Isolation and purification of N-formylmethionine aminopeptidase from rat intestine.

The intestinal mucosal epithelium is exposed to products of intestinal bacteria including potent inflammatory N-formylmethionyl oligopeptides. An N-formylmethionine aminopeptidase has been purified 2300-fold from rat intestine and was shown to degrade natural fMet oligopeptides from Escherichia coli culture supernatants with loss of bioactivity (release of specific granule constituents from human polymorphonuclear leucocytes) and immuno-reactivity (assessed using a polyclonal anti-fMet-Leu-Phe antiserum). The enzyme which was specific for N-terminal acyl-methionine residues had a native Mr of 340,000 and comprised four sub-units of Mr 82,000. The presence of this enzyme in intestinal mucosa could prevent absorption of intact bioactive fMet peptides produced by commensal bacteria in the gut lumen.

Assessment of neutrophil leukocyte secretory response to fMLP in whole blood in vitro.

A simple, precise method has been developed for assessing neutrophil secretory responses (release of vitamin B12 binding protein from specific granules) to challenge of aliquots of whole blood with the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Dose-response studies performed on blood from normal healthy volunteers showed higher maximal secretory responses in males than females (33.3 +/- SEM 2.2 vs. 27.4 +/- 2.5, P less than .005) a left shift in dose-response curves after feeding compared to fasting (P less than .005), spontaneous up-regulation of responses in blood incubated at 37 degrees C for 1 h, and marked upregulation in response to preincubation with endotoxin. This whole blood challenge method may be used to study neutrophil responses in groups of individuals or patients without the confounding effects of changes in cell responses resulting from cell isolation procedures. The method may also be used as a bioassay for neutrophil-activating factors.

Flow cytometry analysis of the expression of neutrophil FMLP receptors.

The number of cell surface FMLP receptors expressed by peripheral blood neutrophils taken from healthy individuals was quantitated using a direct fluorescence label analysed on the FACScan. Receptor numbers, assayed on cells from heparinised blood prepared at 4 degrees C or 37 degrees C or in the presence of LPS (30 ng/ml) were 12,540 +/- SD 2413, 18,588 +/- 4938 and 45,041 +/- 10,744 respectively. Anticoagulation with EGTA resulted in 25-30% greater basal receptor numbers but had no effect on receptor level after maximal stimulation with LPS. Double labelling with a neutrophil-specific mAb showed that the entire neutrophil population expressed FMLP receptors.

Immunodeficiency, malabsorption and secretory diarrhea. A new syndrome.

Described here is a patient with severe watery diarrhea associated with common variable immunodeficiency. Malabsorption for fat, bile acids, vitamin B12 and xylose was demonstrated, but the patient failed to respond to all the usual therapeutic maneuvers. The diarrhea responded only to high dose steroid therapy. Intestinal perfusion studies showed a hitherto undescribed, presumably acquired, glucose-stimulated water, sodium and chloride secretion in the jejunum and ileum, whereas normal fluid and electrolyte transport occurred from bicarbonate and mannitol solutions. Glucose absorption itself was normal and no hormonal, morphologic or biochemical defect was demonstrated to account for the phenomenon. The patient was also interesting when compared with other patients with common variable immunodeficiency in having normal plasma cells in the intestinal mucosa and an extensive family involvement.

Malabsorption and macroamylasemia. Response to gluten withdrawal.

A 36 year old woman presented with malabsorption and macroamylasemia. The macroamylase was characterized and shown to be a complex of pancreatic amylase and immunoglobulin A(IgA). The patient had the clinical and histologic features of adult celiac disease, and responded to a gluten-free diet. The macroamylase complex disappeared from the serum after gluten withdrawal, a hitherto unreported finding in the syndrome of malabsorption and hyperamylasemia.

Involvement of M1 cholinergic receptors and enteric nerves in the spasmogenic activity of bacterial N-formyl oligopeptides on guinea-pig ileum.

Bacterial N-formyl-methionyl oligopeptides are spasmogenic for guinea-pig ileum in vitro but the mechanism of this effect is not understood. To investigate this phenomenon further, we have determined pA2 values (the negative logarithm of the concentration of an antagonist reducing a double-dose agonist response to a single-dose response) for a number of potential antagonists of N-formyl-met-leu-phe (F-met-leu-phe) using histamine, acetylcholine, 5HT and substance P as control agonists. Atropine, pirenzepine and tetrodotoxin were potent inhibitors of F-met-leu-phe induced contraction (pA2's 8.4, 8.0 and 7.9, respectively) suggesting involvement of neural and cholinergic pathways in the response. Sulphasalazine, known to block the F-met-leu-phe receptor on neutrophil leucocytes, was also a potent inhibitor. Tachyphylaxis induced by either 5HT, or substance P, did not diminish the response to F-met-leu-phe, suggesting that these potential mediators were not involved. These studies indicate that bacterially synthesized formyl-methionyl oligopeptides bind to cells bearing receptors in guinea-pig ileum and produce muscle contraction via enteric cholinergic (M1) neural pathways.

