Predictors of Raised Viral Load during Antiretroviral Therapy in Patients with and without Prior Antiretroviral Use: A Cross-Sectional Study

Objectives

In Lagos, Nigeria, Médecins Sans Frontières (MSF) and the Ministry of Health (MoH) commenced free antiretroviral treatment (ART) in a hospital-based clinic. We performed a cross-sectional study to compare factors associated with raised viral load between patients with (“experienced”) and without (“naïve”) prior antiretroviral (ARV) exposure at commencement of ART at the clinic. We also examined factors influencing ARV adherence in experienced patients prior to clinic entry.

Methods

We included adult patients receiving ART from MSF who answered a questionnaire about previous antiretroviral use. Multivariate logistic regression was used to estimate odds ratios (OR) for raised viral load (≥1000 copies/mL).

Results

1246 (96%) patients answered: 1075 (86%) reported no, and 171 (14%) some, prior ARV exposure. ARV-naïve patients were more immunosuppressed at baseline: 65% vs 37% (p<0.001) had CD4<200; 17% vs 9% (p = 0.013) were WHO stage 4. Proportionately more experienced than naïve patients had raised viral loads (20% vs 9%, p<0.001) on ART in the MSF/MoH clinic. Raised viral load was associated with prior ARV experience (adjusted OR = 3.74, 95%CI 2.09–6.70, p<0.001) and complete interruption of current ART (adjusted OR = 3.71, 95%CI 2.06–6.68, p<0.001). Higher CD4 at time of VL and a higher self-rated score of recent adherence were associated with lower OR of a raised viral load. Among experienced patients who missed pills before joining MSF/MoH, most common reasons were because ARVS were not affordable (58%) or available (33%), with raised viral load associated with being unsure how to take them (OR = 3.16, 95%CI 1.10–9.12, p = 0.033).

Conclusions

Patients previously exposed to ARVs had increased OR of raised viral load. The cost and availability of ARVs were common reasons for missing ARVs before joining the MSF/MoH clinic, and inadequate patient knowledge was associated with raised viral load.     

Exploration of in vitro pro-drug activation and futile cycling by glutathione S-transferases: thiol ester hydrolysis and inhibitor maturation

In addition to glutathione (GSH) conjugating activity, glutathione S-transferases (GSTs) catalyze "reverse" reactions, such as the hydrolysis of GSH thiol esters. Reverse reactions are of interest as potential tumor-directed pro-drug activation strategies and as mechanisms for tissue redistribution of carboxylate-containing drugs. However, the mechanism and specificity of GST-mediated GSH thiol ester hydrolysis are uncharacterized. Here, the GSH thiol esters of ethacrynic acid (E-SG) and several nonsteroidal antiinflammatory agents have been tested as substrates with human GSTs. The catalytic hydrolysis of these thiol esters appears to be a general property of GSTs. The hydrolysis of the thiol ester of E-SG was studied further with GSTA1-1 and GSTP1-1, as a model pro-drug with several possible fates for the hydrolysis products: competitive inhibition, covalent enzyme adduction, and sequential metabolism. In contrast to hydrolysis rates, significant isoform-dependent differences in the subsequent fate of the products ethacrynic acid and GSH were observed. At low [E-SG], only the GSTP1-1 efficiently catalyzed sequential metabolism, via a dissociative mechanism.     

Effects of copper ions released from metallic copper on CHO-K1 cells

The aim of this study was to investigate the cytotoxic and genotoxic effect of copper extracts obtained from metallic copper in Chinese hamster ovary (CHO-K1) cell line using neutral red (NR), sister chromatid exchange (SCE), chromosomal aberrations (CA) and cell-cycle kinetics tests. Cells were cultured in Ham-F10 with different copper-containing extracts obtained after the immersion of copper disks for 1, 2, 3, 9, 12, 24, 48 and 72 h in culture medium. Results from cytotoxicity assay showed an inverted U-shape response evidenced in changes in lysosomal activity and mitotic index. The analysis of CA revealed an increase of abnormal metaphases for copper concentration (cCu) in the 5.67-7.42 mg/L dose-range (p<0.001). In addition, SCE frequencies were higher for treated cells when compared with controls in the 1.56-7.42 mg/L concentration range (p<0.001). The absence of metaphases indicated cytotoxicity for cCu≥10.85 mg/L. Results show that cells close to copper-containing materials releasing copper ions are susceptible to cytotoxic and genotoxic effects.     

