Transmission of HIV-1 drug resistance mutations within partner-pairs: A cross-sectional study of a primary HIV infection cohort.

BACKGROUND: Transmission of human immunodeficiency virus type 1 (HIV-1) drug resistance mutations, particularly that of minority drug-resistant variants, remains poorly understood. Population-based studies suggest that drug-resistant HIV-1 is less transmissible than drug-susceptible viruses. We compared HIV-1 drug-resistant genotypes among partner-pairs in order to assess the likelihood of transmission of drug resistance mutations and investigate the role of minority variants in HIV transmission. METHODS AND FINDINGS: From 1992-2010, 340 persons with primary HIV-1 infection and their partners were enrolled into observational research studies at the University of Washington Primary Infection Clinic (UWPIC). Out of 50 partner-pairs enrolled, 36 (72%) transmission relationships were confirmed by phylogenetic distance analysis of HIV-1 envelope (env) sequences, and 31 partner-pairs enrolled after 1995 met criteria for this study. Drug resistance mutations in the region of the HIV-1 polymerase gene (pol) that encodes protease and reverse transcriptase were assessed by 454-pyrosequencing. In 25 partner-pairs where the transmission direction could be determined, 12 (48%) transmitters had 1-4 drug resistance mutations (23 total) detected in their HIV-1 populations at a median frequency of 6.0% (IQR 1.5%-98.7%, range 1.0%-99.6%). Of 10 major mutations detected in five transmitters at a frequency >95%, 100% (95% CI 69.2%-100%) were detected in recipients. All of these transmitters were antiretroviral (ARV)-naïve at the time of specimen collection. Fourteen mutations (eight major mutations and six accessory mutations) were detected in nine transmitters at low frequencies (1.0%-11.8%); four of these transmitters had previously received ARV therapy. Two (14% [95% CI 1.8%-42.8%]) G73S accessory mutations were detected in both transmitter and recipient. This number is not significantly different from the number expected based on the observed frequencies of drug-resistant viruses in transmitting partners. Limitations of this study include the small sample size and uncertainties in determining the timing of virus transmission and mutation history. CONCLUSIONS: Drug-resistant majority variants appeared to be commonly transmitted by ARV-naïve participants in our analysis and may contribute significantly to transmitted drug resistance on a population level. When present at low frequency, no major mutation was observed to be shared between partner-pairs; identification of accessory mutations shared within a pair could be due to transmission, laboratory artifact, or apolipoprotein B mRNA-editing enzyme, catalytic polypeptides (APOBECs), and warrants further study.

Prevalence of Pre-antiretroviral-Treatment Drug Resistance by Gender, Age, and Other Factors in HIV-Infected Individuals Initiating Therapy in Kenya, 2013-2014.

Background: Pre-antiretroviral-treatment drug resistance (PDR) is a predictor of human immunodeficiency virus (HIV) treatment failure. We determined PDR prevalence and correlates in a Kenyan cohort. Methods: We conducted a cross-sectional analysis of antiretroviral (ARV) treatment-eligible HIV-infected participants. PDR was defined as ≥2% mutant frequency in a participant's HIV quasispecies at pol codons K103N, Y181C, G190A, M184 V, or K65R by oligonucleotide ligation assay and Illumina sequencing. PDR prevalence was calculated by demographics and codon, stratifying by prior ARV experience. Poisson regression was used to estimate prevalence ratios. Results: PDR prevalences (95% confidence interval [CI]) in 815 ARV-naive adults, 136 ARV-experienced adults, and 36 predominantly ARV-naive children were 9.4% (7.5%-11.7%), 12.5% (7.5%-19.3%), and 2.8% (0.1%-14.5%), respectively. Median mutant frequency within an individual's HIV quasispecies was 67%. PDR prevalence in ARV-naive women 18-24 years old was 21.9% (9.3%-40.0%). Only age in females associated with PDR: A 5-year age decrease was associated with adjusted PDR prevalence ratio 1.20 (95% CI, 1.06-1.36; P = .004). Conclusions: The high PDR prevalence may warrant resistance testing and/or alternative ARVs in high HIV prevalence settings, with attention to young women, likely to have recent infection and higher rates of resistance. Clinical Trials Registration: NCT01898754.

Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies.

BACKGROUND & AIMS: Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. METHODS: We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell culture-derived HCV-producing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell culture-derived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein E2 and HCV virions by immunoprecipitation. RESULTS: Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein E2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. CONCLUSIONS: In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines.

Methylglyoxal-derived advanced glycation end products contribute to negative cardiac remodeling and dysfunction post-myocardial infarction.

Advanced glycation end-products (AGEs) have been associated with poorer outcomes after myocardial infarction (MI), and linked with heart failure. Methylglyoxal (MG) is considered the most important AGE precursor, but its role in MI is unknown. In this study, we investigated the involvement of MG-derived AGEs (MG-AGEs) in MI using transgenic mice that over-express the MG-metabolizing enzyme glyoxalase-1 (GLO1). MI was induced in GLO1 mice and wild-type (WT) littermates. At 6 h post-MI, mass spectrometry revealed that MG-H1 (a principal MG-AGE) was increased in the hearts of WT mice, and immunohistochemistry demonstrated that this persisted for 4 weeks. GLO1 over-expression reduced MG-AGE levels at 6 h and 4 weeks, and GLO1 mice exhibited superior cardiac function at 4 weeks post-MI compared to WT mice. Immunohistochemistry revealed greater vascular density and reduced cardiomyocyte apoptosis in GLO1 vs. WT mice. The recruitment of c-kit+ cells and their incorporation into the vasculature (c-kit+CD31+ cells) was higher in the infarcted myocardium of GLO1 mice. MG-AGEs appeared to accumulate in type I collagen surrounding arterioles, prompting investigation in vitro. In culture, the interaction of angiogenic bone marrow cells with MG-modified collagen resulted in reduced cell adhesion, increased susceptibility to apoptosis, fewer progenitor cells, and reduced angiogenic potential. This study reveals that MG-AGEs are produced post-MI and identifies a causative role for their accumulation in the cellular changes, adverse remodeling and functional loss of the heart after MI. MG may represent a novel target for preventing damage and improving function of the infarcted heart.

Hyperglycemia inhibits cardiac stem cell-mediated cardiac repair and angiogenic capacity.

BACKGROUND: The impact of diabetes mellitus on the cardiac regenerative potential of cardiac stem cells (CSCs) is unknown yet critical, given that individuals with diabetes mellitus may well require CSC therapy in the future. Using human and murine CSCs from diabetic cardiac tissue, we tested the hypothesis that hyperglycemic conditions impair CSC function. METHODS AND RESULTS: CSCs cultured from the cardiac biopsies of patients with diabetes mellitus (hemoglobin A1c, 10±2%) demonstrated reduced overall cell numbers compared with nondiabetic sourced biopsies (P=0.04). When injected into the infarct border zone of immunodeficient mice 1 week after myocardial infarction, CSCs from patients with diabetes mellitus demonstrated reduced cardiac repair compared with nondiabetic patients. Conditioned medium from CSCs of patients with diabetes mellitus displayed a reduced ability to promote in vitro blood vessel formation (P=0.02). Similarly, conditioned medium from CSCs cultured from the cardiac biopsies of streptozotocin-induced diabetic mice displayed impaired angiogenic capacity (P=0.0008). Somatic gene transfer of the methylglyoxal detoxification enzyme, glyoxalase-1, restored the angiogenic capacity of diabetic CSCs (diabetic transgenic versus nondiabetic transgenic; P=0.8). Culture of nondiabetic murine cardiac biopsies under high (25 mmol/L) glucose conditions reduced CSC yield (P=0.003), impaired angiogenic (P=0.02) and chemotactic (P=0.003) response, and reduced CSC-mediated cardiac repair (P<0.05). CONCLUSIONS: Diabetes mellitus reduces the ability of CSCs to repair injured myocardium. Both diabetes mellitus and preconditioning CSCs in high glucose attenuated the proangiogenic capacity of CSCs. Increased expression of glyoxalase-1 restored the proangiogenic capacity of diabetic CSCs, suggesting a means of reversing diabetic CSC dysfunction by interfering with the accumulation of reactive dicarbonyls.

Reducing methylglyoxal as a therapeutic target for diabetic heart disease.

