Biomedical subjects

W M Gold

Publications and source records attributed to W M Gold.

At least 37 records · Page 2Linked to original sources

PubMed1989

Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs.

Supernatants obtained by degranulation of dog mastocytoma cells greatly increased the sensitivity and the magnitude of the contractile response of isolated dog bronchial smooth muscle to histamine. The enhanced contractile response was reversed completely by H1-receptor antagonists and was prevented by an inhibitor of tryptase (a major protease released with histamine from secretory granules of mast cells). The potentiation of histamine-induced contractions was reproduced by active tryptase in pure form. The contractions due to the combination of histamine and purified tryptase were abolished by the Ca2+ channel blockers nifedipine and verapamil. The bronchoconstricting effects of KCl and serotonin, which, like histamine, contract airway smooth muscle by a mechanism predominantly involving membrane potential-dependent Ca2+ transport, were also potentiated by tryptase. However, the contractile effects of acetylcholine, which contracts dog airway smooth muscle by a mechanism independent of Ca2+ channels, were unaffected by tryptase. These findings show a striking promotion of agonist-induced bronchial smooth muscle contraction by mast cell tryptase, via direct or indirect effects on Ca2+ channels, and the findings therefore suggest a novel potential mechanism of hyperresponsiveness in dog bronchi.

Airway Resistance↗

PubMed1988

Dog mastocytoma proteoglycans: occurrence of heparin and oversulfated chondroitin sulfates, containing trisulfated disaccharides, in three cell lines.

The cell-associated proteoglycans synthesized by three dog mastocytoma cell lines were isolated and their structural features compared. The lines were propagated as subcutaneous tumors in athymic mice for over 25 generations. In primary cell culture, all three lines incorporated [35S]sulfate into high molecular weight proteoglycans which were heterogeneous in size and glycosaminoglycan content. Two lines, BR and G, synthesized both a heparin proteoglycan (HPG) and a chondroitin sulfate proteoglycan (ChSPG) in different proportions. The third line, C2, synthesized predominantly a ChSPG with little or no detectable heparin. Gel filtration of the 35S-labeled HPG and ChSPG from the BR line on Sepharose CL-4B in dissociative conditions (4 M guanidine, Triton X-100) yielded a major polydisperse peak (Kav = 0.22) accounting for 70% of 35S activity. Under aggregating conditions (0.1 M sodium acetate) on Sepharose CL-4B, the BR proteoglycans eluted in the excluded volume. Proteoglycans from lines G and C2 also eluted in the void volume under nondissociative conditions, however the C2 line yielded additional fractions of smaller hydrodynamic size (Kav = 0.81) suggesting the presence of intracellular proteoglycan cleavage products or incompletely processed proteoglycans. As assessed by dissociative chromatography on Sepharose CL-4B, proteoglycans from the BR line were resistant to proteinase cleavage under conditions which degraded a rat chondrosarcoma proteoglycan. For all lines, glycosaminoglycans released by pronase/alkaline-borohydride had molecular weights ranging from 20,000 to 50,000 on gel filtration. For line BR, 75% of 35S-labeled glycosaminoglycans were degraded to oligosaccharides by nitrous acid, and the remaining 25% were degraded by chondroitinase ABC. Corresponding percentages for line G were 89% and 11%, and for line C2, 2% and 98%. Paper chromatography of the chondroitinase digestion products from lines BR and C2 showed products corresponding to unsaturated standards delta Di-diSB and delta Di-diSE, derived from the disaccharides IdoUA-2-SO4----GalNAc-4-SO4 and GlcUA----GalNAc-4,6-diSO4 respectively, in addition to smaller amounts of monosulfated disaccharides. Glycans from lines C2 and BR contained small quantities of a trisulfated disaccharide which was degraded to delta Di-diSB upon incubation with chondro-6-sulfatase. The results demonstrate the simultaneous presence of heparin and polysulfated chondroitin sulfate in dog mast cells of clonal origin.

Animals↗

PubMed1988

Selective adhesion of mast cells to tracheal epithelial cells in vitro.

