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Background: Human herpesvirus 8 (HHV-8), the cause of Kaposi's sarcoma, is common among HIV-infected persons. The exact route of transmission of HHV-8 in various populations is still debated. Goal: The goal was to define the correlates of HHV-8 infection among men recently infected with human immunodeficiency virus. Study Design: Three hundred forty-two HIV-infected U.S. military men were evaluated using a questionnaire regarding potential risk factors and laboratory data, including HHV-8, herpes simplex virus 2 (HSV-2), syphilis, hepatitis B, and hepatitis C serologies. Results: The seroprevalence of HHV-8 was 32%. HHV-8 was significantly associated with hepatitis B seropositivity (odds ratio [OR], 2.44; 95% confidence interval [CI], 1.5-4.1), and black ethnicity was negatively associated with HHV-8 (OR, 0.6; 95% CI, 0.3-0.9) in the multivariate analysis. HHV-8 was not associated with drug use or hepatitis C seropositivity. Among men who have sex with men (MSM), HHV-8 infection correlated with hepatitis B seropositivity (OR, 2.2; 95% CI, 1.1-4.3) and HSV-2 (OR, 2.6; 95% CI, 1.4-4.9). Among heterosexuals, the correlates of HHV-8 were different; blacks as compared with whites (OR, 0.3; 95% CI, 0.1-0.8) and married versus single status (OR, 0.4; 95% CI, 0.2-0.9) were associated with a lower rate of HHV-8 infection. Among heterosexuals, hepatitis B, HSV-2, and sexual behaviors were not associated with HHV-8. Conclusion: This study suggests that the seroprevalence of HHV-8 is increased in both MSM and heterosexual men with HIV infection, and that the route(s) of HHV-8 acquisition might be different between

Compulsion-like alcohol drinking (CLAD), where consumption continues despite negative consequences, is a major obstacle to treating alcohol use disorder. The locus coeruleus area in the brainstem and norepinephrine receptor (NER) signaling in forebrain cortical regions have been implicated in adaptive responding under stress, which is conceptually similar to compulsion-like responding (adaptive responding despite the presence of stress or conflict). Thus, we examined whether anterior insula (aINS)-to-brainstem connections and alpha-1 NERs regulated compulsion-like intake and alcohol-only drinking (AOD). Halorhodopsin inhibition of aINS–brainstem significantly reduced CLAD, with no effect on alcohol-only or saccharin intake, suggesting a specific aINS–brainstem role in aversion-resistant drinking. In contrast, prazosin inhibition of alpha-1 NERs systemically reduced both CLAD and AOD. Similar to systemic inhibition, intra-aINS alpha-1-NER antagonism reduced both CLAD and AOD. Global aINS inhibition with GABAR agonists also strongly reduced both CLAD and AOD, without impacting saccharin intake or locomotion, while aINS inhibition of calcium-permeable AMPARs (with NASPM) reduced CLAD without impacting AOD. Finally, prazosin inhibition of CLAD and AOD was not correlated with each other, systemically or within aINS, suggesting the possibility that different aINS pathways regulate CLAD versus AOD, which will require further study to definitively address. Together, our results provide important new information showing that some aINS pathways (aINS–brainstem and NASPM-sensitive) specifically regulate compulsion-like alcohol consumption, while aINS more generally may contain parallel pathways promoting CLAD versus AOD. These findings also support the importance of the adaptive stress response system for multiple forms of alcohol drinking.

Lymph node fine-needle aspiration (LNFNA) may be a potentially more accurate, less painful, and more cost-effective way of sampling and monitoring lymph tissue than biopsy in patients with human immunodeficiency virus (HIV). Routine LNFNA would sample lymph tissue directly from the patient through a small needle. Doctors surgically removed lymph nodes from 5 patients with HIV and sampled half of each lymph node by LNFNA. They processed the other half into either a cell mixture or into slices to be examined microscopically. Comparisons of viral concentrations and viral RNA and DNA levels between the samples did not show significant variation. Microscopic examination of the tissue slices showed that more than 95% of the viral RNA was in the areas of the lymph tissue that produce immune cells. Tests used to detect viral RNA in all of the samples showed that more than 80% of the viral RNA was in this same area of the tissue.

