Telomere Length Shortening in Microglia: Implication for Accelerated Senescence and Neurocognitive Deficits in HIV.

The widespread use of combination antiretroviral therapy (cART) has led to the accelerated aging of the HIV-infected population, and these patients continue to have a range of mild to moderate HIV-associated neurocognitive disorders (HAND). Infection results in altered mitochondrial function. The HIV-1 viral protein Tat significantly alters mtDNA content and enhances oxidative stress in immune cells. Microglia are the immune cells of the central nervous system (CNS) that exhibit a significant mitotic potential and are thus susceptible to telomere shortening. HIV disrupts the normal interplay between microglia and neurons, thereby inducing neurodegeneration. HIV cART contributes to the inhibition of telomerase activity and premature telomere shortening in activated peripheral blood mononuclear cells (PBMC). However, limited information is available on the effect of cART on telomere length (TL) in microglia. Although it is well established that telomere shortening induces cell senescence and contributes to the development of age-related neuro-pathologies, the effect of HIV-Tat on telomere length in human microglial cells and its potential contribution to HAND are not well understood. It is speculated that in HAND intrinsic molecular mechanisms that control energy production underlie microglia-mediated neuronal injury. TL, telomerase and mtDNA expression were quantified in microglial cells using real time PCR. Cellular energetics were measured using the Seahorse assay. The changes in mitochondrial function were examined by Raman Spectroscopy. We have also examined TL in the PBMC obtained from HIV-1 infected rapid progressors (RP) on cART and those who were cART naïve, and observed a significant decrease in telomere length in RP on cART as compared to RP's who were cART naïve. We observed a significant decrease in telomerase activity, telomere length and mitochondrial function, and an increase in oxidative stress in human microglial cells treated with HIV Tat. Neurocognitive impairment in HIV disease may in part be due to accelerated neuro-pathogenesis in microglial cells, which is attributable to increased oxidative stress and mitochondrial dysfunction.

Assessment of Antibody Interference of Enfuvirtide (T20) Function Shows Assay Dependent Variability.

BACKGROUND: During HIV infection, fusion of the viral and cellular membranes is dependent on folding of the gp41 trimer into a six-helix bundle. Fusion inhibitors, such as the antiretroviral Enfuvirtide (T20), interfere with the formation of the gp41 six-helix bundle. Recent in vitro studies reveal that the gp41 immunodominant region one targeting antibody 3D6 can block T20 interference, but the clinical and pathophysiologic significance of this finding is unclear. OBJECTIVE/METHOD: We have previously characterized a number of antibodies that target conformational epitopes on gp41and herein characterized their ability to interfere with T20 in multiple assays and assess their prevalence in HIV infected subjects. RESULTS: The T20 interference by antibody 3D6 was confirmed in a CHO-HXB2 envelope/ HeLaT4+ cell culture assay. Antibodies that target an immunodominant region one epitope, as well as a gp41 discontinuous epitope, also interfered in this assay, however, not all antibodies that targeted these epitopes showed T20 interference. This response was not due to the direct binding of T20 by the antibodies and could not be replicated utilizing TZM-bl and HL2/3 cells. Notably, serum competition studies on a panel of HIV subjects demonstrate that these conformational targeting antibodies are common in the HIV population. CONCLUSION: The relatively common nature of antibodies targeting these epitopes, the disparate in vitro results, and lack of reported clinical failures ascribed to such antibodies leads us to conclude that antibody interference of T20 is likely not clinically relevant. However, this warrants continued consideration with the advancement of other fusion inhibitors.

Cutaneous alternariosis in a renal transplant patient successfully treated with posaconazole: Case report and literature review.

Cutaneous alternariosis is an uncommon fungal infection that most commonly presents in organ transplant patients on immunosuppressive therapy. There are no clinical trials or guidelines to guide treatment of this condition, however itraconazole is the most commonly used antifungal in published cases. Here we report on a case of cutaneous alternariosis in a renal transplant recipient treated with a newer antifungal, posaconazole. A review of published reports of cutaneous alternariosis since 2008 is also discussed.

New Insights into the Disease Progression Control Mechanisms by Comparing Long-Term-Nonprogressors versus Normal-Progressors among HIV-1-Positive Patients Using an Ion Current-Based MS1 Proteomic Profiling.

