Biomedical subjects

H R Miller

Publications and source records attributed to H R Miller.

At least 55 records · Page 3Linked to original sources

PubMed1995

Mast cell tryptase levels in normal canine tissues.

Levels of canine tryptase from various tissues were quantified using a competition enzyme-linked immunosorbent assay (ELISA). The assay utilises an affinity-purified rabbit anti-tryptase antibody in the solid phase and alkaline-phosphatase conjugated tryptase together with unlabelled tryptase in the fluid phase. The assay will rapidly quantify 40-5000 ng ml-1 of tryptase in tissue extracts. Tissues from the skin, gut, liver and lung were studied, of which canine gut appeared to contain the highest levels of tryptase per milligram wet weight, which may suggest an important role for this enzyme at this site. This assay may prove valuable in assessing the role of mast cells in various disease states in the dog.

Animals↗

PubMed1995

Ovine mast cell heterogeneity is defined by the distribution of sheep mast cell proteinase.

The presence or absence of the granule chymase, sheep mast cell proteinase (SMCP), was determined in trachea, bronchus, bronchial lymph node, lung, thymus, spleen, liver, flank skin, abomasum, duodenum, jejunum, ileum, colon and mesenteric lymph node by immunohistochemistry and by enzyme-linked immunosorbent assay using a polyclonal, affinity purified anti-SMCP antibody. Additionally, the presence of putative ovine mast cell subsets was investigated by comparing the number of mast cells identified histochemically (toluidine blue pH 0.5) with the number detected by immunostaining. The thymus had the greatest density of mast cells (225.7 +/- 23.4 cells mm-2, histochemically) and the highest concentration of SMCP (19.7 +/- 9.3 micrograms SMCP g-1 wet tissue). There was a high degree of correlation between toluidine blue and anti-SMCP cell counts for all tissues (r2 = 0.96, P < 0.001) with the exception of skin and liver. On the basis of reactivity to the anti-SMCP antibody, two populations of mast cells were defined, notably those in gastrointestinal tissues (analogs to the mucosal mast cell subset) and those present in skin (the putative ovine connective tissue mast cell subset). Ovine mast cell heterogeneity, resulting from differential expression of SMCP, was thus confirmed.

Animals↗

PubMed1995

Quantitation of transforming growth factor-beta in plasma and pulmonary epithelial lining fluid of sheep experimentally infected with maedi-visna virus.

A model of experimental infection with EV1, a lytic British isolate of maedi-visna virus (MVV), was developed. Ten Texel sheep were allocated to two groups and inoculated by the respiratory route with different inocula. Six of the animals received 10(7.2) TCID50 (tissue culture infective dose) of EV1 strain, while four sheep were sham-inoculated with identically prepared virus-free buffer solution. Experimental infection was followed for 8 weeks post-inoculation (PI), with development of precipitating antibodies to MVV developed in the MVV-inoculated animals during the first 4 weeks PI. Transforming growth factor-beta (TGF-beta) levels, in both bronchoalveolar lavage fluid supernatant and plasma samples, were measured. Concentrations of pulmonary epithelial lining fluid (PELF) TGF-beta were calculated. TGF-beta concentrations in PELF were approximately 165-fold higher than in plasma. No significant differences in the concentrations of plasma or PELF TGF-beta, either within or between groups, were observed.

Animals↗

PubMed1995

In vitro response of lymphocytes from bronchoalveolar lavage fluid and peripheral blood to mitogen stimulation during natural maedi-visna virus infection.

