Biomedical subjects

A D Befus

Publications and source records attributed to A D Befus.

At least 19 recordsLinked to original sources

PubMed1995

Proteinases of rat mast cells. Peritoneal but not intestinal mucosal mast cells express mast cell proteinase 5 and carboxypeptidase A.

Six basic proteins of 26 to 38 kDa with isoelectric points (pI) > or = 8.5 were abundant in proteins separated by two-dimensional SDS-PAGE from adult rat peritoneal mast cells (MC). One was identified previously as rat mast cell proteinase (RMCP) 1, a chymase of 26 to 28 kDa, pI > 9.0. Microsequence analyses showed that two polypeptides of about 29 and 30 kDa had NH2 terminal amino acid sequences homologous to mouse MC proteinase 5 (MCP-5), whereas the amino terminals of the 33, 35, and 36 kDa proteins were homologous to MC carboxypeptidase A (MC-CPA). Rabbit Abs produced against synthetic peptides of the identified NH2 terminal sequences were used in immunoblot studies. At least three proteins reacted with Abs to MC-CPA, whereas Abs to MCP-5 detected three adjacent polypeptides, rather than just the two identified by using microsequence analysis. Removal of oligosaccharide side chains using peptide:N-glycosidase F reduced the heterogeneity of each set of three polypeptides (MCP-5 and MC-CPA) to a band of each protein of a lower M(r). The serine proteinase inhibitor [3H]diisopropylfluorophosphate ([3H]DFP) bound to a proteinase of 30 to 35 kDa, which is probably MC tryptase (pI < or = 6.0). Immunoblot analysis of proteins from intestinal mucosal mast cells showed RMCP-2, but not RMCP-1, MCP-5, or MC-CPA. This is the first report of MCP-5 in the rat and of clearly distinguishable glycosylated forms of MC CPA. These proteinases appear to be restricted in their distribution to selected MC populations, but little is known about their functions.

Amidohydrolases↗

PubMed1995

Inhibition of tumour necrosis factor-alpha (TNF-alpha) release from mast cells by the anti-inflammatory drugs, sodium cromoglycate and nedocromil sodium.

TNF-alpha is a cytokine thought to be involved in the pathogenesis of asthma and in several other inflammatory conditions. Given recent evidence that mast cells (MC) are an important source of TNF-alpha, we investigated the effects of two anti-inflammatory drugs, nedocromil sodium (NED) and sodium cromoglycate (SCG), on rat MC-derived TNF-alpha. We established that at least 2 h pretreatment with NED or SCG followed by washing was required to inhibit TNF-alpha-dependent cytotoxicity by rat peritoneal MC (PMC). A maximum inhibition of TNF-alpha occurred after 6 h treatment. The inhibitory effect of NED and SCG (10(-5)-10(-3)M) was concentration-dependent (20-37% for NED and 16-37% for SCG). The time-course analysis and the use of cycloheximide, an inhibitor of protein synthesis, provided strong evidence that new protein synthesis by the MC is required for this inhibitory effect. Furthermore, 24 h treatment with 1 mM NED inhibited the levels of mRNA for TNF-alpha by 59-83%. In addition to the effect on TNF-alpha-dependent cytotoxicity by MC, 20 min pretreatment with 10(-4) M NED and SCG inhibited antigen-stimulated TNF-alpha release (6h) by 42% and 48%, respectively. Interestingly, the functionally distinct intestinal mucosal MC (IMMC) is unresponsive to these drugs with regard to histamine secretion. However, as with PMC, 2h pretreatment with NED or SCG inhibited TNF-alpha-dependent cytotoxicity by IMMC. These effects may be important in the action of these drugs in vivo in the late phase reaction in asthma or other inflammatory conditions.

Animals↗

PubMed1995

Interferons differentially regulate histamine and TNF-alpha in rat intestinal mucosal mast cells.

