In situ generation of nitric oxide by myenteric neurons but not by mononuclear cells of the human colon.

1. Production of nitric oxide (NO) is implicated in the pathogenesis of inflammatory bowel disease. However, the cells responsible for the production of NO in situ in the human colon remain unknown. 2. Surgical samples from 12 patients with ulcerative colitis, eight patients with Crohn's disease and 10 controls were studied. Possible generation of NO was visualized by reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity in human colon. Immunohistological staining for various NO synthase (NOS) isoforms (endothelial, neuronal and inducible), nitrotyrosine and interleukin-2 was also performed. 3. Reduced NADPH diaphorase activity was not found in lamina propria mononuclear cells, but was found in colonic epithelium, endothelium and myenteric neurons and their processes. 4. The NADPH-diaphorase activity positive processes were significantly less common in colon from patients with Crohn's disease compared with control colon. 5. Endothelial NOS was constitutively expressed on colonic endothelium. 6. Neuronal NOS was constitutively expressed on myenteric neurons. 7. Expression of inducible NOS (iNOS) was increased in the epithelium and endothelium of the colon of patients with ulcerative colitis. 8. No correlation was found between expression of iNOS and NADPH diaphorase activity. 9. Nitrotyrosine was expressed by lamina propria leucocytes, but not by epithelium. 10. Interleukin-2 was expressed on both leucocytes and myenteric neurons. 11. Colonic epithelium, endothelium and myenteric neurons synthesize NO. Myenteric neurons were principally responsible for NO production and NO may act as a neurotransmitter in the enteric nervous system.

Endoscopic resection of small rectal carcinoid tumours using an aspiration method with a transparent overcap.

Rectal carcinoid tumours are often small and rarely metastatic. Endoscopic resection may, therefore, be the best treatment for small tumours. We diagnosed rectal carcinoid tumours in four women and three men (mean age 53 years; range, 43 - 63) between 1994 and 1999. Tumour depth was evaluated using a high-frequency ultrasonographic probe in five of the seven patients. All tumours were resected by endoscopic mucosal resection using an aspiration method with a transparent overcap. Histologically, all tumours were located within the submucosal layer. Tumour cells were found at the resected margin of the lateral side in one patient, and at the bottom of the margin in another. Both patients were followed up with frequent endoscopy, and no local recurrences have been detected at 1-year or 4-year follow-ups. Ultrasonographic examination before resection is useful to estimate tumour depth. Endoscopic resection by an aspiration method with a transparent overcap is safe and effective for the treatment of small rectal carcinoid tumours.

Prediction of short-term outcome for patients with active ulcerative colitis.

We report results of a retrospective chart review to evaluate factors predicting short-term outcome of patients with ulcerative colitis treated by corticosteroids. Between January 1992 and December 1997, we treated 71 patients with ulcerative colitis (44 with severe and 27 with moderately severe disease). Forty-nine patients were treated by conventional prednisolone therapy and 22 patients by steroid pulse therapy. There were no differences in clinical or endoscopic improvement between the two treatments. Clinical examination showed that 41 patients entered remission, 17 patients improved, and 13 patients did not respond. Endoscopically, 26 patients entered remission, 30 patients improved, and 15 patients did not respond. Extent of disease, type of disease (first attack, relapsing, or chronic active type), and endoscopic findings were factors useful in predicting short-term outcome of medical treatment.

Decreased anti-Saccharomyces cerevisiae antibody titer by mesalazine in patients with Crohn's disease.

