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Biomedical subjects

B H Hirst

Publications and source records attributed to B H Hirst.

At least 19 recordsLinked to original sources
PubMed2004

Identification of chicken lysozyme g2 and its expression in the intestine.

Lysozyme is an important component of the innate immune system, protecting the gastrointestinal tract from infection. The aim of the present study was to determine if lysozyme is expressed in the chicken ( Gallus gallus) intestine and to characterise the molecular forms expressed. Immunohistochemical staining localised lysozyme to epithelial cells of the villous epithelium along the length of the small intestine. There was no evidence for lysozyme expression in crypt epithelium and no evidence for Paneth cells. Immunoblots of chicken intestinal protein revealed three proteins: a 14-kDa band consistent with lysozyme c, and two additional bands of approximately 21 and 23 kDa, the latter consistent with lysozyme g. RT-PCR analyses confirmed that lysozyme c mRNA is expressed in 4-day, but not older chicken intestine and lysozyme g in 4- to 35-day chicken intestine. A novel chicken lysozyme g2 gene was identified by in silico analyses and mRNA for this lysozyme g2 was identified in the intestine from chickens of all ages. Chicken lysozyme g2 shows similarity with fish lysozyme g, including the absence of a signal peptide and cysteines involved in disulphide bond formation of the mammalian and bird lysozyme g proteins. Analyses using SecretomeP predict that chicken lysozyme g2 may be secreted by the non-classical secretory pathway. We conclude that lysozyme is expressed in the chicken small intestine by villous enterocytes. Lysozyme c, lysozyme g and g2 may fulfil complimentary roles in protecting the intestine.

Amino Acid Sequence↗
PubMed2001

Targeting polymerised liposome vaccine carriers to intestinal M cells.

Due to their transcytotic capability, intestinal M cells may represent an efficient potential route for oral vaccine delivery. We previously demonstrated that the lectin Ulex europaeus agglutinin 1 (UEA1, specific for alpha-L-fucose residues) selectively binds to mouse Peyer's patch M cells and targets 0.5 microm polystyrene microparticles to these cells. Using a gut loop model we now demonstrate that covalently-membrane-bound UEA1 similarly targets polymerised liposomes (Orasomes, approximately 200 nm diameter), potential biocompatable oral vaccine delivery vehicles, to mouse M cells. Targeting was inhibited by alpha-L-fucose while the co-entrapped adjuvant, monophosphoryl Lipid A (MPL), failed to exert any detrimental effect on UEA1-mediated M cell targeting. Lectin-mediated M cell targeting may thus permit the efficacy of mucosal vaccines to be enhanced if cellular relationship between particle binding and immune outcome can be established.

Animals↗
PubMed2001

Exploiting M cells for drug and vaccine delivery.

The specialised antigen sampling M cells represent an efficient portal for mucosal drug and vaccine delivery. Delivery may be achieved using synthetic particulate delivery vehicles including poly(DL-lactide-co-glycolide) microparticles and liposomes. M cell interaction of these delivery vehicles is highly variable, and is determined by the physical properties of both particles and M cells. Delivery may be enhanced by coating with reagents including appropriate lectins, microbial adhesins and immunoglobulins which selectively bind to M cell surfaces. Live attenuated microorganisms are also suitable as vaccines and mucosal vectors and many, including Salmonella typhimurium, innately target to M cells. After cell surface adhesion, delivery vehicles are rapidly transported across the M cell cytoplasm to underlying lymphoid cells and may subsequently disseminate via the lymphatics. Further definition of M cell development and function should permit exploitation of their high transcytotic capacity for safe and reliable mucosal delivery.

Animals↗
PubMed2001

Glycine supply to human enterocytes mediated by high-affinity basolateral GLYT1.

BACKGROUND & AIMS: Intestinal glycine transport is involved in nutrient absorption and enterocyte homeostasis, particularly for glutathione synthesis. The primary aim of this study was to characterize the mechanism of postabsorptive (basolateral) glycine acquisition by the enterocyte. METHODS: Assimilation of [(14)C]glycine was studied in human enterocytic Caco-2 cells, and expression of the glycine transporter GLYT1 was examined in Caco-2 cells and human intestine by reverse-transcription polymerase chain reaction, immunoblotting, and immunohistochemistry. The regulation of glycine transport in Caco-2 cells by phorbol-ester-induced protein kinase C activation was investigated. RESULTS: Basolateral glycine uptake into Caco-2 cells is predominantly Na(+) and Cl(-) dependent and is 4-fold greater than apical uptake. The dominant Na(+)- and Cl(-)-dependent mechanism was characterized by a restricted inhibition profile, selectively sensitive to sarcosine, with an apparent Michaelis constant of 40-80 micromol/L, indicating system GLY. Consistent with these functional data, molecular techniques detected expression of GLYT1 messenger RNA and protein in the human intestine and Caco-2 cells. Protein kinase C activation reduced maximum velocity for GLYT1-mediated glycine uptake without effect on the Michaelis constant. The reduction in functional activity was independent of a measured protein kinase C-induced decrease in GLYT1 messenger RNA levels. CONCLUSIONS: Enterocytes express GLYT1 along the length of the crypt-villus axis, where it mediates high-affinity basolateral glycine uptake.