Mast cell (MC) heterogeneity has been well characterized in the rat where it has been shown that connective tissue MC, often represented by peritoneal MC (PMC), and the intestinal mucosal MC (IMMC) exhibit many differences in mediator content and responsiveness to secretagogues and anti-allergic drugs. Pretreatment (20 hr) of PMC with interferon (IFN)-alpha/beta or IFN-gamma, significantly reduced antigen-stimulated histamine release. By contrast, for IMMC, the same IFN treatment did not modify antigen-stimulated histamine secretion. Although IFN treatment differentially modulates histamine secretion from PMC and IMMC, pretreatment of both MC types with IFN-alpha/beta or IFN-gamma inhibited their tumour-necrosis factor-alpha (TNF-alpha)-dependent cytotoxicity. In 20 hr of culture, IMMC spontaneously released 98 pg/10(6) MC of TNF-alpha, whereas PMC released about threefold more TNF-alpha, 282 pg/10(6) MC. In addition, direct assessment of stored TNF-alpha established that IMMC store less TNF-alpha (68 pg/10(6) MC) than PMC (404 pg/10(6) MC). In summary, TNF-alpha content of PMC and IMMC was different, but IFN inhibited TNF-alpha-dependent cytotoxicity by both MC types. By contrast, treatment with IFN-alpha/beta or IFN-gamma inhibits antigen-induced histamine secretion by PMC, but does not modify antigen-induced histamine secretion by IMMC. Thus, IFN differentially regulate the secretion of histamine and TNF-alpha in PMC and IMMC.

Animals↗

PubMed1994

Neuroendocrine regulation of inflammation and tissue repair by submandibular gland factors.

Interactions between the immune, nervous and endocrine systems are important in inflammation and tissue repair. One neuroendocrine pathway involves polypeptide factors derived from the submandibular glands, whose synthesis and release are controlled by cervical sympathetic nerves. This novel pathway of immune-neuroendocrine communication is the cervical sympathetic trunk-submandibular gland (CST-SMG) axis. Here, Ronald Mathison, Joseph Davison and Dean Befus discuss the contributions of this axis to the neuroendocrine regulation of inflammation and tissue repair.

Animals↗

PubMed1994

Possible protective immunity in human opisthorchiasis.

Chronic infections with the liver flukes Opisthorchis viverrini and Clonorchis sinensis affect over 30 million people in southeastern Asia. With ongoing exposure, reinfection readily occurs following curative treatment and cumulative infections result in significant morbidity and a predisposition to cholangiocarcinoma. Though protective immunity has never been described in human opisthorchiasis, heterogeneity in worm burden occurs and a small number of exposed residents of endemic areas remain apparently uninfected. To explore the nature of this heterogeneity, we compared levels of serum antibody (Ab) to O. viverrini measured by an enzyme-linked immunosorbent assay in 83 stool egg-positive and 49 stool egg-negative residents of an O. viverrini-endemic area in Thailand. Compared to the egg-positive residents, the egg-negative group had significantly higher levels of immunoglobulin (Ig)G, IgA and IgM to adult worm homogenate (AWH) and total Ab to metacercaria homogenate (MH). Furthermore, immunoblot analyses revealed that a significantly higher proportion of sera from the egg-negative residents had IgA reactivity against a 38-kDa AWH antigen and IgM reactivity against carbohydrate epitopes of a 42-kDa AWH glycoprotein antigen. These findings support a hypothesis that the egg-negative group includes individuals who may be immunologically resistant to this usually chronic infection.

Adolescent↗

PubMed1994

Temporal analysis of the anti-inflammatory effects of decentralization of the rat superior cervical ganglia.

Bilateral decentralization of the superior cervical ganglia (SCG) reduced the pulmonary inflammation that develops 4-8 h after induction of anaphylaxis in Nippostrongylus brasiliensis-sensitized rats. Histamine levels in peritoneal lavage fluid and rat mast cell protease type II in serum were increased to comparable levels in sham-operated and decentralized rats. In vitro stimulation of alveolar macrophages (ALM) with lipopolysaccharide (LPS) provoked tumor necrosis factor-alpha (TNF-alpha) release that was two to three times greater with unchallenged decentralized rats than with sham-operated rats. However, after allergen challenge LPS-stimulated TNF-alpha release from ALM of sham-operated rats increased threefold and lasted at least 24 h, whereas with decentralized rats release of this cytokine actually decreased at 4 and 8 h. The increase in the phagocytosis and respiratory burst of circulating neutrophils seen at 4 and 8 h after allergen challenge in sham-operated rats was reduced significantly by decentralization. These results suggest that the attenuation of anaphylaxis-induced pulmonary inflammation that occurs with decentralization of the SCG is primarily associated with downregulation of neutrophil and macrophage functions.