The effect of pirenzepine on gallbladder emptying in humans.

The effect of the selective antimuscarinic agent, pirenzepine, on gallbladder function was studied in six healthy volunteers, using 99mTc HIDA (N-[2,6-diethylthenyl] carbamoylmethyl iminodiacetic acid) hepatobiliary scanning. Pirenzepine, in doses that inhibit gastric acid secretion, did not alter gallbladder emptying responses to sham feeding stimulation or to a test meal.

Large and small forms of cholecystokinin in human plasma: measurement using high pressure liquid chromatography and radioimmunoassay.

Defining the role of cholecystokinin (CCK) in gut physiology and disease has proved difficult because of problems with development of radioimmunoassays and because CCK exists in several different molecular forms which have different biological actions. In order to measure small (8 amino acid residues, CCK 8) and large (33 and 39 residues) forms of CCK in plasma we have developed high pressure liquid chromatographic (HPLC) fractionation of plasma prior to radioimmunoassay. Fasting plasma CCK 8 and CCK 33/39 levels were usually undetectable (less than 3 and less than 6 pmol/1, respectively). After a liquid fat meal both CCK 8 and CCK 33/39 levels were significantly elevated at 5 min (11.3 +/- 3.3 and 11.6 +/- 2.6 pmol/1, respectively). Peak CCK 8 levels occurred at 30 min (15.0 +/- 4.4 pmol/1) while peak CCK 33/39 levels occurred at 120 min (16.7 +/- 4.9 pmol/1). Total CCK levels showed a biphasic response to the meal. These CCK 8 and CCK 33/39 responses to oral fat are consistent with a role for these hormones in the regulation of gallbladder emptying and pancreatic secretion.

Differential distribution of molecular forms of cholecystokinin in human and porcine small intestinal mucosa.

To examine the distribution of cholecystokinins (CCKs) along the small intestine we examined the nature of CCKs in samples of jejunum, mid-intestine and ileum from human and porcine intestine. CCKs in intestinal mucosa were extracted by boiling in both neutral and acid conditions, and subjected to high pressure liquid chromatography (HPLC) to separate the forms of CCK followed by radioimmunoassay of separate fractions. In neutral extracts of human intestine CCK immunoreactivity totalled 119.4, 22.9 and less than 1 ng/g in jejunum, mid-intestine and ileum, whilst in acid extracts the corresponding values were 65.3, 47.4 and less than 1 ng/g. Amounts of CCK extracted from porcine mucosa were of similar magnitude. In neutral extracts material co-chromatographing on HPLC with synthetic porcine CCK 8 predominated, whilst in acid extracts material co-chromatographing with CCKs 33/39 was the major form. These forms of human and porcine CCKs extracted from the mucosa behaved similarly to CCK 8 and CCK 33/39 standards on HPLC, in the radioimmunoassay and on molecular exclusion chromatography - suggesting marked similarity of the CCKs in the two species. In both species there was a marked change in the ratios of CCK 8: CCK 33/39 down the intestine from 1: 0.8 in human jejunum to 1: 5.6 in mid-intestine and from 1: 1.5 in porcine jejunum to 1: 5.8 in mid-intestine. These observations may explain the changing patterns of CCKs in circulation with time after ingestion of a fat meal and the greater impairment of CCK 8 than CCK 33/39 release observed in coeliac disease.

The effect of some anti-inflammatory agents on elastase release from neutrophils in-vitro.

In view of the potential role of released polymorphonuclear leucocyte elastase in causing tissue damage, the effect of commonly used anti-inflammatory drugs on elastase release from neutrophils has been studied in-vitro. Elastase release from neutrophils exposed to the synthetic bacterial cell wall peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (10(-6) M) was quantitated using a radiometric immunoassay and a functional assay of elastase. Prednisolone and non-steroidal anti-inflammatory drugs inhibited elastase release at concentrations from 0.1 mM-0.1 nM. No inhibition by sulphosalicylic acid, D-penicillamine or chloroquine sulphate was observed. The clinical relevance of these findings is discussed.

Carriage, quantification, and predominance of methanogens and sulfate-reducing bacteria in faecal samples.

