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Biomedical subjects

H R Miller

Publications and source records attributed to H R Miller.

At least 91 records · Page 5Linked to original sources
PubMed1990

Distribution of intestinal mast cell proteinase in blood and tissues of normal and Trichinella-infected mice.

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed for mouse intestinal mast cell proteinase (IMCP). Specificity was demonstrated by the absence of immunoreactivity with extracts of isolated serosal mast cells (SMC), or with high concentrations (50 micrograms/ml) of the antigenically similar rat mast cell proteinases I or II. The small and large intestines in normal mice were the major sources of IMCP, there being little or no IMCP in non-mucosal tissues. Concentrations of IMCP in normal (non-parasitized) mice were low, but were increased 100-1000-fold intestines of mice infected 10 days earlier with Trichinella spiralis. The kinetic response of secreted IMCP into the blood of mice following infection with T. spiralis was also studied. Systemic release of IMCP coincided with the immune expulsion of adult worms from the intestine, and peak concentrations (9.45 micrograms/ml IMCP) occurred 9 days after infection. The tissue distribution of IMCP, its secretion into blood, and its enteric accumulation during parasite infection, are consistent with a mucosal mast cell (MMC) source for IMCP. The results are discussed in the context of similar findings for rat mast cell proteinase II.

Animals↗
PubMed1990

Functional correlations between mucosal mast cell activity and immunity to Trichinella spiralis in high and low responder mice.

Levels of intestinal mast cell protease (IMCP) were quantified in serum, gut tissue and in intestinal fluids taken from mice infected with Trichinella spiralis during primary and secondary infections. The ability to generate a mast cell response was dependent on the response phenotype of the mouse strain used. The mast cell response in rapid responder mice (NIH) occurred sooner and was more pronounced than in either intermediate (SWR) and low responder (B1O) mice. This pattern was also reflected in the concentration of IMCP found in various tissues examined. The correlations between IMCP concentrations in blood, and worm expulsion, are discussed.

Animals↗
PubMed1990

Mapping of the rat mast cell granule proteinases RMCPI and II by enzyme-linked immunosorbent assay and paired immunofluorescence.

The distribution of the rat mast cell granule proteinases, rat mast cell proteinase I and II (RMCPI and II respectively) has been determined in rat tissues with the aid of highly sensitive and specific enzyme-linked immunosorbent assays (ELISA) and paired immunofluorescence. The major source of RMCPII is the gastrointestinal tract, although low concentrations were also detected in non-mucosal sites including thymus, mesenteric lymph nodes, liver, bone marrow, heart, kidney and spleen. Cellular localization by paired immunofluorescence showed that most cells contained either RMCPI or RMCPII, although a minor subpopulation in which individual cells contained both proteinases was also identified in a few tissues. RMCPII-containing cells predominated at mucosal surfaces but were also found in non-mucosal tissues. Individual cells expressing both RMCPI and II were present in lung, liver mesenteric lymph node and submucosa of stomach and were occasionally represented amongst serosal cells from the peritoneal cavity. Connective tissue mast cells of skin and tongue were identified as major sources of RMCPI, although this proteinase was widely distributed in all tissues examined. The present study demonstrates the heterogeneity of mast cell proteinase phenotypes in the rat and emphasises the difficulties in determining mast cell subtypes on tissue location alone.

Animals↗
PubMed1990

Role of mucosal mast cells in intestinal graft-versus-host reaction in the mouse.

Hyperplasia of mucosal mast cells (MMC) is found in many enteropathies which are caused by T lymphocytes, but their exact role is unknown. In this study we have investigated whether MMC play a part in the immunologically mediated enteropathy which occurs in mice with graft-versus-host reaction (GvHR). There were simultaneous increases in the numbers of jejunal MMC and in the concentrations of mouse intestinal mast cell proteinase both in serum and in the intestine in two separate models of GvHR. Although these changes developed in parallel with the evolving GvHR, there was no correlation between the degree of MMC activation and the severity of the intestinal pathology. In addition, mast cell deficient W/Wv mice developed systemic and intestinal GvHR as severe as their normal congenic littermates, despite a markedly deficient MMC response. We conclude that the role of MMC in enteropathy may be to regulate or repair T lymphocyte mediated immunopathology.