Anaphylaxis↗

PubMed1994

Bronchoalveolar leucocyte responses to trickle infections with Nippostrongylus brasiliensis in rats.

There is a need for experimental systems allowing study of host responses generated by continuous, low-level exposures to parasites. To assess the pulmonary inflammatory responses in different types of exposure to infection we used bronchoalveolar lavage (BAL). Rats sensitized by 500 Nippostrongylus brasiliensis larvae given in 20 doses over a 4-wk period (group T) and challenged with 500 larvae 33 days after the initial exposure were compared to rats initially given 1 sensitizing dose of 500 larvae (group B) and also to naive, sham-treated (group S) rats, subsequently challenged with 500 larvae. BAL performed prior to final challenge revealed markedly increased numbers of macrophages and eosinophils in group T, but there were only minor changes in numbers of these cells in group B. After final challenge, numbers of BAL macrophages and eosinophils were greater in group T than in group B, although in group B there was a rapid increase in numbers of these cells. Changes in numbers of BAL neutrophils were not correlated with previous sensitization to N. brasiliensis. Thus, there was a pronounced influx of leucocytes into the pulmonary lumen after secondary challenge in rats sensitized by repeated exposures to low doses of larvae.

Animals↗

PubMed1993

Analysis of human skin mast cell proteins by two-dimensional gel electrophoresis. Identification of tryptase as a sialylated glycoprotein.

Proteins of mast cells purified from human foreskin were separated by 2-D polyacrylamide gel electrophoresis using either nonequilibrium pH gradient electrophoresis or isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. Silver staining showed that a major feature of skin mast cell 2-D protein maps was a variety of relatively abundant proteins in the m.w. range of 29 to 37 kDa and covering a broad pH range from 5.0 to 8.5. Tryptase was identified on Western blots of 2-D-separated proteins by its binding of mAb and of 3H-diisopropylfluorophosphate. The precise distribution of tryptase varied among individuals but this protein generally occupied a continuum of molecular weights between 28 and 37 kDa and ranged in isoelectric point between 5.0 and 6.5. Tryptase was one of a number of mast cell proteins that bound the lectin concanavalin A as well as lectins specific for sialic acid, demonstrating that this enzyme is a sialylated glycoprotein. The diffuse m.w. distribution of skin mast cell tryptase (31 to 36 kDa) observed after SDS-PAGE was reduced to a single band of 30 kDa after treatment with protein-N-glycosidase F to remove asparagine-linked oligosaccharides. This finding suggests that intrinsic m.w. heterogeneity of tryptase in skin mast cells is largely a result of the addition of variable amounts of oligosaccharide to the tryptase polypeptide.

Child, Preschool↗

PubMed1993

Decentralization of the superior cervical ganglia inhibits mast cell mediated TNF alpha-dependent cytotoxicity. 1. Potential role of salivary glands.

Decentralization or ganglionectomy of the superior cervical ganglia (SCG) reduces pulmonary inflammation, as well as chemotaxis and activation of circulating neutrophils. However, the protective effect of decentralization was abolished when combined with removal of the submandibular glands (sialadenectomy) in the same animals. Thus, it has been postulated that the submandibular glands (SMG) release an anti-inflammatory factor(s) that is controlled by cervical sympathetic nerves. Decentralization of SCG did not modify in vitro histamine release or in vivo levels of rat mast cell protease II, but it reduced mast cell (MC)-mediated tumor necrosis factor alpha (TNF alpha)-dependent cytotoxicity. Combined decentralization/sialadenectomy abrogated the inhibition of MC cytotoxic activity, as we have shown previously for pulmonary inflammation and neutrophil functions. However, sialadenectomy alone inhibited MC-mediated TNF alpha-dependent cytotoxicity, an observation which suggests that SMG produce a factor(s) that can potentiate MC cytotoxic activity. Studies of the effects of SMG-derived factors, such as epidermal growth factor, nerve growth factor, and transforming growth factor beta (TGF beta), showed that only pretreatment of MCs with TGF beta 10(-8) g/ml inhibited MC-mediated TNF alpha-dependent cytotoxicity. Thus, the modulation of MC-mediated TNF alpha-dependent cytotoxicity by cervical sympathetic innervation and SMG is complex and distinct from the modulation of pulmonary inflammation and neutrophil functions identified previously.