For decades, epidemiological studies have found significant differences in the susceptibility to disease progression among HIV-carrying patients. One unique group of HIV-1-positive patients, the long-term-nonprogressors (LTNP), exhibits far superior ability in virus control compared with normal-progressors (NP), which proceed to Acquired Immune Deficiency Syndrome (AIDS) much more rapidly. Nonetheless, elucidation of the underlying mechanisms of virus control in LTNP is highly valuable in disease management and treatment but remains poorly understood. Peripheral blood mononuclear cells (PBMC) have been known to play important roles in innate immune responses and thereby would be of great interest for the investigation of the mechanisms of virus defense in LTNP. Here, we described the first comparative proteome analysis of PBMC from LTNP (n = 10) and NP (n = 10) patients using a reproducible ion-current-based MS1 approach, which includes efficient and reproducible sample preparation and chromatographic separation followed by an optimized pipeline for protein identification and quantification. This strategy enables analysis of many biological samples in one set with high quantitative precision and extremely low missing data. In total, 925 unique proteins were quantified under stringent criteria without missing value in any of the 20 subjects, and 87 proteins showed altered expressions between the two patient groups. These proteins are implicated in key processes such as cytoskeleton organization, defense response, apoptosis regulation, intracellular transport, etc., which provided novel insights into the control of disease progressions in LTNP versus NP, and the expression and phosphorylation states of key regulators were further validated by immunoassay. For instance, (1) SAMH1, a potent and "hot" molecule facilitating HIV-1 defense, was for the first time found elevated in LTNP compared with NP or healthy controls; elevated proteins from IFN-α response pathway may also contribute to viral control in LTNP; (2) decreased proapoptotic protein ASC along with the elevation of antiapoptotic proteins may contribute to the less apoptotic profile in PBMC of LTNP; and (3) elevated actin polymerization and less microtubule assembly that impede viral protein transport were first observed in LTNP. These results not only enhanced the understanding of the mechanisms for nonprogression of LTNP, but also may afford highly valuable clues to direct therapeutic efforts. Moreover, this work also demonstrated the ion-current-based MS1 approach as a reliable tool for large-scale clinical research.

Transmitted antiretroviral drug resistance in the men who have sex with men HIV patient cohort, Beijing, China, 2008-2011.

Transmitted drug resistance (TDR) is an ongoing public health problem in HIV disease treatment. However, little is known about TDR among men who have sex with men (MSM) patients in China. In addition, TDR prevalence among patients with acute HIV infection (AHI) or early HIV infection (EHI) was believed higher than that of patients with chronic HIV infection (CHI), but as AHI is typically either unidentified or crudely defined in large populations, very few direct comparisons have been made. We did a retrospective analysis of TDR in 536 antiretroviral-naive MSM patients from our immunodeficiency clinics at You'an Hospital, Capital Medical University (CMU), in Beijing, China, 2008-2011. The cohort included 266 patients with AHI/EHI and 270 patients with CHI. We analyzed the subtype, estimated the TDR prevalence, and characterized the model of TDR and the predicted drug sensitivity. Additionally, we made a comparison of TDR between the patients with AHI/EHI and patients with CHI. Our results indicated that among the 536 patients, HIV-1 subtype CRF01_AE accounted for 52.1%, subtype B accounted for 24.8%, CRF07_BC/ CRF08_BC accounted for 21.6% (116/536), and 1.3% were denoted as unique recombinant forms (URFs). A total of 7.8% patients had one or more transmitted HIV-1 drug resistance mutations, representing 6.2% for PI-related mutations, 0.9% for NRTI-related mutations, and 1.7% for NNRTI-related mutations. Although patients with AHI/EHI had a higher TDR prevalence as compared to that of patients with CHI, the difference was not statistically significant. There was no significant difference in TDR model and predicted drug susceptibility between the two groups of patients either. This study provides important strategic information for public health planning by healthcare officials in China and warrants a comprehensive study with larger patient cohorts from various healthcare centers within China.

Comparative Effectiveness of Darunavir 1,200 mg Daily and Approved Dosing Strategies for Protease Inhibitor-Experienced Patients.

Background. HIV protease inhibitors exhibit concentration-dependent viral inhibition. Higher once daily doses of darunavir boosted with ritonavir (DRV/r) may achieve viral suppression in place of twice daily dosing. International antiretroviral adherence guidelines recommend once daily regimens whenever possible. We present data on virologic suppression achieved with DRV 1,200 mg and ritonavir 100 mg once daily compared to approved DRV regimens. Methods. This retrospective observational study included all patients treated with DRV after documented use of another protease inhibitor at an urban immunodeficiency clinic. Data collection from inception of DRV use in August 2006 through March 2012 included patient demographics, viral loads, CD4+ cell counts, and resistance test results. The primary outcome of virologic suppression was defined as <50 copies/mL at 24 weeks. