Modelling the gastric epithelium for testing of new chemical entities

A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. A successful model should exhibit tight junction formation, maintenance of differentiation and polarity. Conditions for primary culture of guinea-pig gastric mucous epithelial cell monolayers on Tissue Culture Plastic (TCP) and membrane insects (Transwells) were established. Tight junction formation for cells grown on Transwells for three days was assessed by measurement of transepithelial resistance (TEER) and permeability of mannitol and fluorescein. Coating the polycarbonate filter with collagen IV, rather with collagen I, enhanced tight junction formation. TEER for cells grown on Transwells coated with collagen IV was close to that obtained with intact guinea-pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [3H] glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on TCP, but no major difference was found between cells grown on collagens I and IV. However, monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide. The proportion of cells, which stained positively for mucin with periodic Schiff reagent, was greater than 95% for all culture conditions. Gastric epithelial monolayers grown on Transwells coated with collagen IV were able to withstand transient (30 min) apical acidification to pH 3, which was associated with a decrease in [3H] mannitol flux and an increase in TEER relative to pH 7.4. The model was used to provide the first direct demonstration that an NSAID (indomethacin) accumulated in gastric epithelial cells exposed to low apical pH. In conclusion, guinea-pig epithelial cells cultured on collagen IV represent a promising model of the gastric surface epithelium suitable for screening procedures.

Interactions between oligoamines and superoxide anion

1- Oligoamines and EDTA inhibited the reduction of cytochrome-C and nitrobule tetrazolium (NBT) induced by the hypoxanthine/xanthine oxidase superoxide anion generating system in the following order of effectiveness: putrescine > diaminopropane > spermidine > EDTA > spermine > cadaverine. 2- Oligoamines and EDTA did not affect the rate of urate formation from the hypoxanthine/xanthine oxidase system. 3- Oligoamines and EDTA inhibited the reduction of cytochrome-C induced by stimulated PMNL's in the same order of effectiveness as mentioned before. 4- Oligoamines and EDTA inhibited luminol dependent stimulated PMNL's chemiluminescence. 5- Oligoamines and EDTA inhibited the aerobic photoreduction of NBT. 6- Oligoamines-copper sulphate complexes inhibited the reduction of cytochrome-C induced by the hypoxanthine/xanthine oxidase system more effectively than oligoamines or copper sulphate individually. 7- Superoxide anion, hydrogen peroxide and hydroxyl radical induced breakdown of isolated intact guinea pig liver lysosomes. 8- Oligoamines and EDTA protected isolated intact guinea pig liver lysosomes from the lytic effect of superoxide anion generated either by the hypoxanthine/xanthine oxidase system or by stimulated PMNL's. 9- Oligoamines and EDTA have no stabilizing effect on isolated intact guinea pig liver lysosomes. 10- The uptake of oligoamines by lysosomes was in the following order: putrescine > spermidine > spermine. 11- Oligoamines were metabolised into aldehyde compounds either by the hypoxanthine/xanthine oxidase system or stimulated PMNL's. 12- Oligoamines and EDTA have no effect on the activities of free lysosomal enzymes (acid phosphatase and -glucosaminidase). 13- Oligoamines and EDTA inhibited lipid peroxidation in guinea pig liver lysosomes induced either by the hypoxanthine/xanthine oxidase or ascorbic acid-ferrous sulphate. 14- Oligoamines and EDTA have no effect on the release of PGE_2 from stimulated peritoneal guinea pig PMNL's. 15- Oligoamines increased the uptake of (^3H)thymidine and (^3H)leucine by stimulated peritoneal guinea pig macrophages in the following order of effectiveness: spermine > spermidine > putrescine > cadaverine. 16- PGE_2, dibutyryl Cyclic AMP, and theophylline inhibited luminol dependent stimulated peritoneal guinea pig PMNL's chemiluminescence.

