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Biomedical subjects

Barry H Hirst

Publications and source records attributed to Barry H Hirst.

14 recordsLinked to original sources
PubMed2005

The novel avian protein, AWAK, contains multiple domains with homology to protease inhibitory modules.

We report the purification of a 3.5kDa peptide with antimicrobial activity from the mucosa and epithelial cells of chicken intestine. The peptide contains a pattern of cysteines characteristic of a whey acidic protein (WAP) domain and was identified as the carboxy terminal fragment of a novel 767 amino acid avian protein which has a proposed molecular weight of 81kDa. Using the conserved domain database (CDD) we identified this 81kDa protein to contain multiple amino acid motifs with homology to WAP domains and an amino acid motif with homology to a Kunitz proteinase inhibitor domain. We propose to call this avian protein AWAK (Avian WAP motif containing, Kunitz domain containing). The presence of WAP and Kunitz modules suggests that AWAK has proteinase inhibitor activity. RT-PCR analyses demonstrated expression of the AWAK gene in the chicken intestine.

Amino Acid Motifs↗
PubMed2005

Exploiting receptor biology for oral vaccination with biodegradable particulates.

The effective delivery of antigens via the oral route is an extremely desirable goal. Mucosal delivery of antigens stimulates mucosal and systemic immunity without affecting maternal antibodies and reduces the need for sterile needles or trained personnel. To date, there are very few commercially available oral vaccines and despite numerous reports in the scientific literature to show the success of biodegradable antigen carriers, none of these have achieved commercial status. Nevertheless, many studies have shown the great potential of biodegradable antigen carriers for oral vaccination in preclinical studies, but a more rational approach may be to specifically target antigen-loaded biodegradable microspheres to cells in the mucosal immune system which transport and process antigens for T cell recognition. Modern cell and molecular biology techniques have unearthed a wealth of information regarding important receptors involved in the capture of luminal antigens by microfold or membranous (M) cells and receptors on dendritic cells (DCs) which may allow future targeting of antigens to specific DC phenotypes, thus directing the immune response appropriately. In this review, we consider the use of currently available biodegradable antigen carriers and speculate on how these may be improved to more efficiently target mucosal effector sites.

Administration, Oral↗
PubMed2005

Antibiotic exposure does not influence MRP2 functional expression in Caco-2 cells.

Multidrug resistance-associated protein 2 (MRP2) is associated with active drug efflux and may influence oral bioavailability of common classes of drugs. MRP2 expression demonstrates plasticity. Caco-2 cells, a routine in vitro model for predicting oral bioavailability, are often cultured in media containing antibiotics. We have investigated whether exposure of Caco-2 cells to two common antibiotic regimes alters MRP2 functional expression. Caco-2 cells were grown in the presence or absence of either gentamicin or penicillin-streptomycin for up to 9 weeks. MRP2 functional activity was assessed by calcein efflux across the apical membrane. MRP2 protein expression was determined by immunoblots. Neither antibiotic regime resulted in consistent changes in calcein efflux across the apical membrane (reflecting MRP2 activity) or basolateral membrane (reflecting MRP3 and possibly MRP6 activity) of Caco-2 cells. MRP2 protein expression also showed no change in response to antibiotic exposure. Routine exposure of Caco-2 cells to penicillin-streptomycin or gentamicin does not affect apical MRP2 functional activity in intestinal enterocytic Caco-2 cells. Extrapolating these results to the situation in vivo suggests that the oral bioavailability of MRP substrates is not predicted to be influenced by recent courses of antibiotics.

Anti-Bacterial Agents↗
PubMed2005

Who's talking to whom? Epithelial-bacterial pathogen interactions.

Our perception that host-bacterial interactions lead to disease comes from rare, unsuccessful interactions resulting in the development of detectable symptoms. In contrast, the majority of host-bacterial interactions go unnoticed as the host and bacteria perceive each other to be no threat. In July 2004, a focused international symposium on epithelial-bacterial pathogen interactions was held in Newcastle upon Tyne (UK). The symposium concentrated on recent advances in our understanding of bacterial interactions at respiratory and gastrointestinal mucosal epithelial layers. For the host these epithelial tissues represent a first line of defence against invading bacterial pathogens. Through the discovery that the innate immune system plays a pivotal role during host-bacterial interactions, it has become clear that epithelia are being utilized by the host to monitor or communicate with both pathogenic and commensal bacteria. Interest in understanding the bacterial perspective of these interactions has lead researchers to realize that the bacteria utilize the same factors associated with disease to establish successful long-term interactions. Here we discuss several common themes and concepts that emerged from recent studies that have allowed physiologists and microbiologists to interact at a common interface similar to their counterparts -- epithelia and bacterial pathogens. These studies highlight the need for further multidisciplinary studies into how the host differentiates between pathogenic and commensal bacteria.

Bacteria↗
PubMed2004

Differential multidrug resistance-associated protein 1 through 6 isoform expression and function in human intestinal epithelial Caco-2 cells.