BACKGROUND: Saccharomyces cerevisiae may contribute to the pathophysiology of Crohn's disease. We determined serum anti-Saccharomyces cerevisiae antibody (ASCA) levels in patients with inflammatory bowel disease. METHODS AND RESULTS: Immunoglobulin G (IgG) ASCA was measured by using an ELISA in serum samples from 19 patients with ulcerative colitis, 18 patients with Crohn's disease and 7 healthy controls. The ASCA level was significantly higher in patients with ulcerative colitis and patients with Crohn's disease than in controls, and was significantly higher in patients with Crohn's disease compared with patients with ulcerative colitis. Age, gender, disease activity, extent of disease and small bowel involvement each did not affect ASCA levels. The use of elemental or polymeric diet therapy for Crohn's disease and administration of corticosteroids to patients with inflammatory bowel disease also did not affect ASCA levels. The ASCA titer was significantly lower in patients with Crohn's disease taking mesalazine than in those not taking it, although, serum IgG levels did not differ between these two groups, which might imply a suppression of IgG production by mesalazine at the intestinal level. CONCLUSIONS: The finding of increased serum ASCA titers in patients with inflammatory bowel disease suggests that Saccharomyces cerevisiae may play a role in the pathophysiology of this condition.

[Differential diagnosis of ulcerative colitis].

Important points during differential diagnosis of ulcerative colitis from other inflammatory disorders are endoscopic examination and microbial studies of stools. In acute phase of enterocolitis in which waterly diarrhea with bloody stool and abdominal pain appeared, infectious enterocolitis by Shigella, Salmonella, Campylobacter and Yersinia, which sometimes causes mucosal edema, hyperemia, erosions and ulceration should be distinguished carefully. Microbial studies of stool would bring helpful information in such situation. In chronic phase of inflammatory diseases of bowel, they often showed chronic diarrhea associated with mucobloody stools and abdominal pain. They often revealed mucosal inflammation mimicking ulcerative colitis during endoscopic evaluation. Among them, most important diseases are amebic colitis, ischemic colitis, radiation colitis and antibiotics associated hemorrhagic colitis.

Type 1 T-helper cell predominance in granulomas of Crohn's disease.

OBJECTIVE: The pathogenesis of Crohn's disease (CD) is thought to be associated with production of several cytokines, especially type-1 cytokines. To elucidate the in situ cytokine profiles in CD, cytokine-containing cells were localized by immunohistochemistry, with special attention to noncaseating granulomas. The results were compared with those from studies of ulcerative colitis (UC). METHODS: We adopted the biotin-streptavidin-peroxidase method on frozen sections obtained at surgery from patients with CD or UC, and we immunohistochemically examined the expression of several cytokines (interferon-gamma, interleukin-2, -4, -10, and -12). RESULTS: In normal colonic tissue, expression of these cytokines was rare except for interleukin-4. In actively inflamed areas of CD, increased expression of all cytokines by mononuclear cells was observed. In contrast, granulomas in CD involved interferon-gamma+ lymphocytes and interleukin-12+ macrophage-lineage cells (epithelioid cells and multinucleated giant cells) but few interleukin-4+ or -10+ cells. Actively inflamed areas of UC also showed an increase in the number of cytokine-containing cells; however, quantitative analysis revealed that there was more expression of interferon-gamma and interleukin-12, and less of interleukin-10, in CD than in UC, indicating the presence of more type 1 T-helper cells in CD tissue than in UC. CONCLUSIONS: The findings of the present study suggest that granulomas of CD are coupled with type 1 T-helper responses; these responses may contribute to the pathogenesis of this disease.

Down-regulation by bucillamine of lamina propria leucocytes in the rat colitis model.

1. The efficacy of bucillamine has been reported in various inflammatory models. 2. In the present study, the effects of 2 weeks administration of bucillamine on hapten-induced experimental rat colitis were analysed. The gross morphological damage score was evaluated and the expression of CD4, CD8, CD11a, CD11b, CD25, CD54 and class II major histocompatibility complex (MHC) antigen was investigated by immunohistochemical methods. 3. Mean bodyweight was significantly increased in colitic rats given 750 micrograms bucillamine compared with colitic rats given vehicle (distilled water). Gross morphological damage was significantly less in colitic rats given 250 or 750 micrograms bucillamine compared with rats given vehicle. 4. Immunohistological studies revealed that CD4, CD11a, CD11b, CD54 and class II MHC antigen expression of infiltrating leucocytes was significantly lower in rat colonic mucosa treated with bucillamine. 5. From these findings, it appears that bucillamine may act through the down-regulation of the activation of lamina propria leucocytes in hapten-induced rat colitis.

Ulcerative colitis with skip lesions at the mouth of the appendix: a clinical study.