In allergic and nonallergic lung diseases, if intraluminal mast cells adhere to airway epithelium, inflammatory mediators released from activated mast cells may reach high local concentrations and thus greatly affect airway function. To determine whether mast cells adhere to airway epithelial cells, radiolabeled or unlabeled dog mastocytoma cells were incubated with cultured dog tracheal epithelial cells, with extracellular matrix substrates, and with cryostat-cut sections of dog trachea. Mast cells adhered well to cultured epithelial cells (35 +/- 13% adhesion, mean +/- 1 SD, n = 23) but adhered poorly to types I and IV collagen or to fibronectin (less than 7.5% mean adhesion in all cases). Similarly, in tracheal tissue sections, mast cells adhered preferentially to epithelial cells in surface epithelium or in submucosal glands but not to basal membrane or connective tissue. Adhesion to cultured epithelial cells was a characteristics of a subpopulation of mast cells, could persist for more than 48 h, did not require energy or the presence of divalent cations, and was not mediated by a known family of leukocyte-associated adhesion glycoproteins. Adhesion was completely abolished by pretreatment of mast cells with pronase E or proteinase K but not with trypsin (up to 10 micrograms/ml at 37 degrees C for 20 min each). In contrast, pretreatment of cultured epithelial cells with any of these proteinases had no effect on adhesion. It is concluded that dog mastocytoma mast cells adhere to dog tracheal epithelial cells and do so selectively. It is suggested that mast cell adhesion to airway epithelium may play a role in the effectiveness of mast cell-epithelial cell interactions, and thus, in certain lung diseases, airway function may be affected by intraluminal mast cells more than is currently appreciated.

Animals↗

PubMed1988

Asbestos exposure-cigarette smoking interactions among shipyard workers.

We studied the roentgenograms, pulmonary function tests, and physical findings of 294 shipyard workers to evaluate asbestos exposure-cigarette smoking interactions. Roentgenographic parenchymal opacities, decreased pulmonary diffusing capacity for carbon monoxide, decreased flow at low lung volume, rales, and clubbing were each significantly related to the number of years elapsed since first exposure to asbestos and cigarette smoking status when analyzed by logistic regression. A dose-dependent cigarette smoking response that was consistent with synergism was present only for parenchymal opacities and decreased flow at low lung volume. These findings suggest that decreased flow at low lung volume, possibly reflecting peribronchiolar fibrosis, may be a functional corollary to smoking-associated parenchymal roentgenographic opacities among some asbestos-exposed individuals.

Asbestos↗

PubMed1988

Cutaneous allergic response in atopic dogs: relationship of cellular and histamine responses.

We studied the cutaneous response to intradermal antigen using clinical, histologic, and physiologic criteria in ragweed-sensitized dogs. The clinical response was measured early (the wheal at 20 minutes) and late (induration at 6 hours). We assessed cutaneous responsiveness to histamine before and 6 hours after injection (intradermally) of ragweed (n = 5, antigen group) and diluent (n = 4, 10% glycerin in 0.9% NaCl, sham group); we measured the wheal in response to histamine (1.0 ng to 0.1 mg, intradermally), constructed a dose-response curve, and interpolated the provocative dose (milligrams) of histamine required to create a wheal 10 mm larger than the response to saline control. Skin biopsy specimens were obtained before and after injection of either ragweed or diluent. Consistent with the human late-phase response, neutrophils and eosinophils were present in the dermis at 1 hour, maximal at 6 hours, and decreased at 24 hours. Mononuclear cells increased significantly at 6 hours and were the predominant cells present at 24 hours after antigen. The late clinical response correlated only with influx of eosinophils (rs = 0.85; p less than 0.005). Histamine responsiveness increased markedly after antigen (p less than 0.0001), did not change after glycerin diluent (sham), and was correlated with the intensity of neutrophil influx at 6 hours (rs = 0.69; p less than 0.05), and to a much greater degree with mononuclear cell influx at 6 hours (rs = 0.85; p less than 0.005).

Animals↗

PubMed1988

Effect of eicosapentaenoic acid in asthma.

The role of arachidonic acid metabolites in the pathogenesis of airway inflammation and clinical asthma is currently unknown. The addition of eicosapentaenoic acid (EPA) to the diet of humans has been shown to generate metabolites that are less potent than their arachidonic acid counterparts. The substitution of EPA for arachidonic acid metabolites in patients might cause a decrease in airway inflammation and an improvement in clinical asthma. We studied the effect of addition of EPA to the diet of twelve asthmatic patients. Standard clinical evaluations and pulmonary function tests were done on weeks 0, 3, 6, 10, 12 and 14. Patients ingested either low-dose EPA (0.1 g/day) or high-dose EPA (4.0 g/day) from weeks 6-14 (total of 8 weeks). There was no difference in clinical status or pulmonary function between groups at the start of the study. There was no change in clinical status or pulmonary function between or within groups at the end of 8 weeks of EPA ingestion.

Adult↗

PubMed1988

Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining.

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.

Animals↗

PubMed1988

Tryptase and chymase: comparison of extraction and release in two dog mastocytoma lines.

