Claire Marriott

The Effect of Some Preservatives Used in Nasal Preparations on Mucociliary Clearance

Abstract— The effect of methyl‐ρ‐hydroxybenzoate, propyl‐ρ‐hydroxybenzoate, chlorbutol, chlorocresol, EDTA, benzalkonium chloride, chlorhexidine, phenylmercuric nitrate and phenylmercuric borate on mucociliary transport rate of the frog palate has been examined. Following a variable number of applications all these preservatives halted transport, the first three reversibly. However, applications of thiomersal (0·01%) were well tolerated. The frog palate possesses a ciliated epithelium protected by mucus, since some of our findings are at variance with those previously reported results where the protective effect of mucus was negligible in the in‐vitro model (usually trachea) employed, it would appear that the contribution of mucus to effective mucociliary clearance should not be underestimated.

Selenium stimulates pancreatic beta-cell gene expression and enhances islet function

The present study investigated the role of selenium in the regulation of pancreatic beta‐cell function. Utilising the mouse beta‐cell line Min6, we have shown that selenium specifically upregulates Ipf1 (insulin promoter factor 1) gene expression, activating the −2715 to −1960 section of the Ipf1 gene promoter. Selenium increased both Ipf1 and insulin mRNA levels in Min6 cells and stimulated increases in insulin content and insulin secretion in isolated primary rat islets of Langerhans. These data are the first to implicate selenium in the regulation of specific beta‐cell target genes and suggest that selenium potentially promotes an overall improvement in islet function.

Preparation of hydrophobic and hydrophilic albumin microspheres and determination of surface carboxylic acid and amino residues

Abstract Albumin microspheres (MS) have been studied extensively as delivery systems for targeting drugs since they are biodegradable, non-toxic, relatively easy to prepare and their size range can be controlled. A method for albumin MS production was developed which was faster, processed larger quantities of starting material than previous methods, and had chemically reactive groupings on the MS surface to which ligands could be attached. Relatively hydrophobic, hydrophilic and also carboxymethylated MS were manufactured. The number of carboxylic acid residues was determined on the surface of these MS using 14 C-glycine ethyl ester hydrochloride as a probe, and the number of amino groups was determined using 14 C-sodium acetate as a probe. The number of carboxylic acid residues per unit surface area for the hydrophobic, hydrophilic and carboxymethylated MS was 2.1 × 10 4 , 4.1 × 10 4 and 8.4 × 10 4 , respectively, and the number of amino acid residues was 2.2 × 10 3 , 5.0 × 10 2 and 5.0 × 10 2 , respectively

Pseudoislets as primary islet replacements for research : report on a symposium at King's College London, London UK

Laboratory-based research aimed at understanding processes regulating insulin secretion and mechanisms underlying β-cell dysfunction and loss in diabetes often makes use of rodents, as these processes are in many respects similar between rats/mice and humans. Indeed, a rough calculation suggests that islets have been isolated from as many as 150,000 rodents to generate the data contained within papers published in 2009 and the first four months of 2010. Rodent use for islet isolation has been mitigated, to a certain extent, by the availability of a variety of insulin-secreting cell lines that are used by researchers world-wide. However, when maintained as monolayers the cell lines do not replicate the robust, sustained secretory responses of primary islets which limits their usefulness as islet surrogates. On the other hand, there have been several reports that configuration of MIN6 β-cells, derived from a mouse insulinoma, as three-dimensional cell clusters termed ‘pseudoislets’ largely recapitulates the function of primary islet β-cells. The Diabetes Research Group at King’s College London has been using the MIN6 pseudoislet model for over a decade and they hosted a symposium on “Pseudoislets as primary islet replacements for research”, which was funded by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), in London on 15th and 16th April 2010. This small, focused meeting was conceived as an opportunity to consolidate information on experiences of working with pseudoislets between different UK labs, and to introduce the theory and practice of pseudoislet culture to laboratories working with islets and/or β-cell lines but who do not currently use pseudoislets. This short review summarizes the background to the development of the cell line-derived pseudoislet model, the key messages arising from the symposium and emerging themes for future pseudoislet research.

Inability to process and store proinsulin in transdifferentiated pancreatic acinar cells lacking the regulated secretory pathway.

