Picotamide inhibition of excess in vitro thromboxane B2 release by colorectal mucosa in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease is associated with increased mucosal release of eicosanoids. Among these, thromboxane A2 has been proposed as a possible inflammatory mediator; its suppression may be a useful therapeutic option. METHODS: Using a tissue incubation technique, we compared release of immunoreactive thromboxane B2 by colonic biopsies from patients with ulcerative colitis, Crohn's disease and controls, and assessed the inhibitory effect of picotamide, a thromboxane synthesis inhibitor-receptor antagonist, which has been widely used in Italy for management of ischaemic heart and cerebrovascular disease. RESULTS: Increased amounts of thromboxane B2 were released from biopsies from patients with active ulcerative colitis (median 238 pg/20 min/mg wet weight (interquartile range 147- 325), n = 12) and active Crohn's disease (252 (174-450), 6) compared with those from patients with quiescent ulcerative colitis (95 (61- 140), 12) or Crohn's disease (105 (57-201), 13), or controls (136 (64- 206), 8). Incubation with picotamide at concentrations between 100 microM and 1 mM reduced thromboxane B2 release (IC50 890 microM). CONCLUSION: Since increased thromboxane A2 production may have pathogenetic importance, thromboxane synthesis inhibitor-receptor antagonists such as picotamide merit therapeutic trial in the management of inflammatory bowel disease.     

Follistatin and activin A production by the male reproductive tract

Follistatin is a binding protein for the activin and inhibin family of hormones, regulating their biological activity. In the male reproductive tract, the interaction of these factors is likely to be involved in the regulation of the proliferation of several cell types. We have investigated the presence of follistatin and activin A in seminal plasma using specific immunoassays and have localized follistatin and activin/inhibin subunits in the adult human testis, prostate and seminal vesicle to establish their likely sources. High concentrations of immunoreactive follistatin were present in seminal plasma in normal men (mean 97.9 ng/ml; 1.43 ng/ml in peripheral plasma) and were similar in men with oligo/azoospermia and following vasectomy. Follistatin immunoreactivity was localized to both Leydig and Sertoli cells of the testis, and to epithelial cells of the prostate gland and seminal vesicle, which are likely to be the predominant sources of the hormone in seminal plasma. Activin A was also present in seminal plasma in normal men but was undetectable following vasectomy, thus deriving from the testis. Consistent with this finding, the betaA-subunit was immunolocalized in Sertoli and Leydig cells but was not present in seminal vesicle or prostate gland. The functional significance of the high concentrations of follistatin secreted into seminal plasma by the prostate gland and/or seminal vesicle is uncertain, but they may regulate the biological activity of testis-derived activin A and inhibin B.      

Neutrophils and host defense

Neutrophils, the predominant phagocytes of circulating blood, are the first cells to arrive at sites of infection. Although neutropenia has long been recognized to predispose to infection, recently other syndromes marked by frequent infections have been shown to be caused by an underlying neutrophil dysfunction. Efforts to define the molecular pathology of such disorders have helped delineate the molecular basis of normal neutrophil function. Advances have been made in defining the roles of the neutrophil's varied receptors in recognition, movement, and adhesive phenomena. Progress in establishing the pathogenesis of chronic granulomatous disease has provided important insights into the enzymatic machinery that normal neutrophils use to produce antimicrobial oxidants. The identification and precise characterization of antimicrobial components, such as defensins, have outlined the potential roles of "natural antibiotics" in neutrophil-mediated host-defense functions. These areas of neutrophil function will be reviewed and placed in a clinical context to guide physicians in evaluating children and adults with frequent or unusual infections.     

Enhancement of murine IgE antibody detection by IgG removal

RATIONALE: Although animal models for the study of allergic reactions are desirable, the use of mice has been hindered by the lack of sufficiently sensitive in vitro immunoglobulin epsilon (IgE) antibody assays. The aim of this study was to enhance IgE antibody measurements by immunoglobulin gamma (IgG) depletion. METHODS: Seven- to eight-week-old female mice of four strains (C3H/HeJ, CBA/J, C57Bl/6J, and Balb/c) were immunized (20 mice/group) with shrimp or peanut extracts using Al(OH)(3) as adjuvant. Following immunization, animals were sacrificed by exsanguination and the sera of each group pooled. Initial measurements of IgE antibody levels by enzyme-linked immunosorbent assay (ELISA) were relatively low; IgG and IgE reactivity patterns by immunoblot were similar. Thus, sera from shrimp or peanut immunized mice were depleted of IgG (absorbed 3-6 times with immobilized protein G) and then tested for IgE antibody to shrimp or peanut allergen. RESULTS: A 3- to 5-fold increase in IgE antibody reactivity as measured by ELISA was demonstrated when >80-90% of the IgG was removed. This increase in detection of allergen-specific IgE occurred in sera from all mouse strains and to all allergens tested. In addition, reactivity of IgE antibodies to peanut or shrimp allergens by immunoblot increased visually approximately 4- to 10-fold. CONCLUSIONS: These studies indicate that allergen-specific IgG antibodies, which may be in more than 100-fold excess to IgE antibodies, interferes with detection of allergen-specific IgE, probably by competitive binding to allergenic epitopes. Substantial depletion of IgG antibodies (>80%) result in a significant increase in the sensitivity of the antibody measurements.     