Mast cell secretory granules contain unique tryptic and chymotryptic serine proteases that differ between species and tissues. Direct comparison of these proteases in single-cell types has been hindered by the difficulty of obtaining adequate numbers of pure mast cells. In this study, we were able to compare tryptic and chymotryptic enzyme activity in cells of presumed monoclonal origin, using two stable lines ('BR' and 'G') of dog mastocytomas. The gel-filtration profiles, inhibitor susceptibilities and substrate preferences of tryptic and chymotryptic mastocytoma protease activities established their close resemblance to the tryptases and chymases of human and rodent mast cells. Striking heterogeneity was observed in the amounts and solubilities of the tryptic and chymotryptic activity in the two different mastocytoma cell lines. Incubation of cells from both lines with calcium ionophore A23187 caused non-cytotoxic release of protease activity. In contrast to chymase from rat connective tissue mast cells, protease activity that was insoluble after extraction at low ionic strength became soluble following ionophore-stimulated release. Neither tryptic nor chymotryptic activity was activated during degranulation, suggesting the absence of inactive precursors. Cells of the 'BR' line released both tryptic and chymotryptic activity in parallel with the granule marker histamine; cells of the 'G' line released a much smaller proportion of tryptic activity than of either chymotryptic activity or histamine. These differences in release of granule constituents from cells of common origin could be explained by developmental variations in the production of performed mediators or by differential regulation of preformed mediator release. We conclude that the differences in protease content, solubility and release in these mastocytoma lines are useful in evaluating the potential pathophysiological significance of the contribution of proteases to mast cell heterogeneity.

Animals↗

PubMed1987

Effect of inhaled ozone on lung histamine in conscious guinea pigs.

The effect of short-term ozone (O3) exposure on pulmonary mast cell function was examined. Guinea pigs were continuously exposed to 1.0 ppm O3 for 2, 4, and 8 hr. O3 exposure produced a significant decrease in lung histamine concentration. Two-hour exposure to O3 caused a 22.4 +/- 7.0% decrease in lung histamine concentration compared with controls. Ozone exposures of 4 and 8 hr caused lung histamine concentrations to decrease by 43.7 +/- 7.7 and 49.0 +/- 7.5% (P less than 0.05), respectively, without significant changes in lung water or protein, or evidence of cytotoxicity. These results suggest that O3 or its metabolites affect pulmonary mast cell function by stimulating the release of histamine from the lung.

Administration, Inhalation↗

PubMed1987

Histamine release and circulating cells after antigen inhalation in allergic dogs.

Histamine can be recovered from the blood of ragweed-sensitized dogs after aerosol antigen challenge, although its source is unknown. Neutrophils and eosinophils have been recovered from bronchoalveolar lavage fluid (BALF) obtained under identical conditions. We investigated the time course of changes in histamine levels in plasma and BALF taken from ragweed-sensitized dogs after aerosol challenge. Changes in the numbers of circulating neutrophils, eosinophils, lymphocytes, monocytes, and platelets were also studied. After 3 min, total pulmonary resistance (RL) was maximally increased and systolic blood pressure was maximally decreased. Histamine levels in plasma and BALF were increased and circulating eosinophils and neutrophils were decreased. After 15 min, platelet numbers were reduced. By 90 min, changes in RL, blood pressure, plasma and BALF histamine concentrations, and circulating neutrophils and eosinophils had returned to base-line values, but platelet numbers remained significantly decreased. Sham challenge caused no significant changes in any of these variables. Intravenous administration of histamine in doses large enough to attain plasma levels comparable with those achieved after aerosol antigen challenge resulted in no concomitant rise in BALF histamine levels. We conclude that antigen challenge in sensitized dogs causes increases in BALF and plasma histamine levels and is associated with a reduction in circulating neutrophils, eosinophils, and platelets. It is likely that antigen causes airway mast cells to release mediators that move down a concentration gradient from the airways to the pulmonary circulation.

Administration, Inhalation↗

PubMed1987

Inflammatory mediators, tachykinins and enkephalinase in airways.

Immunologic and nonimmunologic stimuli evoke responses in the airways by complex mechanisms, often involving mast cells and mast cell-derived mediators. These mechanisms can be investigated using isolated mast cell lines for biochemical and physiologic studies. Neuropeptides as well as the enzymes that degrade neuropeptides are present in airway tissues. In the case of substance P, metabolism in the airway has demonstrated physiologic significance. The physiologic response to a stimulus depends upon the interaction between locally released inflammatory mediators and neuropeptides and the enzymes which degrade them.

Endopeptidases↗

PubMed1986

Alterations in human leukocyte function induced by ingestion of eicosapentaenoic acid.