OBJECTIVE: We examined the clinical characteristics of ulcerative colitis patients who demonstrated endoscopically discontinuous lesions at the mouth of the appendix. METHODS: Of patients with initial or recurrent active ulcerative colitis who underwent total colonoscopy during the past 3 yr at Osaka City General Hospital, we selected those who had skip lesions in the mouth of the appendix before treatment, and examined their gender, age, disease type, sites of lesions, inflammatory reaction, severity of disease, effects of treatment, and posttreatment course. RESULTS: Discontinuous lesions at the mouth of the appendix were found in 10 patients, who had the following common clinical features: the major lesion was usually present in the lower part of the large bowel including the rectum, many of the patients had suffered an initial attack only, all patients had mild disease, and many of the patients responded quite satisfactorily to treatment with salicylazosulfapyridine. CONCLUSION: Numerous patients with ulcerative colitis with discontinuous lesions at the mouth of the appendix were observed and their clinical characteristics were examined. Determination of the clinical significance of skip lesions in the appendix will contribute to elucidation of the pathogenesis of ulcerative colitis.

Separation of disaccharides by affinity capillary electrophoresis in lectin-containing electrophoretic solutions.

Separation of the 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of simple disaccharides (maltose, cellobiose, gentiobiose, lactose, and melibiose) by affinity capillary electrophoresis was investigated using lectin-containing neutral phosphate buffers, filled in a linear polyacrylamide-coated capillary. When Lens culinaris agglutinin (LCA) was added, the derivatives of glucobioses were retarded with varying magnitudes depending on the amount of LCA and were well separated from each other and from galactosyl glucose under optimized conditions. Addition of Ricinus communis 60 kDa agglutinin (RCA60) to the phosphate buffer gave a different migration profile, in which the derivatives of galactosyl glucoses were more retarded than those of glucobioses. However, addition of either lectin did not accomplish complete separation of the derivatives of all these disaccharides even under optimum conditions. The addition of two kinds of lectins in appropriate proportions improved separation. Thus, the binary system composed of LCA and RCA60, as well as LCA and soybean agglutinin from Glycine max (SBA), gave better separation of these derivatives, giving peak tops for all derivatives.

Functional diversity of infiltrating macrophages in inflamed human colonic mucosa ulcerative colitis.

1. Phenotypical heterogeneity of macrophages infiltrating the colonic mucosa of patients with ulcerative colitis has been shown in many reports. The functional diversity of macrophages in inflamed colonic mucosa remains unclear. 2. Intestinal macrophages have been characterized by immunohistochemical staining and their ability to generate free oxygen radicals in inflamed and non-inflamed mucosa. 3. No correlation was found between RFD1 (activated dendritic cells), RFD7 (tissue macrophages) or RFD9 (epithelioid cells, giant cells in association with granuloma) positivity and either CD68 or human histocompatibility complex class II antigen (HLA-DR) positivity. The proportions of RFD7- and RFD9-positive cells were significantly increased in inflamed colonic mucosa. Nitroblue tetrazolium-reducing mononuclear cells were CD68 or RFD7 positive but they were rarely positive for HLA-DR and RFD1. 4. These results suggest that nitroblue tetrazolium-reducing macrophages are (scavenger) macrophages.

Platelet ionized magnesium, cyclic AMP, and cyclic GMP levels in migraine and tension-type headache.

Decreased serum and intracellular levels of magnesium have been reported in patients with migraine. It has been suggested that magnesium may play an important role in the attacks and pathogenesis of headaches. We measured ionized magnesium, cyclic AMP (adenosine monophosphate), and cyclic GMP (guanosine monophosphate) in platelets of patients with migraine, in patients with tension-type headache, and in healthy controls. The platelet level of ionized magnesium from patients with tension-type headache was significantly lower than the levels from the other two groups. The platelet level of cyclic AMP from patients with migraine was higher than those from the other groups. We found no significant differences in the platelet cyclic GMP levels among the three groups. It is suggested that reduced platelet ionized magnesium in patients with tension-type headache is related to abnormal platelet function, and that increased platelet cyclic AMP in patients with migraine is related to alteration of neurotransmitters in the platelet.