Animals↗

PubMed1993

Further studies on the effect of nitrogen dioxide on mast cells: the effect of the metabolite, nitrite.

To evaluate the relationship between atmospheric nitrogen dioxide exposure and the development of allergic diseases, the effects of nitrite as a chemical product of inhaled nitrogen dioxide on mast cell functions were investigated. We have studied nitrite-induced histamine release from two functionally distinct mast cell populations, namely peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC) of Nippostrongylus brasiliensis-infected rats. High concentrations of nitrite alone (10, 20, and 50 mM) induced histamine release from IMMC, but not from PMC. Moreover, histamine release from PMC and IMMC stimulated with sensitizing antigen was significantly enhanced by pretreatment with 50 mM nitrite or nitrate. No differences in histamine release from nitrite-treated and control PMC were seen below 1 mM. To investigate the effect of nitrite on tumor cell cytotoxic activity, PMC were incubated with various concentrations of nitrite. Pretreatment with 5 and 50 mM nitrite markedly depressed tumor necrosis factor (TNF)-alpha-dependent natural cytotoxicity of PMC for the tumor target WEHI-164. Thus, high concentrations of nitrite enhanced mast cell histamine release, but depressed TNF-alpha-dependent cytotoxicity. However, low concentrations of nitrite (< 1 mM) that would normally be produced by short-term atmospheric exposure to nitrogen dioxide may have no significant effects on mast cell functions.

Animals↗

PubMed1993

Prostaglandins inhibit inflammatory mediator release from rat mast cells.

BACKGROUND: Mast cells have been implicated in the pathogenesis of gastric ulceration. It is possible that prostaglandins exert cytoprotective effects by inhibiting the release of proulcerogenic mediators from mast cells. METHODS: The effects of three prostaglandins on the release of platelet-activating factor, tumor necrosis factor, and histamine from rat mast cells (peritoneal and intestinal mucosal) activated with calcium ionophore or antigen were assessed. RESULTS: Upon stimulation with either agonist, intestinal mucosal and peritoneal mast cells released significant quantities of platelet-activating factor. Preincubation for 5 minutes with misoprostol, prostaglandin (PG)E2, 16,16-dimethyl PGE2, ketotifen, or PF-5901 concentration-dependently reduced ionophore-stimulated platelet-activating factor release; significant effects were observed with picomolar to nanomolar concentrations of the prostaglandins and micromolar concentrations of the other compounds. Tumor necrosis factor release from peritoneal and mucosal mast cells was also significantly inhibited by the prostaglandins in picomolar to nanomolar concentrations. Misoprostol and PGE2 at concentrations of 5-50 nmol/L significantly inhibited histamine release from peritoneal mast cells stimulated with ionophore but did not affect histamine release stimulated by antigen. CONCLUSIONS: These results show potent inhibitory effects of prostaglandins on the release of pro-ulcerogenic inflammatory mediators from mast cells. Such effects may contribute to the protective and anti-inflammatory effects of prostaglandins in the gastrointestinal tract and elsewhere.

Animals↗

PubMed1993

Neural regulation of neutrophil involvement in pulmonary inflammation.

1. The high viscoelastic property of neutrophils is the major factor contributing to their extensive accumulation (more than 50% of circulating neutrophils) in the pulmonary microvasculature. 2. The cholinergic parasympathetic and adrenergic sympathetic nerves modulate the size of the pulmonary neutrophil pool by regulating arterial and venous pressures, increases in which promote or reduce neutrophil transit times, respectively. 3. Biochemical factors, such as the cytokines and complement, which act upon the neutrophils to increase their viscoelasticity and promote the interaction of neutrophil cell adhesion molecules with counter ligands on the endothelial cell, are the primary factors regulating the size of the pulmonary pool of vascular neutrophils. 4. The primary afferent nerves, through their release of substance P, are the most important neural elements regulating neutrophil accumulation and function. Substance P facilitates the actions of other inflammatory agents (e.g. LTB4, platelet activating factor) on neutrophil adhesion, migration and biochemical reactivity. 5. The sympathetic nervous system indirectly regulates neutrophil functions by regulating the release of an immunosuppressive factor from submandibular glands. 6. With continued, study of nervous system regulation of neutrophil function, the mechanisms by which psychological factors affect these cells will eventually be revealed.