Animals↗
PubMed1990

Immunity to internal parasites.

This review of internal parasitic diseases discusses methods of immune diagnosis, immune mechanisms of the host, and immunoprophylaxis against both protozoan and metazoan parasites of livestock. The limiting factors for immunodiagnosis are specificity and whether the host animals respond sufficiently to produce detectable serological responses. Because the array of antigens is so complex, the method of choice for helminths is to use excretory/secretory products of the parasite as antigens for microplate ELISA. Similarly, for protozoa, immunodiagnosis is facilitated by using a few, selected, immunodominant antigens rather than extracts of whole parasite. However, serodiagnosis does not always identify parasitised animals especially in the young, malnourished or sick host. Similarly, parasites may themselves modulate the response of the host and remain serologically undetectable. The production of recombinant, pure, parasite proteins is already facilitating diagnostic serology for several parasite species and, using recombinant DNA probes, it is now possible to distinguish inter- and intra-species differences. The central role of inflammatory cytokines, the regulatory proteins produced by activated T helper cells, in modulating both protective and potentially damaging inflammatory responses is discussed. Finally, the genetic control of the immune response and its relevance to immunoprophylaxis is considered. Several interesting developments in the production of sub-unit vaccines and the potential methods of delivery are appraised.

Animals↗
PubMed1990

Interaction of murine intestinal mast cell proteinase with inhibitors (serpins) in blood; analysis by SDS-PAGE and western blotting.

The interaction of mouse intestinal mast cell proteinase (IMCP) with serine proteinase inhibitors (serpins) in blood was analysed: (i) by examining the capacity of the inhibitors in blood to block the binding of the irreversible serine esterase inhibitor [3H]diisopropyl fluorophosphate (DFP); (ii) by Western blotting. The binding of [3H]DFP to IMCP was blocked very rapidly by inhibitors in mouse serum and, by Western blotting, this inhibition was associated with the appearance of a 73,000 MW proteinase/inhibitor complex together with a series of higher (greater than 100,000) MW complexes. IMCP was not dissociated from these complexes when electrophoresed under reducing conditions, although prior heat treatment of mouse serum (60 for 30-160 min) abolished the formation of all proteinase/inhibitor complexes. Similarly, the activity of a 48,000 MW inhibitor of chymotrypsin was abolished by heat treatment. A titration experiment established that between 0.5 and 5 mg IMCP were inhibited per ml of serum. The properties and MW of the IMCP inhibitor complexes are typical of serpins and suggest that IMCP secreted during intestinal immunological reactions would be rapidly and irreversibly inactivated by plasma-derived inhibitors.

Animals↗
PubMed1989

Amino acid sequence of a mouse mucosal mast cell protease.

The amino acid sequence has been determined of a mouse mucosal mast cell protease isolated from the small intestines of mice infected with Trichinella spiralis. The active protease contains 226 residues. Those corresponding to the catalytic triad of the active site of mammalian serine proteases (His-57, Asp-102, and Ser-195 in chymotrypsin) occur in identical positions. A computer search for homology indicates 74.3% and 74.1% sequence identity of the mouse mast cell protease compared to those of rat mast cell proteases I and II (RMCP I and II), respectively. The six half-cystine residues in the mouse mast cell protease are located in the same positions as in the rat mast cell proteases, cathepsin G, and the lymphocyte proteases, suggesting that they all have identical disulfide bond arrangements. At physiological pH, the mouse and rat mucosal mast cell proteases have net charges of +3 and +4, respectively, as compared to +18 for the protease (RMCP I) from rat connective tissue mast cells. This observation is consistent with the difference in solubility between the mucosal and connective tissue mast cell proteases when the enzymes are extracted from their granules under physiological conditions.