Prolyl aminopeptidase from Serratia marcescens: cloning of the enzyme gene and crystallization of the expressed enzyme.

We cloned and sequenced the Serratia marcescens prolyl aminopeptidase (SPAP) gene. Nucleotide sequence analysis revealed an open reading frame of 951 bp, encoding a protein of 317 amino acids with a predicted molecular weight of 36,083. The expressed enzyme was purified about 90-fold on columns of Toyopearl HW65C and DEAE-Toyopearl, with an activity recovery of 30%. The apparent molecular weight of the purified enzyme was 36,000 and 38,000 as estimated by SDS-PAGE and gel filtration, respectively. The enzyme was not inhibited by diisopropyl phosphofluoridate (DFP) or phenylmethylsulfonyl fluoride (PMSF), but was markedly inhibited by 3,4-dichloroisocoumarin (DCIC). Crystals of the enzyme were grown by the hanging drop vapor diffusion method using PEG6000 as a precipitant at pH 6.5. The crystals are tetragonal with cell dimensions a= b =65.6 A, and c=169.8 A, a space group P4(1)2(1)2 or P4(3)2(1)2, and probably contain one monomer in the asymmetric unit. They diffract to at least 2.22 A resolution.

Expression of costimulatory molecules B7-1 and B7-2 in macrophages and granulomas of Crohn's disease: demonstration of cell-to-cell contact with T lymphocytes.

The pathogenesis of Crohn's disease, an intractable inflammatory disease, involves impaired and/or excessive activation of mucosal macrophages and T lymphocytes. B7-1 (CD80) and B7-2 (CD86) molecules are costimulatory molecules that are indispensable to T-cell activation by antigen-presenting cells. To elucidate the roles and characteristics of these antigen-presenting cells in Crohn's disease, in situ localization of B7-1 and B7-2 (in relation to the distribution of T cells) was clarified by light and electron microscopic immunohistochemistry. The results were compared with those from a study of ulcerative colitis. Normal colonic tissue expressed B7-1 or B7-2 only sporadically. In active Crohn's disease, however, an increase in the number of B7-1/B7-2+ cells correlated with an increase in expression of HLA-DR and intercellular adhesion molecule-1. Most B7-1/B7-2+ cells were identified as noncaseating granulomas or as macrophages, which tended to form an aggregate especially in ulcer bases. In active ulcerative colitis, the increase of B7-1/B7-2+ cells was not as prominent as that in Crohn's disease. Double immunohistochemistry revealed a close cellular distribution between noncaseating granulomas and T cells. Immunoelectron microscopy confirmed the expression of B7-1/B7-2 along the plasma membranes of cytoplasmic processes of granuloma cells, where lymphocytes were closely attached. The present study suggested that granuloma formation in Crohn's disease is coupled with antigen presentation via a B7-1/B7-2-CD28 pathway, which may contribute to the pathogenesis of the disease.

Effect of exogenous growth hormone on in vitro proliferation of thymic lymphocytes from the hypophysectomized rats.

Thymus weight and total thymic lymphocyte (TL) number decreased markedly after hypophysectomy (HX). Serum level of insulin-like growth factor-I (IGF-I), one of mediators brought about by growth hormone (GH), reduced considerably after the operation. Exogenous GH stimulation enhanced significantly DNA synthetic activity of TLs from HX group only at lower cell concentrations, less than 5 x 10(5) cell/ml. However, there was not a significant difference in the percentage increase of the TL-DNA synthetic activity with exogenous GH between HX and sham-operated (SO) groups. Since GH-responding TLs appeared to be mature cells and to exist in thymus medulla, the results suggest that rate of the responding cells in the thymus medulla of HX animals is similar to that of SO ones. Furthermore, there was not a significant difference in the percentages of rosette-forming cells and non-rosette-forming cells, reflecting nearly the maturation steps of TLs, between groups. It is difficult to explain such an aspect in spite of severe reduction of TLs after hypophysectomy, since the proliferative-responding rate of TLS to exogenous GH stimulation and the constitutive ratio of each maturation step of TLs in the HX group were almost same as those of SO group. The contradictory data found in thymus after hypophysectomy are discussed from the point of view of the compensatory effects of growth hormone-releasing hormone on TLs from the operated animals.