Animals↗

PubMed1993

Extracts of mosquito salivary gland inhibit tumour necrosis factor alpha release from mast cells.

Extracts of salivary glands of the yellow fever mosquito Aedes aegypti inhibit tumour cell-stimulated release of tumour necrosis factor alpha (TNF alpha) from rat mast cells, but do not inhibit antigen-induced histamine secretion. This inhibitory activity for TNF alpha is found in salivary glands of female but not in male mosquitoes. This inhibition is not mediated by bacterial contamination (LPS), by calcitonin gene related peptide (CGRP), nerve growth factor (NGF), epidermal growth factor (EGF) or transforming growth factor beta (TGF beta). The factor(s) has a molecular weight > 10 kDa and is neutralized by boiling for 10 min or heating at 56 degrees C for 30 min. The modulation of this proinflammatory mediator, TNF alpha, produced by mast cells in sites of blood feeding may facilitate completion of the blood meal, and as reported for certain vector-transmitted parasites, may enhance infectivity.

Aedes↗

PubMed1992

Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds.

1. Peritoneal mast cells from rat were co-incubated in vitro in a platelet aggregometer cuvette with washed rabbit platelets. In response to stimulation with calcium ionophore (A23187; 1-5 microM), the mast cells released a substance which stimulated the platelets to aggregate. These concentrations of ionophore did not stimulate platelet aggregation in the absence of mast cells, nor affect the responsiveness of the platelets to aggregation induced by thrombin or PAF. Release of a PAF-like substance was also observed in response to stimulation of the mast cells with antigen. 2. This pro-aggregatory activity is attributable to the release of PAF by the mast cells, since the activity could be abolished by preincubating the platelets with a specific PAF receptor antagonist (WEB 2086; 10 microM). Furthermore, the platelet-aggregating factor co-migrated with PAF on thin-layer chromatographs and could be abolished by incubation with phospholipase A2 (20 micrograms ml-1) or a specific antibody directed against PAF. 3. The release of PAF by peritoneal mast cells could be inhibited, in a concentration-dependent manner, by PF-5901 (IC50 of 3.9 microM) or Wy-50,295 (IC50 of 1.2 microM), two structurally similar compounds with inhibitory effects on leukotriene synthesis, as well as leukotriene D4 (LTD4) receptor antagonist properties. 4. Inhibition of PAF synthesis was not observed when the mast cells were incubated with a structurally unrelated 5-lipoxygenase inhibitor (A-64077), a structurally dissimilar inhibitor of 5-lipoxygenase activating protein (MK-886) or with a structurally related LTD4 receptor antagonist (MK-571) which lacks inhibitory effects on leukotriene synthesis, each at concentrations of up to 100 microM.5. Neither PF-5901 nor Wy-50,295 (1 or 10 microM) significantly affected histamine release or prostaglandin D2 synthesis by peritoneal mast cells in response to calcium ionophore stimulation.6. These results demonstrate the ability of a class of quinoline-based compounds to inhibit PAF synthesis by peritoneal mast cells. This activity does not appear to be related to effects of these compounds on leukotriene synthesis or LTD4 receptors. The ability of these compounds to inhibit PAF synthesis may contribute to their anti-inflammatory properties.

Animals↗

PubMed1992

Platelet activating factor and systemic anaphylaxis in Nippostrongylus brasiliensis-sensitized rats: differential effects of PAF antagonists.

1. The effects of two platelet-activating factor (PAF) antagonists, WEB 2086 and BN 52021, in reducing the changes in extravasation (Evans blue technique) and blood flow (radiolabelled microsphere method) to various organs and tissues following anaphylactic shock in the Nippostrongylus brasiliensis-sensitized rat were investigated. 2. Both antagonists attenuated anaphylaxis-induced increases in plasma protein leak in the trachea, stomach and small intestine, although they did not block extravasation in the colon and kidneys. 3. Anaphylaxis-induced decreases in blood flow to the adrenals were effectively antagonized by WEB 2086, although this antagonist did not reverse blood flow decreases to any other tissues. BN 52021, on the other hand, did not alter anaphylaxis-induced decreases in blood flow to the adrenals, but effectively prevented dramatic decreases in blood flow to the large and small bowel and spleen. 4. Anaphylactic shock produced marked reduction in blood pressure that was partly reversed by WEB 2086, whereas BN 52021 effectively blocked the decreases in cardiac output. 5. Thus, PAF is responsible for some of the haemodynamic and extravasation of protein changes associated with systemic anaphylaxis in the rat, although the differential inhibition observed with the two antagonists suggests that PAF alters vascular responsiveness through different mechanisms in selected tissues.

Anaphylaxis↗

PubMed1992

Capsaicin-induced hyperemia in the stomach: possible contribution of mast cells.

Topical application of capsaicin to the gastric mucosa results in marked hyperemia as a consequence of the release of vasoactive neuropeptides from sensory afferent neurons. Because many of these neuropeptides have the capacity to induce mast cell degranulation, we investigated the possible contribution of mast cells to capsaicin-induced hyperemia. Application of capsaicin to the gastric mucosa of normal rats resulted in a concentration-dependent increase in blood flow. In rats in which mastocytosis was induced by prior infection with Nippostrongylus brasiliensis, the hyperemic responses to capsaicin were significantly greater than in control rats. This augmented hyperemic response could be significantly attenuated by pretreatment with a histamine H1-receptor antagonist (pyrilamine) or with a mast cell stabilizer (doxantrazole). Depletion of mucosal mast cells through treatment with dexamethasone also significantly reduced the hyperemic response to capsaicin. Hyperemic response to capsaicin in normal rats and in rats with mucosal mastocytosis could be completely abolished by pretreatment with ruthenium red or prior ablation of the sensory afferent neurons with capsaicin. These results suggest that in rats with gastric mastocytosis, sensory neuron-dependent activation of mast cells contributes to the hyperemic response to topical capsaicin. These findings are therefore consistent with the hypothesis that there is communication between nerves and mast cells in the gastric mucosa, at least in rats previously infected with N. brasiliensis.

Animals↗

PubMed1992

Separation and characterization of adult worm proteins and glycoproteins from the liver fluke Opisthorchis viverrini.

Detailed studies of liver fluke proteins and antigens are necessary to facilitate further investigation of the human immune responses to these parasites. Accordingly, Opisthorchis viverrini antigens were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. We initially encountered excessive background smearing, vertical streaking, and indistinct bands that were similar to problems previously described by investigators studying this and other trematodes including Schistosoma mansoni. These problems were especially evident with silver staining of proteins and occurred despite the extensive use of protease inhibitors. They were minimized by using mini (vs. large) SDS-PAGE and Coomassie blue protein staining. With the latter 2 techniques, adult worm somatic proteins and excretory-secretory products were separated and characterized. Immunoblots using rabbit anti-adult worm sera demonstrated that some of these proteins were antigens common to both the adult and metacercarial stages. Several of these antigens also corresponded (according to molecular weight) to glycoproteins, detected by concanavalin A blotting. These findings form a base for subsequent studies of the human immune response to liver fluke infection.

Animals↗

PubMed1992

Role for the cervical sympathetic trunk in regulating anaphylactic and endotoxic shock.

This study tests the hypothesis that spinal nerves projecting down the cervical sympathetic trunk contribute to the regulation of systemic immune responses. Decentralization or ablation (ganglionectomy) of the superior cervical ganglia (SCG), which receive innervation from spinal segments C8-T8, were found to reduce the pulmonary inflammatory response consequent to induction of anaphylaxis in rats sensitized to the nematode Nippostrongylus brasiliensis. Furthermore, the hypotensive responses to IV endotoxin were attenuated in sensitized rats by these operations, whereas decentralization without ganglionectomy protected against endotoxic shock in normal (unsensitized) rats. These results suggest that systemic inflammatory events are regulated by the cervical sympathetic nervous system at a level superior to the superior cervical ganglia. Further studies are warranted to investigate the role of the cervical and thoracic sympathetic nerves in the regulation of systemic immunological function.

Anaphylaxis↗