Colonic IgA producing cells and macrophages are reduced in recurrent and non-recurrent Clostridium difficile associated diarrhoea.

BACKGROUND: In Clostridium difficile associated diarrhoea (CDAD), histological changes in the colonic mucosa range from minimal inflammation to pseudomembranous colitis (PMC). The disease also recurs in a considerable proportion of patients. AIM: To investigate mucosal immune system cells in colonic biopsies of patients with CDAD. METHODS: Colonic biopsies were obtained from 12 control patients with diarrhoea, six patients with CDAD and minimal inflammation, and 10 patients with CDAD with pseudomembranous colitis (samples obtained from areas with and without inflammatory exudate). Immunohistochemical studies were performed using antibodies to T cells (CD3), macrophages (CD68), B/plasma cells (CD79alpha), and to IgA, IgM, and IgG. Labelled cells in lamina propria were quantified. RESULTS: In contrast to T cells, there were significant reductions in B/plasma cell and macrophage counts in all biopsies from patients with CDAD compared with controls (p<0.001). Studies using anti-immunoglobulin antibodies showed significant reductions in IgA producing cells in CDAD biopsies (p<0.05), with the greatest reduction in samples from patients with PMC. In contrast, there was a significant increase (p<0.05) in IgG producing cells in CDAD biopsies. Only patients with PMC relapsed. In these patients, B/plasma cell and IgA producing cell counts (in biopsies with and without inflammatory exudates) were significantly lower (p<0.01) in mucosal samples from those who subsequently relapsed (five) than those who did not. CONCLUSIONS: A selective reduction in mucosal IgA producing cells and macrophages is associated with colonic disease in C difficile infected patients. Severe reduction in colonic IgA producing cells may predispose to recurrence of CDAD.

Clostridium difficile associated diarrhoea in hospitalised patients: onset in the community and hospital and role of flexible sigmoidoscopy.

OBJECTIVES: Clostridium difficile associated diarrhoea (CDAD) is a hospital acquired infection in which optimal methods for diagnosis and the scale of the problem in the community remain to be determined. In hospitalised patients with CDAD, we aimed to (i) study patients in whom the onset of diarrhoea was in the community and (ii) investigate the role of bedside flexible sigmoidoscopy in diagnosis. METHODS: Patients with CDAD (onset in hospital or community) were studied prospectively. In those with diarrhoea of unknown aetiology, flexible sigmoidoscopy was compared with stool assay for C difficile cytotoxin. RESULTS: Of 136 patients with CDAD (which was associated with antibiotic exposure in 96%), diarrhoea started in the community in 38 (28%; majority in own home) and while an inpatient in 98 (72%). The majority with CDAD onset in the community had been hospitalised over the preceding 12 months (86.8% v 57.1% in the hospital onset group; p<0.001). In 56 patients with pseudomembranous colitis at sigmoidoscopy, the stool C difficile cytotoxin test was negative in 29 (52%) but toxigenic C difficile was isolated from all of nine stool samples cultured. Of patients with pseudomembranous colitis, 30.4% relapsed over the subsequent 57.7(4.2) days. CONCLUSIONS: In a significant proportion of hospitalised patients with CDAD, diarrhoea started in the community. However, the majority of these had been hospital inpatients previously when they may have acquired C difficile, with the subsequent onset of diarrhoea in the community following exposure to antibiotics. Flexible sigmoidoscopy is superior to the stool C difficile cytotoxin test in a subgroup of patients with pseudomembranous colitis. Sigmoidoscopy should therefore be considered in all hospitalised patients with diarrhoea in whom the stool test for C difficile cytotoxin and enteric pathogens is negative.

Written disclosure: a way of coping with traumatic stress.

Verbal debriefing after a traumatic incident may interrupt the natural adaptive processes that help people to deal with such incidents. This article examines the effectiveness of written interventions to help people experiencing distress after trauma experienced at work. As an alternative to critical incident debriefing, the authors recommend a staff education programme in tandem with written disclosure.

Second-harmonic generation in Langmuir Blodgett waveguide overlays on single-mode optical fiber.

Second-harmonic radiation has been obtained from Langmuir-Blodgett films of E-N -octadecyl-4-[2-(4-dibutylaminophenyl)ethenyl]quinolinium octadecylsulfate, deposited as a waveguide overlay upon optical fiber that is single mode at the pump wavelength (lambda=1064 nm) . A quadratic relationship between the pump power and second-harmonic intensity was observed.

Systemic multifocal fibrosclerosis.