Two groups of six adults with persistent asthma, who were identical clinically, received 0.1 or 4 g of purified eicosapentaenoic acid ethyl ester (EPA) daily for 8 weeks. Both doses increased significantly the generation of leukotriene B5 (LTB5) from EPA by polymorphonuclear (PMN) and mononuclear leukocytes, while only the high dose decreased leukocyte arachidonic acid (AA) and the generation of LTB4 and prostaglandin E2 from AA. Only the high dose led to inhibition of PMN leukocyte chemotaxis to multiple stimuli by a mean of 57-70% (P less than 0.01), without altering monocyte chemotaxis, the production of platelet-activating factor by mononuclear leukocytes, or the IgE-dependent release of histamine from basophils. Both doses of EPA increased the responses of T lymphocytes to phytohemagglutinin by a mean of 73% or more (P less than 0.01) without modifying the numbers of helper and suppressor T lymphocytes. EPA affects the functions of several types of leukocytes critical to inflammation and immunity.

Administration, Oral↗

PubMed1986

Purification of Ascaris suum antigen: its allergenic activity in vitro and in vivo.

Crude aqueous extracts of Ascaris suum (CE) have been used widely to study IgE-mediated reactions in various experimental preparations. Because some CE may contain a polypeptide, a mast cell degranulating peptide (MCDP), that degranulates mast cells by nonimmunologic mechanisms, various protocols have been used to ensure that the Ascaris preparation used did not contain MCPD. In general, these protocols have assumed MCDP had been without providing proof. Even protocols designed to isolate the major antigenic determinants from CE have usually been designed to evaluate immunogenic characteristics of the purified Ascaris; thus, few systematic comparisons of CE with purified Ascaris exist concerning mast cell degranulation, and few studies have demonstrated that MCDP has been removed during purification. Since Ascaris has proved to be useful in a variety of studies of IgE-mediated reactions, particularly in large animals (dog and sheep), we have developed a protocol to purify CE and MCDP and characterize their physiochemical and immunologic properties. We compared the allergenic activity of our purified Ascaris to that of CE and MCDP in skin and lung of natively sensitized dogs and in unsensitized rat peritoneal mast cells. Our results indicate that MCDP probably contaminates CE by less than 1.0%. However, the biologic activity of MCDP in dog lung appears insignificant and probably contributes little to CE-induced reactions in doses of CE commonly used (less than or equal to 100 mg injected). If a purified Ascaris preparation is essential, our protocol will yield an Ascaris preparation that has potent IgE-mediated effects in dog preparations with insignificant contamination by MCDP.

Allergens↗

PubMed1986

Cutaneous mast cell heterogeneity: response to antigen in atopic dogs.

Because mast cells (MCs) in skin of atopic dogs are heterogeneous with respect to tissue fixation and staining properties, we determined the effect of antigen on each type of MC in vivo. Skin biopsies were done in anesthetized, ragweed-sensitized dogs before and at 1, 3, 6, and 24 hours after intradermal injections of ragweed antigen (n = 5) or glycerin diluent (n = 4). In each case, one biopsy specimen was fixed with formalin, and a second specimen from an adjacent abdominal site was fixed with basic lead acetate. In sections stained with Alcian blue, 49.7% more MCs (p less than 0.05) were detected in tissue fixed with basic lead acetate ("typical" plus "atypical" MCs: 2916 +/- 581/mm3; mean +/- SEM) than in tissue fixed with formalin ("typical" MCs: 1955 +/- 537/mm3). After antigen, the number of "typical" MCs detectable in tissue sections progressively decreased during the 24-hour period, whereas the number of "atypical" MCs was lowest at 1 hour and had increased at 24 hours. After diluent, MC numbers did not change significantly over time. A late-phase response (LPR), detected clinically as induration and edema, was present 6 hours after antigen in four of five dogs, but LPR was not detected after diluent. The size of LPR was correlated (r = 0.85; p less than 0.05) with the decrease in the number of "typical" MCs at 6 hours. We conclude that the response of the "typical" and "atypical" MC to antigen in vivo differs markedly. The "atypical" MCs participate in the early, acute response to antigen, and the "typical" MCs may be associated with the development of the LPR.

Animals↗

PubMed1986

cAMP immunocytochemistry provides evidence for functional VIP receptors in trachea.

