptation to this agent, which entails the increase in mucosal blood flow, the rise in neutrophil activation, and the enhancement in mucosal growth.

After intravenous injections, we have found the immunological half-life of purified porcine proinsulin to be more prolonged than purified porcine single-component insulin in both swine (22 and 9 min, respectively) and in baboons (18 and 8 min, respectively). Studies in humans have also indicated a longer half-life of proinsulin than insulin (1). These findings have prompted the present investigation of the in vitro degradation of insulin, proinsulin, and the connecting peptide that links the A and B chains of insulin in the proinsulin molecule, using an isolated liver perfusion system.Methods. Intact livers, weighing 4.5-6.8 g, from male Wistar rats, fasted for 48 hr, were cyclically perfused by the method of Hems et al. ( 2 ) . The perfusion fluid consisted of washed human erythrocytes, 2 g/lOO ml bovine serum albumin (Cohn, Fraction V ) , and Krebs-Henseleit buffer, pH 7.4, with a hemoglobin concentration of approximately 3 g/lOO ml. After complete isolation from the circulation, the livers were perfused in situ by adjusting the hydrostatic pressure of the perfusate to give maximal perfusion rates without liver swelling. This was approximately 2-3 ml/g wet weight of liver. The total perfusion volume was 150 ml at the beginning of the experiment. After a 40-min equilibration period, purified porcine single-component insulin (Lilly ), proinsulin (Lilly ), or con-

Abstract. MRP8, MRP14 and their heterodimer MRP8/14 (27E10 antigen) are myeloic related proteins which have been shown to have a major role in inflammatory and immunological responses. In the present study monospecific antibodies against MRPs were used to investigate immunohistochemically the distribution of these proteins in routinely processed bowel tissues from 23 patients with ulcerative colitis (UC). MRP8, MRP14 and their heterocomplex MRP8/l4 were demonstrated in the majority of granulocytes and macrophages in tissues of patients with active UC. Furthermore by employing the ELISA technique we measured MRP8/14 serum levels in 62 patients with UC and the results were compared with those for healthy controls. Disease activities were determined by established clinical activity indices. Serum MRP8/14 concentrations were significantly ( P < 0.0001) increased in patients with active ulcerative colitis. No enhancement of serum levels were found for MRP14 and MRP8 alone, respectively. The follow-up of individual patients with initially active disease showed a decrease of MRP8/14 serum levels in parallel with clinical improvement following the start of therapy. It is thus concluded that MRP8/14 accurately reflects the degree of disease activity in UC. Further, possible biological function of MRPs seems to be associated with the heterodimeric form (27E10 antigen) rather than with individual proteins. Our morphological results confirm the finding of enhanced MRP8/14 serum levels in patients with active UC.

Renal brush-border membrane vesicles isolated from rats kept for 6-8 weeks on a low-phosphate diet (0.15% of dry matter) showed a markedly faster Na(+)-dependent phosphate uptake than did membrane vesicles isolated from animals kept on a high-phosphate diet (2% of dry matter). Phosphate-uptake rate by brush-border membrane vesicles isolated from animals on a low-phosphate diet remained significantly increased after acute parathyroidectomy. Dietary adaptation was also observed in animals that had been parathyroidectomized before exposure to the different diets. In animals on the low-phosphate diet parathyrin administration inhibited phosphate uptake by brush-border vesicles only if the animals were repleted with P(i) (5ml of 20mm-NaH(2)PO(4)) 1h before being killed. After acute phosphate loading and parathyrin administration the difference in the transport rate between the two dietary groups remained statistically significant. The results suggest that the adaptation of proximal-tubule phosphate transport to dietary intake of phosphate is reflected in the Na(+)/phosphate co-transport system located in the luminal membrane of the proximal-tubule cell. Since the dietary effects on phosphate transport by brush-border membranes are only partially reversed by acute changes in parathyrin concentration and are also observed in chronically parathyroidectomized animals, the adaptation of the Na(+)/phosphate co-transport system to dietary phosphate intake seems to involve an additional mechanism independent of parathyrin.