Diabetes is a well-known risk factor for the development of cardiovascular diseases. Diabetes affects cardiac tissue through several different, yet interconnected, pathways. Damage to endothelial cells from direct exposure to high blood glucose is a primary cause of deregulated heart function. Toxic by-products of non-enzymatic glycolysis, mainly methylglyoxal, have been shown to contribute to the endothelial cell damage. Methylglyoxal is a precursor for advanced glycation end-products, and, although it is detoxified by the glyoxalase system, this protection mechanism fails in diabetes. Recent work has identified methylglyoxal as a therapeutic target for the prevention of cardiovascular complications in diabetes. A better understanding of the glyoxalase system and the effects of methylglyoxal may lead to more advanced strategies for treating cardiovascular complications associated with diabetes.

The intracellular redox state is a core determinant of mitochondrial fusion.

Mitochondrial hyperfusion has recently been shown to function as a cellular stress response, providing transient protection against apoptosis and mitophagy. However, the mechanisms that mediate this response remain poorly understood. In this study, we demonstrate that oxidized glutathione (GSSG), the core cellular stress indicator, strongly induces mitochondrial fusion. Biochemical and functional experiments show that GSSG induces the generation of disulphide-mediated mitofusin oligomers, in a process that also requires GTP hydrolysis. Our data outline the molecular events that prime the fusion machinery, providing new insights into the coupling of mitochondrial fusion with the cellular stress response.

Role of renin-angiotensin system in activation of macrophages by modified lipoproteins.

Angiotensin II favors the development of atherosclerosis. Our goal was to determine if foam cell formation increases angiotensin II generation by the endogenous renin-angiotensin system (RAS) and if endogenously produced angiotensin II promotes lipid accumulation in macrophages. Differentiated THP-1 cells were treated with acetylated low-density lipoproteins (ac-LDL), native LDL (n-LDL), or no LDL. Expression of RAS genes was assessed and angiotensin I/II levels were quantified in media and cell lysate. Ac-LDL increased angiotensin I/II levels and the angiotensin II/I ratio in cells and media after foam cell formation. Renin mRNA or activity did not change, but renin blockade completely inhibited the increase in angiotensin II. Angiotensinogen mRNA but not protein level was increased. Angiotensin-converting enzyme (ACE) and cathepsin G mRNA and activities were enhanced by ac-LDL. Inhibition of renin, ACE, or the angiotensin II receptor 1 (AT1-receptor) largely abolished cholesteryl ester formation in cells exposed to ac-LDL and decreased scavenger receptor A (SR-A) and acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT-1) protein levels. Inhibition of renin or the AT1-receptor in cells treated with oxidized LDL also decreased SR-A and ACAT-1 protein and foam cell formation. ac-LDL also increased angiotensin II by human peripheral blood monocyte-derived macrophages, whereas blockade of renin decreased cholesterol ester formation in these macrophages. These findings indicate that, during foam cell formation, angiotensin II generation by the endogenous RAS is stimulated and that endogenously generated angiotensin II is crucial for cholesterol ester accumulation in macrophages exposed to modified LDL.

Cathepsin G deficiency decreases complexity of atherosclerotic lesions in apolipoprotein E-deficient mice.

Cathepsin G is a serine protease with a broad range of catalytic activities, including production of angiotensin II, degradation of extracellular matrix and cell-cell junctions, modulation of chemotactic responses, and induction of apoptosis. Cathepsin G mRNA expression is increased in human coronary atheroma vs. the normal vessel. To assess whether cathepsin G modulates atherosclerosis, cathepsin G knockout (Cstg(-/-)) mice were bred with apolipoprotein E knockout (Apoe(-/-)) mice to obtain Ctsg(+/-)Apoe(-/-) and Ctsg(+/+)Apoe(-/-) mice. Heterozygous cathepsin G deficiency led to a 70% decrease in cathepsin G activity in bone marrow cells, but this reduced activity did not impair generation of angiotensin II in bone marrow-derived macrophages (BMDM). Atherosclerotic lesions were compared in male Cstg(+/-)Apoe(-/-) and Cstg(+/+)Apoe(-/-) mice after 8 wk on a high-fat diet. Plasma cholesterol levels and cholesterol distribution within serum lipoprotein fractions did not differ between genotypes nor did the atherosclerotic lesion areas in either the aortic root or aortic arch. Cstg(+/-)Apoe(-/-) mice, however, showed a lower percentage of complex lesions within the aortic root and a smaller number of apoptotic cells compared with Cstg(+/+)Apoe(-/-) littermates. Furthermore, apoptotic Cstg(-/-) BMDM were more efficiently engulfed by phagocytic BMDM than were apoptotic Ctsg(+/+) BMDM. Thus cathepsin G activity may impair efferocytosis, which could lead to an accumulation of lesion-associated apoptotic cells and the accelerated progression of early atherosclerotic lesions to more complex lesions in Apoe(-/-) mice.