Amino Acid Sequence↗
PubMed1989

Rapid detection of influenza virus infections in human fetal lung diploid cell cultures.

Haemadsorbing foci were found in human fetal lung (HFL) diploid cell cultures 12 h after inoculation with influenza viruses A and B. The size and number of the foci were maximal after 48 h of incubation, being limited by production of an unidentified inhibitor. By contrast, inoculation with parainfluenza virus type 3 led to haemadsorption which increased during 10 days of incubation. For the detection of influenza viruses A and B maximum sensitivity was achieved by changing the medium, the day before use to one that was serum free. The number of foci at 15.5 h post-infection and infectivity for primary African green monkey kidney (AGMK) cultures were similar. Virus infectivity and production of haemagglutinin in HFL cells were entirely cell-associated; they were not affected by treatment with trypsin. Nevertheless, influenza viruses A and B antigens were identified in the infected cells by means of immunofluorescence at 15.5 h and virus was recovered by passage of frozen and thawed cells in AGMK cultures. For rapid routine diagnosis of viral infections, the early haemadsorption test was shown to have the same sensitivity as immunofluorescence tests on specimens and virus detection by the shell-vial technique but was cheaper and simpler to perform.

Cells, Cultured↗
PubMed1989

Effect of cyclosporin A treatment on the enteropathy of graft-versus-host reaction in the rat: a quantitative study of intestinal morphology, epithelial cell kinetics and mucosal immune activity.

Mucosal graft-versus-host reaction (GvHR) of the small intestine exemplifies an immunologically mediated enteropathy that is associated with expansion of mucosal mast cells (MMC). Quantitative measures of intestinal morphology, epithelial cell kinetics and mucosal immune activity were used to assess the effect of the immunosuppressive agent, cyclosporin A (CyA), in ameliorating this enteropathy and on increased activity of MMC in the jejunum. GvHR was induced in two groups of PVGU x PVGC rats by irradiation (4.50 Gy) and intravenous injection of PVGC spleen cells (150 x 10(6)). One group remained untreated, while a second group of eight rats was treated with a 50 mg/kg dose of CyA subcutaneously given daily for the first 3 days and then every second day, and which had commenced the day before induction of GvHR. On day 14, all animals were killed. Treatment with CyA prevented intestinal crypt hyperplasia but did not affect villus length, and normalized the crypt cell production rate (CCPR) from 38 to 15 cells/crypt/h (P less than 0.0001). CyA reduced the number of MMC and jejunal content of the MMC associated protease, rat mucosal mast cell protease II (RMCPII). Mean serum RMCPII concentration was reduced from 302 (s.d. = 112) in GvHR animals to 10 (s.d. = 6) ng/mL in GvHR/CyA-treated rats (P less than 0.0001). We conclude that CyA ameliorates the enteropathy of GvHR and depresses the activation of MMC, as evident by the strongly depressed serum RMCPII concentration.

Animals↗
PubMed1989

Separate effects of irradiation and of graft-versus-host reaction on rat mucosal mast cells.

