Chronic treatment with a stable obestatin analogue significantly alters plasma triglyceride levels but fails to influence food intake, fluid intake, body weight, or body composition in rats.

Obestatin (OB(1-23) is a 23 amino acid peptide encoded on the preproghrelin gene, originally reported to have metabolic actions related to food intake, gastric emptying and body weight. The biological instability of OB(1-23) has recently been highlighted by studies demonstrating its rapid enzymatic cleavage in a number of biological matrices. We assessed the stability of both OB(1-23) and an N-terminally PEGylated analogue (PEG-OB(1-23)) before conducting chronic in vivo studies. Peptides were incubated in rat liver homogenate and degradation monitored by LC-MS. PEG-OB(1-23) was approximately 3-times more stable than OB(1-23). Following a 14 day infusion of Sprague Dawley rats with 50 mol/kg/day of OB(1-23) or a N-terminally PEGylated analogue (PEG-OB(1-23)), we found no changes in food/fluid intake, body weight and plasma glucose or cholesterol between groups. Furthermore, morphometric liver, muscle and white adipose tissue (WAT) weights and tissue triglyceride concentrations remained unaltered between groups. However, with stabilised PEG-OB(1-23) we observed a 40% reduction in plasma triglycerides. These findings indicate that PEG-OB(1-23) is an OB(1-23) analogue with significantly enhanced stability and suggest that obestatin could play a role in modulating physiological lipid metabolism, although it does not appear to be involved in regulation of food/fluid intake, body weight or fat deposition.

Intravaginal immunization using the recombinant HIV-1 clade-C trimeric envelope glycoprotein CN54gp140 formulated within lyophilized solid dosage forms

Vaccine-mediated prevention of primary HIV-1 infection at the heterosexual mucosal portal of entry may be facilitated by highly optimised formulations or drug delivery devices for intravaginal (i.vag) immunization. Previously we described hydroxyethylcellulose (HEC)-based rheologically structured gel vehicles (RSVs) for vaginal immunization of an HIV-1 vaccine candidate, a soluble recombinant trimeric HIV-1 clade-C envelope glycoprotein designated CN54gp140. Here we investigated the efficacy of lyophilized solid dosage formulations (LSDFs) for prolonging antigen stability and as i.vag delivery modalities. LSDFs were designed and developed that upon i.vag administration they would reconstitute with the imbibing of vaginal fluid to mucoadhesive, site-retentive semi-solids. Mice were immunized with lyophilized equivalents of (i) RSVs, (ii) modified versions of the RSVs more suited to lyophilization (sodium carboxymethyl cellulose (NaCMC)-based gels) and (iii) Carbopol® gel, all containing CN54gp140. NaCMC-based LSDFs provided significantly enhanced antigen stability compared to aqueous-based RSVs. Rheological analysis indicated the NaCMC-based LSDFs would offer enhanced vaginal retention in woman compared to more conventional vaginal gel formulations. All LSDFs were well tolerated in the mouse model. Following i.vag administration, all LSDFs boosted systemic CN54gp140-specific antibody responses in sub-cutaneously primed mice. Induction of CN54gp140-specific antibody responses in the female genital tract was evident. Of all the LSDFs the fastest releasing which was lyophilized Carbopol® gel elicited immune responses comparable to buffer instillation of antigen suggesting that rather than slower sustained release, initial high burst release from the LSDFs may suffice. The boosting of specific immune responses upon i.vag administration indicates that LSDFs are viable mucosal vaccine delivery modalities promoting antigen stability and facilitating intimate exposure of CN54gp140 to the mucosal-associated lymphoid tissue of the female genital tract.

Exchange protein directly activated by cAMP 1 (Epac1) is expressed in human neutrophils and mediates cAMP-dependent activation of the monomeric GTPase Rap1

