Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality

Antenatal HIV testing in rural eastern Uganda in 2003: incomplete rollout of the prevention of mother-to-child transmission of HIV programme?

BACKGROUND: Uganda began to implement the prevention of mother-to-child transmission (PMTCT) of HIV programme in 2000, and by the end of 2003 it had expanded to cover 38 of the 56 districts including Mbale District. However, reports from Mbale Hospital showed that less than 10% of pregnant women accepted antenatal HIV testing. We therefore conducted a study to determine the proportion of pregnant women who tested for HIV and the gaps and barriers in PMTCT implementation. METHODS: The study was a cross sectional household survey of women aged 18 years or more, with children aged one year or less, who resided in Mbale Town or in the surrounding Bungokho County. We also conducted in-depth interviews with six health workers in Mbale Hospital. RESULTS: In 2003, we interviewed 457 women with a median age of 24 years. The prevalence of antenatal HIV testing was 10 percent. The barriers to antenatal HIV testing were unavailability of voluntary counselling and testing services (44%), lack of HIV counselling (42%) and perceived lack of benefits for HIV infected women and their infants. Primipara (OR 2.6, 95% CI 1.2–5.8), urban dwellers (OR 2.7, 95% CI 1.3–5.8), women having been counselled on HIV (OR 6.2, 95% CI 2.9–13.2), and women with husbands being their primary confidant (OR 2.3, 95% CI 1.0–5.5) were independently associated with HIV testing. CONCLUSION: The major barriers to PMTCT implementation were unavailability of PMTCT services, particularly in rural clinics, and poor antenatal counselling and HIV testing services. We recommend that the focus of the prevention of mother-to-child transmission of HIV programme should shift to the district and sub-district levels, strengthen community mobilization, improve the quality of antenatal voluntary counselling and HIV testing services, use professional and peer counsellors to augment HIV counselling, and ensure follow-up care and support for HIV positive women and their infants

Demographic and microbial characteristics of extrapulmonary tuberculosis cases diagnosed in Malatya, Turkey, 2001-2007

<p>Abstract</p> <p>Background</p> <p>Extrapulmonary tuberculosis (EPTB) has an increasing rate in Turkey. The reason remains largely unknown. A better understanding of the demographic and microbial characteristics of EPTB in the Turkish population would extend the knowledgebase of EPTB and allow us to develop better strategies to control tuberculosis (TB).</p> <p><b>Methods</b></p> <p>We retrospectively evaluated clinical and laboratory data of 397 bacteriologically-confirmed TB cases diagnosed during an eight year-period using by chi-square analysis and multivariate logistic regression model.</p> <p>Results</p> <p>Of the 397 study patients, 103 (25.9%) had EPTB and 294 (74.1%) had pulmonary tuberculosis (PTB). The most commonly seen two types of EPTB were genitourinary TB (27.2%) and meningeal TB (19.4%). TB in bone/joints, pleural cavity, lymph nodes, skin, and peritoneal cavity occurred at a frequency ranging from 9.7% to 10.7%. The age distribution was significantly different (P < 0.01) between PTB and EPTB, with patients older than 45 years tending to have an increased risk of EPTB. Furthermore, the distribution of different types of EPTB differed significantly among age groups (P = 0.03). Meningeal and bone and/or joint TB were more commonly observed among the male patients, while lymphatic, genitourinary, and peritoneal TB cases were more frequently seen among females. Unique strain infection was statistically significantly associated with EPTB (OR: 2.82, 95% CI [1.59, 5.00])</p> <p>Conclusions</p> <p>EPTB accounted for a significant proportion of TB cases in Malatya, Turkey between 2001 and 2007. The current study has provided an insight into the dynamics of EPTB in Malatya, Turkey. However, the risk factors for having EPTB in Malatya, Turkey remain to be assessed in future studies using population-based or randomly selected sample.</p

Effect of Low Temperature on Growth and Ultra-Structure of Staphylococcus spp

The effect of temperature fluctuation is an important factor in bacterial growth especially for pathogens such as the staphylococci that have to remain viable during potentially harsh and prolonged transfer conditions between hosts. The aim of this study was to investigate the response of S. aureus, S. epidermidis, and S. lugdunensis when exposed to low temperature (4°C) for prolonged periods, and how this factor affected their subsequent growth, colony morphology, cellular ultra-structure, and amino acid composition in the non-cytoplasmic hydrolysate fraction. Clinical isolates were grown under optimal conditions and then subjected to 4°C conditions for a period of 8 wks. Cold-stressed and reference control samples were assessed under transmission electron microscopy (TEM) to identify potential ultra-structural changes. To determine changes in amino acid composition, cells were fractured to remove the lipid and cytoplasmic components and the remaining structural components were hydrolysed. Amino acid profiles for the hydrolysis fraction were then analysed for changes by using principal component analysis (PCA). Exposure of the three staphylococci to prolonged low temperature stress resulted in the formation of increasing proportions of small colony variant (SCV) phenotypes. TEM revealed that SCV cells had significantly thicker and more diffuse cell-walls than their corresponding WT samples for both S. aureus and S. epidermidis, but the changes were not significant for S. lugdunensis. Substantial species-specific alterations in the amino acid composition of the structural hydrolysate fraction were also observed in the cold-treated cells. The data indicated that the staphylococci responded over prolonged periods of cold-stress treatment by transforming into SCV populations. The observed ultra-structural and amino acid changes were proposed to represent response mechanisms for staphylococcal survival amidst hostile conditions, thus maintaining the viability of the species until favourable conditions arise again