AIMS: To determine carriage rates and densities of methanogens and sulfate-reducing bacteria in adults and children using molecular methods, and to also determine if a reciprocal relationship exists between these organisms. METHODS AND RESULTS: Real-time PCR was used to detect and quantify methanogens and sulfate-reducing bacteria. Real-time PCR was more sensitive than breath methane measurements. Real-time PCR assays were applied to faecal DNA samples collected from 40 children and 12 adults. Methanogens were present in 25% of the children and 42% of the adults studied, and sulfate-reducing bacteria were detected in 15% of the children and 58% of the adults. High levels of sulfate-reducing bacteria were found in two methanogenic adults. CONCLUSIONS: Carriage rates and densities of methanogens and sulfate-reducing bacteria are greater in adults than in children. Competition does not necessarily lead to the predominance of one group in the faecal microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes sensitive, molecular assays that could be used to monitor these organisms in gastrointestinal disease. Therapeutic exclusion of one group from the bowel would not necessarily lead to the expansion of the other, as there does not appear to be a reciprocal relationship between these groups.

Liver and gastrointestinal function in pregnancy.

Difficulties arise in the interpretation of liver tests in the pregnant subject, since some values increase (alkaline phosphatase) whilst others remain unchanged (transaminases) or fall during pregnancy. The diagnosis and management of some causes of jaundice in pregnancy, such as viral hepatitis, gall stones, benign intrahepatic cholestasis and acute fatty liver of pregnancy are discussed. Little is known about the commonest symptoms of pregnancy (nausea, vomiting and constipation) other than that they might be due to hormonally induced alteration of sphincter tone. However, pre-existing bowel disease has a greater effect on pregnancy. Fertility is reduced in poor nutritional states (e.g. coeliac and Crohn's diseases) and an increased occurrence of spontaneous abortion has been noted. For inflammatory bowel diseases, the time of onset is important in determining the outcome of pregnancy. Relapse in the disease is commonest in the first trimester and in the puerperium. Treatment of these conditions is essentially as in the non-pregnant subject. The controversial subject of sulphasalazine and steroid usage in pregnancy is discussed.

Pharmacological inhibition of chenodeoxycholate-induced fluid and mucus secretion and mucosal injury in the rabbit colon.

The effects of various pharmacological agents on bile acid-induced fluid secretion, mucus secretion, and mucosal injury were investigated using a perfusion technique in rabbit colon. Atropine markedly reduced and carbachol potentiated the fluid secretion, mucus output, and mucosal damage observed during bile acid perfusion. In contrast, pretreatment of the colonic mucosa with lignocaine and parenteral administration of methysergide and somatostatin produced a modest reduction in the fluid secretory response without apparent effects on mucus output or mucosal damage. These results suggested that cholinergic agonists and antagonists influence the mucosal resistance to bile acid-induced injury possibly through their effects on mucus secretion. Increasing or decreasing mucosal resistance to the detergent effects of bile acids appeared to have marked effects on the magnitude of induced fluid secretion. A minor reduction in overall secretory response to bile acids was also apparent with agents not influencing mucus secretion or mucosal injury.

Physiochemical studies of indocyanine green (ICG): absorbance/concentration relationship, pH tolerance and assay precision in various solvents.

Indocyanine green (ICG) obeyed the Beer-Lambert law within the concentration range 1.25 micrograms/ml-10.0 micrograms/ml in distilled water, methanol, dimethylformamide (DMF), 1.2-propanediol and aqueous buffers (pH 9.0), but only up to 7.5 micrograms/ml in human bile and 0.5% human albumin, and only to 5.0 micrograms/ml in human duodenal fluid. ICG was rapidly (less than 1 h) decomposed to a colorless derivative at pH less than 5 and greater than 11, but remained relatively stable for 48 h at pH 8-10. ICG is an indicator and a weak acid with a pKa of 3.27. In bile stabilized with 25% methanol, the precision of the method (CV) is 5% and the accuracy is 106-127%.

Anti-inflammatory effects of LPS, MDP and FMLP on carrageenan pleurisy in the rat.

Bacterial products fmet-leu-phe (FMLP), muramyl dipeptide (MDP) and lipopolysaccharide (LPS) were assayed for their ability to alter the inflammatory response to lambda carrageenan-induced pleurisy in Hooded Surgery rats. Continuously infused FMLP, or one initial i.v. dose of FMLP, MDP or LPS either ablated or partially suppressed the pleurisy. Total circulating leucocytes and neutrophils were suppressed by 55-65% when compared to the normal circulating leucocyte response to carrageenan pleurisy, excepting the protocol incorporating a single i.v. dose of FMLP where suppression was intermediate at 30%. There were also significant changes in the expression of FMLP receptors on circulating neutrophils. MDP and LPS induced a receptor number increase of 2 and 1.7 times initial value respectively, whilst a continuous FMLP infusion caused a receptor decrease to 0.3 times the initial value. The introduction of bacterial products at an alternative site to that of the pleurisy had an anti-inflammatory effect and the pleurisy was reduced.

Coeliac disease presenting with a leuco-erythroblastic anaemia.

A 49-year-old Irishman presented as an emergency with watery diarrhoea and a leuco-erythroblastic anaemia. Investigations showed that he had coeliac disease but no evidence of bone marrow infiltration. His leuco-erythroblastic picture disappeared on treatment with iron and folic acid.