Intracellar signalling in the HGT-1 gastric cell line and in rat isolated parietal cells

The work presented in this thesis was undertaken to increase understanding of the intracellular mechanisms regulating acid secretion by gastric parietal cells. Investigation of the effects of protein kinase C on secretory activity induced by a variety of agents was a major objective. A further aim was to establish the sites at which epidermal growth factor (EGF) acts to stimulate prostaglandin E2 (PGE2) production and to inhibit acid secretion. These investigations were carried out by using the HGT-1 human gastric cancer cell line and freshly isolated rat parietal cells. In HGT-1 cells, the cyclic AMP response to histamine and to truncated glucagon-like peptide 1 (TGLP-1) was reduced when protein kinase C was activated by 12-0-tetradecanoylphorbol 13-acetate (TPA). Receptor-binding studies and experiments in which cyclic AMP production in HGT-1 cells was stimulated by gastric inhibitory polypeptide, cholera toxin and forskolin suggested that the effect of TPA was mediated by uncoupling of the histamine H2 receptor from the guanine nucleotide regulatory protein Gs, possibly by phosphorylation of the receptor. An involvement of protein kinase C α in this effect was suggested because an antibody to this isoform specifically prevented the inhibitory effects of TPA on histamine-stimulated adenylate cyclase activity in a membrane fraction prepared from HGT-1 cells. Carbachol-stimulated secretory activity in parietal cells was specifically inhibited by Ro 31-8220, a bisindolylmaleimide inhibitor of protein kinase C. Thus protein kinase C may play a role in the activation of the secretory response to carbachol. In parietal cells prelabelled with [3H]-arachidonic acid or [3H]myristic acid, EGF did not affect [3H]-fatty acid or [3H] - diacylglycerol content. No evidence for effects of EGF on phosphatidylinositol glycan-specific phospholipase C, phospholipase A2 or on low Km cyclic AMP phosphodiesterase activities were found.

Regulation of mucus secretion by cells isolated from the rat gastric mucosa

     This study was undertaken to further understanding of the mechanisms which regulate mucus secretion by rat stomach cells. Particular objectives were: (i) to develop and use a radiochemical assay to estimate the secretion of mucin by a suspension of gastric mucosal cells in vitro, (ii) to develop and use a solid-phase enzyme immunoassay (EIA) to study the regulation of the release of bulk gastric mucin from the isolated cells and (iii) to compare the results obtained with the two procedures.      Cells were isolated by exposure of gastric mucosa to pronase and EDTA. Cell suspensions were preincubated with D-[6-3H]glucosamine. [3H]-labelled material of high molecular mass released into the incubation medium, was purified by Fast Protein Liquid Chromatography, and appeared to be gastric mucin. Some unidentified [3H]-labelled material of lower molecular mass was also found in the medium. Release of [3H]-labelled high molecular mass material was essentially linearly related to time. Secretin, isoprenaline and carbachol stimulated release of [3H]-labelled high molecular mass material. The half-maximally effective concentrations of secretin and isoprenaline were 2.3nM and 34nM respectively. Histamine, gastrin and epidermal growth factor were without effect.      A rabbit polyclonal antibody was raised by using purified 'native' rat gastric mucin as immunogen. The antibody preparation appeared specific for rat gastric mucin and was used to establish a quantitative solid-phase EIA. Release of bulk mucin was essentially linearly related to time. Phorbol-12-myristate-13-acetate (PMA), forskolin and A23187 dose-dependently stimulated bulk mucin release. Synergistic interactions were observed between PMA and forskolin, and PMA and A23187. Secretin and isoprenaline were confirmed as mucin secretogogues.      In conclusion gastric mucin release was investigated for the first time by using a suspension of gastric mucosal cells. Two different assay procedures were developed. Some pathways and agents responsible for controlling mucin secretion were identified.

