Pharmacological studies on nitro‐naproxen (naproxen‐2‐nitrooxyethylester)

Naproxen‐2‐nitrooxyethylester (S‐(+)‐2‐(6‐methoxy‐2‐naphthyl)propanoic acid‐2‐nitrooxyethylester, LE‐EK06) was synthesized from naproxen and 2‐nitrooxyethylbromide as a novel nitric oxide–releasing derivative of naproxen. Molar equivalents of LE‐EK06 (6.93–27.73 mg/kg, p.o.) to naproxen dose‐dependently exhibited greater antinociceptive activity in comparison to naproxen in a writhing assay. The compound (5.54–22.18 mg/kg, p.o.) showed greater anti‐inflammatory activity at 2 h after as comparable to its effect at 4 h after carrageenan challenge in rats. Further, LE‐EK06 (9.45 mg/kg, p.o.) was more potent in the carrageenan‐evoked hyperalgesia. LE‐EK06 (11.09 mg/kg, p.o.) and naproxen (8.0 mg/kg, p.o.) showed a comparable inhibitory effect on exudate formation and migration of polymorphonuclear leukocytes (PMNs) in a carrageenan‐induced pleurisy test. Further, the compound (11.09 mg/kg, p.o.) significantly reduced myeloperoxidase activity in carrageenan‐treated paw and demonstrated significantly less gastrotoxicity in acute and chronic (21 days) studies. The scanning electron microscopy revealed that LE‐EK06 showed only mild gastric damage (slight disruption of mucus layer) in comparison to naproxen. The present study suggested that naproxen‐2‐nitrooxyethylester (LE‐EK06) represents a novel gastric sparing NSAID. Drug Dev. Res. 61:66–78, 2004. © 2004 Wiley‐Liss, Inc.     

Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

Virological failure (VF) has been identified as the earliest, most predictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to the high cost and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to determine VF, in accordance with current WHO guideline recommendations for therapy switching in RLS. The assay described here is an internally controlled qualitative real-time PCR targeting the conserved long terminal repeat domain of HIV-1. This assay was applied to HIV-1 subtypes A to H and further evaluated on HIV-1 clinical plasma samples from South Africa (n = 191) and Tanzania (n = 42). Field evaluation was performed in Uganda using local clinical plasma samples (n = 176). Furthermore, assay performance was evaluated for DBS. This assay is able to identify VF for all major HIV-1 group M subtypes with equal specificity and has a lower detection limit of 1.00E+03 copies/ml for plasma samples and 5.00E+03 copies/ml for DBS. Comparative testing yielded accurate VF determination for therapy switching in 89% to 96% of samples compared to gold standards. The assay is robust and flexible, allowing for “open platform” applications and producing results comparable to those of commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings.     

Building capacity for the assessment of HIV drug resistance: experiences from the PharmAccess African Studies to Evaluate Resistance network

The PharmAccess African Studies to Evaluate Resistance (PASER) network was established as a collaborative partnership of clinical sites, laboratories, and research groups in 6 African countries; its purpose is to build research and laboratory capacity in support of a coordinated effort to assess population-level acquired and transmitted human immunodeficiency virus type-1 drug resistance (HIVDR), thus contributing to the goals of the World Health Organization Global HIV Drug Resistance Network. PASER disseminates information to medical professionals and policy makers and conducts observational research related to HIVDR. The sustainability of the network is challenged by funding limitations, constraints in human resources, a vulnerable general health infrastructure, and high cost and complexity of molecular diagnostic testing. This report highlights experiences and challenges in the PASER network from 2006 to 2010.     