Advanced glycation end products and diabetic retinopathy.

Retinopathy is a serious microvascular complication of diabetes and a major cause of blindness in young adults, worldwide. Early diabetic retinopathy is characterized by a loss of pericytes from retinal capillaries, the appearance of acellular capillaries and microaneurysms, and a breakdown of the blood-retinal barrier. In later stages, this can evolve into the proliferative phase in which there is neovascularization of the retina, which greatly increases the probability of vision loss. Advanced glycation end products (AGEs) which accumulate under hyperglycemic conditions are thought to play an important role in the pathogenesis of diabetic retinopathy. AGEs arise primarily by the modification of amine groups of proteins by reactive dicarbonyls such as methylglyoxal. Intracellular proteins including anti-oxidant enzymes, transcription factors and mitochondrial proteins are targets of dicarbonyl modification and this can modify their functional properties and thus compromise cellular physiology. Likewise, modification of extracellular proteins by dicarbonyls can impair cell adhesion and can generate ligands that can potentially bind to cell surface AGE receptors that activate pro-inflammatory signaling pathways. AGE inhibitors have been shown to provide protection in animal models of diabetic retinopathy and currently are being evaluated in clinical trials.

Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH.

During atherogenesis, the extracellular pH of atherosclerotic lesions decreases. Here, we examined the effect of low, but physiologically plausible pH on aggregation of modified LDL, one of the key processes in atherogenesis. LDL was treated with SMase, and aggregation of the SMase-treated LDL was followed at pH 5.5-7.5. The lower the pH, the more extensive was the aggregation of identically prelipolyzed LDL particles. At pH 5.5-6.0, the aggregates were much larger (size >1 µm) than those formed at neutral pH (100-200 nm). SMase treatment was found to lead to a dramatic decrease in α-helix and concomitant increase in ß-sheet structures of apoB-100. Particle aggregation was caused by interactions between newly exposed segments of apoB-100. LDL-derived lipid microemulsions lacking apoB-100 failed to form large aggregates. SMase-induced LDL aggregation could be blocked by lowering the incubation temperature to 15°C, which also inhibited the changes in the conformation of apoB-100, by proteolytic degradation of apoB-100 after SMase-treatment, and by HDL particles. Taken together, sphingomyelin hydrolysis induces exposure of protease-sensitive sites of apoB-100, whose interactions govern subsequent particle aggregation. The supersized LDL aggregates may contribute to the retention of LDL lipids in acidic areas of atherosclerosis-susceptible sites in the arterial intima.

Immunochemical analysis of the electronegative LDL subfraction shows that abnormal N-terminal apolipoprotein B conformation is involved in increased binding to proteoglycans.