Effect of bucillamine on the rat trinitrobenzene sulfonic acid induced model of colitis.

Salicylazosulphapyridine and corticosteroids are the only remedies for inflammatory bowel disease currently in clinical use. They do not, however, necessarily bring about satisfactory therapeutic benefits, so that new agents are needed. In this study, we evaluated the effect of bucillamine, a new antirheumatic agent, in experimental rat colitis induced by trinitrobenzene sulfonic acid enema. Wistar rats were given vehicle alone (n = 16) or treated with 50 (n = 20) or 150 (n = 20) mg/kg of bucillamine daily for three weeks after induction of colitis. Conventional histological sections of the colon stained by haematoxylin and eosin were prepared and observed under a light microscope to determine colonic damage scores. The determinations were significantly lower in the group treated with 50 mg/kg of bucillamine and tended to be lower in the group treated with 150 mg/kg of bucillamine than in the untreated group, which implied that the experimentally induced colonic inflammatory changes and ulcerations were alleviated by bucillamine. Blood tests showed no abnormal values at the end of the treatment. The present observations suggest that bucillamine should be developed further as a possible therapy for inflammatory bowel disease.

Distribution and anti-inflammatory effect of mesalazine on carrageenan-induced colitis in the rabbit.

1. A controlled-release preparation of mesalazine microgranules (PentasaR; Ferring AS, Vanlose, Denmark) releases the active ingredient over a wide area from the small intestine to the rectum and is consequently expected to bring about therapeutic benefits to patients with ulcerative colitis and Crohn's disease. 2. Mesalazine microgranules (50 or 150 mg/kg per day) were administered orally to each rabbit with carrageenan-induced colitis for six weeks. Its inhibitory effect on colonic mucosal damage was assessed in terms of the microscopic damage scores, leukotriene B4 concentrations and concentrations of mesalazine derivatives. 3. At the end of the experiment, the mesalazine 150 mg group had gained a significantly greater bodyweight than the control group. Microscopic damage was significantly lower in the 150 mg group than in the untreated control group. Tissue concentrations of 5-aminosalicylic acid and acetyl-5-amino-salicylic acid in the small and large intestine were higher in the 150 mg group than in the 50 mg group. Mucosal leukotriene B4 levels tended to be lower in rabbits receiving the larger dose of mesalazine. 4. The present study indicates that slow release 5-amino-salicylic acid at the larger dose reaches the large bowel in sufficiently high concentrations following oral administration and significantly reduces carrageenan-induced colitis in the rabbit.

Prolyl aminopeptidase is also present in Enterobacteriaceae: cloning and sequencing of the Hafnia alvei enzyme-gene and characterization of the expressed enzyme.

The Hafnia alvei prolyl aminopeptidase gene (hpap) was cloned and sequenced, the expressed enzyme (HPAP) was purified to homogeneity and thoroughly characterized. An open reading frame of 1,281 bp was found to code for the enzyme, resulting in a protein of 427 amino acids with a molecular weight of 48,577. HPAP resembles the Aeromonas sobria enzyme, having 45% identity and the same distinctive properties with respect to size and substrate specificities. Both enzyme show similar chromatographic behavior, and HPAP could be purified following the procedure previously described for the Aeromonas enzyme. HPAP was found to be resistant to diisopropylphosphofluoridate as are most of the prolyl aminopeptidases hitherto described. In spite of this similarity, no inhibition by 1 mM p-chloromercuribenzoate solution could be detected. Significant inhibition was, however, observed when the enzyme was incubated with 3,4-dichloroisocoumarin. This study confirms the presence of two types of prolyl aminopeptidases, of which the Hafnia and Aeromonas enzymes constitute one group and the Bacillus, Neisseria, and Lactobacillus enzymes the other, and describes the cloning of the first prolyl aminopeptidase gene from an Enterobacteriaceae.