To investigate the effects of maedi-visna virus (MVV) infection on cell-mediated immunity, the in vitro response of bronchoalveolar lavage fluid (BALF) and peripheral blood (BP) lymphocytes (PBL) to exogenous mitogen was analysed. BALF and PBL from control (n = 9) and MVV-infected (n = 7) animals were cultured fro 3 days in the presence and absence of concanavalin A (Con A). Lymphocyte expression of the interleukin-2 receptor (IL-2R) antigen, a parameter of lymphocyte activation, was quantified by dual-colour flow cytometry using the bovine anti-IL-2R monoclonal antibody IL-A111. IL-2R expression by lymphocytes in BALF and PB from control and MVV-infected animals, with and without Con A stimulation, were compared. In the absence of Con A stimulation, the proportion of cultured BALF CD8+ and gamma delta T cells expressing IL-2R was significantly (P < 0.05) lower for MVV-infected animals than for controls. After Con A stimulation the proportion of BALF CD4+ lymphocytes from MVV-infected animals that expressed IL-2R remained significantly (P < 0.05) lower than for controls. Comparisons within group showed that, after Con A stimulation, the proportion of all the T cell subsets in the control group expressing IL-2R, namely CD4+ (P < 0.001), CD8+ (P < 0.001) and gamma delta T cells (P < 0.05), was significantly increased. In the MVV-infected group, this increase was significant (P < 0.05) for CD4+ and CD8+ T cells, but not for gamma delta T cells. In vitro mitogen stimulation of PB T lymphocytes from both control and MVV-infected animals induced a significant elevation in the proportion of all T cell subsets expressing IL-2R when compared to cultured unstimulated control cells. However, there was considerable heterogeneity in the response to Con A of PB T cells from both groups of animals. The expression of IL-2R followed a different pattern to that of BALF lymphocytes, the proportion of unstimulated gamma delta / IL-2R+ T cells from MVV-infected animals being significantly (P < 0.05) higher than that of controls, and the proportion of cultured unstimulated CD8+ / IL-2R+ T cells from MVV-infected animals being significantly (P < 0.05) lower than that from controls. From these studies it can be concluded that the BALF T lymphocyte immune dysfunction observed during natural MVV infection, characterized by impaired IL-2R expression, is maintained under in vitro conditions.

Animals↗

PubMed1995

Basal secretion and anaphylactic release of rat mast cell protease-II (RMCP-II) from ex vivo perfused rat jejunum: translocation of RMCP-II into the gut lumen and its relation to mucosal histology.

The kinetics of the release of rat mast cell protease-II (RMCP-II) from mucosal mast cells in the jejunum of Nippostrongylus brasiliensis primed (immune) rats was investigated using ex vivo perfusion of a segment of jejunum through the cranial mesenteric artery. The aim of the study was to assess the role of the protease in anaphylaxis and in particular to ascertain whether it is responsible for the histological changes, which include widespread epithelial shedding, seen in the mucosa in in vivo models of anaphylaxis. Perfusion of the jejunal vasculature with a Krebs-Ringer buffer showed that there was basal secretion of RMCP-II by jejunal mast cells in all rats studied. The baseline concentration of RMCP-II was significantly greater (p < 0.05) in immune rats (> 7 ng/ml) previously exposed to nippostrongylus infection than in control, naive animals (< 2 ng/ml). Challenge of immune rats with 100 or 400 worm equivalents of whole worm antigen resulted in an immediate (within 40 seconds) and significant (p < 0.02) increase in the concentration of RMCP-II (to > 3 micrograms/ml) in the vascular perfusate, which was not seen in naive rats or immune rats challenged with an irrelevant antigen. Greater amounts of RMCP-II were also recovered from the jejunal lumen of immune rats compared with naive rats after challenge of both groups with worm antigen. Despite the release of microgram quantities of RMCP-II into the gut lumen and vascular perfusate, however, there were no significant changes seen in the mucosal histology. These results suggest that RMCP-II alone is not responsible fore the loss of gut epithelium seen during anaphylaxis in the rat.

Anaphylaxis↗

PubMed1994

Characterization of cultured mast cells derived from Ws/Ws mast cell-deficient rats with a small deletion at tyrosine kinase domain of c-kit.