We describe a case of hydronephrosis as a result of retroperitoneal fibrosis in a patient who had previous sclerosing lobulitis of the breast. To the best of our knowledge this is the first reported association between these two conditions in the english literature. We presume these conditions are linked and unify them under the general heading of systemic multifocal fibrosclerosis.

Effect of specificity on ligand conformation in acyl-chymotrypsins.

I.r. difference spectroscopy combined with 13C and 18O double-isotope substitution was used to examine the ester acyl carbonyl stretching vibration of hydrocinnamoyl-chymotrypsin. A single acyl carbonyl stretching band was observed at 1731 cm-1. This contrasts with previous i.r. and resonance Raman spectroscopic studies of a number of trans-3-arylacryloyl-chymotrypsins which showed two acyl carbonyl stretching bands in the region of 1700 cm-1, which were proposed to represent productive and non-productive conformations of the acyl-enzyme. The single acyl carbonyl band for hydrocinnamoyl-chymotrypsin suggests only a single conformation, and the comparatively high frequency of this band implies little or no hydrogen-bonding to this carbonyl group. Enzymic hydrogen-bonding to the acyl carbonyl is believed to give bond polarization and thereby catalytic-rate acceleration. Thus, in view of the apparent lack of such hydrogen-bonding in hydrocinnamoyl-chymotrypsin, it should be the case that this acyl-chymotrypsin is less specific than trans-3-arylacryloyl-chymotrypsins, whereas the opposite is true. It is therefore proposed that there may be a productive acyl carbonyl population of lower stretching frequency for hydrocinnamoyl-chymotrypsin, but that this is too small to be discerned because of either a relatively high deacylation rate or an unfavourable conformational equilibrium. The single acyl carbonyl band for hydrocinnamoyl-chymotrypsin is significantly broader than those for trans-3-arylacryloyl-chymotrypsins, indicating that this group is more conformationally mobile and dispersed in the former. This can be correlated with the absence of acyl carbonyl hydrogen-bonding in hydrocinnamoyl-chymotrypsin, and with the much greater flexibility of the saturated hydrocinnamoyl group than unsaturated trans-3-arylacryloyl. This flexibility is presumably the reason why hydrocinnamoyl-chymotrypsin is more specific than trans-3-arylacryloyl-chymotrypsins. Resonance Raman spectroscopy is limited to the non-specific trans-3-arylacryloyl-chymotrypsins because of its chromophoric requirement, whereas i.r. may be used to examine non-chromophoric more specific acyl-enzymes such as hydrocinnamoyl-chymotrypsin. The results presented in this paper suggest that trans-3-arylacryloyl-chymotrypsins are atypical.

A sensitive double-sandwich ELISA for neutrophil elastase. Assay results in unconcentrated bronchoalveolar lavage fluid.

We developed a sensitive double-sandwich ELISA assay for neutrophil elastase (NE) using affinity-purified NE antibody. The assay was capable of detecting NE levels of 0.2 ng/ml and was used to determine NE in bronchoalveolar lavages (BAL) of 12 healthy subjects (6 nonsmokers and 6 smokers) with a mean age of about 27 yr. NE levels in the unconcentrated cell-free supernatant of BAL, subjected to high-speed centrifugation (17,000 x g for 30 min) to sediment subcellular debris, were similar in the smokers who abstained overnight from smoking and in the nonsmokers (24.4 +/- 13.9 versus 23.7 +/- 12.3 ng/mg [SD] albumin). NE levels were significantly higher in lavage fluid not subjected to high-speed centrifugation, reflecting the presence of NE bound to subcellular debris that was sedimented by high-speed centrifugation. Concentration by ultrafiltration through a Millipore CX-10 filter was accompanied by loss of protein with a relatively greater loss of NE than albumin, resulting in lower NE/albumin ratios in concentrated than in unconcentrated lavage. It is therefore recommended that NE levels be determined on unconcentrated BAL after high-speed centrifugation to sediment subcellular debris.

Bronchial leukocyte proteinase inhibitor levels in bronchial washings in asthma patients.

To evaluate whether epithelial damage of airways in asthma could be related to diminished levels of the low molecular weight bronchial leukocyte proteinase inhibitor (BLPI) in airways, we determined BLPI in bronchial washings of 13 asthma patients and 13 healthy subjects, using a sensitive enzyme-linked immunoassay. The patients had asthma due to western red cedar and had bronchial washings done 24 to 48 hours after a mild to moderate asthmatic reaction induced by inhalation challenge. We did not find significant differences in BLPI concentrations in lavage fluid of asthma patients and healthy control subjects. The ratios of BLPI to albumin levels in bronchial washings appeared to be lower among asthmatic patients, but this difference was mainly due to an increase in albumin levels in lavage fluid in asthma. In addition, there were no significant differences in BLPI levels in washings obtained from main and segmental bronchi in both control subjects and asthma patients.