T cell mediated immune responses in the gut can produce enteropathy and malabsorption. We have investigated the relevance of mucosal mast cells (MMC) to the mechanisms of this enteropathy by using graft-versus-host reaction (GvHR) in the rat as a model of mucosal delayed type hypersensitivity. Measurements of mucosal architecture, intraepithelial lymphocytes (IEL) and MMC counts were performed in control and experimental rats, and release of rat mast cell protease II (RMCPII) into the bloodstream was used as an index of MMC activation. In unirradiated rats, jejunal MMC count was increased on day 14 of the GvHR (mean 272/mm2 v 182 in controls, p less than 0.01), as was serum RMCPII (p less than 0.01). Irradiated rats (4.5 Gy, reconstituted with isogeneic spleen cells) had low counts of IEL and crypt hyperplasia seven to 14 days after irradiation. Irradiated rats with GvHR (induced by ip injection of parental strain spleen cells) and studied on days 7, 10 and 14, had significant enteropathy with longer crypts and higher CCPR than matched irradiated animals (p less than 0.05 on day 14 when compared with irradiation alone). Intraepithelial lymphocytes counts, however, reflected only the effect of radiation. Irradiation, with or without GvHR, led to the virtual disappearance of jejunal MMC, undetectable jejunal RMCPII and very low levels of RMCPII in serum (all p less than 0.01 when compared with unirradiated controls). These experiments show that there is a modest expansion in jejunal MMC in unirradiated rats with semiallogeneic GvHR, whereas irradiation, alone or associated with GvHR, profoundly depletes MMC for at least two weeks. The enteropathy of GvHR can evolve in the virtual absence of MMC.

Animals↗
PubMed1989

IgE-mediated release of rat mast cell protease II, beta-hexosaminidase and leukotriene C4 from cultured bone marrow-derived rat mast cells.

Functional characteristics of cultured bone marrow-derived rat mast cells (BMMC) were studied. BMMC were shown to release in a time- and dose-dependent fashion the mucosal mast cell (MMC)-specific enzyme, rat mast cell protease II (RMCPII), following IgE-mediated activation in vitro. RMCPII release was temporally associated with that of the mast cell granule-derived enzyme, beta-hexosaminidase (beta-hex). Release of the pre-formed granule constituents, RMCPII and beta-hex, was associated with the generation of the membrane-derived lipid mediator, leukotriene C4 (LTC4) and, in older cultures, substantial amounts were generated (25.2 ng/10(6) BMMC). Absolute amounts of RMCPII, beta-hex and LTC4 released were dependent upon the age of the BMMC. These results extend our previous observations on the staining properties and protease content of rat BMMC and provide evidence that these cells are functionally, as well as histochemically, analogous to the MMC subset, which is so prominent during intestinal nematode infections in rats.

Animals↗
PubMed1989

Lymphocyte subpopulations in the blood of sheep persistently infected with border disease virus.

The surface phenotypes of peripheral blood lymphocytes in groups of lambs and adult sheep persistently infected with Border disease virus (P-I BD) were compared with those of healthy controls. The proportion and number of lymphocytes bearing surface immunoglobulin (sIg+) and expressing class II MHC antigen (B cells) were significantly increased. A significant increase in CD1+ lymphocytes was also evident. Conversely, the proportion of T lymphocytes in P-I BD lambs was reduced. A marked reduction in the proportion of circulating lymphocytes expressing class I MHC antigen was also observed. These findings were not affected by differences in the strain of the virus responsible for the persistent infection.

Aging↗
PubMed1988

Mast cell protease release and mucosal ultrastructure during intestinal anaphylaxis in the rat.

Intestinal anaphylaxis is associated with disturbances in gut function that are antigen-specific and dependent on mast cell degranulation. Using an animal model of intestinal anaphylaxis, we have correlated alterations in water and electrolyte transport, associated with intraluminal challenge, with specific intestinal mucosal mast cell degranulation by following systemic as well as local release of rat mast cell protease II. This protease is specific for intestinal mucosal mast cells and is known to selectively attack type IV collagen, which is found in basement membranes. Intraluminal antigen challenge in sensitized animals dramatically increased serum and intraluminal levels of rat mast cell protease II. Serum levels continued to rise throughout the duration of antigen challenge. Although light microscopy of challenged intestine demonstrated little distortion of mucosal architecture, ultrastructural examination revealed significant disruption to the basement membrane and underlying collagenous matrix of the intestinal mucosa. Our findings indicate that during mucosal immunoglobulin E-mediated reactions, rat mast cell protease II is released and is associated with ultrastructural changes in the intestinal mucosa. The systemic appearance of this specific protease provides a serum marker of intestinal anaphylaxis.