Delivery of phosphonoformate prodrugs

AIDS dementia complex is a common neurological syndrome thought to result from the invasion of the CNS by HIV. Phosphonoformate has anti-HIV activity but due to its charged nature is excluded from the CNS by the blood-brain barrier. Lipophilic triesters of phosphonoformate designed to improve transport properties are unsuitable prodrugs due to their rapid and complicated hydrolysis, involving competitive P-O and P-C bond cleavage. Diesters, though hydrolytically stable, are considered too polar to passively diffuse into the CNS. Hydrophilic drugs mimicking endogenous nutrients are known to be actively transported across the blood-brain barrier. In this thesis the possibility that diesters of phosphonoformate may be actively transported is investigated. Triesters of phosphonoformate with labile aryl carboxyl esterrs were synthesised and their hydrolysis followed by 31P NMR spectroscopy. The triesters were found to undergo rapid hydrolysis via P-C bond cleavage to the phosphite. Phosphonoformate diesters designed to be analogues of actively transported -keto acids have been synthesised and fully characterised. Tyrosine-phosphonoformate and lipid-phosphonoformate conjugates have also been synthesised and characterised. An in vitro model of the blood-brain barrier utilising confluent monolayers of porcine brain microvessel endothelial cells grown on a permeable support has been established. The presence of enzyme and antigen markers specific to the blood-brain barrier has been demonstrated for the endothelial cells and the diffusional properties of the model investigated with hydrophilic and lipophilic compounds. Active transport systems for -keto acids and large amino acids have been identified in the endothelial cell monolayers using 14C-pyruvate and 3H-L-tyrosine respectively. Temperature and concentration dependence of the two systems have been demonstrated and transport constants calculated. Competition with 14C-pyruvate transport was shown with other monocarboxylic acids including the anti-epileptic drug valproate. Stereospecificity was shown in that L-lactate inhibited pyruvate transport while D-lactate did not. Sodium methyl methoxycarbonylphosphonate, a phosphonoformate diester was shown not to compete for 14C-pyruvate transport indicating that this compound has no affinity for the carrier. Competition with 3H-L-tyrosine transport was shown with other large amino acids, including the anti-Parkinsonian agent L-dopa. Stereospecificity was shown using L- and D-tyrosine and L- and D-dopa. The tyrosine-phosphonoformate conjugate, which was stable under the experimental conditions, was shown to compete with 3H-Ltyrosine transport indicating that it may be actively transported at the blood-brain barrier. Thirty two triesters, diesters and monoesters of phosphonoformate, showed no activity in an anti-HIV screen above that attributable to hydrolysis to the parent compound.

Investigations of the site and mechanisms of drug-induced reductions in the reabsorption of divalent cations by the kidney

Disturbances in electrolyte homeostasis are a frequent adverse side-effect of the administration of aminoglycoside antibiotics such as gentamicin, and the antineoplastic agent cis-platinum. The aims of this work were to further elucidate the site(s) and mechanism(s) by which these drugs may produce disturbances in the renal reabsorption of calcium and magnesium. These investigations were undertaken using a range of in vivo and in vitro techniques and models. Initially, a series of in vivo studies was conducted to delineate aspects of the acute and chronic effects of both drugs on renal electrolyte handling and to select and evaluate an appropriate animal model: subsequent investigations were focused on gentamicin. In a study of the acute and chronic effects of cis-platinum administration, there were pronounced acute changes in a variety of indices of nephrotoxic injury, including electrolyte excretion. Most effects resolved but there were chronic increases in the urinary excretion of calcium and magnesium. The renal response of three strains of rat (Fischer 344, Sprague-Dawley (SD), and Wistar) to a ranges of doses of gentamicin was also investigated. Drug administration produced substantially different responses between strains, in particular marked differences in calcium and magnesium excretion. The results suggested that the SD rat was an appropriately sensitive strain for use in further investigations. Acute infusion of gentamicin in the anaesthetised SD rat produced rapid, substantial increases in the fractional excretion of calcium and magnesium, while sodium and potassium output were unaffected, confirming previous results of similar experiments using F344 rats. Studies using lithium clearance measurements in the anaesthetised SD rat were undertaken to investigate the effects of gentamicin on proximal tubular calcium reabsorption. Lithium clearance was unaffected by acute gentamicin infusion, suggesting that the site of acute gentamicin-induced hypercalciuria may not be located in the proximal tubule. Inhibition of Ca2+ ATPase activity was investigated as a potential mechanism by which calcium reabsorption could be affected after aminoglycoside administration. In vitro, both Ca2+ ATPase and Na+/K+ ATPase activity could be similarly inhibited by the presence of aminoglycosides, in a dose-related manner. Whilst inhibition of Na+/K+ ATPase could be demonstrated biochemically after in vivo administration of gentamicin, there were no concurrent effects on Ca2+ ATPase activity, suggesting that inhibition of Ca2+ ATPase activity is unlikely to be a primary mechanism of aminoglycoside-induced reductions of calcium reabsorption. Histochemical studies could not discern inhibition of either Na+/K+ ATPase or Ca2+ ATPase activity after in vivo administration of gentamicin. Selection of renal cell lines for further investigative in vitro studies on the mechanisms of altered cation reabsorption was considered using MTT (3-(4,5,-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Neutral Red cytotoxicity assays. The ability of LLC-PK1 and LLC-RK1 cell lines to correctly rank a series of nephrotoxic compounds with their known nephrotoxic potency in vivo was studied. Using these cell lines grown on semi-permeable inserts, alterations in the paracellular transport of 45Ca was investigated as a possible mechanism by which gentamicin could alter calcium reabsorption in vivo. Short term exposure (I h) of LLC-RK1 cells to gentamicin, via both cell surfaces, resulted in a reduction in paracellular permeability to both transepithelial 3H-mannitol and 45Ca fluxes. When LLC-RK1 cells were exposed via the apical surface only, similar dose-related reductions were seen to those observed when cells were exposed to the drug from both sides. Short-term basal exposure to gentamicin appeared to contribute less to the observed reductions in 3H-mannitol and 45Ca fluxes. Experiments investigating transepithelial movement of 45Ca and 3H-mannitol on LLC-PK1 cells after acute gentamicin exposure were inconclusive. Longer exposure (48 h) to gentamicin caused an increase in the permeability of the monolayer and a consequent increase in transepithelial 45Ca flux in the LLC-RK1 cell line; increases in permeability of LLC-PK1 cells to 45Ca and 3H-mannitol were not apparent under the same conditions. The site and mechanism at which gentamicin, in particular, alters calcium reabsorption cannot be definitively described from these studies. However, indirect evidence from lithium clearance studies suggests that the site of the lesion is unlikely to be located in the proximal tubule. The mechanism by which gentamicin exposure alters calcium reabsorption may be by reducing paracellular permeability to calcium rather than by altering active calcium transport processes.

The Role of protein kinase C modulation in the antiproliferative effects of bistratene A, bryostatin 1 and phorbol esters

PKC-mediated signalling pathways are important in cell growth and differentiation, and aberrations in these pathways are implicated in tumourigenesis. The objective of this project was to clarify the link between cell growth inhibition and PKC modulation.The PKC activators bryostatin 1 and 12-0-tetradecanoylphorbol-13-acetate (TPA) inhibited growth in A549 and MCF-7 adenocarcinoma cells with great potency, and induced HL-60 leukaemia cell differentiation. Bistratene A affected these cells similarly. Experiments were conducted to test the hypotheses that bistratene A exerts its effects via PKC modulation and that characteristics of cytostasis induced by bryostatin 1 and TPA depend upon PKC isozyme-specific events. After incubation of A549 cells with TPA or bistratene A, 2D phosphoprotein electrophoretograrns revealed three proteins phosphorylated by both agents. However, bistratene A was unable to induce the formation of cellular networks on the basement membrane substitute Matrigel, and staurosporine was unable to reverse bistratene A-induced [3H]thymidine uptake inhibition, unlike TPA. Bistratene A did not induce PKC translocation or downregulation, activate or inhibit A549 and MCF-7 cell cytosolic PKC or compete for phorbol ester receptors. Western blot analysis and hydroxylapatite chromatography identified PKC α, ε and ζ in these cells. Bistratene A was unable to activate any of these isoforms. Therefore the agent does not exert its antiproliferative effects by modulation of PKC activity. The abilities of bryostatin 1 and TPA (10nM-1μM) to induce PKC isoform translocation and downregulation were compared with antiproliferative effects. Both agents induced dose-dependent downregulation and translocation of PKC α and ε to particulate and nuclear cell fractions. PKC ζ was translocated to the particulate fraction by both agents in MCF-7 cells. The similarity of PKC isoform redistribution by these agents did not explain their divergent effects on cell growth, and the role of nuclear translocation of PKC in cytostasis was not confirmed by these studies. Alternative factors governing the characteristics of growth inhibition induced by these agents are discussed.

Antitumour imidazotetrazines: probes for the major groove of DNA

The antitumour imidazotetrazinones are believed to act as prodrugs for the triazene series of alkylating agents, showing a marked pteference for the alkylation of the middle guanine residue in a run of three or more contiguous guanines. However, the. exact nature of the interactions of imidazotetrazinones within the micro~environment of DNA are; as yet unknown. In order to examine such interactions a three pronged approach involving molecular modelling, synthetic chemistry and biological analysis has been undertaken during the course of this project. . Molecular modelling studies have shown that for the 8-carboxamido substituted imidazotetrazinones antitumour activity is dependent upon the. presence of a free NH group which can be involved in the formation of both intramolecular and intermolecular hydrogen bonds, and the presence of a non-bulky substituent with a small negative potential . volume. Modelling studies involving the docking of .mitozolomide into the major groove of DNA in the region of a triguanine sequence has shown that a number of hydrogen bonding interactions are feasible. A series of 8-substituted carboxamide derivatives of mitozolomide have been synthesised via the 8-acid chloride and 8-carboxylic acid derivatives including a number of peptide analogues. The peptide derivatives were based upon the key structural features of the helix-turn-helix motif of DNA-binding proteins with a view to developing agents that are capable of binding to DNA with greater selectivity. An examination of the importance of intramolecular hydrogen bonding in influencing the antitumour activity:of :the imidazotetrazinones has led to the synthesis of the novel pyrimido[4',5' :4,3]pyrazolo[5,1-d]-1,2,3,5-tetrazine ring system. In general, in vitro cytotoxicity assays showed that the new derivatives were less active against the TLX5 lymphoma cell line. than the parent compound mitozolomide despite an increased potential for hydrogen bonding interactions. Due to the high reactivity of the: tetrazinone ring system it is difficult to study the interactions between the imidazotetrazinones and DNA. Consequently a number of structural analogues that are stable under physiological conditions have been. prepared based upon the 1,2,3 triazin-4(3H)-one ring system fused with both benzene and pyrazole rings. Although the 3-methylbenzotriazinones failed to antagonise the cytotoxic activity of temozolomide encouraging results with a 3-methylpyrazolotriazinone may suggest the existence of an imidazotetrazinone receptor site within DNA. The potential of guanine rich sequences to promote the alkylating selectivity of imidazotetrazinones by acting as a catalyst for ring cleavage and thereby generation of the alkylating agent was examined. Experiments involving the monitoring: of the rate of breakdown of mitozolomide incubated in the presence of synthetic oIigonucleotides did not reveal any catalytic effect resulting from the DNA. However, it was noted that the breakdown of mitozolomide was dependent upon the type of buffer used in the incubations and this may indeed mask any catalysis by the oligonucleotides.

DNA methylation at cytosine position 5

DNA methylation appears to be involved in the regulation of gene expression. Transcriptionally inactive (silenced) genes normally contain a high proportion of 5-methyl-2'-deoxycytosine residues whereas transcriptionally active genes show much reduced levels. There appears good reason to believe that chemical agents capable of methylating 2'-deoxycytosine might affect gene expression and as a result of hypermethylating promoter regions of cytosine-guanine rich oncogenic sequences, cancer related genes may be silenced. This thesis describes the synthesis of a number of `electrophilic' S-methylsulphonium compounds and assesses their ability to act as molecules capable of methylating cytosine at position 5 and also considers their potential as cytotoxic agents. DNA is methylated in vivo by DNA methyltransferase utilising S-adenoxylmethionine as the methyl donor. This thesis addresses the theory that S-adenoxylmethionine may be replaced as the methyl donor for DNA methytransferase by other sulphonium compounds. S-[3H-methyl]methionine sulphonium iodide was synthesised and experiments to assess the ability of this compounds to transfer methyl groups to cytosine in the presence of DNA methyltransferase were unsuccessful. A proline residue adjacent to a cysteine residue has been identified to a highly conserved feature of the active site region of a large number of prokaryotic DNA methyltransferases. The thesis examines the possibility that short peptides containing the Pro-Cys fragment may be able to facilitate the alkylation of cytosine position 5 by sulphonium compounds. Peptides were synthesised up to 9 amino acids in length but none were shown to exhibit significant activity. Molecular modelling techniques, including Chem-X, Quanta, BIPED and protein structure prediction programs were used to assess any structural similarities that may exist between short peptides containing a Pro-Cys fragment and similar sequences present in proteins. A number of similar structural features were observed.