Multidrug resistance-associated protein (MRP) isoforms 1 through 6 mRNA are expressed in the human intestine and Caco-2 cells. In Caco-2 cells, the rank order for mRNA expression was MRP2 > or = MRP6 > MRP4 > or = MRP3 > MRP1 = MRP5. The functional expression of MRP-like activity was quantified as the efflux of the fluorescent probe calcein from confluent, polarized monolayers of Caco-2 cells. Calcein efflux was sensitive to temperature, energy depletion, and the MRP antagonist MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]. Calcein efflux across the apical membrane of Caco-2 cells exceeded that across the basolateral by approximately 2-fold, correlating with the apical localization of MRP2 visualized by immunocytochemical staining. T84 cells do not express MRP2 and show a predominance of basolateral calcein efflux over apical efflux. MRP3 was localized by immunocytochemical staining to the basolateral membrane. MRP1 staining was not localized to either membrane domain and MRP5 staining was not detected. Thus, basolateral calcein efflux may reflect a function of MRP3 or MRP4 and 6 inferred by their basolateral localization in other tissues. Basolateral, but not apical, calcein efflux was sensitive to glutathione depletion with buthioninesulfoximine, indicating that whereas MRP2-mediated apical efflux is independent of glutathione, basolateral efflux is glutathione-dependent. Benzbromarone, probenecid, pravastatin, and diclofenac were able to inhibit both apical and basolateral calcein efflux. The apical calcein efflux in Caco-2 cells was selectively sensitive to indomethacin and propranolol, but not verapamil or erythromycin, whereas the converse was observed for basal efflux. The differential pharmacological sensitivity of apical (MRP2) and basolateral calcein efflux provides tools for dissecting MRP isoform functional roles.

Antimetabolites↗
PubMed2004

M cell targeting by lectins: a strategy for mucosal vaccination and drug delivery.

Bioadhesins are a recognised method of enhancing the absorption of drugs and vaccines at mucosal surfaces. Additionally, bioadhesins allow for cell specific targeting. Lectin-mediated targeting and delivery exploits unique surface carbohydrates on mucosal epithelial cells. The antigen-sampling M cells offer a portal for absorption of colloidal and particulate delivery vehicles, including bacteria, viruses and inert microparticles. We review work supporting the use of lectins to aid targeting to intestinal M cells. Consideration is also given to lectin-mediated targeting in non-intestinal sites and to the potential application of other bioadhesins to enhance M cell transport. While substantial hurdles must be overcome before mucosal bioadhesins can guarantee consistent, safe, effective mucosal delivery, this strategy offers novel opportunities for drug and vaccine formulation.

Drug Delivery Systems↗
PubMed2004

The ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability.

Many orally administered drugs must overcome several barriers before reaching their target site. The first major obstacle to cross is the intestinal epithelium. Although lipophilic compounds may readily diffuse across the apical plasma membrane, their subsequent passage across the basolateral membrane and into blood is by no means guaranteed. Efflux proteins located at the apical membrane, which include P-glycoprotein (Pgp; MDR1) and MRP2, may drive compounds from inside the cell back into the intestinal lumen, preventing their absorption into blood. Drugs may also be modified by intracellular phase I and phase II metabolising enzymes. This process may not only render the drug ineffective, but it may also produce metabolites that are themselves substrates for Pgp and/or MRP2. Drugs that reach the blood are then passed to the liver, where they are subject to further metabolism and biliary excretion, often by a similar system of ATP-binding cassette (ABC) transporters and enzymes to that present in the intestine. Thus a synergistic relationship exists between intestinal drug metabolising enzymes and apical efflux transporters, a partnership that proves to be a critical determinant of oral bioavailability. The effectiveness of this system is optimised through dynamic regulation of transporter and enzyme expression; tissues have a remarkable capacity to regulate the amounts of protein both at transcriptional and post-transcriptional levels in order to maintain homeostasis. This review addresses the progress to date on what is known about the role and regulation of drug efflux mechanisms in the intestine and liver.

ATP-Binding Cassette Transporters↗
PubMed2004

P-glycoprotein potentiates CYP3A4-mediated drug disappearance during Caco-2 intestinal secretory detoxification.