We have developed an open-source workflow that allows for quantitative single-cell analysis of organelle morphology, distribution, and inter-organelle contacts with an emphasis on the analysis of mitochondria and mitochondria-endoplasmic reticulum (mito-ER) contact sites. As the importance of inter-organelle contacts becomes more widely recognized, there is a concomitant increase in demand for tools to analyze subcellular architecture. Here, we describe a workflow we call MitER (pronounced “mightier”), which allows for automated calculation of organelle morphology, distribution, and inter-organelle contacts from 3D renderings by employing the animation software Blender. We then use MitER to quantify the variations in the mito-ER networks of Saccharomyces cerevisiae, revealing significantly more mito-ER contacts within respiring cells compared to fermenting cells. We then demonstrate how this workflow can be applied to mammalian systems and used to monitor mitochondrial dynamics and inter-organelle contact in time-lapse studies.

Liver damage is a serious and sometimes fatal consequence of long‐term alcohol intake, which progresses from early‐stage fatty liver (steatosis) to later‐stage steatohepatitis with inflammation and fibrosis/necrosis. However, very little is known about earlier stages of liver disruption that may occur in problem drinkers, those who drink excessively but are not dependent on alcohol. We examined how repeated binge‐like alcohol drinking in C57BL/6 mice altered liver function, as compared with a single binge‐intake session and with repeated moderate alcohol consumption. We measured a number of markers associated with early‐ and later‐stage liver disruption, including liver steatosis, measures of liver cytochrome P4502E1 (CYP2E1) and alcohol dehydrogenase (ADH), alcohol metabolism, expression of cytokine mRNA, accumulation of 4‐hydroxynonenal (4‐HNE) as an indicator of oxidative stress, and alanine transaminase/aspartate transaminase as a measure of hepatocyte injury. Importantly, repeated binge‐like alcohol drinking increased triglyceride levels in the liver and plasma, and increased lipid droplets in the liver, indicators of steatosis. In contrast, a single binge‐intake session or repeated moderate alcohol consumption did not alter triglyceride levels. In addition, alcohol exposure can increase rates of alcohol metabolism through CYP2E1 and ADH, which can potentially increase oxidative stress and liver dysfunction. Intermittent, excessive alcohol intake increased liver CYP2E1 mRNA, protein, and activity, as well as ADH mRNAand activity. Furthermore, repeated, binge‐like drinking, but not a single binge or moderate drinking, increased alcohol metabolism. Finally, repeated, excessive intake transiently elevated mRNAfor the proinflammatory cytokine IL‐1B and 4‐HNElevels, but did not alter markers of later‐stage liver hepatocyte injury. Together, we provide data suggesting that even relatively limited binge‐like alcohol drinking can lead to disruptions in liver function, which might facilitate the transition to more severe forms of liver damage. The present study discovered that even limited voluntary binge‐like intake can damage the liver. Using different drinking models we were able to directly compare the effects of excessive versus more moderate levels of alcohol consumption. Excessive alcohol consumption produced multiple signs of liver dysfunction, including increased liver triglycerides, lipogenesis, and alcohol clearance, which were not present after moderate alcohol consumption. Importantly, our study demonstrates that even a short history of excessive drinking can result in multiple symptoms of early stage liver pathology.

Introduction When used in combination, antiretroviral drugs are highly effective for suppressing HIV replication. Nevertheless, treatment failure commonly occurs and is generally associated with viral drug resistance. The choice of an alternative regimen may be guided by a drug-resistance test. However, interpretation of resistance from genotypic data poses a major challenge.Methods As an alternative to current interpretation systems, we have developed artificial neural network (ANN) models to predict virological response to combination therapy from HIV genotype and other clinical information.Results ANN models trained with genotype, baseline viral load and time to follow-up viral load (1,154 treatment change episodes from multiple clinics), produced predictions of virological response that were highly significantly correlated with actual responses (r2=0.53; P<0.00001) using independent test data from clinics that contributed training data. Augmented models, trained with the additional variables of baseline CD4+T-cell count and four treatment history variables, were more accurate, explaining 69% of the variance in virological response. Models trained with the full input dataset, but only those data involving highly active antiretroviral therapy (three or more full-dose antiretroviral drugs in combination), performed at an intermediate level, explaining 61% of the variance. The augmented models performed less well when tested with data from unfamiliar clinics that had not contributed data to the training dataset, explaining 46% of the variance in response.Conclusion These data indicate that ANN models can be quite accurate predictors of virological response to HIV therapy even for patients from unfamiliar clinics. ANN models therefore warrant further development as a potential tool to aid treatment selection.

ABSTRACTWe describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4- and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.