Folate metabolism in the female weanling rat

The metabolism of a mixture of [2-14C] and [3',5',7,9-3H] folic acid was studied in female weanling rats. Intact folates and folate catabolites were excreted in the urine. Folate polyglutamates were found in the tissues. Rats treated with the oestrogen diethylstilbestrol and 17 -ethynyloestradiol exhibited marked changes in the metabolic handling of folic acid and folate catabolism was greatly increased compared to controls. Allopurinol treatment gave greater label retention in the gut, with a substantial increase in catabolism compared to normals. A dose response relationship was illustrated between allopurinol dose and folate catabolism. After lead acetate dosing there was little radioactivity in the urine and tissues over 72h and more radioactivity was retained in the faeces compared to normals. Excretion of intact folates was depressed, especially 5MeTHF and 10CHOTHF. A tenfold increase in both lead and folic acid dosage resulted in an even further decrease of radioactivity in the tissues and urine over 72h. Excretion in the faeces was further elevated. Ferrous sulphate administration resulted in increased catabolism. The retention of radioactivity in the liver, kidney and gut was greatly reduced. A new method of folate analysis; Sephadex LH-20 was introduced. In vitro superoxide anion formation was illustrated using an allopurinol/xanthine oxidase system. Histological studies were employed to qualitatively and quantitatively illustrate the oxidative status in livers and brains of allopurinol and ferrous sulphate dosed rats. Increased dose related formazan deposition was observed when livers of pretreated animals were incubated with nitroblue tetrazolium. Formazan deposition was reduced in pretreated animals also treated with the anti-oxidants vitamin E, mannitol or 4-hydroxy-methyl-4,6-ditertiary-butylphenol. A possible route of folate catabolism is scission by a non-enzymic oxidation involving active oxygen species.

Design, synthesis and development of PDE5 inhibitors

This research project is concerned with the design, synthesis and development of new phosphodiesterase 5 (PDE5) inhibitors with improved selectivities and lower toxicities. Two series of a 5 member and a 6 member ring fused heterocyclic compounds were designed, and synthesized. By alteration of starting materials and fragments, two virtual libraries, each is consisted of close to hundred compounds, were obtained successfully. The screening of sexual stimulation activity with rabbits demonstrated both groups of compounds were able to stimulate rabbit penile erection significantly. The following toxicity studies revealed 2-(substituted-sulfonylphenyl)-imidazo [1,5-a]-1,3,5-triazine-4-(3H)-one group possessed an unacceptable toxicity with oral LD50 about 200mg/kg; while 2-(substituted-sulfonylphenyl)-pyrrolo[2,3-d]pyrimidin-4-one group showed an acceptable toxicity with oral LD50 over 2000mg/kg. The continued bioactivity studies showed yonkenafil, the representative of 2-(substituted-sulfonylphenyl)-pyrrolo[2,3-d]pyrimidin-4-one group, has a better selectivity towards PDE5 and PDE6 than sildenafil and a better overall profile of sexual stimulation on animals than sildenafil. Chronic toxicity studies of yonkenafil further confirmed yonkenafil did not cause any serious side effect and damage on animal models and most actions were explainable. Based on evidences of the above studies, yonkenafil were recommended to enter clinical trials by the regulation authority of China, SFDA. Currently yonkenafil has been through the Phase I clinical trials and ready to progress into Phase II. Hopefully, yonkenafil will provide an alternative to the ED patients in the future.