Electronegative LDL (LDL(-)) is a minor subfraction of modified LDL present in plasma. Among its atherogenic characteristics, low affinity to the LDL receptor and high binding to arterial proteoglycans (PGs) could be related to abnormalities in the conformation of its main protein, apolipoprotein B-100 (apoB-100). In the current study, we have performed an immunochemical analysis using monoclonal antibody (mAb) probes to analyze the conformation of apoB-100 in LDL(-). The study, performed with 28 anti-apoB-100 mAbs, showed that major differences of apoB-100 immunoreactivity between native LDL and LDL(-) concentrate in both terminal extremes. The mAbs Bsol 10, Bsol 14 (which recognize the amino-terminal region), Bsol 2, and Bsol 7 (carboxyl-terminal region) showed increased immunoreactivity in LDL(-), suggesting that both terminal extremes are more accessible in LDL(-) than in native LDL. The analysis of in vitro-modified LDLs, including LDL lipolyzed with sphingomyelinase (SMase-LDL) or phospholipase A(2) (PLA(2)-LDL) and oxidized LDL (oxLDL), suggested that increased amino-terminal immunoreactivity was related to altered conformation due to aggregation. This was confirmed when the aggregated subfractions of LDL(-) (agLDL(-)) and oxLDL (ag-oxLDL) were isolated and analyzed. Thus, Bsol 10 and Bsol 14 immunoreactivity was high in SMase-LDL, ag-oxLDL, and agLDL(-). The altered amino-terminal apoB-100 conformation was involved in the increased PG binding affinity of agLDL(-) because Bsol 10 and Bsol 14 blocked its high PG-binding. These observations suggest that an abnormal conformation of the amino-terminal region of apoB-100 is responsible for the increased PG binding affinity of agLDL(-).

Assessment of minority frequency pretreatment HIV drug-resistant variants in pregnant women and associations with virologic non-suppression at term.

OBJECTIVE: To assess in ART-naïve pregnant women randomized to efavirenz- versus raltegravir-based ART (IMPAACT P1081) whether pretreatment drug resistance (PDR) with minority frequency variants (<20% of individual's viral quasispecies) affects antiretroviral treatment (ART)-suppression at term. DESIGN: A case-control study design compared PDR minority variants in cases with virologic non-suppression (plasma HIV RNA >200 copies/mL) at delivery to randomly selected ART-suppressed controls. METHODS: HIV pol genotypes were derived from pretreatment plasma specimens by Illumina sequencing. Resistance mutations were assessed using the HIV Stanford Database, and the proportion of cases versus controls with PDR to their ART regimens was compared. RESULTS: PDR was observed in 7 participants (11.3%; 95% CI 4.7, 21.9) and did not differ between 21 cases and 41 controls (4.8% vs 14.6%, p = 0.4061). PDR detected only as minority variants was less common (3.2%; 95% CI 0.2, 11.7) and also did not differ between groups (0% vs. 4.9%; p = 0.5447). Cases' median plasma HIV RNA at delivery was 347c/mL, with most (n = 19/22) showing progressive diminution of viral load but not ≤200c/mL. Among cases with viral rebound (n = 3/22), none had PDR detected. Virologic non-suppression at term was associated with higher plasma HIV RNA at study entry (p<0.0001), a shorter duration of ART prior to delivery (p<0.0001), and randomization to efavirenz- (versus raltegravir-) based ART (p = 0.0085). CONCLUSIONS: We observed a moderate frequency of PDR that did not significantly contribute to virologic non-suppression at term. Rather, higher pretreatment plasma HIV RNA, randomization to efavirenz-based ART, and shorter duration of ART were associated with non-suppression. These findings support early prenatal care engagement of pregnant women and initiation of integrase inhibitor-based ART due to its association with more rapid suppression of plasma RNA levels. Furthermore, because minority variants appeared infrequent in ART-naïve pregnant women and inconsequential to ART-suppression, testing for minority variants may be unwarranted.

Low-frequency pre-treatment HIV drug resistance: effects on 2-year outcome of first-line efavirenz-based antiretroviral therapy.

OBJECTIVES: Assess the impact of pre-treatment high-frequency and low-frequency drug-resistant HIV variants on long-term outcomes of first-line efavirenz-based antiretroviral therapy (ART). DESIGN: Prospective observational study. METHODS: Participants' pre-treatment plasma RNA had two sections of HIV pol encoding reverse transcriptase sequenced (Illumina, MiSeq) using unique molecular identifiers to detect wild-type (pre-treatment drug-resistant variants less than 1% of viral quasispecies), low-frequency (1-9%) or high-frequency drug-resistant variants (10-100%). Associations between pre-treatment drug resistance and virologic outcomes over 24 months of efavirenz-based ART were assessed for the number and frequency of mutations by drug class and other resistance parameters. RESULTS: Virologic failure was detected in 30 of 352 (9%) and pre-treatment drug-resistant variants were detected in the viral quasispecies of 31 of 352 (9%) participants prescribed efavirenz-based ART. Survival analyses revealed statistically significant associations between pre-treatment drug resistance at low (P < 0.0001) and high (P < 0.001) frequencies, at oligonucleotide ligation assay (OLA) (P < 0.00001) and non-OLA (P < 0.01) codons, to a single-antiretroviral class (P < 0.00001), and a shorter time to virologic failure of efavirenz-based ART. Regression analyses detected independent effects across resistance categories, including both low-frequency (P < 0.01) and high-frequency (P < 0.001) drug-resistant variants. CONCLUSION: We observed that pre-treatment HIV drug resistance detected at low frequencies increased the risk of virologic failure over 24 months of efavirenz-based ART, but that most failures, regardless of drug-resistant variants' frequencies, were detected within a year of ART initiation. These observations suggest that when efavirenz-based ART is prescribed, screening for pre-treatment drug resistance by an assay capable of detecting low-frequency variants, including OLA, may guide clinicians to prescribe more effective ART.