The Ws mutant allele of rats represents a 12-base deletion at the tyrosine kinase domain of the c-kit gene. Although homozygous Ws/Ws rats were deficient in both connective tissue-type mast cells (CTMC) and mucosal-type mast cells (MMC), mast cells did develop when bone marrow cells of Ws/Ws rats were cultured in the presence of concanavalin A-stimulated spleen cell conditioned medium (ConA-SCM). Although the proliferative response of rat cultured mast cells (RCMC) derived from Ws/Ws rats to ConA-SCM was comparable to that of RCMC derived from control normal (+/+) rats, the proliferative response of Ws/Ws RCMC to rat recombinant stem cell factor (rrSCF; a ligand for the c-kit receptor tyrosine kinase) was much lower than that of +/+ RCMC. However, a slight c-kit kinase activity was detectable in Ws/Ws RCMC, and the proliferation of Ws/Ws RCMC was accelerated when rrSCF was added to ConA-SCM. Because CTMC contain rat mast cell protease-I (RMCP-I) and MMC contain RMCP-II, the phenotype of +/+ and Ws/Ws RCMC in various culture conditions was evaluated by immunohistochemistry of RMCPs. Both +/+ and Ws/Ws RCMC showed the MMC-like phenotype (RMCP-I-/II+) when they were cultured with ConA-SCM alone. Most +/+ RCMC and about half of Ws/Ws RCMC acquired a novel protease (RMCP-I+/II+) phenotype when they were cultured with rrSCF alone. However, because the number of Ws/Ws RCMC dropped to one-tenth in the medium containing rrSCF alone, the absolute number of Ws/Ws RCMC with the RMCP-I+/II+ phenotype did not increase significantly. The effect of rrSCF in inducing the novel phenotype was suppressed when ConA-SCM was added to rrSCF. In contrast, +/+ and Ws/Ws RCMC cocultured with +/+ fibroblasts showed the RMCP-I+/II+ phenotype even in the presence of ConA-SCM. Moreover, a fibroblast cell line derived from SI/SI mouse embryos that did not produce SCF did not support the survival of both +/+ and Ws/Ws RCMC but did induce the RMCP-I+/II+ phenotype in about half of +/+ and Ws/Ws RCMC when their survival was supported by the addition of ConA-SCM. The normal signal transduction through the c-kit receptor did not appear to be prerequisite for the acquisition of the RMCP-I+/II+ phenotype.

Animals↗

PubMed1994

Effects of stem cell factor (kit-ligand) and interleukin-3 on the growth and serine proteinase expression of rat bone-marrow-derived or serosal mast cells.

The effects of rat stem-cell factor (SCF) and interleukin-3 (IL-3), alone or in combination, on the in vitro growth and serine proteinase expression of rat serosal/connective-tissue mast cells (CTMC) or bone marrow-derived mast cells (BMMC) were examined. Rat SCF stimulated the growth of both CTMC and BMMC. IL-3 stimulated BMMC growth to a lesser extent than did SCF, whereas CTMC numbers did not increase in IL-3. However, SCF and IL-3 had synergistic effects on the growth of both BMMC and CTMC. SCF favoured the maintenance of rat mast cell proteinase-I (RMCP-I) in CTMC, but did not induce detectable production of RMCP-I in BMMC. In contrast, when IL-3 or lymph node-conditioned medium (LNCM) was added to SCF, a subpopulation of CTMC expressed and stored the soluble proteinase RMCP-II. In BMMC, the RMCP-II content of cells maintained in SCF was significantly less than that of cells maintained in IL-3 or LNCM. RMCP-II also appeared in the supernatants of BMMC, especially when BMMC numbers were increasing rapidly in SCF with or without IL-3 or LNCM. Thus, SCF and IL-3 can regulate the growth of rat BMMC and CTMC, as well as influence their production and release of proteinases.

Animals↗

PubMed1994

Development and validation of the Stirling Eating Disorder Scales.

The development and reliability/validity check of an 80-item, 8-scale measure for use with eating disorder patients is presented. The Stirling Eating Disorder Scales (SEDS) assess anorexic dietary behavior, anorexic dietary cognitions, bulimic dietary behavior, bulimic dietary cognitions, high perceived external control, low assertiveness, low self-esteem, and self-directed hostility. The SEDS were administered to 82 eating disorder patients and 85 controls. Results indicate that the SEDS are acceptable in terms of internal consistency, reliability, group validity, and concurrent validity.

Adult↗

PubMed1994

Fasting or dexamethasone treatment reduce protease content in rat lung mast cells and modulation of histamine synthesis by H3 receptors.

