Opposing Effects of Low Molecular Weight Heparins on the Release of Inflammatory Cytokines from Peripheral Blood Mononuclear Cells of Asthmatics

Background

T-cell-mediated inflammatory cytokines, such as interleukin (IL)-4, IL-5, IL-13 and tumor necrosis factor-alpha (TNF-α), play an important role in the initiation and progression of inflammatory airways diseases. Low-molecular-weight heparins (LMWHs), widely used anticoagulants, possess anti-inflammatory properties making them potential treatment options for inflammatory diseases, including asthma. In the current study, we investigated the modulating effects of two LMWHs (enoxaparin and dalteparin) on the release of cytokines from stimulated peripheral blood mononuclear cells (PBMCs) of asthmatic subjects to identify the specific components responsible for the effects.

Methods

PBMCs from asthmatic subjects (consist of ~75% of T-cells) were isolated from blood taken from ten asthmatic subjects. The PBMCs were pre-treated in the presence or absence of different concentrations of LMWHs, and were then stimulated by phytohaemagglutinin for the release of IL-4, IL-5, IL-13 and TNF-α. LMWHs were completely or selectively desulfated and their anticoagulant effect, as well as the ability to modulate cytokine release, was determined. LMWHs were chromatographically fractionated and each fraction was tested for molecular weight determination along with an assessment of anticoagulant potency and effect on cytokine release.

Results

Enoxaparin inhibited cytokine release by more than 48%, whereas dalteparin increased their release by more than 25%. The observed anti-inflammatory effects of enoxaparin were independent of their anticoagulant activities. Smaller fractions, in particular dp4 (four saccharide units), were responsible for the inhibitory effect of enoxaparin. Whereas, the larger fractions, in particular dp22 (twenty two saccharide units), were associated with the stimulatory effect of dalteparin.

Conclusion

Enoxaparin and dalteparin demonstrated opposing effects on inflammatory markers. These observed effects could be due to the presence of structurally different components in the two LMWHs arising from different methods of depolymerisation. This study provides a platform for further studies investigating the usefulness of enoxaparin in various inflammatory diseases.     

Tracking Cholera through Surveillance of Oral Rehydration Solution Sales at Pharmacies: Insights from Urban Bangladesh

Background

In Bangladesh, pharmacy-purchased oral rehydration solution (ORS) is often used to treat diarrhea, including cholera. Over-the-counter sales have been used for epidemiologic surveillance in the past, but rarely, if ever, in low-income countries. With few early indicators for cholera outbreaks in endemic areas, diarrhea-related product sales may serve as a useful surveillance tool.

Methodology/Principal Findings

We tracked daily ORS sales at 50 pharmacies and drug-sellers in an urban Bangladesh community of 129,000 for 6-months while simultaneously conducting surveillance for diarrhea hospitalizations among residents. We developed a mobile phone based system to track the sales of ORS and deployed it in parallel with a paper-based system. Our objectives were to determine if the mobile phone system was practical and acceptable to pharmacists and drug sellers, whether data were reported accurately compared to a paper-based system, and whether ORS sales were associated with future incidence of cholera hospitalizations within the community. We recorded 47,215 customers purchasing ORS, and 315 hospitalized diarrhea cases, 22% of which had culture-confirmed cholera. ORS sales and diarrhea incidence were independently associated with the mean daily temperature; therefore both unadjusted and adjusted models were explored. Through unadjusted cross-correlation statistics and generalized linear models, we found increases in ORS sales were significantly associated with increases in hospitalized diarrhea cases up to 9-days later and hospitalized cholera cases up to one day later. After adjusting for mean daily temperature, ORS was significantly associated with hospitalized diarrhea two days later and hospitalized cholera one day later.

Conclusions/Significance

Pharmacy sales data may serve as a feasible and useful surveillance tool. Given the relatively short lagged correlation between ORS sales and diarrhea, rapid and accurate sales data are key. More work is needed in creating actionable algorithms that make use of this data and in understanding the generalizability of our findings to other settings.     