Vasoactive intestinal peptide (VIP), first isolated from porcine intestine (S. I. Said and V. Mutt, Science Wash. DC 169: 1217-1218, 1970), has been identified in postganglionic autonomic axons of many tissues. VIP has potent regulatory effects on the function of various cell types within these tissues, ranging from relaxation of smooth muscle to ion transport. Recently, VIP has been implicated in the regulation of mucus secretion in the respiratory tract, a process involving release of macromolecules from exocrine cells and transport of ions and water across the airway mucosa. However, because airway glands and mucosa both consist of mixed cell populations, it was unclear which specific cells contained VIP receptors and contributed to VIP-evoked responses. We identified these specific cells by using immunocytochemical techniques to monitor concentration changes in adenosine 3',5'-cyclic monophosphate (cAMP), the intracellular compound known to mediate VIP responses. Serous and mucous cells of ferret tracheal submucosal glands and ciliated and basal cells of dog tracheal mucosa all increased cAMP in response to VIP stimulation. We conclude that these cell types possess VIP receptors and thus participate in VIP-stimulated responses. In contrast, ferret tracheal epithelium and dog epithelial goblet cells showed little or no reactivity after VIP, and thus we believe that these cells lack VIP receptors.

Animals↗

PubMed1986

Effects of mast cell-derived mediators on epithelial cells in canine trachea.

We examined the interaction between mast cell-derived mediators and the electrical and ion transport properties of canine tracheal epithelium. We compared the effect of mediators released by immunologic challenge of sensitized lung parenchyma with that of mediators released from canine mastocytoma cells challenged with calcium ionophore A23187. Short-circuit current (Isc) increased by 19.2 +/- 3.0 microA/cm2 in response to mediators released from sensitized lung fragments challenged with ragweed antigen. This effect was not due to histamine. When the epithelial tissues were pretreated with indomethacin, the same mediator supernatant increased Isc by only 3.8 +/- 4.3 microA/cm2. The mediators released from 10(7) mastocytoma cells challenged with calcium ionophore increased Isc by 25.1 +/- 13.6 microA/cm2. In the presence of indomethacin, the Isc increased by 2.0 +/- 0.4 microA/cm2. Mastocytoma-derived mediators produced an increase in net chloride secretion without a significant effect on net sodium absorption. This study provides direct evidence that mast cell-derived mediators can stimulate epithelial ion transport in canine trachea and suggests that the effect is indirect and dependent on intact cyclooxygenase pathways in the tracheal epithelium.

Animals↗

PubMed1986

Isolated canine mastocytoma cells: propagation and characterization of two cell lines.

Five different dog mastocytoma tumors were successfully transplanted and propagated in BALB/c nude mice. Cells from two of these tumors were passaged serially through at least four generations of mice without morphological or functional change. The average yield from a 2-cm tumor harvested from a mouse was 1.2 +/- 2.8 X 10(9) mast cells with greater than 90% viability. Cells of one line were larger and more heavily granulated than the other, and contained 1.29 +/- 0.74 pg histamine/cell (mean +/- SD). Calcium ionophore A23187 and compound 48/80 caused dose dependent histamine release with no significant difference in release from generation to generation. The smaller cells contained 0.06 +/- 0.06 pg histamine/cell. Histamine release after calcium ionophore or compound 48/80 was dose dependent and unchanged through serial passages. Following passive sensitization antigen caused dose-dependent histamine release confirming the presence of IgE receptors on these cells. In both cell lines histamine release was inhibited by terbutaline, dibutyryl adenosine 3',5'-cyclic monophosphate, or isobutylmethylxanthine. These methods provide a morphologically and functionally stable population of nearly pure canine mast cells for biochemical and physiological studies.

Animals↗

PubMed1986

Immunologic and nonimmunologic responsiveness in ragweed-sensitized dogs.

The relationship between airway responsiveness to inhaled antigen and histamine, immunologic release of lung histamine, immunologic responsiveness of skin, and specific immunoglobulin E (IgE) antibodies were examined in 11 inbred allergic dogs immunized with extracts of ragweed and grass and 5 nonimmunized control dogs from the same colony. Airway responsiveness to antigen and histamine was characterized by the doses that increased the airflow resistance of the total respiratory system to twice the control values (ED200). Highly significant correlations were found between airway responsiveness and cutaneous responsiveness to antigen and other immunologic characteristics (e.g., IgE and histamine released from lung by inhaled antigen) in all dogs. In ragweed-sensitized dogs, there was an inverse correlation between immunologic responsiveness (reflected by the cutaneous response to antigen and histamine released from lung by inhaled antigen) and nonimmunologic responsiveness of airways (histamine ED200: r = 0.73, P less than 0.05 and r = 0.75, P less than 0.01, respectively). Antigen ED200 was also correlated with histamine release from lung after antigen inhalation (r = 0.74; P less than 0.01). We conclude that airway reactions to inhaled antigen in allergic dogs are dependent not only on immunologic factors but also on the degree of nonimmunologic airway responsiveness to histamine and that these factors are correlated inversely.

Animals↗