The sensitivity of mast cells to H3-receptor modulation was studied in rat lung under various hormonal conditions. The heterogeneity of mast cell sub-populations in rat lung was assessed by the tissue content of rat mast cell protease I (RMCP I) and rat mast cell protease II (RMCP II). After 24 h fasting, concentrations of RMCP I were unchanged whereas the concentration of RMCP II was significantly reduced by 49%. The [3H]histamine (HA) synthesis was concomitantly decreased by 35%. In addition, the modulation of [3H]HA synthesis by the H3 receptor agonist, (R)alpha-methylHA and by the antagonist, thioperamide, observed in control rats, was lost in fasted rats. Single and repeated administrations of dexamethasone did not influence RMCPI concentrations, but decreased the concentrations of RMCP II with a parallel decrease in [3H]HA synthesis. The inhibitory effect of (R)alpha-methylHA on [3H]HA synthesis was also reduced. These results suggest that a subpopulation of RMCP II-containing mast cells, very sensitive to environmental factors, could be the mast cells synthesizing HA in an H3-receptor-dependent manner.

Animals↗

PubMed1994

Quantitation of alpha-1 proteinase inhibitor in the pulmonary epithelial lining fluid of horses with chronic obstructive pulmonary disease.

The concentration of alpha-1 proteinase inhibitor (API) was measured in the pulmonary epithelial lining fluid (PELF) of horses with chronic obstructive pulmonary disease (COPD) while they had clinical signs and while they had none. The concentrations of total protein, albumin and API were significantly higher in the PELF of animals with clinical signs of COPD. The correlation between albumin and API in the PELF suggested that most of the API was derived from the serum.

Animals↗

PubMed1994

Measurement by ELISA of equine alpha-1-proteinase inhibitor in uterine flushings from mares.

An enzyme linked immunosorbent assay (ELISA) was developed and used to estimate the concentrations of the serine proteinase inhibitor, alpha-1 proteinase inhibitor (API), in uterine flushings recovered from mares at different stages of the oestrous cycle and before and after the induction of experimental endometritis. There was a significant increase in the concentrations of API and albumin relative to total protein in flushings recovered during oestrus compared with dioestrus but no difference was observed in the concentrations of these proteins relative to total protein before and after the induction of endometritis. A regression analysis revealed a significant correlation between the concentrations of albumin and API in the flushings examined, suggesting that the API was derived entirely from serum and was not produced locally in the uterus.

Animals↗

PubMed1994

Functional relationship between mast cells and C-sensitive nerve fibres evidenced by histamine H3-receptor modulation in rat lung and spleen.

1. Mast cell populations in rat lung and spleen were characterized by the presence of two specific protease markers, rat mast cell protease I and II, using both histochemical and radioimmunoassay techniques. Three mast cell populations with different size, morphology, and localization were found in lung and spleen and were identified according to the expression of rat mast cell protease I (RMCPI+) or rat mast cell protease II (RMCPII+) or of both proteases (RMCPI/II+). 2. All three mast cell types were in the vicinity of calcitonin-gene-related-peptide-immunoreactive (CGRP+) nerve fibres in controls as well as in rats infected by Nippostrongylus brasiliensis in which a large increase in the number of both RMCPII+ and RMCPI/II+ mast cells was found. Ablation of the CGRP+ fibres by neonatal treatment with capsaicin resulted in a marked increase in the number of RMCPII+ and RMCPI/II+ cells in lung and, even more, in spleen of adult rats. 3. The interaction of mast cells with CGRP+ C-fibres was assessed pharmacologically by evaluation of the effects of histamine H3-receptor ligands known to act on various types of nerve endings, including those of C-fibres. The effects of H3-receptor ligands were assessed in controls, nematode-infected rats and neonatally capsaicinized rats. Mast cell activity was evaluated by measurement of [3H]histamine synthesis from [3H]histidine. In control rats, administration of the H3-receptor agonist (R)-alpha-methylhistamine and antagonist thioperamide, decreased and enhanced respectively [3H]histamine synthesis in lung and spleen, indicating a tonic control of mast cell activity by histamine via H3-receptors. Such effects were not found in the jejunum, although RMCPII+ mast cells are in close apposition with neuropeptide-containing fibres. The effects of the H3-receptor agents were maintained in lung and spleen of nematode-infected rats, but were almost suppressed in capsaicinized rats. 4. It is concluded that the control of mast cells by histamine acting at H3-receptors involves neuropeptide-containing nerves and presumably reflects the operation of a local neuron-mast cell feedback loop controlling processes such as 'neurogenic inflammation'. This loop still functions when mast cells proliferate in an inflammatory condition. These observations suggest that the use of histamine H3-receptor agonists may constitute a novel therapeutic approach to limit excessive inflammatory responses resulting from dysregulation of this feedback loop.