Amino Acid Transport Systems, Neutral↗
PubMed2000

Lectin-mediated mucosal delivery of drugs and microparticles.

Absorption of drugs and vaccines at mucosal surfaces may be enhanced by conjugation to appropriate bioadhesins which bind to mucosal epithelia. Bioadhesins might also permit cell- and site-selective targeting. One approach is to exploit surface carbohydrates on mucosal epithelial cells for lectin-mediated delivery. We review work supporting the use of lectins as mucosal bioadhesins in the gastrointestinal and respiratory tracts, the oral cavity and the eye. The gastrointestinal tract is particularly favoured for mucosal delivery. Many studies have demonstrated that the antigen sampling intestinal M cells offer a portal for absorption of colloidal delivery vehicles. Evidence is presented that M cell targeting may be achieved using M cell-specific lectins, microbial adhesins or immunoglobulins. While many hurdles must be overcome before mucosal bioadhesins can guarantee consistent, safe, effective mucosal delivery, this is an exciting area of research that has important implications for future drug and vaccine formulation.

Animals↗
PubMed2000

Differential cytokeratin and glycoconjugate expression by the surface and crypt epithelia of human palatine tonsils.

Human palatine tonsils are clinically important due to their susceptibility to tonsillitis and association with other local and systemic diseases. Paradoxically, the tonsils function as antigen sampling sites of the mucosal immune system and, consequently, the tonsil epithelia perform both protective and antigen sampling roles. These epithelia are divided into stratified squamous epithelium overlying the tonsil surface and crypt epithelium lining the tonsil crypts, the latter of which includes reticular areas which are infiltrated by lymphocytes and are responsible for antigen sampling. In this study we characterised cytokeratin and glycoconjugate expression by healthy epithelia of human palatine tonsils. We identified pan-epithelial tonsil markers and also demonstrated that the surface and reticular crypt epithelia are differentiated by the expression of multiple cytokeratins. The latter finding supports the hypothesis that these epithelia undergo alternate differentiation pathways and possess different functional roles. In addition, we identified cell subpopulations in the tonsil epithelia which may represent distinct cell subtypes including specialised antigen sampling cells. These findings establish a basis for future studies to investigate histochemical changes in tonsil epithelia that are associated with or predispose to local and/or systemic disease.

Adolescent↗
PubMed1999

Bacterial xylanase expression in mammalian cells and transgenic mice.

The energy which simple-stomached livestock can derive from dietary plant material is limited by the lack of plant polysaccharide degrading enzymes in their gastro-intestinal (GI) tract and the inefficient microbial fermentation of such material in their hind-gut. In poultry the non-starch polysaccharides found in cereal grains can also impair normal digestive function as they form viscous gels in the GI tract inhibiting the breakdown and absorption of nutrients. The nutrition of such livestock could, therefore, be improved by the introduction of enzymes able to degrade plant polysaccharides in the small intestine. We describe the expression of a xylanase, XYLY', from the bacterium Clostridium thermocellum in mammalian cells and the exocrine pancreas of transgenic mice. The enzyme is synthesised, secreted and functionally active in the eukaryote system. This work demonstrates the feasibility of generating animals with the endogenous capacity to depolymerise the xylan component of hemi-cellulose.

Animals↗
PubMed1998

Absorptive apical amiloride-sensitive Na+ conductance in human endometrial epithelium.