Intravascular transfer contributes to postprandial increase in numbers of very-low-density hepatitis C virus particles.

BACKGROUND & AIMS: The physical association of hepatitis C virus (HCV) particles with lipoproteins in plasma results in distribution of HCV in a broad range of buoyant densities. This association is thought to increase virion infectivity by mediating cell entry via lipoprotein receptors. We sought to determine if factors that affect triglyceride-rich lipoprotein (TRL) metabolism alter the density and dynamics of HCV particles in the plasma of patients with chronic HCV infection. METHODS: Fasting patients (n = 10) consumed a high-fat milkshake; plasma was collected and fractionated by density gradients. HCV- RNA was measured in the very-low-density fraction (VLDF, d < 1.025 g/mL) before and at 7 serial time points postprandially. RESULTS: The amount of HCV RNA in the VLDF (HCV(VLDF)) increased a mean of 26-fold, peaking 180 minutes after the meal (P < .01). Quantification of HCV RNA throughout the density gradient fractions revealed that HCV(VLDF) rapidly disappeared, rather than migrating into the adjacent density fraction. Immuno-affinity separation of the VLDF, using antibodies that recognize apolipoprotein B-100 and not apolipoprotein B-48, showed that HCV(VLDF) is composed of chylomicron- and VLDL-associated HCV particles; peaking 120 and 180 minutes after the meal, respectively. Plasma from fasting HCV-infected patients mixed with uninfected plasma increased the quantity of HCV(VLDF), compared with that mixed with phosphate-buffered saline, showing extracellular assembly of HCV(VLDF). CONCLUSIONS: Dietary triglyceride alters the density and dynamics of HCV in plasma. The rapid clearance rate of HCV(VLDF) indicates that association with TRL is important for HCV infectivity. HCV particles, such as exchangeable apolipoproteins, appear to reassociate with TRLs in the vascular compartment.

Greater dietary fat oxidation in obese compared with lean men: an adaptive mechanism to prevent liver fat accumulation?

Liver fat represents a balance between input, secretion, and oxidation of fatty acids. As humans spend the majority of a 24-h period in a postprandial state, dietary fatty acids make an important contribution to liver fat metabolism. We compared hepatic fatty acid partitioning in healthy lean (n = 9) and abdominally obese (n = 10) males over 24 h. Volunteers received three mixed meals adjusted for basal metabolic rate. U-13C-labeled fatty acids were incorporated into the meals, and [2H2]palmitate was infused intravenously to distinguish between sources of fatty acids incorporated into VLDL-TG. Immunoaffinity chromatography was used to isolate VLDL-TG of hepatic origin. Liver and whole body fatty acid oxidation was assessed by isotopic enrichment of 3-hydoxybutyrate and breath CO2. We found a similar contribution of dietary fatty acids to VLDL-TG in the two groups over 24 h. The contribution of fatty acids from splanchnic sources was higher (P < 0.05) in the abdominally obese group. Ketogenesis occurred to a significantly greater extent in abdominally obese compared with lean males, largely due to lessened downregulation of postprandial ketogenesis (P < 0.001). The appearance of 13C in breath CO2 was also greater (P < 0.001) in abdominally obese compared with lean men. Hepatic elongation and desaturation of palmitic acid were higher (P < 0.05) in abdominally obese than in lean males. Oxidation of dietary fatty acids and hepatic desaturation and elongation of palmitic acid occurred to a greater extent in abdominally obese men. These alterations may represent further pathways for redirection of fatty acids into export from the liver or oxidation to prevent liver fat accumulation.