Animals↗

PubMed1994

Hepatic recruitment of mast cells occurs in rats but not mice infected with Schistosoma mansoni.

The pathogenesis of infection with Schistosoma mansoni in rats is distinct from that in mice. Rats are non-permissive hosts and infection is terminated in the liver before egg laying commences whereas the parasites completes its life cycle in mice. Comparison of the mast cell responses in the two species reveals that a pronounced hepatic mastocytosis occurs in the rat and this is concomitant with the demise of the parasite. The majority of recruited hepatic mast cells contain the highly soluble granule chymase, rat mast cell protease-II, which is released systemically into blood during the period of parasite elimination. In contrast, very few mast cells are found in livers of parasitized mice and none contain the soluble granule chymase mouse mast cell protease-1. However, during egg deposition in the gut, an intraepithelial mastocytosis occurs in parasitized mice. These intraepithelial cells are typical mucosal mast cells as determined by their content of mouse mast cell protease-1. Recruitment of mucosal mast cells occurs in the intestinal lamina propria of infected rats soon after the parasites migrate to the liver. These findings suggest that mast cells of the mucosal phenotype are involved in the pathogenesis of the hepatic response to infection in the rat but that, in the mouse, mucosal mastocytosis is associated with intestinal sensitization by egg antigens.

Animals↗

PubMed1994

Decrease in the alpha 1-proteinase inhibitor Spi3 in equine bronchoalveolar lavage fluid.

The alpha 1-proteinase inhibitors of trypsin, Spi1, Spi3A, and Spi3B, in bronchoalveolar lavage fluid (BALF) and serum of horses were separated by electrophoresis, and their proportions were quantified in 12 control horses and 12 with chronic obstructive pulmonary disease (COPD). A significantly lower proportion of Spi3B (P < 0.05) and higher proportion of Spi1 (P < 0.02 to P < 0.01) were detected in BALF, compared with serum, in control and COPD-affected horses and appeared to be attributable to reduced Spi3 activity in BALF. There was no significant difference between the control and COPD groups in this respect, indicating that the decrease in Spi3 may be a physiologic phenomenon. The differences observed may be associated with proteolytic damage to or preferential complex formation by Spi3.

Animals↗

PubMed1993

Biochemical and immunological characterization of multiple glycoforms of mouse mast cell protease 1: comparison with an isolated murine serosal mast cell protease (MMCP-4).

Five highly soluble, chymotrypsin-like, neutral serine proteases, with molecular masses in the range 30-33 kDa, were isolated from Trichinella spiralis-infected mouse small intestine. These enzymes were closely related antigenically on Western blotting and by Ouchterlony double diffusion using a polyclonal, cross-absorbed, sheep antibody raised against mouse mast cell protease-1 (MMCP-1) and on the basis of N-terminal amino acid sequence analysis, were identified as variant forms of MMCP-1. Substrate and inhibitor analysis confirmed that the five variants (MMCP-1 A-E) had similar characteristics, although highly significant (P = 0.025 to P < 0.0001) variations in Km and kcat, were detected. Against human alpha 1-proteinase inhibitor the Ki for MMCP-1C (45 pM) was significantly (P < 0.0001) greater than those for the other proteases (0.76-2.2 pM). The differences in electrophoretic mobility are probably a result of variable glycosylation, since removal of N-linked carbohydrate produced a polypeptide of approx. 28 kDa in each case which was, like the native enzyme, immunoreactive on Western blotting. A much less soluble 28 kDa enzyme was isolated from serosal mast cells and identified as MMCP-4 by N-terminal amino acid sequencing. Like MMCP-1 it has chymotrypsin-like substrate specificities with activity at neutral pH. However, it was antigenically distinct from MMCP-1 and, using sheep anti-MMCP-1, was not detected on Western blotting or by Ouchterlony double diffusion, e.l.i.s.a. or immunohistochemistry. This last technique established that the MMCP-1 variants were uniquely present in enteric mast cells, thereby providing a highly selective means of distinguishing the mucosal and connective tissue mast cell subsets in the mouse.

