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Biomedical subjects

A D Befus

Publications and source records attributed to A D Befus.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1991

Potentiation of tumor necrosis factor-alpha-mediated cytotoxicity of mast cells by their production of nitric oxide.

Nitric oxide (NO or endothelium-derived relaxing factor) has many of biologic actions, including the maintenance of blood pressure, inhibition of platelet aggregation, and cytotoxicity by phagocytic cells. Several cell types produce NO from L-arginine. Given recent emphasis on mast cell (MC)-dependent TNF-alpha-mediated cytotoxicity, we investigated the role of NO in rat peritoneal MC (PMC)-and intestinal mucosal mast cell-mediated cytotoxicity. MC cytotoxicity against the TNF alpha-sensitive target, WEHI-164, was potentiated by L-arginine. The NO competitive inhibitors, N omega-nitro-L-arginine and NG-methyl-L-arginine, diminished the cytotoxicity of rat PMC by 27 and 17%, respectively. However, hemoglobin, which binds to NO, inhibited the cytotoxic activity of PMC by 49% in the presence of 1 mM L-arginine and by 24% in L-arginine-free medium. The latter suggests that PMC use intracellular stores of L-arginine to produce NO. Neither hemoglobin nor NO metabolites affected human rTNF-alpha cytotoxicity. Furthermore, sodium nitroprusside, with its free radical NO group, restored PMC cytotoxicity in L-arginine-free medium to the level observed in 1 mM L-arginine medium. Studies with a platelet aggregation bioassay and various NO inhibitors confirmed that PMC produce NO. In addition, increased levels of NO2- were observed in medium of A23187, TNF-alpha, or WEHI-164-stimulated PMC.

Animals↗
PubMed1991

Tumor necrosis factor-alpha dependent cytotoxicity of human skin mast cells is enhanced by anti-IgE antibodies.

Mast cells dispersed from human skin and purified by density-gradient centrifugation were cytotoxic toward the mouse fibrosarcoma cell line WEHI-164. Skin mast cells were not cytotoxic toward the NK cell-sensitive cell line K562. Killing of WEHI-164 occurred over a prolonged (greater than 18 h) period of incubation with mast cells and was effectively inhibited by polyclonal antibodies and mAb against TNF-alpha suggesting that this cytokine plays an important role in mast cell-mediated cytotoxicity. Whereas lysates of rat peritoneal mast cells exhibited cytotoxicity toward WEHI-164, this was not found with lysates of unstimulated skin mast cells suggesting that TNF-alpha is not stored preformed in the latter. Killing of WEHI-164 cells by skin mast cells was enhanced by anti-IgE and there was a significant correlation between histamine release and cytotoxicity after activation with this stimulus. We conclude that human skin mast cells are a potential source of TNF-alpha and suggest that these cells, particularly after activation, might contribute to the synthesis of this multifunctional cytokine in inflammatory sites.

Animals↗
PubMed1990

Inhibition of mast cell-mediated cytotoxicity by IFN-alpha/beta and -gamma.

Although IFN enhance the cytotoxic activity of NK cells, K cells, and monocytes, IFN-alpha/beta and IFN-gamma did not stimulate the cytotoxic activity of rat peritoneal mast cells (PMC), but had an inhibitory effect. Preincubation for 2 h with 100 and 200 U/ml of IFN-gamma and IFN-alpha/beta, respectively, inhibited PMC cytotoxicity against WEHI-164 target cells. Lower concentrations of IFN-gamma (12.5 U/ml) and IFN-alpha/beta (25 U/ml) inhibited cytotoxicity of PMC after 8 h preincubation. The inhibitory effect of IFN was concentration and time dependent. In contrast to cytotoxicity, the release of histamine by PMC was not stimulated by the target cells WEHI-164 and there was no correlation between histamine release and cytotoxic activity of PMC. Specific antibody to subclasses of IFN prevented the inhibition of PMC cytotoxic activity, but preincubation with antibodies to the alternate subclass of IFN did not affect the observed inhibition. Moreover, the presence of both subclasses of IFN showed an additive inhibition of PMC cytotoxicity. The cytotoxic activity of PMC can be completely inhibited by the addition of anti-TNF during the assay. At high concentrations (400 U/ml), IFN inhibited the release of TNF from PMC. In the presence of RNA or protein synthesis inhibitors, IFN did not inhibit cytotoxicity of PMC further. We postulate that IFN may alter gene expression in mast cells in a manner that down-regulates their functions.

Animals↗
PubMed1990

Mast cell heterogeneity: two-dimensional gel electrophoretic analyses of rat peritoneal and intestinal mucosal mast cells.

Peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC) were purified from rats infected with the nematode Nippostrongylus brasiliensis. Overall protein constituents of both mast cell subtypes were analyzed by two-dimensional gel electrophoresis using either nonequilibrium pH gradient electrophoresis (NEPHGE) or isoelectric focusing (IEF) in the first dimension and SDS-PAGE (10%) in the second dimension followed by silver staining. PMC had seven dominant basic proteins (PB2-8; pI 9-9.5) with estimated molecular masses of 26 to 37 kDa, as well as 80 to 90 neutral or acidic proteins, most of which had pI 6 to 7.5 and estimated molecular masses of 20 to 100 kDa. All the basic proteins were granule-associated. Three basic proteins, PB6 (29 kDa), PB7 (28 kDa) and PB8 (RMCP I, 26 kDa), bound [3H]diisopropyl fluorophosphate (DFP), suggesting that they are serine proteases. However, only PB8 was reactive with antibodies to RMCP I. Another basic component (less than 14 kDa), perhaps a degradation product of PB6, PB7 or PB8, also bound [3H]DFP. By comparison, IMMC possessed nine basic proteins (IB1-9) and, in general, they were more acidic (pI about 8.5-9) than those of PMC. Four major basic proteins (IB6-9) were all 24 kDa but were slightly different in isoelectric points. These and another 46-kDa basic component (IB2) were reactive with antibodies to RMCP II and bound [3H]DFP. There were no other DFP-binding proteins in IMMC. In spite of remarkable differences between basic granule-associated proteins in PMC and basic proteins in IMMC, spots in the neutral-acidic range were for the most part similar in the two mast cell subsets, although quantitative differences were evident for some spots. Thus, rat mast cell populations from the peritoneal cavity and intestinal mucosa exhibit marked heterogeneity in their protein constituents with basic pI, including in their granule-associated proteins with serine protease activity.

Animals↗
PubMed1990

Alterations in regional blood flow in rats following sensitization to the nematode Nippostrongylus brasiliensis: effects of PAF antagonists.

1. Changes in tissue and organ blood flow associated with sensitization of rats to the nematode parasite, Nippostrongylus brasiliensis, were studied 30 to 35 days after infection, a time when very few worms remain in the animal. 2. Neither active nor passive sensitization modified heart rate, mean arterial blood pressure, cardiac output or total peripheral resistance. Passive sensitization and administration of non-immune sera did not modify blood flow to any of the tissues studied. 3. Active sensitization increased hepatic arterial blood flow, but decreased blood flow to the stomach, duodenum, jejunum and the submandibular glands. These effects cannot be attributed to residual nematode infections as treatment with the anthelmintic, thiabendazole, did not alter blood flow relative to untreated, actively sensitized rats. 4. The effects of active sensitization on blood flow were probably due to an action of platelet-activating factor (PAF) since treatment of actively sensitized animals with the selective antagonists, WEB-2086 and BN 52021, reversed the decrease in flow seen to the intestinal regions. The PAF antagonists increased blood flow to the kidneys and the trachea of sensitized animals. 5. These results suggest that the PAF released from undetermined sources in nematode-sensitized rats, produces altered blood flow, primarily to the stomach and proximal small bowel.

Animals↗
PubMed1990

Hemodynamic changes associated with anaphylaxis in parasite-sensitized rats.

Thirty days after infection with the parasite Nippostrongylus brasiliensis, microcirculatory changes associated with worm allergen-induced anaphylactic shock in pentobarbital-anesthetized rats were studied. Allergen challenge decreased heart rate, mean arterial blood pressure, and cardiac output (CO) for 60 min, although CO increased markedly by 240 min. Blood flow (ml.min-1.g wet tissue-1) was reduced to most tissues, with the exception of the kidneys, hepatic arterial, heart, and brain, for 60 min. Depressed flow to the adrenals and enhanced flow to the kidneys, trachea, stomach, and mesentery were observed at 240 min. The percent distribution of cardiac output per gram wet tissue (%CO/g) increased initially to the kidneys, heart, liver, and cervical spinal cord, decreased to the spleen and cecum, and remained unchanged to other gastrointestinal tissues. The %CO/g to most tissues returned to normal by 60 min but decreased to the adrenals at 240 min. The intestine is an important organ affected by anaphylaxis in the rat, and the liver may participate in alleviating anaphylaxis.

Anaphylaxis↗
PubMed1990

Characterization of nonspecific esterase activity in macrophages and intestinal epithelium of the rat.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.

Animals↗
PubMed1990

Trickle infections with Nippostrongylus brasiliensis in rats: larval migration through the lungs.