Human intestinal Caco-2 cell monolayers grown in the presence of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) were used to test the hypothesis that drugs which interact with the apical efflux pump P-glycoprotein (Pgp) may enhance CYP3A4-mediated disappearance of substrates. 6beta-hydroxytestosterone production, a marker of CYP3A4 activity, was approximately 3- and 7-fold greater in 1,25(OH)2D3-treated cells compared to untreated cells when incubated with 50 and 500 microM testosterone, respectively, and was unaffected by the addition of digoxin to reduce Pgp activity. In the presence of digoxin, secretory transport of vinblastine and erythromycin, substrates for both Pgp and cytochrome P450 3A4 (CYP3A4), was significantly reduced, whereas absorptive transport was unaffected. In contrast, no directional transport of testosterone, a substrate for CYP3A4 only, was observed, either in the presence or absence of digoxin. Over 2 h, disappearance of erythromycin and vinblastine from the incubation medium was significantly greater from the basolateral than from the apical compartments. In the presence of digoxin, disappearance of both compounds from the basolateral, but not from the apical compartments, was significantly reduced. In contrast, disappearance of testosterone was unaffected by the addition of digoxin, demonstrating that the effect of digoxin on erythromycin and vinblastine disappearance was via inhibition of Pgp function, rather than on CYP3A4 activity. Thus, evidence is provided for Pgp/CYP3A4 co-substrates, Pgp potentiates CYP3A4-mediated drug disappearance during intestinal secretory detoxification.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Increased expression of specific intestinal amino acid and peptide transporter mRNA in rats fed by TPN is reversed by GLP-2.

Intestinal function depends on the presence of luminal nutrients and is altered during starvation and refeeding. Amino acids are essential for enterocytes, but the luminal supply is compromised with changes in dietary intake. To test the hypothesis that during periods of restricted luminal nutrient availability mucosal cells undergo adaptations aimed toward preserving amino acid supply, the expression of amino acid and peptide transporter mRNAs was quantified in rats with no oral intake, whose nutritional status was maintained with total parenteral nutrition (TPN). The role of the intestinotrophic hormone glucagon-like peptide-2 (GLP-2) was investigated in the adaptive responses. Rats were administered TPN with or without exogenous GLP-2. Amino acid and peptide transporter mRNAs in small intestine mucosa were measured by semiquantitative RT-PCR. Compared with orally fed rats, removal of luminal nutrition increased the expression of ASCT1, SAT2, and GLYT1 mRNAs in the duodenum and of ASCT2, EAAC1, NBAT, and PepT1 mRNAs in the ileum. CAT1, PAT1, and SN2 mRNA abundances were unaffected. GLP-2 reversed these effects. Three subgroups of transporters were identified by regional differences in response to TPN. This may reflect differing roles for substrates of transporters located apically and basally and along the proximal-distal axis of the intestine. The importance of maintaining amino acid supply for intestinal mucosal cells is illustrated.

Amino Acid Transport Systems↗
PubMed2002

Expression of the peptide transporter hPepT1 in human colon: a potential route for colonic protein nitrogen and drug absorption.

Substrates of the proton-coupled peptide transporter, hPepT1, include dietary di- and tripeptides plus therapeutically important drugs such as the beta-lactam antibiotics and angiotensin-converting enzyme inhibitors. Expression and function of hPepT1 in the small bowel is well established. We have compared levels of hPepT1 mRNA expression in regions of human gut by RT-PCR methods and examined the expression of hPepT1 in normal human colon using an anti-hPepT1 antipeptide antibody. hPepT1 mRNA was expressed in the large intestine, although at lower levels than in the small intestine. Quantitatively, expression in ileum was 4.6-fold greater than in sigmoid colon. Immunoreactive hPepT1 was detected in human colon at lower levels than in ileum. The pattern of expression differed between the two tissues: whilst expression in the ileum was localised to the apical enterocyte membrane along the length of the crypt-villus axis, expression in the colonocyte was detected at the apical membrane towards the luminal surface but predominantly at the basal membrane towards the base of the crypt. We conclude that distal regions of the bowel express hPepT1, which may provide a mechanism for colonic protein-nitrogen absorption and for absorption of therapeutically important peptidomimetic drugs.

Biological Transport↗
PubMed2002

Expression of junction-associated proteins differentiates mouse intestinal M cells from enterocytes.

The intestinal lymphoid follicles and associated structures are specialised antigen sampling and inductive sites of the mucosal immune system. The overlying follicle-associated epithelium (FAE) includes the specialised antigen sampling M cells that are also exploited as a route of pathogen invasion. In this immunohistochemical study we analysed the junctional complexes of the mouse intestinal FAE. Protein expression at this site resembled that of other simple epithelia. Specifically, claudin-1/3 and ZO-1 were detected in the tight junctions, E-cadherin, alpha-, beta- and gamma-catenin, vinculin, alpha-actinin and polymerised actin were associated with the adherens junctions and the desmosomes were labelled with a desmosomal protein probe. These markers failed to reveal cell type-associated variations in the tight junctions and desmosomes. In contrast, M cell adherens junctions were distinguished by enhanced expression of beta-catenin, alpha-actinin, polymerised actin and, in some areas, E-cadherin. In addition, M cell junctions exhibited increased expression of intercellular adhesion molecule-1 and phosphotyrosine, and the M cell apical surfaces displayed characteristic patterns of beta-catenin, alpha-actinin and actin expression. We have thus partially defined the junctional complexes of mouse intestinal FAE and identified M cell-specific characteristics that may further explain the biology and function of this unique cell type.

Adherens Junctions↗