Anaphylaxis↗
PubMed1988

Proteinase phenotypes and fixation properties of rat mast cells in parasitic lesions caused by Mesocestoides corti: selective and site-specific recruitment of mast cell subsets.

The distribution, fixation properties, and protease phenotypes of mast cells populating lesions caused by the metacestode stage of the cestode Mesocestoides corti in the rat were characterized. Intraperitoneal infection with M. corti induced severe granulomatous types of reactions around the pancreas and further lesions in the liver. These sites were infiltrated with mast cells which contained either rat mast cell protease I or II derived respectively from connective tissue (CTMC) or mucosal mast cells (MMC). A proportion of cells in pancreatic granulomas had staining and fixation properties identical to those of intestinal mucosal mast cells; others were typical connective tissue mast cells. Subcutaneous inoculation of parasites was associated with nodular dermal reactions, and all of the infiltrating mast cells had the fixation and staining properties of CTMC and contained RMCPI uniquely. Increased numbers of RMCPII-containing mast cells were present in the intestines of rats infected intraperitoneally. Significant quantities of RMCPII were present in homogenates of pancreatic granulomas and in livers of rats harbouring intraperitoneal infections but none was detected in skin. These findings suggest that mast cells of different phenotypes are selectively recruited to some, but not all, lesions.

Animals↗
PubMed1988

Lymphocyte phenotypes in the abomasal mucosa of sheep infected with Haemonchus contortus.

Lymphocyte subpopulations in the abomasal mucosa of worm-free and parasitized sheep were assessed in situ. A preponderance of T-lymphocytes, with approximately equal numbers of cells expressing CD5, CD4 and CD8 antigens, was found. Most of the lymphocytes expressing CD8 lacked CD5. Using a panel of 15 monoclonal antibodies to ovine leucocyte antigens, abomasal lymphoid follicles in the mucosa were shown to resemble lymph node follicles phenotypically. Abomasal epithelial cells contained major histocompatibility complex (MHC) class II antigen. Infection or hyperimmunization of pasture-reared sheep with the gastric nematode Haemonchus contortus increased the numbers of mucosal mast cells and eosinophils but did not alter the phenotypic composition or number of mucosal lymphocytes or the pattern of expression of MHC class II antigens.

Abomasum↗
PubMed1988

Granule proteinases define mast cell heterogeneity in the serosa and the gastrointestinal mucosa of the mouse.

In order to define further mast cell heterogeneity in the mouse, affinity-purified antibodies against a 28,000 MW serine proteinase from mouse intestinal mast cells (IMCP) and against rat mast cell proteinase I (RMCPI) were used to characterize mast cell cytoplasmic granules immunohistochemically. On Western blot, anti-IMCP cross-reacted with RMCPI and with a 25,000 MW antigen from isolated mouse serosal mast cells (SMC). Anti-RMCPI did not react with IMCP, although it identified the same 25,000 MW antigen from SMC. Isolated SMC (85-90% pure) lacked the 28,000 MW IMCP on Western blot, even though, immunohistochemically, the cells were stained with both anti-RMCPI and anti-IMCP. Anti-IMCP stained the granules of more than 85% of all mast cells detected with toluidine blue in the tongue or gastrointestinal mucosa. The specificity of anti-RMCPI which, in the rat, detects very few mucosal mast cells was almost identical to that of anti-IMCP for murine tongue and gastric and large intestinal mucosae, but a significant proportion of cells in distal jejunal, ileal and caecal mucosae were not stained with this antibody. The immunohistochemistry of the large numbers of mast cells recruited to jejunum following infection 10 days previously with 300 Trichinella spiralis muscle larvae was similar to that of uninfected control mice. The results show that considerable mast cell heterogeneity exists within the gastrointestinal mucosa of the mouse and indicate that there are both similarities and differences between mouse and rat in the distribution of mast cells and of their granule proteinases.

Animals↗