Amino Acid Sequence↗

PubMed1993

Infection of Nippostrongylus brasiliensis induces development of mucosal-type but not connective tissue-type mast cells in genetically mast cell-deficient Ws/Ws rats.

Ws/Ws rats have a small deletion at the tyrosine kinase domain of the c-kit gene and are deficient in both mucosal mast cells (MMC) and connective tissue-type mast cells (CTMC). The role of the c-kit receptor in the development of MMC and CTMC was investigated by infecting Ws/Ws and control +/+ rats with Nippostrongylus brasiliensis (NB), which induces T-cell-dependent mast cell proliferation. Although mast cells did not develop in the skin of Ws/Ws rats, a significant number of mast cells developed in the jejunum after NB infection. These mast cells had the MMC protease phenotype (rat mast cell protease [RMCP] I-/II+) and lacked heparin because they were not stained with berberine sulfate. Globule leukocytes were also detected in the mucosal epithelium of these rats. However, the number of MMC and the serum concentration of RMCP II in NB-infected Ws/Ws rats were only 13% and 7% of those of NB-infected +/+ rats, respectively. A small number of mast cells also developed in the lung, liver, and mesenteric lymph nodes of Ws/Ws rats after NB infection. Although mast cells in these tissues had the MMC phenotype throughout the observation period, the increased mast cells in the lung and liver of +/+ rats acquired a CTMC-like phenotype and were RMCP I+/II+, berberine sulfate+, and formalin resistant. These results indicate that the need for the stimulus through the c-kit receptor appears to be greater in the development of CTMC in the skin as well as for CTMC-like mast cells in the lung and liver than for the development of MMC.

Animals↗

PubMed1993

Comparative studies of the Spi1 proteins of three equine alpha-1-proteinase inhibitor haplotypes following isolation by affinity chromatography.

1. Antiproteinase deficiency can result in excessive proteinase-induced tissue damage. The major anti-elastase (Spi1) protein of equine alpha 1-proteinase inhibitor (alpha 1-PI) was isolated from the plasma/serum of three common haplotypes (I, L and U). 2. The N-terminal amino acid sequences of the three inhibitors were identical, but were only approx 65-77% homologous with two other published equine Spi1 sequences. 3. All three inhibitors complexed quickly and irreversibly with equine leucocyte proteinase 2A (kass = 2 x 10(7) M-1 sec-1). They were also efficient inhibitors of chymase (rat mast cell proteinase-II; kass = 2 x 10(5) M-1 sec-1; Ki = 2 x 10(-10) M). There was therefore no evidence of deficient inhibition in the Spi1 variants of the I,L and U haplotypes.

Amino Acid Sequence↗

PubMed1993

Altered expression of mast cell proteases in the rat. Quantitative and immunohistochemical analysis of the distribution of rat mast cell proteases I and II during helminth infection.

Expression of mast cell granule protease is regulated in a tissue-specific fashion in the rat. The granule chymases rat mast cell proteases I and II (RMCP I and II) predominate in non-mucosal and mucosal sites, respectively. Intestinal mastocytosis, a T cell-mediated phenomenon associated with enteric nematodiasis, is accompanied by massive local expression of RMCP II and by release of this protease systemically into blood. The present observations, where both RMCP I and II have been quantified by ELISA and immunolocalized by paired fluorescence, show that the expression of both proteases in parasitized rats is profoundly altered at sites distant from infection. Thus, RMCP II-containing cells are recruited to liver and thymus, and in the thymus there is a > 2-fold increase in concentration of RMCP I. The latter protease is depleted from bone marrow and mesenteric lymph node early during infection, but concentrations of RMCP I in trachea/larynx, lung, and skeletal and cardiac muscle are increased. Increased mast cell counts in intestine, lung and liver are highly correlated with tissue concentrations of RMCP II.

Animals↗