1. Human endometrial epithelial cells cultured on porous tissue culture supports formed tight, polarized epithelial monolayers with features characteristic of tight epithelia. Endometrial epithelial layers generated significant transepithelial electrical resistance (750 Omega cm2) and potential difference (15.3 mV), with an inward short-circuit current (Isc; 20.5 microA cm-2). 2. The Isc was linearly proportional to the external Na+ concentration and was abolished in the absence of Na+. The Isc was sensitive to apical amiloride. Net 22Na+ flux was in the absorptive apical to basolateral direction and fully accounted for the inward Isc. In addition, apical to basolateral and net 22Na+ transport were reduced in the presence of amiloride. 3. The Isc was also sensitive to addition of ouabain and Ba2+ to the basal solution, consistent with a role for basolateral Na+-K+-ATPase and K+ channels in generation of the current. 4. These data demonstrate that human endometrial epithelial cells in primary culture produce tight, functional monolayers on permeable supports. We provide the first evidence that human endometrial epithelial cells have an inward Isc accounted for by an amiloride-sensitive Na+ conductance. The Na+-absorptive function of the endometrium may provide an appropriate environment for sperm function and embryo growth.

Absorption↗
PubMed1998

Substrate upregulation of the human small intestinal peptide transporter, hPepT1.

1. Molecular mechanisms underlying physiological adaptation to increased levels of dietary peptides have been elucidated by studying the response to the substrate glycyl-L-glutamine (Gly-Gln) of the proton-coupled peptide transporter, hPepT1, in the Caco-2 human intestinal cell line. Vmax for apical uptake of [14C]glycyl-[14C]sarcosine was increased 1.64 (+/- 0.34)-fold after incubation of Caco-2 cells for 3 days in a peptide-rich medium (4 mM Gly-Gln replacing 4 mM Gln). 2. A full-length Caco-2 hPepT1 cDNA clone was identical to human small intestinal hPepT1 with the exception of a single amino acid substitution Ile-662 to Val. Transcript sizes, on Northern blots of Caco-2 poly(A)+ RNA probed with a 630 bp 5' hPepT1 cDNA probe, correspond to the reported band pattern seen with human small intestinal RNA. The dipeptide-induced increase in substrate transport was accompanied by a parallel increase of 1.92 (+/- 0.30)-fold (n = 9) in hPepT1 mRNA. This was in part due to an increase in hPepT1 mRNA half-life from 8.9 +/- 1.1 to 12.5 +/- 1.6 h (n = 3), but the increase in half-life does not account fully for the observed increase in mRNA levels, suggesting that there was also a dipeptide-mediated increase in hPepT1 transcription. 3. Anti-hPepT1-specific antipeptide antibodies localized hPepT1 exclusively to the apical membrane of human small intestinal enterocytes and Caco-2 cells. Gly-Gln supplementation of media resulted in a 1.72 (+/- 0.26)-fold (n = 5) increase in staining intensity of Caco-2 cells. 4. We conclude that Caco-2 cells provide an appropriate model for the study of adaptation of intestinal hPepT1, at the molecular level, to increased levels of dietary peptide. The magnitude of functional increase in apical peptide transport activity in response to Gly-Gln can be fully accounted for by the increased levels of hPepT1 protein and mRNA, the latter mediated by both enhanced hPepT1 mRNA stability and increased transcription. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte, independent of hormonal and/or neural control.

Adenocarcinoma↗
PubMed1998

Co-culture of two MDCK strains with distinct junctional protein expression: a model for intercellular junction rearrangement and cell sorting.

Distinct epithelial MDCK cell strains displaying extremes in transepithelial electrical resistance (paracellular permeability) have been established in co-culture and the subsequent cellular behaviour and formation of junctional complexes investigated. After high-density seeding, MDCK strain I and II cells in co-culture are initially randomly distributed but subsequently sort themselves out in a time-dependent manner to form separate homotypic aggregates. The final pattern of cell arrangement of homotypic aggregates depends on the relative seeding proportion of each cell type. Immunostaining of established marker proteins for junctional complexes has revealed that MDCK I and II cells differ in the degree of expression of the zonula-adherens-associated protein, E-cadherin, their cytoskeletal architecture and the junctional distribution of a desmosomal protein, and by showing subtle differences in tight junction staining for the zona-occludens-associated proteins, ZO-1 and occludin. The distinct pattern of junctional protein expression is maintained when the two MDCK strains are co-cultured; however, morphologically atypical intercellular junctions between heterotypic cells at the boundary of homotypic cell aggregates have been observed. It has been suggested that cell sorting, a phenomenon yet to be completely understood, is involved in important morphogenetic processes. We propose that co-culture of strains of the well-characterised MDCK cell line may be a novel but well-defined cell system for studying epithelial cell rearrangement and sorting in intact epithelial sheets.

Animals↗
PubMed1998

Increased tyrosine phosphorylation causes redistribution of adherens junction and tight junction proteins and perturbs paracellular barrier function in MDCK epithelia.