Epac1 and Epac2 bind cAMP and mediate cAMP-dependent activation of Rap1. cAMP is produced in neutrophils in response to many chemoattractants. This second messenger plays a key role in the regulation of the functions of neutrophils. However, it is still not known whether Epacs are expressed in human neutrophils. We found that stimulation of PLB-985 cells differentiated into neutrophil-like cells, human neutrophils with 8CPT-2Me-cAMP (a selective activator of Epacs), or FK (a diterpene that augments the intracellular level of cAMP) led to GTP-loading of Rap1. Epac1 mRNA was expressed in UND and DF PLB-985 cells, but Epac1 protein was only detected in DF PLB-985 cells. In human neutrophils, the Epac1 transcript was present, and Epac1 protein could be detected by Western blot analysis if the cells had been treated with the serine protease inhibitor PMSF. FK induced adhesion of PLB-985 cells and human neutrophils on fibrinogen, a ligand for beta 2 integrins. Interestingly, in DF PLB-985 cells, but not in human neutrophils, 8CPT-2Me-cAMP induced beta 2 integrin-dependent adhesion. The failure of 8CPT-2Me-cAMP to induce beta 2 integrin-dependent human neutrophil adhesion could be explained by the fact that this compound did not induce a switch of the beta 2 integrins from a low-affinity to a high-affinity ligand-binding conformation. We concluded that Epac1 is expressed in human neutrophils and is involved in cAMP-dependent regulation of Rap1. However, the loading of GTP on Rap1 per se is not sufficient to promote activation of beta 2 integrins. J. Leukoc. Biol. 90: 741-749; 2011.

Molecular investigations into vaginal immunization with HIV gp41 antigenic construct H4A in a quick release solid dosage form

A robust vaginal immune response is considered essential for an effective prophylactic vaccine that prevents transmission of HIV and other sexually acquired diseases. Considerable attention has recently focused on the potential of vaginally administered vaccines as a means to induce such local immunity. However, the potential for vaccination at this site remains in doubt as the vaginal mucosa is generally considered to have low immune inductive potential. In the current study, we explored for the first time the use of a quick release, freeze-dried, solid dosage system for practical vaginal administration of a protein antigen. These solid dosage forms overcome the common problem associated with leakage and poor retention of vaginally administered antigen solutions. Mice were immunized vaginally with H4A, an HIV gp41 envelope based recombinant protein, using quick release, freeze-dried solid rods, and the immune responses compared to a control group immunized via subcutaneous H4A injection. Vaginally immunized mice failed to elicit robust immune responses. Our detailed investigations, involving cytokine analysis, the stability of H4A in mouse cervicovaginal lavage, and elucidation of the state of H4A protein in the immediate-release dosage form, revealed that antigen instability in vaginal fluid, the state of the antigen in the dosage form, and the cytokine profile induced are all likely to have contributed to the observed lack of immunogenicity. These are important factors affecting vaginal immunization and provide a rational basis for explaining the typically poor and variable elicitation of immunity at this site, despite the presence of immune responsive cells within the vaginal mucosae. In future mucosal vaccine studies, a more explicit focus on antigen stability in the dosage form and the immune potential of available antigen-responsive cells is recommended. © 2012 Elsevier Ltd. All rights reserved.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis:in vitro evidence in cultured epithelial cells

BACKGROUND:
Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR.
OBJECTIVES:
The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects.
METHODS:
Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function.
RESULTS:
Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway.
CONCLUSIONS:
This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

Oxidative stress and increased type-IV collagenase levels in bronchoalveolar lavage fluid from newborn babies

Oxidative stress may increase lung permeability by upregulation of matrix-metalloproteinase-9 (MMP-8), a type-IV collagenase that can disrupt alveolar basement membranes. We have compared a marker of oxidative stress (protein carbonyl residues) with levels of MMP-8 and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), in bronchoalveolar lavage samples from newborn babies. Bronchoalveolar lavage samples (n = 87, two from each time point) were taken in the first 6 postnatal days from 41 ventilated babies: 18 of

ROLE OF INTRACELLULAR AND EXTRACELLULAR CALCIUM IN HISTAMINE-RELEASE FROM RAT PERITONEAL MAST-CELLS

The present study provides evidence for a number of calcium pools important in histamine secretion from the mast cell. Firstly, calcium loosely bound to the cell membrane, and in rapid equilibrium with the extracellular environment, may be utilized for histamine release induced by most secretagogues. Secondly, all inducers are able to mobilize deeply buried or internal stores of calcium to initiate exocytosis. Finally, calcium bound to regulatory sites in the membrane may modulate the secretory process, Removal of calcium from the latter sites by brief treatment with chelating agents markedly enhances the secretory response in the absence of extracellular calcium, probably by facilitating the mobilization of bound stores of the ion, Saturation of these sites in the presence of excess calcium inhibits the release process and may restrict influx of the cation.