The effects of exposure of rats to repeated low-level (trickle) infections with Nippostrongylus brasiliensis were assessed by measuring intestinal and lung worm burdens. Worm recoveries from the intestine, made during a period of trickle infection in rats of different ages, showed a virtually complete rejection of intestinal worms in old rats and a partial rejection in young rats. Recoveries from lungs were made in young rats after challenge infection with 500 third-stage (L3) larvae, given after a 2- or 4-wk period of sensitization, during which rats were infected with 10 or 20 doses of 25 larvae. Such trickle infections elicited a strong host response to a challenge infection, manifested by low recoveries of larvae and an increased duration of larval retention in lungs. In another group of rats sensitized by a single dose of 250 L3 larvae, the recovery of larvae from challenge infection and their clearance from the lungs were similar to these observed in rats uninfected prior to challenge. The effect of trickle infections on preintestinal stages was most pronounced and consistent in rats exposed to larvae the greater numbers of times and over the longest period.

Animals↗
PubMed1990

Characterization of Nippostrongylus brasiliensis infection in different strains of mice.

Five mouse strains, CBA/J, BALB/c, C3H/HeJ, A/J, and C57Bl/6J-bg-bg, all showed similar expulsion kinetics for Nippostrongylus brasiliensis (infective dose = 500 L3). Typically, parasite recovery was maximal on day 2 in the lungs and by day 4 in the small intestine. Few worms (less than 5% infective dose) were recovered on day 14 in all strains. These same mouse strains exhibited immune depression on day 5 of infection with mesenteric lymph node cells (MLN) showing reduced (10-30% normal) IgM, IgG, and IgA responses against heterologous antigen. The intestinal mast cell numbers and tissue histamine levels were examined in CBA/J mice. Mast cell numbers increased (normal = less than 1/villous crypt unit; VCU) from day 5 and peaked on day 12 (greater than 15/VCU). Intestinal histamine levels did not completely correlate with mast cell numbers with maximum concentrations (240 +/- 73 ng/g, 2-fold over normal) reached by day 8. Histamine concentrations in the intestine returned to normal levels by day 20.

Animals↗
PubMed1989

Isolation and characterization of lung mast cells from rats with bleomycin-induced pulmonary fibrosis.

To study the nature and extent of mast cell heterogeneity within a single species, we have developed methodologies to isolate rat lung mast cells (LMC) and have compared these to peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC). In normal and athymic nude (rnu/rnu) rats, a single intratracheal administration of bleomycin (5 U/kg) leads to pulmonary fibrosis accompanied by parenchymal hyperplasia of mast cells that are histochemically like PMC rather than IMMC. Using collagenase digestion of fibrotic rat lungs (30-80 days after bleomycin treatment), we recovered an average of 58.1 x 10(6) viable cells per rat, containing 2.5% mast cells. Control experiments in which PMC were subjected to the isolation procedure used for LMC showed that there was no qualitative effect on PMC, but that a reduction of 26-60% in responsiveness to secretagogues occurred. Isolated LMC secreted histamine in response to 48/80, A23187, substance P, VIP and somatostatin and bradykinin, but at lower levels than PMC. The anti-allergic compound theophylline, which does not inhibit antigen-induced histamine secretion by IMMC, was effective against both LMC and PMC. Taken together, the thymus independence of pulmonary mast cell hyperplasia, the histochemical characteristics and the responsiveness to secretagogues and anti-allergic compounds indicate that the majority of dispersed LMC are similar to PMC rather than to IMMC. Whether LMC should be considered analogous to PMC or, because of their size, histamine content and responsiveness to many secretagogues, intermediate between PMC and IMMC, remains to be determined through additional studies.

Animals↗
PubMed1988

Isolation and in vitro translation of mRNA from rat peritoneal mast cells and rat basophilic leukemia cells.

In the absence of any specific literature on the isolation of RNA from mast cells, our initial attempts established that unusual measures would be needed to prepare acceptable yields of high quality RNA from peritoneal mast cells of normal adult rats. Accordingly, we developed procedures for the isolation and characterization of RNA from rat peritoneal mast cells (PMC) and basophilic leukemia cells (RBL). The significant components of the procedures include: separation and removal of mast cell granules to minimize contamination of RNA with proteins and proteoglycans; use of bentonite in phenol extractions; and repetition of extractions and precipitation. The amounts of total RNA extracted from PMC were about 15% of those from RBL, although the percentage mRNA of total RNA in PMC and RBL was similar (1.8 and 2.0%). Ribosomal RNA banding patterns in agarose gel electrophoresis and in vitro translation experiments indicate that the isolated RNA can be employed for analysis of molecular mechanisms of mast cell function and heterogeneity.

Animals↗
PubMed1988

Generation of leukotriene C4, leukotriene B4, and prostaglandin D2 by immunologically activated rat intestinal mucosa mast cells.