Polarized monolayers of strain II Madin-Darby canine kidney cells (MDCK II) were treated with vanadate/H2O2, known inhibitors of protein tyrosine phosphatase activity. Vanadate/H2O2 treatment resulted in a rapid increase in paracellular permeability as revealed by decreased transepithelial resistance and increased permeability to inulin. These alterations in epithelial barrier function coincided with increased phosphotyrosine immunofluorescence in the vicinity of intercellular junctions and with redistribution of F-actin, the adherens junction protein E-cadherin and the tight junction protein ZO-1. The effects of vanadate/H2O2 on intercellular junction permeability and protein distribution were completely blocked by the specific protein tyrosine kinase (PTK) inhibitor tyrphostin 25 and partially inhibited by the alternative PTK inhibitor genistein. The relative potency of these two inhibitors in blocking the effects of vanadate/H2O2 on intercellular junctions correlated with their abilities to inhibit tyrosine phosphorylation. The potent ser/thr protein kinase inhibitor staurosporine had only a small influence on the vanadate/H2O2-induced increase in paracellular permeability and did not affect the observed redistribution of intercellular junction proteins or phosphotyrosine immunofluorescence. The relative potencies of these distinct protein kinase inhibitors in reversing the effects of vanadate/H2O2 indicate that these effects are directly related to tyrosine phosphorylation. In conclusion, our data provide evidence that enhanced tyrosine phosphorylation of intercellular junction proteins in MDCK epithelia increases paracellular permeability and can also induce prominent reorganization of the junctional complex.

Actins↗
PubMed1998

The rat mucosal mast cell chymase, RMCP-II, alters epithelial cell monolayer permeability in association with altered distribution of the tight junction proteins ZO-1 and occludin.

Mucosal mast cells undergo hyperplasia in a variety of inflammatory bowel diseases including nematode infection in man and animals. The intra-epithelial localization of these cells make their soluble mediators prime candidates for modulators of epithelial function. In particular previous in vivo and ex vivo studies have established a link between the release of the highly soluble mast cell granule chymases and increased mucosal permeability. The hypothesis that the rat mast cell protease, RMCP-II, directly increases permeability to macromolecules via the paracellular route is tested in this study. Monolayers of epithelial cells (Madin-Darby canine kidney cell line) were exposed to varying concentrations of RMCP-II in vitro, in the absence of other cell types or mediators, and the effect on permeability and tight junction associated proteins was investigated. Basolateral, but not apical, exposure of polarized MDCK monolayers on porous supports to RMCP-II led to concentration- (> 100 microg/ml) and time-dependent increases in electrical conductance and permeability to mannitol (MW182) and inulin (MW5000), which was accompanied by decreases in the immunostaining of the tight junction-associated proteins occludin and ZO-1. Furthermore, prolonged exposure to RMCP-II (> 12 hours) resulted in the formation of identifiable gaps separating adjacent epithelial cells, in the absence of evidence of cytotoxicity. Inhibition of RMCP-II with Soya bean trypsin inhibitor completely abrogated the response, demonstrating that proteolysis was required. These data provide direct evidence that the rat mast cell chymase RMCP-II can, in the absence of other inflammatory mediators, increase epithelial permeability via an effect on the paracellular route.

Animals↗
PubMed1998

Ulex europaeus 1 lectin targets microspheres to mouse Peyer's patch M-cells in vivo.

The interaction of latex microspheres with mouse Peyer's patch membranous M-cells was studied in a mouse gut loop model after the microspheres were coated with a variety of agents. Carboxylated microspheres (diameter 0.5 micron) were covalently coated with lectins Ulex europaeus 1, Concanavalin A, Euonymus europaeus and Bandeiraea simplicifolia 1 isolectin-B4, human immunoglobulin A or bovine serum albumin. Of the treatments examined, only Ulex europaeus (UEA1) resulted in significant selective binding of microspheres to M-cells. UEA1-coated microspheres bound to M-cells at a level 100-fold greater than BSA-coated microspheres, but binding to enterocytes was unaffected. Incubation of UEA1-coated microspheres with alpha-L-fucose reduced M-cell binding to a level comparable with BSA-coated microspheres. This indicated that targeting by UEA1 was via a carbohydrate receptor on the M-cell surface. Adherence of UEA1-coated microspheres to M-cells occurred within 10 min of inoculation into mouse gut loops and UEA1-coated microspheres were transported to 10 microns below the apical surface of M-cells within 60 min of inoculation. UEA1-coated microspheres also targeted mouse Peyer's patch M-cells after intragastric administration. These results demonstrated that altering the surface chemistry of carboxylated polystyrene microspheres increased M-cell targeting, suggesting a strategy to enhance delivery of vaccine antigens to the mucosal immune system.