Cryptdins: antimicrobial defensins of the murine small intestine.

Paneth cells are specialized small intestine epithelial cells that contain lysozyme, possess phagocytic properties, and secrete cytoplasmic granules into the intestinal crypt lumen after the entry of bacteria. Recent studies by Ouellette and associates (A. J. Ouellette, R. M. Greco, M. James, D. Frederick, J. Naftilan, and J. T. Fallon, J. Cell Biol. 108:1687-1695, 1989) indicated that murine Paneth cells produce prodefensin mRNA, but the properties of its peptide product were not reported. We purified two closely related defensins, cryptdin 1 and cryptdin 2, from a subcellular fraction of murine small intestine cells that was enriched in Paneth cells. Both peptides contained 35 amino acid residues, including the characteristic defensin "signature" of six invariantly conserved cysteines. Cryptdins 1 and 2 were approximately 90 to 95% homologous to each other and to the carboxy-terminal domain of the 93-amino-acid defensin precursor, cryptdin A, described by Ouellette and associates (Ouellette et al., J. Cell Biol. 108:1687-1695, 1989). Both cryptdins exerted bactericidal activity against Listeria monocytogenes EGD and Escherichia coli ML-35p in vitro. Their potency exceeded that of human neutrophil defensin HNP-1 but was considerably lower than that of NP-1, a defensin produced by rabbit neutrophils and alveolar macrophages. Both cryptdins killed mouse-avirulent Salmonella typhimurium 7953S (phoP) much more effectively than its phoP+, mouse-virulent, isogenic counterpart, S. typhimurium 14028S. Our data indicate that mouse intestinal prodefensins are processed into 35-amino-acid mature defensins (cryptdins) with broad-spectrum antimicrobial properties. The production of defensins and lysozyme by Paneth cells may enable them to protect the small intestine from bacterial overgrowth by autochthonous flora and from invasion by potential pathogens that cause infection via the peroral route, such as L. monocytogenes and Salmonella species.     

Antimicrobial activity of rabbit leukocyte defensins against Treponema pallidum subsp. pallidum.

Defensins, which are peptides with broad antimicrobial activity, are major constituents of rabbit neutrophils and certain macrophages. We tested six rabbit defensins, NP-1, NP-2, NP-3a, NP-3b, NP-4, and NP-5, for activity against Treponema pallidum subsp. pallidum. Mixtures of T. pallidum and defensin in 10% normal rabbit serum (NRS) or heat-inactivated NRS (HI-NRS) were incubated anaerobically for various time periods ranging between 0 and 16 h and then examined by dark-field microscopy for treponemal motility or inoculated intradermally into rabbits to assess treponemal virulence. Immobilization of T. pallidum by NP-1 (400 micrograms/ml) occurred after 4 and 8 h of coincubation in mixtures containing NRS and HI-NRS, respectively. Similarly, neutralization of T. pallidum by NP-1 occurred more rapidly and was complete when incubations were performed in NRS as compared with that in HI-NRS. Endpoint titration confirmed the augmentation of NP-1 antitreponemal activity by heat-labile serum factors; NP-1 showed neutralizing activity at 4 micrograms/ml (about 1 microM) in NRS and at 40 micrograms/ml in HI-NRS. When NP-1 was tested in serum that was deficient in C6, the T. pallidum neutralizing activity of NP-1 was reduced to levels slightly greater than that observed in HI-NRS. NP-1 that had been reduced and alkylated was inactive against T. pallidum. When NP-2, NP-3a, NP-3b, NP-4, and NP-5 were tested at 400 micrograms/ml, all exerted potent treponemicidal activity, manifested by abrogation or delayed development of cutaneous lesions relative to that of controls. These data suggest that defensins may equip certain macrophages and neutrophils to participate in host defense against T. pallidum, that the direct activity of defensins against T. pallidum is enhanced by heat-labile serum factors (presumably complement), and that conformational factors influence the biological activity of the defensin molecule.     

Double-blind, placebo-controlled food challenge (DBPCFC) as an office procedure: a manual

There is now enough experience with the use of double-blind, placebo-controlled, food challenge (DBPCFC) to recommend its use as an office procedure for most patients complaining of adverse reactions to foods. This manual discusses the practical methods required for the allergist to undertake DBPCFC in the office. Thorough histories supplemented by food allergen skin testing are used to design a DBPCFC that carefully attempts to reproduce the history of food-induced symptoms described by the patient. Precautions that must be taken are delineated before challenge, as is treatment that may be required if a reaction occurs. For those foods to which challenges are positive, longitudinal evaluation with repeated challenge at appropriate intervals help to determine whether or not the problem will resolve over a period of time.