ABSTRACTThe performance of a silica chip-based resequencing method, the Affymetrix HIV PRT 440 assay (hereafter referred to as the Affymetrix assay), was evaluated on a panel of well-characterized nonclade B viral isolates and on isolates exhibiting length polymorphisms. Sequencing of human immunodeficiency virus type 1 (HIV-1) polcDNAs from clades A, C, D, E, and F resulted in clade-specific regions of base-calling ambiguities in regions not known to be associated with resistance polymorphisms, as well as a small number of spurious resistance polymorphisms. The Affymetrix assay failed to detect the presence of additional serine codons distal to reverse transcriptase (RT) codon 68 that are associated with multinucleoside RT inhibitor resistance. The increasing prevalence of non-clade B HIV-1 strains in the United States and Europe and the identification of clinically relevant polgene length polymorphisms will impact the generalizability of the Affymetrix assay, emphasizing the need to accommodate this expanding pool of polgenotypes in future assay versions.

Vascular endothelial growth factor (VEGF), a potent angiogenic factor and endothelial cell-specific mitogen, is up-regulated by hypoxia. However, the mechanism(s) responsible for hypoxic induction of VEGF has not been clearly delineated. We report that the steady state VEGF mRNA levels are increased 12 ± 0.6-fold, but the transcriptional rate for VEGF is increased only 3.1 ± 0.6-fold by hypoxia in PC12 cells. In order to investigate cis-regulatory sequences which mediate this response to hypoxia, we cloned the rat genomic sequences encoding VEGF and identified a 28-base pair element in the 5′ promoter that mediates hypoxia-inducible transcription in transient expression assays. This element has sequence and protein binding similarities to the hypoxia-inducible factor 1 binding site within the erythropoietin 3′ enhancer. Post-transcriptional mechanisms have also been suggested to play a role in the hypoxic induction of VEGF. Evidence is provided that a frequently used polyadenylation site is 1.9 kilobases downstream from the translation termination codon for rat VEGF. This site is 1.5 kilobases further downstream from the polyadenylation site previously reported for VEGF. This new finding reveals sequence motifs in the 3′-untranslated region that may mediate VEGF mRNA stability.

Clofazimine (Lamprene) is an antimycobacterial drug that has antiinflammatory activity in a number of chronic autoimmune skin disorders. We report 22 patients treated with clofazimine for chronic graft-versus-host disease (cGVHD). The initial dose was 300 mg orally in a single daily dose for 90 days. After 90 days, the dose was lowered to 100 mg orally each day and the medication continued indefinitely as tolerated. Treatment courses lasted 7 to 835 days and were generally well tolerated. Gastrointestinal side effects occurred in eight of 22 patients (36%) and hyperpigmentation was noted in 12 of 22 patients (55%), which resolved upon decrease or discontinuation of the drug. Over 50% of patients with skin involvement, flexion contractures, or oral manifestations achieved complete or partial responses. Seven of 22 patients (32%) were able to reduce other immunosuppressive medications. Thus, clofazimine is safe and has encouraging efficacy in cGVHD, particularly if sclerodermatous skin, joint contractures, or oral manifestations are present. The mechanism by which clofazimine induces a response is unknown, but might be secondary to suppression of alloreactive T-cell function in cGVHD target organs. Clofazimine deserves further study for the treatment of cGVHD.

Several letters to the editor are presented in response to articles including an article about a girlfriend who broke off a relationship because of her partner's tri training, the issue on swimsuit featuring triathletes and an article about Amanda Beard, an icon of athleticism and feminine beauty.

Two previously healthy, immunocompetent men had persistent Rochalimaea henselae bacteremia with clinical relapses after courses of antibiotics to which the isolates were ultimately demonstrated susceptible in vitro. Both had sustained tick bites prior to their illnesses, thus demonstrating an association not previously identified, although suspected. The first patient had relapsing fever, constitutional symptoms, and an episode of aseptic meningitis despite therapy with amoxicillin, then with doxycycline, and then with ceftriaxone. Thereafter, he spontaneously became asymptomatic during a span of 2 months of persistent bacteremia. Finally, after 2 weeks of therapy with ceftriaxone plus gentamicin, followed by 4 weeks of therapy with oral ciprofloxacin, his bacteremia was cured. The second man had relapsing fever and constitutional symptoms after courses of tetracycline, then of chloramphenicol, and then of doxycycline. He became permanently asymptomatic after serial 2-week courses of chloramphenicol and erythromycin. The greater efficacy of lysis-centrifugation blood cultures in the recovery of R. henselae was noted.