Mucosal mast cells (MMC) were isolated from the intestine of Nippostrongylus brasiliensis-infected rats and then activated with Ag or with anti-IgE in order to assess their metabolism of arachidonic acid to leukotriene (LT) C4, LTB4, and prostaglandin D2 (PGD2). After challenge of MMC preparations of 19 +/- 1% purity with five worm equivalents of N. brasiliensis Ag, the net formation of immunoreactive equivalents of LTC4, LTB4, and PGD2 was 58 +/- 8.3, 22 +/- 4.5, and 22 +/- 3.4 ng/10(6) mast cells, respectively (mean +/- SE, n = 7). When MMC preparations of 56 +/- 9% purity were activated by Ag, the net generation of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) MMC was 107 +/- 15, 17 +/- 5.4, and 35 +/- 18 ng, respectively. These data indicate that the three eicosanoids originated from the MMC rather than from a contaminating cell. Analysis by reverse phase HPLC of the C-6 sulfidopeptide leukotrienes present in the supernatants of the activated MMC preparations of lower purity revealed LTC4, LTD4, and LTE4. In a higher purity MMC preparation only LTC4 was present, suggesting that other cell types in the mucosa are able to metabolize LTC4 to LTD4 and LTE4. The release of histamine and the generation of eicosanoids from intestinal MMC and from peritoneal cavity-derived connective tissue-type mast cells (CTMC) isolated from the same N. brasiliensis-infected rats were compared. When challenged with anti-IgE, these MMC released 165 +/- 41 ng of histamine/10(6) mast cells, and generated 29 +/- 3.6, 12 +/- 4.2, and 4.7 +/- 1.0 ng (mean +/- SE, n = 3) of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) mast cells, respectively. In contrast, CTMC isolated from the same animals and activated with the same dose of anti-IgE released approximately 35 times more histamine (5700 +/- 650 ng/10(6) CTMC), generated 7.5 +/- 2.3 ng of PGD2/10(6) mast cells, and failed to release LTC4 or LTB4. These studies establish, that upon immunologic activation, rat MMC and CTMC differ in their quantitative release of histamine and in their metabolism of arachidonic acid to LTC4 and LTB4.

Animals↗
PubMed1988

Effect of histamine on proliferation of normal human adult lung fibroblasts.

Fibrotic lung tissue shows increased connective tissue deposition and fibroblast proliferation and in addition a substantial increase in mast cell numbers in and around the fibrotic area. To elucidate the question of whether products of mast cells affect the proliferative behaviour of structural cells in the lung and thereby contribute to fibrogenesis, the effect of histamine, a prominent mast cell derived mediator, on the in vitro proliferation of primary cultures of normal adult human lung fibroblasts was studied. Histamine enhanced fibroblast proliferation in a dose dependent manner, with an optimum effect at a physiological concentration of 10(-7) mol/l. This effect occurred when cells were exposed to histamine at restricted times during cell growth and was shown to depend in part on the stage of the cell cycle reached by the fibroblasts. The histamine induced proliferation was mediated through an H2 histamine receptor on the fibroblast, being inhibited by cimetidine, an H2 antagonist, and not by pyrilamine maleate, an antagonist of the H1 receptor. Mast cell products such as histamine may interact with and promote the increased fibroblast proliferation found in pulmonary fibrosis.

Adult↗
PubMed1987

Mast cells from the human intestinal lamina propria. Isolation, histochemical subtypes, and functional characterization.

With the use of a collagenase dispersion technique, cells were isolated from the lamina propria of the human small and large intestine. The cell suspensions contained 8% mast cells, which on average contained 1 to 2 pg of histamine/cell. With the use of histochemical procedures based upon fixative sensitivity and dye binding, which identify functionally distinct mast cell subtypes in the rat, dispersed human intestinal mast cells contained approximately equal proportions of two histochemical subtypes analogous to those in the rat. Whether these are functionally distinct as in the rat remains to be determined. The histochemically mixed mast cell populations from the human intestinal mucosa secreted histamine in a dose- and energy-dependent manner in response to anti-IgE and A23187, but not 48/80. Theophylline, doxantrazole, quercetin, and salbutamol all significantly inhibited anti-IgE-induced histamine secretion by human intestinal mast cells, but cromolyn sodium and the experimental antisecretory drugs, nedocromil sodium and FPL 52694, did not inhibit histamine secretion by the mast cell mixture to a statistically significant extent. Cromolyn sodium inhibited histamine secretion by 15 to 30%, and whether this reflected inhibition of one of the two histochemical mast cell subtypes to a greater extent than the other or all the cells to a minimal degree remains to be established. Control investigations of the intestinal cell isolation procedure indicated that these qualities did not reflect effects of the cell dispersal procedure. Further characterization and analysis of intestinal mast cells is essential to determine if functionally distinct mast cell subtypes exist in human tissues.

Antibodies, Anti-Idiotypic↗