Risk factors in smoking habits of high school students

Although the hazards of smoking is well known, the number of smokers among high school students is still high. There are many factors influencing these students to start smoking. This study was conducted to discover the size of the population of smokers among high school students is still high. There are many factors influencing these students to start smoking. This study was conducted to discover the size of the population of smokers among high school students, their smoking behaviour, and what factors might be associated as risk factors. Questionnaires were filled by 1627 respondents between 12 and 22 years of age of which 955 (58.7%) are boys and 672 (41.3%) are girls. Among all male respondents there were found 622 (86.2%) experimental smokers; among all the girls there were only 99 (13.7%) experimental smokers. From the total number of experimental smokers, 377 (95.2%) boys and 19 (4.8%) girls eventually became smokers. One hundred and seventeen (16.2%) students tried smoking before 13 years old, 65 of these students became smokers. This study found that socio-economic status, parents, friends, and siblings who smoked, and social environment, have significant influence on high school students' smoking habits. To prevent and lower the number of smokers among students in high schools, health and smoking education must be started in the schools as early as possible.     

Direct fluorochrome labeling of phage display library clones for studying binding specificities: applications in flow cytometry and fluorescence microscopy

Phage display technology is increasingly employed to identify high-affinity peptides and single-chain antibodies with binding specificities for a diversity of target types. The analysis of phage-binding sensitivity and specificity typically employs directly labeled secondary antiphage antibodies and potentially tertiary labels, such as fluorochromes and enzymes, when biotinylated antibodies are used. However, secondary or tertiary reagents may not be feasible or desirable for some target types and applications. Here, we present a simple approach for directly labeling phage clones with two common amine-reactive fluorochromes. We show that these fluorochromes label the pVIII major coat protein and that the binding selectivity of peptides displayed on the pIII protein of several well-characterized phage clones is maintained in flow cytometric analysis and immunofluorescence microscopy. Uniquely, such labeled phage, in part, represent self-propagating reagents because conjugation does not impair the ability to efficiently reproduce in bacteria, although relabeling with fluorochrome would be necessary. Our data suggest that primary labeled phage clones may be used similarly to primary antibody conjugates.     

Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure

Most patients failing antiretroviral treatment in Uganda continue to fail their treatment regimen even if a dominant drug-resistant HIV-1 genotype is not detected. In a recent retrospective study, we observed that approximately 30% of HIV-infected individuals in the Joint Clinical Research Centre (Kampala, Uganda) experienced virologic failure with a susceptible HIV-1 genotype based on standard Sanger sequencing. Selection of minority drug-resistant HIV-1 variants (not detectable by Sanger sequencing) under antiretroviral therapy pressure can lead to a shift in the viral quasispecies distribution, becoming dominant members of the virus population and eventually causing treatment failure. Here, we used a novel HIV-1 genotyping assay based on deep sequencing (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drug-resistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more sensitive methods to detect minority HIV-1 variants.     

T cell activation in HIV-seropositive Ugandans: differential associations with viral load, CD4+ T cell depletion, and coinfection

Immune activation is thought to play a major role in the pathogenesis of human immunodeficiency virus (HIV). This effect may be particularly relevant in Africa, where endemic coinfections may contribute to disease progression, perhaps as a consequence of enhanced immune activation. We investigated the expression of CD38 and human leukocyte antigen (HLA)-DR on T cells in 168 HIV-seropositive volunteers in Uganda. We observed higher levels of CD4(+) and CD8(+) T cell activation in Uganda, compared with those reported in previous studies from Western countries. Coexpression of CD38 and HLA-DR on both CD4(+) and CD8(+) T cell subsets was directly correlated with viral load and inversely correlated with CD4(+) T cell counts. In antiretroviral therapy (ART)-naive volunteers, viral load and CD4(+) T cell count had stronger associations with CD8(+) and CD4(+) T cell activation, respectively. Virus suppression by ART was associated with a reduction in T cell activation, with a stronger observed effect on reducing CD8(+) compared with CD4(+) T cell activation. The presence of coinfection was associated with increased CD4(+) T cell activation but, interestingly, not with increased CD8(+) T cell activation. Our results suggest that distinct mechanisms differentially drive activation in CD4(+) and CD8(+) T cell subsets, which may impact the clinical prognostic values of T cell activation in HIV infection.