Effect of smoking on functional activity of plasma alpha 1-protease inhibitor.

We determined the levels of alpha 1-protease inhibitor, the plasma trypsin-inhibiting capacity (TIC), and elastase-inhibiting capacity (EIC) in 29 nonsmokers and 30 smokers, who were healthy volunteers matched for age (mean age, 39 +/- 12 years [+/- SD]). The functional activity of plasma alpha 1-protease inhibitor (in micrograms of enzyme inhibited per microgram of alpha 1-protease inhibitor) was slightly but significantly lower in smokers, compared with nonsmokers, both for TIC and EIC (smokers' TIC and EIC were 88.0 +/- 16.2 percent (+/- SD) and 90.4 +/- 17.9 percent of the respective mean values in nonsmokers; p less than 0.05). Among smokers, there was a significant negative correlation (r = -0.37; p less than 0.05) between the average number of cigarettes smoked per day and the functional activity of plasma alpha 1-protease inhibitor; the seven subjects who smoked 40 or more cigarettes per day had significantly lower EIC and TIC than the remaining smokers. In 12 smokers tested before and after a two-hour period of intense smoking of eight cigarettes, there was a statistically significant decrease (p less than 0.05) in EIC one hour after smoking to 93.9 +/- 2.5 percent (+/- SE) of the initial value prior to smoking. It is concluded that there is a slight but significant decrease in the functional activity of plasma alpha 1-protease inhibitor in smokers, both for TIC and EIC.

Methylene blue decreases the functional activity of alpha-1-protease inhibitor in bronchoalveolar lavage.

Methylene blue, used as an external marker for bronchoalveolar lavage (BAL) in a concentration of 1 microgram/ml, reduced the functional activity of BAL alpha 1-protease inhibitor (alpha 1-PI) in 2 healing nonsmokers who each had 2 BAL done, one with and the other without methylene blue. Inactivation of alpha 1-PI by methylene blue was confirmed by in vitro experiments on diluted serum and concentrated BAL incubated with methylene blue. Inactivation of alpha 1-PI is likely due to its oxidation by methylene blue, as suggested by the in vitro protective effect of dithiothreitol, a reducing agent. These observations indicate a need for caution when considering the use of methylene blue as an external marker for bronchoalveolar lavage.

Acute effect of smoking on elastaselike esterase activity and immunologic neutrophil elastase levels in bronchoalveolar lavage fluid.

In order to determine the acute effect of smoking on elastase in bronchoalveolar lavage fluid (BAL), we obtained BAL from 30 smokers twice, the first before smoking (after 8 h of abstinence) and the second 2 min to 1 h after the subjects had smoked either 2 or 4 cigarettes. Bronchoalveolar lavage was concentrated 100-fold and was assayed for elastaselike activity against succinyl-trialanyl-p-nitroanilide (SLAPN) as substrate. Elastolytic activity against insoluble 3H-elastin and immunologic neutrophil elastase levels tested with a sensitive enzyme-linked immunosorbent assay were determined in some subjects. No activity against insoluble 3H-elastin was detected using a sensitive assay capable of detecting subnanogram quantities of elastase. Total elastaselike activity in BAL against SLAPN was significantly (p less than 0.02) increased in smokers prior to smoking (47.4 +/- SD 20.2 ng human neutrophil elastase (HNE) equivalents) when compared with BAL from 7 nonsmokers (26.3 +/- SD 13.4 ng HNE); however, there was no significant change in enzyme activity in smokers' BAL after smoking. Assays with EDTA and phenyl-methyl-sulfonyl-fluoride as inhibitors suggested that approximately two thirds of elastaselike activity was due to a metalloprotease and that there was negligible serine protease activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactions of nitrosamines with nucleophiles in acid solution.

The kinetics of denitrosation of N-alkyl (and aryl) N-nitrosoaniline derivatives in ethanolic HCl have been studied. In contrast with the results for the corresponding reactions in water, there is no catalysis by added nucleophiles and the solvent isotope effect is 3.8. Similar results were obtained for reactions in water at high nucleophile concentrations and also for N-methyl-N-nitrosourea in water in the absence of added nucleophiles. All the results are consistent with a change in rate-determining step which can be brought about in three independent ways. This also explains the absence of nucleophilic catalysis for N-nitrosation of amides. Results are presented for the nitrosation of N-methyl urea.