Pre-treatment HIV-drug resistance associated with virologic outcome of first-line NNRTI-antiretroviral therapy: A cohort study in Kenya.

BACKGROUND: Pre-treatment HIV-drug-resistance (PDR) to WHO-recommended 1st-line non-nucleoside reverse transcriptase inhibitors (NNRTI)-based antiretroviral treatment (ART) is increasing in low-resource communities. We evaluated the risk of PDR on treatment failure if detected at single or multiple codons, at minority (2-9%) or higher (≥10%) frequencies during efavirenz- vs. nevirapine-ART. METHODS: We conducted a pooled analysis across three cohorts of Kenyans initiating 1st-line NNRTI-ART between 2006 and 2014. Mutations K103N, Y181C, G190A, M184V and K65R were detected by an oligonucleotide ligation assay (OLA) and confirmed by Sanger and next-generation sequencing (NGS). PDR was defined as detection of any mutation by OLA when confirmed by NGS. Treatment failure, defined as plasma HIV RNA ≥400 copies/mL at month-12 of ART, was compared by PDR genotypes. FINDINGS: PDR was detected in 59/1231 (4·8%) participants. Compared to wild-type genotypes, PDR in participants prescribed nevirapine-ART was associated with increased treatment failure [PDR 69·2% (27/39) vs. wild-type 10·4% (70/674); p = 0·0001], whether detected as minority [66·7% (4/6)] or higher [69·7% (23/33)] frequencies in an individual's HIV quasispecies (p = 0·002 and p < 0·0001, respectively), or mutations at single [50·0% (12/24)] or multiple [100·0% (15/15)] codons (p < 0·0001). During efavirenz-ART, PDR was also associated with increased virologic failure [PDR 25·0% (5/20) vs. wild-type 5·0% (25/498); p = 0·005], but only if detected at multiple drug-resistant codons [50·0% (3/6); p = 0·003] or high frequencies PDR [33·3% (5/15); p = 0·001]. INTERPRETATION: The risk that PDR confers for treatment failure varies by number of mutant codons and their frequency in the quasispecies, with a lower risk for efavirenz- compared to nevirapine-based regimens. PDR detection and management could extend the effective use of efavirenz-ART in low-resource settings. FUNDING: NIH, PEPFAR.

Evaluation of the management of pretreatment HIV drug resistance by oligonucleotide ligation assay: a randomised controlled trial.

BACKGROUND: Although experts have recommended testing for pretreatment drug resistance (PDR) before antiretroviral therapy (ART) initiation, there is little evidence to support its implementation. We aimed to establish whether an inexpensive point mutation assay can improve virological suppression by identifying PDR to guide drug selection for ART in a lower-middle income country. METHODS: Investigators did an open-label, randomised controlled trial at three HIV treatment sites in Kenya: two in Nairobi and one in rural Maseno. Individuals (aged ≥2 years) were eligible to participate if they were confirmed HIV-seropositive, qualified for first-line ART, planned to reside in the area for more than 1 year, and provided informed consent. We randomly assigned participants (1:1) to either PDR testing by oligonucleotide ligation assay (OLA) to guide selection of ART or to standard of care, which did not include OLA testing. The OLA-guided therapy group had pre-ART peripheral blood mononuclear cells evaluated for drug resistance to non-nucleoside reverse transcriptase inhibitor (NNRTI) at codons Lys103Asn, Tyr181Cys, Gly190Ala, and to lamivudine at Met184Val, and when at least one drug-resistant codon was detected in a participant's pre-ART specimen, clinicians were directed to prescribe protease inhibitor-based second-line ART. Those without detected resistance and those who were randomised to standard of care received NNRTI-based first-line ART. The primary outcome was plasma HIV-1 RNA of at least 400 copies per mL at 4, 8, or 12 months after ART initiation, which defined virological failure, assessed in all participants who received treatment (data were censored for those lost-to-follow-up or who died). The study has been completed and is registered with ClinicalTrials.gov, NCT01898754. FINDINGS: We screened 1198 participants between May 28, 2013, and Nov 4, 2014, of whom 991 (83%) were enrolled (492 received OLA and 495 received standard of care; four did not begin treatment). 93 participants (prevalence 9·4%) had PDR (95% CI 7·7-11·4). 34 (8·5%) of 400 participants in the OLA group had virological failure at month 12 of ART (95% CI 6·0-11·7) compared with 39 (9·7%) of 402 (7·0-13·0) in the standard-of-care group (log-rank p=0·26). Among participants with PDR, virological failure was lower in the OLA-guided therapy group than in the standard-of-care group: five (14%) of 35 compared with 13 (50%) of 26; p=0·0020). Among those prescribed NNRTI-based ART, participants given efavirenz were less likely to have virological failure than were those receiving nevirapine (odds ratio 0·37, 95% CI 0·22-0·62; p<0·0001). The OLA-guided therapy group had 39 serious non-lethal adverse events and 34 deaths. The standard-of-care group had 34 severe adverse events and 43 deaths, differences that were not significant. Adverse events judged to potentially be due to ART were few and similar between groups, with 17 (16%) in the OLA-guided therapy group and 16 (16%) in the standard-of-care group (p=0·90). INTERPRETATION: Our finding that OLA testing for PDR reduced virological failure in only those with specific PDR mutations suggests that PDR poses less of a risk for virological failure than that predicted by past prevalence estimates, and that the value of PDR testing to reduce virological failure should be assessed for antiretroviral treatment regimens. FUNDING: US National Institutes of Health.