Bacillus enclensis sp. nov., isolated from sediment sample

A novel bacterial strain, designated SGD-1123^T was isolated from Chorao Island, in Goa Province, India. The strain was found to be able to grow at 15–42 °C, pH 5–12 and 0–12 % (w/v) NaCl. The whole cell hydrolysates were found to contain meso-diaminopimelic acid, galactose and arabinose. The major fatty acids were identified as iso-C_15:0 and anteiso-C_15:0, MK-7 was identified as the predominant menaquinone and the predominant polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid. The genomic DNA G+C content was determined to be 44.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolate within the genus Bacillus and further revealed that strain SGD-1123^T had highest sequence similarity with Bacillus aquimaris, and forms a separate clade with its closest relatives i.e. B. aquimaris, Bacillus vietnamensis and Bacillus marisflavi, with which it shares 94.5, 94.1 and 94.1 % similarity respectively. The phylogenetic, chemotaxonomic and phenotypic analyses indicated that strain SGD-1123^T represents a novel species within the genus Bacillus, for which the name Bacillus enclensis is proposed. The type strain is SGD-1123^T (NCIM 5450^T=CCTCC AB 2011125^T).     

Identification of a recurrent insertion mutation in the LDLR gene in a Pakistani family with autosomal dominant hypercholesterolemia

Familial Hypercholesterolemia (FH) results in elevated levels of blood lipids including total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) with normal triglycerides (TG). This disease is one of the major contributors towards an early onset of coronary heart disease (CHD). The aim of the present study was to identify the genes responsible for causing FH in Pakistani population, for this purpose a large consanguineous FH family was selected for genetic analysis. Serum lipid levels, including TC, TG, LDL-C and high density lipoprotein cholesterol (HDL-C), were determined in patients and healthy controls. In order to find the causative mutation in this family, direct sequencing of the low density lipoprotein receptor (LDLR) gene was performed. In addition the part of the Apolipoprotein-B (APOB) gene containing the mutations R3500Q and R3500W was also sequenced. Affected individuals of the family were found to have raised TC and LDL-C levels. Sequencing revealed an insertion mutation (c.2416_2417InsG) in exon 17 of the LDLR gene in all the affected individuals of the family. Common FH causing APOB mutations were not present in this family. Heterozygous individuals had TC levels ranging from ~300–500 mg/dl and the only homozygous individual with typical xanthomas had TC levels exceeding 900 mg/dl. This is the first report of a known LDLR gene mutation causing FH in the Pakistani population. Despite a large heterogeneity of LDLR mutations there are still some common mutations which are responsible for FH throughout the world.     

Plant growth-promoting activity in newly isolated <Emphasis Type="Italic">Bacillus thioparus</Emphasis> (NII-0902) from Western <Emphasis Type="Italic">ghat</Emphasis> forest,India

A Gram-positive rod-shaped bacterium isolated on nutrient agar plates incubated at 28 ± 2°C. The identity of the bacterium was confirmed by sequencing of the 16S rRNA gene and it reveals that it shares highest similarity with Bacillus thioparus CECT 7196^T (99.08%). It was capable of growing at temperatures ranging from 4 to 40°C, but optimum growth was observed at 28 ± 2°C. Strain NII-0902 is endowed with multiple plant growth promotion attributes such as phosphate solubilization, Indole acetic acid (IAA), siderophore and HCN production, which were expressed differentially at sub-optimal temperatures (5–40°C). It was able to solubilize phosphate (17.7 μg ml⁻¹), and produce IAA (139.7 μg ml⁻¹) at 28 ± 2°C. Qualitative detection of siderophore production and HCN were also observed. At 5°C it was found to express all the plant growth promotion attributes except HCN production. The ability to colonize roots is a sine qua non condition for a rhizobacteria to be considered a true plant growth-promoting rhizobacteria (PGPR). Bacillus sp. NII-0902 has a potential ability to colonize roots visualized by transparency, bacterial growth (turbid, milky and narrow zone) along and around roots and truly supported by scanning electron micrograph. Hence, it is proposed that, Bacillus thioparus sp. NII-0902 could be deployed as an inoculant to attain the desired results of bacterization.     