Generation of new beta-cells from the adult pancreas or the embryonic stem cells is being pursued by research groups worldwide. Success will be dependent on confirmation of true beta-cell phenotype evidenced by capacity to process and store proinsulin. The aim of these studies was to robustly determine endocrine characteristics of the AR42J rat pancreatic acinar cell line before and after in vitro transdifferentiation. beta-cell phenotypic marker expression was characterised by RT-PCR, immunostaining, western blotting, ELISA and in human preproinsulin transgene over-expression studies in wild-type AR42J cells and after culture on Matrigel basement membrane matrix with and without growth/differentiation factor supplementation. Pancreatic duodenal homeobox 1 (PDX1), forkhead box transcription factor a2 (Foxa2), glucokinase, pancreatic polypeptide and low-level insulin gene transcription in wild-type AR42J cells were confirmed by RT-PCR. Culture on Matrigel-coated plates and supplementation of medium with glucagon-like peptide 1 induced expression of the beta-cell Glut 2 with maintained expression of insulin and PDX1. Increased biosynthesis and secretion of proinsulin were confirmed by immunocytochemical staining and sensitive ELISA. Absence of the regulated secretory pathway was demonstrated by undetectable prohormone convertase expression. In addition, inability to process and store endogenous proinsulin or human proinsulin translated from a constitutively over-expressed preproinsulin transgene was confirmed. The importance of robust phenotypic characterisation at the protein level in attempted beta-cell transdifferentiation studies has been confirmed. Rodent and human sensitive/specific differential proinsulin/insulin ELISA in combination with human preproinsulin over-expression enables detailed elucidatation of core endocrine functions of proinsulin processing and storage in putative new beta-cells.

Tumor suppressor PDCD4 is a major transcript that is upregulated during in vivo pancreatic islet neogenesis and is expressed in both beta-cell and ductal cell lines

Objectives: We wished to identify a major transcript that is upregulated during in vivo pancreatic islet neogenesis and examine the expression of the gene in beta and ductal cells. Methods: Differential display polymerase chain reaction was used to identify upregulated transcripts after islet neogenesis was stimulated in the rat by brief occlusion of the main pancreatic duct. The expression of this major transcript, namely PDCD4 (programmed cell death gene 4), was measured in beta and ductal cells after stimulation with the incretin hormone glucagon-like peptide 1, mitogenic insulin, the thiazolidinedione rosiglitazone, and by high glucose concentrations. The subcellular location of the protein was also examined. Results: The expression of the Pdcd4 gene in pancreatic beta and ductal cells was found to be stimulated in a comparable manner by either glucagon-like peptide 1, insulin, and by high glucose concentrations. However, intracellular localisation of the PDCD4 protein was shown to be differentially regulated by these stimuli in beta and ductal cells. Furthermore, the thiazolidinedione rosiglitazone specifically upregulates Pdcd4 gene expression in beta cells in a time-dependent manner. Conclusion: This is the first study showing Pdcd4 expression in pancreatic cells. Our data indicate that Pdcd4 expression may be integral in the function of the adult pancreas.

The preservation of nasal preparations

The intranasal route of administration provides an effective and convenient means of delivering a number of compounds to the systemic circulation when the more usual oral or parenteral routes are inappropriate. The preservation of multi-dose nasal drops and sprays is essential. However, preservatives should not impair any of the normal functions of the nasal cavity, such as mucociliary clearance, since patients with compromised clearance can suffer extensively from respiratory tract infections. This paper reviews the results of a number of investigations into the effects of a range of commonly used preservatives on the mucociliary clearance apparatus.

The role of tumour suppressor PDCD4 in beta cell death in hypoxia

Objective Hypoxia is known to induce pancreatic beta cell dysfunction and apoptosis. Changes in Programmed Cell Death Gene 4 (PDCD4) expression have previously been linked with beta cell neogenesis and function. Our aim was to investigate the effects of hypoxia on cell viability, PDCD4 expression and subcellular localisation. Methods MIN6 beta cells and ARIP ductal cells were exposed to 1% (hypoxia) or 21% O2 (normoxia) for 12 or 24 hours. MTT assay, HPI staining, scanning electron microscopy, western blotting and immunocytochemistry analyses were performed to determine the effect of hypoxia on cell viability, morphology and PDCD4 expression. Results 24 hour exposure to hypoxia resulted in ~70% loss of beta cell viability (P<0.001) compared to normoxia. Both HPI staining and SEM analysis demonstrated beta cell apoptosis and necrosis after 12 hours exposure to hypoxia. ARIP cells also displayed hypoxia-induced apoptosis and altered surface morphology after 24 hours, but no significant growth difference (p>0.05) was observed between hypoxic and normoxic conditions. Significantly higher expression of PDCD4 was observed in both beta cells (P<0.001) and ductal (P<0.01) cells under hypoxic conditions compared to controls. PDCD4 expression was localised to the cytoplasm of both beta cells and ductal cells, with no observed effects of hypoxia, normoxia or serum free conditions on intracellular shuttling of PDCD4. Conclusion These findings indicate that hypoxia-induced expression of PDCD4 is associated with increased beta cell death and suggests that PDCD4 may be an important factor in regulating beta cell survival during hypoxic stress.