Analysis of Variance↗
PubMed1998

Inoculum composition and Salmonella pathogenicity island 1 regulate M-cell invasion and epithelial destruction by Salmonella typhimurium.

In the mouse model of Salmonella typhimurium infection, the specialized antigen-sampling intestinal M cells are the primary route of Salmonella invasion during the early stages of infection. Under certain experimental conditions, M-cell invasion is accompanied by M-cell destruction and loss of adjacent regions of the follicle-associated epithelium (FAE), although the conditions responsible for expression of the cytotoxic phenotype in a proportion of previous studies have not been defined. In the present study, we have demonstrated that the cytotoxic effect exerted by wild-type S. typhimurium on mouse Peyer's patch FAE is dependent on the inoculum composition. We have also demonstrated that the extent of FAE destruction correlates with the extent of M-cell invasion. Bacteria inoculated in Luria-Bertani (LB) broth induce extensive FAE loss and exhibit efficient M-cell invasion, whereas bacteria inoculated in phosphate-buffered saline fail to induce significant FAE disruption and invade M cells at significantly lower levels. Similarly, inoculation in LB significantly enhances invasion of Madin-Darby canine kidney cells by wild-type S. typhimurium. Mutants defective for expression of invA, a component of Salmonella pathogenicity island 1 which is vital for efficient invasion of cultured cells, fail to induce FAE destruction and, when inoculated in LB, are attenuated for M-cell invasion. Variation in inv gene expression is, therefore, one possible mechanism by which inoculate composition may regulate the virulence of wild-type S. typhimurium. Our findings suggest that the composition of the gut luminal contents may be critical in determining the outcome of naturally acquired Salmonella infections and that both vaccine formulation and dietary status of vaccine recipients may significantly affect the efficacy and safety of live Salmonella oral vaccine delivery systems.

Animals↗
PubMed1998

M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells.

Quantitative analysis of Yersinia pseudotuberculosis infection of murine gut loops revealed that significantly more wild-type bacteria associated with Peyer's patch M cells than with dome enterocytes or goblet cells. An invasin-deficient mutant was significantly attenuated for M-cell invasion, while beta1 integrin expression was demonstrated in the apical membranes of M cells but not enterocytes. M-cell targeting by Yersinia pseudotuberculosis in vivo may, therefore, be mediated primarily by the interaction of invasin with cell surface beta1 integrins.

Adhesins, Bacterial↗
PubMed1998

Cell-contact-stimulated formation of filamentous appendages by Salmonella typhimurium does not depend on the type III secretion system encoded by Salmonella pathogenicity island 1.

The formation of filamentous appendages on Salmonella typhimurium has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717-724, 1994). We have examined the roles of cell contact and Salmonella pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of S. typhimurium strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type S. typhimurium bacteria adhering to both cultured epithelial cells and to murine Peyer's patch follicle-associated epithelia. Wild-type S. typhimurium adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient S. typhimurium strains carrying mutations in components of SPI1 (invA, invG, sspC, and prgH) exhibited filamentous appendages similar to those on wild-type S. typhimurium when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, an S. typhimurium invG mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on S. typhimurium strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.

Animals↗
PubMed1997

Co-integration and expression of bacterial and genomic transgenes in the pancreatic and intestinal tissues of transgenic mice.

Previous studies in the mammary gland have reported the 'rescue' of poorly expressed cDNA transgenes by their co-integration with a genomic sequence specifically expressed in the mammary tissue. To determine whether a highly expressed genomic sequence co-integrated with a cDNA sequence can rescue expression in other tissues, the expression of a bacterial gene, celE', encoding endoglucanase E' (EGE'), was investigated in the pancreatic and intestinal epithelia of transgenic mice. To rescue pancreatic expression, the human growth hormone genomic sequence was co-integrated with the bacterial gene, whereas to rescue intestinal expression, the genomic sequence encoding the intestinal fatty acid binding protein was used. In both studies the number of transgenics expressing celE' was significantly increased (60%) by the use of a genomic sequence, but only in the intestinal tissues was the level of celE' expression improved. However, this improvement was modest, representing at maximum only a doubling in the levels of EGE'. Thus permissive integration or rescue may be general, but the overall level of rescue is often insubstantial compared to the endogenous expression of the transgene genomic DNA.

Animals↗