Discovery of novel,orally available benzimidazoles as melanin concentrating hormone receptor 1 (MCHR1) antagonists

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays role in several disorders such as obesity, stress, depression and anxiety. The synthesis and biological evaluation of novel benzimidazole derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified.     

Identification of cultivation condition to produce correctly folded form of a malaria vaccine based on Plasmodium falciparum merozoite surface protein-1 in Escherichia coli

The C-terminal, 19-kDa domain of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1₁₉) is among the leading vaccine candidate for malaria due to its essential role in erythrocyte invasion by the parasite. We designed a synthetic gene for optimal expression of recombinant PfMSP-1₁₉ in Escherichia coli and developed a scalable process to obtain high-quality PfMSP-1₁₉. The synthetic gene construct yielded a fourfold higher expression level of PfMSP-1₁₉ in comparison to the native gene construct. Optimization of cultivation conditions in the bioreactor indicated important role of yeast extract and substrate feeding strategy for obtaining enhanced expression of soluble and correctly folded PfMSP-1₁₉. It was observed that the higher expression level of PfMSP-1₁₉ was essentially associated with the generation of higher level of incorrectly folded PfMSP-1₁₉. A simple purification procedure comprising metal affinity and ion exchange chromatography was developed to purify correctly folded form of PfMSP-1₁₉ from cell lysate. Biochemical and biophysical characterization of purified PfMSP-1₁₉ suggested that it was highly pure, homogeneous, and correctly folded.     

Estradiol enhances cell-associated paraoxonase 1 (PON1) activity in vitro without altering PON1 expression

PON1 is a high density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. In vivo animal and human studies have indicated that estradiol (E2) supplementation enhances serum PON1 activity. In this study, we sought to determine if E2 directly up-regulates cell-associated PON1 activity in vitro and to characterize the mechanism of regulation. In vitro E2 treatment of both the human hepatoma cell line Huh7 and normal rat hepatocytes resulted in a 2- to 3-fold increase in cell-associated PON1 catalytic activity. E2 potently induced PON1 activity with average EC₅₀ values of 15 nM for normal hepatocytes and 68 nM for Huh7. The enhancement of PON1 activity by E2 was blocked by the estrogen receptor (ER) antagonist ICI 182,780 indicating that E2 was acting through the ER. The up-regulation of PON1 activity by E2 did not involve enhancement of PON1 mRNA or protein levels and did not promote secretion of PON1. Thus, E2 can enhance cell-associated PON1 activity in vitro without altering PON1 gene expression or protein level. Our data suggest that E2 may regulate the specific activity and/or stability of cell surface PON1.     

Mental health literacy towards depression among non-medical students at a Malaysian university

Background The aim of the present study was to evaluate the knowledge and perception of depression among students of University Sains Malaysia (USM), in Penang, Peninsular Malaysia.Method Face-to-face interviews were conducted using a pre-validated 21-item questionnaire among students at USM.Results A total of 500 respondents participated in the survey comprising 24.6% (n=123) males and 75.4% (n=377) females. Half (50.0%, n=250) were Malays, followed by Chinese (44.0%, n=220) and Indians (6.0%, n=30). Whilst exploring the respondents' knowledge of the symptoms of depression, it was found that Chinese females had a comparatively better knowledge (P=0.058) of the symptoms of depression in comparison with Malays and Indians. Overall, social issues were attributed as the possible cause of depression. A cursory knowledge level was observed regarding medication for depression. Female students were more inclined towards the use of alternative and traditional medicines. However, with regard to seeking professional help, consultation with a psychiatrist was preferred by the majority.Conclusion Overall, a moderate level of knowledge about the symptoms of depression and a cursory knowledge of its therapy were observed. Those with personal experience of depression had better knowledge of the symptoms and therapy. Alternative treatments and traditional medicines were also favoured. There is a risk that this may affect the ability of Malaysian youths to seek evidence-based mental health care.