A Cholera Conjugate Vaccine Containing O-specific Polysaccharide (OSP) of V. cholerae O1 Inaba and Recombinant Fragment of Tetanus Toxin Heavy Chain (OSP:rTTHc) Induces Serum,Memory and Lamina Proprial Responses against OSP and Is Protective in Mice

Background

Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS).

Methodology

Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization.

Principle Findings

Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 μg). We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model.

Conclusion

We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens.     

Development and Validation of a HPLC Method for Dissolution and Stability Assay of Liquid-Filled Cyclosporine Capsule Drug Products

To assay the dissolution samples of a drug product from several sources, a simple but broadly applicable analytical method is always desired. For the liquid-filled cyclosporine capsules, while analyzing the dissolution samples, the current compendial and literature HPLC methods have been found to be inadequate to provide satisfactory separation of the drug and the excipient peaks. Accordingly, a suitable isocratic reverse-phase HPLC method was developed for the analysis of dissolution samples of liquid-filled cyclosporine capsules. The method successfully separated the cyclosporine peak from the interfering chromatographic peaks of the excipients. The method was validated according to the ICH and FDA guidelines. Specificity, selectivity, linearity, accuracy, precision, and robustness were established over 3 days as part of method validation. Additionally, the degradation kinetics of cyclosporine in dissolution media was determined. Cyclosporine degradation followed a zero-order kinetics in the dissolution media with the respective rate constants of −3.5, −1.5, and −0.3%/h at 37°C, 25°C, and 10°C.Key words: cyclosporine, degradation, dissolution, HPLC, liquid-filled capsules     

Search and Destroy: ER Quality Control and ER-Associated Protein Degradation

Abstract

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms. The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.     

Population structure and gene flow of the syntopic turtles Emys and Mauremys from coastal and inland regions of Anatolia (Turkey): results from mitochondrial and microsatellite data

Revealing the genetic basis of the existence of different species living together in different geographic regions provides clarification of this phylogeographic differentiation. In this study, we investigated the population genetics and evaluated the level of genetic variation of inland and coastal populations of Mauremys and Emys in Turkey. Tissue samples of 196 terrapins were studied which were collected from syntopic coastal (Gölbent-Söke/Ayd?n; M. rivulata and E. orbicularis) and inland populations (Bahçesaray/Aksaray; M. caspica and E. orbicularis). DNA was isolated using the InnuPREP DNA Mini Kit. Mitochondrial DNA sequences and allelic variation at 13 microsatellite loci for Mauremys and 12 microsatellite loci for Emys were examined.  Three haplotypes were found for Emys orbicularis (Im, Ip and Iw) collected from the coastal region and two haplotypes for Emys orbicularis (Ig and Im) collected from inland. Two haplotypes were identified for M. caspica (Cmt8 and Cmt9) and three haplotypes were identified for M. rivulata (Rmt3, Rmt24 and Rmt26). Using microsatellites and the software STRUCTURE the most probable value for K was revealed as two 2 for both species. The F_ST value between M. rivulata and M. caspica was 0.39, and between the coastal and inland populations of E. orbicularis 0.09. It can be concluded that Emys populations tend to evolve by somehow preserving the allelic richness they have and Mauremys populations continue to differentiate so that new species emerge in the evolutionary process to reach the ideal allelic structure.

     

Progesterone inhibits the growth of human neuroblastoma: in vitro and in vivo evidence

We investigated the antitumorogenic effects of progesterone (P4) in a human neuroblastoma (SK-N-AS) cell line in vitro and in a mouse xenograft model of neuroblastoma. The safety of P4 was tested in rat primary cortical neurons and human foreskin fibroblasts (HFF-1). At high doses, P4 significantly (P < 0.05) decreased SK-N-AS cell viability in vitro, and this effect was not blocked either by 5α-reductase inhibitor, finasteride or the P4 receptor antagonist RU486. Even at very high doses, P4 did not induce any cell death in healthy primary cortical neurons or HFF-1. The bioavailability of P4 24 h after the last injection in the serum of treated animals was significantly (P < 0.05) higher (10-33 μg/mL) than in untreated animals. In nude mice, P4 (50 and 100 mg/kg) inhibited neuroblastoma growth by ~50% over 8 d of treatment. No drug toxicity was observed in the mice, as measured by body weight and activity. P4 suppressed the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP-9, MMP-2), which are involved in tumor vascular development. High-dose P4 inhibited tumor growth by suppressing cell proliferation and inducing apoptosis, as evidenced by the expression of proliferating cell nuclear antigen and cleaved caspase-3. P4 significantly increased the expression of P4 receptor isoform-A and suppressed phospho-Akt (Ser437) expression. In conclusion, at high doses, P4 effectively inhibits the growth of solid neuroblastoma tumor and has high bioavailability, selective toxicity and a high margin of safety, making it a possible candidate for further study as a potential clinical treatment of neuroblastoma.     

Timing of enteral feeding in cerebral malaria in resource-poor settings: a randomized trial

Background

Early start of enteral feeding is an established treatment strategy in intubated patients in intensive care since it reduces invasive bacterial infections and length of hospital stay. There is equipoise whether early enteral feeding is also beneficial in non-intubated patients with cerebral malaria in resource poor settings. We hypothesized that the risk of aspiration pneumonia might outweigh the potential benefits of earlier recovery and prevention of hypoglycaemia.