Interactions among secretory immunoglobulin A, CD71, and transglutaminase-2 affect permeability of intestinal epithelial cells to gliadin peptides

BACKGROUND & AIMS: The transferrin receptor (CD71) is up-regulated in duodenal biopsy samples from patients with active celiac disease and promotes retrotransport of secretory immunolglobulin A (SIgA)-gliadin complexes. We studied intestinal epithelial cell lines that overexpress CD71 to determine how interactions between SIgA and CD71 promote transepithelial transport of gliadin peptides. METHODS: We analyzed duodenal biopsy specimens from 8 adults and 1 child with active celiac disease. Caco-2 and HT29-19A epithelial cell lines were transfected with fluorescence-labeled small interfering RNAs against CD71. Interactions among IgA, CD71, and transglutaminase 2 (Tgase2) were analyzed by flow cytometry, immunoprecipitation, and confocal microscopy. Transcytosis of SIgACD71 complexes and intestinal permeability to the gliadin 3H-p3149 peptide were analyzed in polarized monolayers of Caco-2 cells. RESULTS: Using fluorescence resonance energy transfer and in situ proximity ligation assays, we observed physical interactions between SIgA and CD71 or CD71 and Tgase2 at the apical surface of enterocytes in biopsy samples and monolayers of Caco-2 cells. CD71 and Tgase2 were co-precipitated with SIgA, bound to the surface of Caco-2 cells. SIgACD71 complexes were internalized and localized in early endosomes and recycling compartments but not in lysosomes. In the presence of celiac IgA or SIgA against p3149, transport of intact 3H-p3149 increased significantly across Caco-2 monolayers; this transport was inhibited by soluble CD71 or Tgase2 inhibitors. CONCLUSIONS: Upon binding to apical CD71, SIgA (with or without gliadin peptides) enters a recycling pathway and avoids lysosomal degradation; this process allows apicalbasal transcytosis of bound peptides. This mechanism is facilitated by Tgase2 and might be involved in the pathogenesis of celiac disease.

Radiolabelling of antigen and liposomes for vaccine biodistribution studies

A relatively simple and effective method to follow the movement of pharmaceutical preparations such as vaccines in biodistribution studies is to radiolabel the components. Whilst single radiolabelling is common practice, in vaccine systems containing adjuvants the ability to follow both the adjuvant and the antigen is favourable. To this end, we have devised a dual-radiolabelling method whereby the adjuvant (liposomes) is labelled with 3H and the antigen (a subunit protein) with 125I. This model is stable and reproducible; we have shown release of the radiolabels to be negligible over periods of up to 1 week in foetal calf serum at 37° C. In this paper we describe the techniques which enable the radiolabelling of various components, assessing stability and processing of samples which all for their application in biodistribution studies. Furthermore we provide examples derived from our studies using this model in tuberculosis vaccine biodistribution studies. © 2010 by the authors.

A collagen IV matrix is required for guinea pig gastric epithelial cell monolayers to provide an optimal model of the stomach surface for biopharmaceutical screening

The surface epithelial cells of the stomach represent a major component of the gastric barrier. A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. Primary cultures of guinea pig gastric mucous epithelial cells were grown on filter inserts (Transwells®) for 3 days. Tight-junction formation, assessed by transepithelial electrical resistance (TEER) and permeability of mannitol and fluorescein, was enhanced when collagen IV rather than collagen I was used to coat the polycarbonate filter. TEER for cells grown on collagen IV was close to that obtained with intact guinea pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [ 3H]glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on plastic culture plates, but no major difference was found between cells grown on collagens I and IV. The proportion of cells, which stained positively for mucin with periodic acid Schiff reagent, was greater than 95% for all culture conditions. Monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide, and were resistant to acidification of the apical medium to pH 3.0 for 30 min. A screen of nonsteroidal anti-inflammatory drugs revealed a novel effect of diclofenac and niflumic acid in reversibly reducing permeability by the paracellular route. In conclusion, the mucous cell preparation grown on collagen IV represents a good model of the gastric surface epithelium suitable for screening procedures. © 2005 The Society for Biomolecular Screening.

An Ins(1,4,5)P3 receptor in Paramecium is associated with the osmoregulatory system

In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.