Activity of spores and extracellular proteins from six Cry+ strains and a Cry- strain of Bacillus thuringiensis subsp. kurstaki against the western spruce budworm, Choristoneura occidentalis (Lepidoptera: Tortricidae).

We characterized insecticidal activity of previously untested strains of Bacillus thuringiensis kurstaki belonging to two crystal serovars (K-1 and K-73) against the western spruce budworm (Choristoneura occidentalis Freeman 1967). By testing various components, we demonstrated that spores play a critical role in the pathogenesis of each strain. Spore-free crystals caused low mortality and purified spores were generally not toxic. The addition of spores to purified protoxin increased toxicity several hundred-fold, regardless of the parental strain from which the spores or protoxins were derived. The crystal and spore components did not account for full insecticidal activity of whole sporulated cultures owing to the toxicity of soluble proteins that are secreted during cell growth. We observed a marked difference in toxicity of secreted proteins between the K-1 and K-73 type strains, with the K-1 preparations causing much higher mortality, mass reduction, and inhibition of pupation. There was a consistent correlation between relative toxicity of secreted protein preparations and the presence and quantity of the Vip3A protein, suggesting that this protein contributes to the virulence of B. thuringiensis subsp. kurstaki in western spruce budworm larvae. However, other virulence factors have to be invoked to explain the synergizing effect of spores from both K-1 and K-73 strains on Cry protein toxicity.

Return to play following anterior cruciate ligament reconstruction: incorporating fatigue into a return to play functional battery. Part A: treadmill running.

BACKGROUND: The risk of reinjury and other sequelae following anterior cruciate ligament reconstruction (ACLR) remains high. Lack of knowledge regarding factors contributing to these risks limits our ability to develop sensitive return to play (RTP) tests. Using a running task, we evaluate whether fatigue induces alterations in foot progression angle (FPA), a proposed biomechanical risk factor and could be used to enhance RTP test sensitivity. METHOD: Transverse plane foot kinematics (FPA) were assessed for 18 post-ACLR subjects during a treadmill running task, before and after a generalised lower limb fatigue protocol. Subject's contralateral limbs were used as a control group. RESULTS: A small but significant difference between FPA for ACLR and contralateral limbs was observed before but not after fatigue. When confounding variables were considered, there was a significant difference in FPA change between ACLR and contralateral limbs from the prefatigue to postfatigue state. CONCLUSIONS: Following ACLR athletes may develop a knee-protective movement strategy that delays the progression of osteoarthritis in the ACL-injured knee. This may, however, increase the risk of ACL reinjury. Following the onset of fatigue this proposed movement strategy, and thus osteoarthritis protection, is lost.