Nanomolar melatonin enhances nNOS expression and controls HaCaT-cells bioenergetics

A novel role of melatonin was unveiled, using immortalized human keratinocyte cells (HaCaT) as a model system. Within a time window compatible with its circadian rhythm, melatonin at nanomolar concentration raised both the expression level of the neuronal nitric oxide synthase mRNA and the nitric oxide oxidation products, nitrite and nitrate. On the same time scale, a depression of the mitochondrial membrane potential was detected together with a decrease of the oxidative phosphorylation efficiency, compensated by glycolysis as testified by an increased production of lactate. The melatonin concentration, ～ nmolar, inducing the bioenergetic effects and their time dependence, both suggest that the observed nitric oxide-induced mitochondrial changes might play a role in the metabolic pathways characterizing the circadian melatonin chemistry.     

Analysis of transmembrane proteins from eukaryotic cells

The topography and properties of plasma membrane proteins from mouse L-929 cells are studied by comparing their availability for enzymatic labeling on the external and internal surfaces of the membrane. In order to study the internal surface, phagolysosomes are prepared from cells after they ingest latex particles. The plasma membrane surrounding these seems to have an “inside-out” orientation. The sugars of the membrane glycoproteins in intact phagolysosomes are not available for interaction with lectins or available for periodate-borotritide labeling. A comparison of the lectin-binding proteins lableled by lactoperoxidase-catalyzed iodination on the external cell surface with those labeled on the internal cell surface suggests that a variety of plasma membrane glycoproteins span the lipid bilayer. Using two-dimensional gel electrophoresis it has been shown that selected proteins are labeled at both the internal and external faces of the plasma membrane. Analysis of the 2-D gel electrophoregrams reveals that there are two distinct prominent proteins at 60,000 and 100,000 daltons which are enzymatically iodinated from both sides of the membrane. The partial hydrolysis of the 100,000 dalton protein reveals that different peptides are iodinated when the iodination is performed on intact cells or on the phagolysosomes. These proteins are extensively phosphorylated in cells incubated with inorganic ³²P. We conclude that the phagolysosome is probably oriented in an “inside-out” configuration and that this membrane preparation can be used to study the topographic organization of membrane proteins. The use of oriented membranes, selective labeling of proteins, and affinity separation of proteins in combination with gel electrophoresis to define the position and properties of proteins is discussed.     

Osservazioni ecologiche preliminari su Petagnia saniculifolia Guss., raro endemismo siculo

Abstract

Preliminary ecological observations on Petagnia saniculifolia Guss., a rare sicilian endemism. — The Authors present the preliminary results of some observations on the anatomy and ecology of Petagnia saniculifolia Guss. and a synthetic phytosociological table in which some species of Molinio-Arrhenatheretea and Querco-Fagetea are present. The particular needs of this species and the precarious environmental conditions are pointed out.     

Conformational origin of the aggregation of recombinant human factor VIII

Aggregation of proteins is a major problem in their use as drugs and is also involved in a variety of pathological diseases. In this study, biophysical techniques were employed to investigate aggregate formation in the pharmaceutically important protein, recombinant human factor VIII (rhFVIII). Recombinant human factor VIII incubated in solution at 37 degrees C formed soluble aggregates as detected by molecular sieve chromatography and dynamic light scattering. This resulted in a corresponding loss of biological activity. Fluorescence and CD spectra of the thermally stressed rhFVIII samples did not, however, suggest significant differences in protein conformation. To identify conformational changes in rhFVIII that may be involved in rhFVIII aggregation, temperature and solutes were used to perturb the native structure of rhFVIII. Far-UV CD and FTIR studies of rhFVIII as a function of temperature revealed conformational changes corresponding to an increase in intermolecular beta-sheet content beginning at approximately 45 degrees C with significant aggregation observed above 60 degrees C. Fluorescence and DSC studies of rhFVIII also indicated conformational changes initiating between 45 and 50 degrees C. An increase in the exposure of hydrophobic surfaces was observed beginning at approximately 40 degrees C, as monitored by increased binding of the fluorescent probe, bis-anilinonaphthalene sulfonic acid (bis-ANS). Perturbation by various solutes produced several transitions prior to extensive unfolding of rhFVIII. In all cases, a common transition, characterized by an increase in the wavelength of the fluorescence emission maximum of rhFVIII from approximately 330 to 335 nm, was observed during thermal and solute perturbation of factor VIII. Moreover, this transition was correlated with an increased association of factor VIII upon incubation at 37 degrees C in the presence of various solutes. These results suggest that association of rhFVIII in solution was initiated by a small transition in the tertiary structure of the protein which produced a nucleating species that led to the formation of inactive soluble aggregates.