A pragmatic approach to resolving technological unfairness: The case of Nike’s Vaporfly & Alphafly running footwear

Background Technology is often introduced into sport to facilitate it or to improve human performance within it. On occasion, some forms of novel technology require regulation or prevention entirely to ensure that a sport remains fair and accessible. Recently, the Nike Vaporfly and Alphafly shoes have received some concerns over their appropriateness for use in competitive distance running. Methods This paper evaluates the use of these shoes against an existing framework for sports technology discourse and adopts a pragmatic approach to attempt to resolve them. Results It is proposed that the three concerns regarding cost, access and coercion cannot be ruled out but likely remain short term issues. As a result, it is proposed that these running shoes are acceptable forms of technology but that ongoing vigilance will be required as such technologies develop further in the future. Conclusions The Nike Vaporfly/Alphafly shoes do push the perceived acceptability of running shoes to the limits of the current sports regulations. However, the alleged gains have not manifested themselves to a level that could be considered excessive when reviewing historical performances or when evaluated against a set of well-cited criteria. The sport will need to adopt a stance of ongoing vigilance as such technologies continue to develop or be optimised in the future

The dopamine β-hydroxylase -1021C/T polymorphism is associated with the risk of Alzheimer's disease in the Epistasis Project

Contains fulltext : 88930.pdf (publisher's version ) (Open Access)BACKGROUND: The loss of noradrenergic neurones of the locus coeruleus is a major feature of Alzheimer's disease (AD). Dopamine beta-hydroxylase (DBH) catalyses the conversion of dopamine to noradrenaline. Interactions have been reported between the low-activity -1021T allele (rs1611115) of DBH and polymorphisms of the pro-inflammatory cytokine genes, IL1A and IL6, contributing to the risk of AD. We therefore examined the associations with AD of the DBH -1021T allele and of the above interactions in the Epistasis Project, with 1757 cases of AD and 6294 elderly controls. METHODS: We genotyped eight single nucleotide polymorphisms (SNPs) in the three genes, DBH, IL1A and IL6. We used logistic regression models and synergy factor analysis to examine potential interactions and associations with AD. RESULTS: We found that the presence of the -1021T allele was associated with AD: odds ratio = 1.2 (95% confidence interval: 1.06-1.4, p = 0.005). This association was nearly restricted to men < 75 years old: odds ratio = 2.2 (1.4-3.3, 0.0004). We also found an interaction between the presence of DBH -1021T and the -889TT genotype (rs1800587) of IL1A: synergy factor = 1.9 (1.2-3.1, 0.005). All these results were consistent between North Europe and North Spain. CONCLUSIONS: Extensive, previous evidence (reviewed here) indicates an important role for noradrenaline in the control of inflammation in the brain. Thus, the -1021T allele with presumed low activity may be associated with misregulation of inflammation, which could contribute to the onset of AD. We suggest that such misregulation is the predominant mechanism of the association we report here

Identifying contextual determinants of problems in tuberculosis care provision in South Africa: a theory-generating case study

Background: Despite progress towards End TB Strategy targets for reducing tuberculosis (TB) incidence and deaths by 2035, South Africa remains among the top ten high-burden tuberculosis countries globally. A large challenge lies in how policies to improve detection, diagnosis and treatment completion interact with social and structural drivers of TB. Detailed understanding and theoretical development of the contextual determinants of problems in TB care is required for developing effective interventions. This article reports findings from the pre-implementation phase of a study of TB care in South Africa, contributing to HeAlth System StrEngThening in Sub-Saharan Africa (ASSET)—a five-year research programme developing and evaluating health system strengthening interventions in sub-Saharan Africa. The study aimed to develop hypothetical propositions regarding contextual determinants of problems in TB care to inform intervention development to reduce TB deaths and incidence whilst ensuring the delivery of quality integrated, person-centred care. Methods Theory-building case study design using the Context and Implementation of Complex Interventions (CICI) framework to identify contextual determinants of problems in TB care. Between February and November 2019, we used mixed methods in six public-sector primary healthcare facilities and one public-sector hospital serving impoverished urban and rural communities in the Amajuba District of KwaZulu-Natal Province, South Africa. Qualitative data included stakeholder interviews, observations and documentary analysis. Quantitative data included routine data on sputum testing and TB deaths. Data were inductively analysed and mapped onto the seven CICI contextual domains. Results: Delayed diagnosis was caused by interactions between fragmented healthcare provision; limited resources; verticalised care; poor TB screening, sputum collection and record-keeping. One nurse responsible for TB care, with limited integration of TB with other conditions, and policy focused on treatment adherence contributed to staff stress and limited consideration of patients’ psychosocial needs. Patients were lost to follow up due to discontinuity of information, poverty, employment restrictions and limited support for treatment side-effects. Infection control measures appeared to be compromised by efforts to integrate care. Conclusions: Delayed diagnosis, limited psychosocial support for patients and staff, patients lost to follow-up and inadequate infection control are caused by an interaction between multiple interacting contextual determinants. TB policy needs to resolve tensions between treating TB as epidemic and individually-experienced social problem, supporting interventions which strengthen case detection, infection control and treatment, and also promote person-centred support for healthcare professionals and patients