Method and Findings

A randomized trial of early (day of admission) versus late (after 60 hours in adults or 36 hours in children) start of enteral feeding was undertaken in patients with cerebral malaria in Chittagong, Bangladesh from May 2008 to August 2009. The primary outcome measures were incidence of aspiration pneumonia, hypoglycaemia and coma recovery time. The trial was terminated after inclusion of 56 patients because of a high incidence of aspiration pneumonia in the early feeding group (9/27 (33%)), compared to the late feeding group (0/29 (0%)), p = 0.001). One patient in the late feeding group, and none in the early group, had hypoglycaemia during admission. There was no significant difference in overall mortality (9/27 (33%) vs 6/29 (21%), p = 0.370), but mortality was 5/9 (56%) in patients with aspiration pneumonia.

Conclusions

In conclusion, early start of enteral feeding is detrimental in non-intubated patients with cerebral malaria in many resource-poor settings. Evidence gathered in resource rich settings is not necessarily transferable to resource-poor settings.

Trial Registration

Controlled-Trials.com ISRCTN57488577     

Increased adipose protein carbonylation in human obesity

Insulin resistance is associated with obesity but mechanisms controlling this relationship in humans are not fully understood. Studies in animal models suggest a linkage between adipose reactive oxygen species (ROS) and insulin resistance. ROS oxidize cellular lipids to produce a variety of lipid hydroperoxides that in turn generate reactive lipid aldehydes that covalently modify cellular proteins in a process termed carbonylation. Mammalian cells defend against reactive lipid aldehydes and protein carbonylation by glutathionylation using glutathione-S-transferase A4 (GSTA4) or carbonyl reduction/oxidation via reductases and/or dehydrogenases. Insulin resistance in mice is linked to ROS production and increased level of protein carbonylation, mitochondrial dysfunction, decreased insulin-stimulated glucose transport, and altered adipokine secretion. To assess protein carbonylation and insulin resistance in humans, eight healthy participants underwent subcutaneous fat biopsy from the periumbilical region for protein analysis and frequently sampled intravenous glucose tolerance testing to measure insulin sensitivity. Soluble proteins from adipose tissue were analyzed using two-dimensional gel electrophoresis and the major carbonylated proteins identified as the adipocyte and epithelial fatty acid-binding proteins. The level of protein carbonylation was directly correlated with adiposity and serum free fatty acids (FFAs). These results suggest that in human obesity oxidative stress is linked to protein carbonylation and such events may contribute to the development of insulin resistance.     

Interferon-γ and proliferation responses to Salmonella enterica Serotype Typhi proteins in patients with S. Typhi Bacteremia in Dhaka, Bangladesh

Background

Salmonella enterica serotype Typhi is a human-restricted intracellular pathogen and the cause of typhoid fever. Cellular immune responses are required to control and clear Salmonella infection. Despite this, there are limited data on cellular immune responses in humans infected with wild type S. Typhi.

Methodology/Principal Findings

For this work, we used an automated approach to purify a subset of S. Typhi proteins identified in previous antibody-based immuno-affinity screens and antigens known to be expressed in vivo, including StaF-putative fimbrial protein-STY0202, StbB-fimbrial chaperone-STY0372, CsgF-involved in curli production-STY1177, CsgD- putative regulatory protein-STY1179, OppA-periplasmic oligopeptide binding protein precursor-STY1304, PagC-outer membrane invasion protein-STY1878, and conserved hypothetical protein-STY2195; we also generated and analyzed a crude membrane preparation of S. Typhi (MP). In comparison to samples collected from uninfected Bangladeshi and North American participants, we detected significant interferon-γ responses in PBMCs stimulated with MP, StaF, StbB, CsgF, CsgD, OppA, STY2195, and PagC in patients bacteremic with S. Typhi in Bangladesh. The majority of interferon-γ expressing T cells were CD4 cells, although CD8 responses also occurred. We also assessed cellular proliferation responses in bacteremic patients, and confirmed increased responses in infected individuals to MP, StaF, STY2195, and PagC in convalescent compared to acute phase samples and compared to controls. StaF is a fimbrial protein homologous to E. coli YadK, and contains a Pfam motif thought to be involved in cellular adhesion. PagC is expressed in vivo under the control of the virulence-associated PhoP-regulon required for intra-macrophage survival of Salmonella. STY2195 is a conserved hypothetical protein of unknown function.

Conclusion/Significance

This is the first analysis of cellular immune responses to purified S. Typhi antigens in patients with typhoid fever. These results indicate that patients generate significant CD4 and CD8 interferon-γ responses to specific S. Typhi antigens during typhoid fever, and that these responses are elevated at the time of clinical presentation. These observations suggest that an interferon-γ based detection system could be used to diagnose individuals with typhoid fever during the acute stage of illness.     

Locomotion Controls Spatial Integration in Mouse Visual Cortex

1. Download : Download high-res image (117KB)
2. Download : Download full-size image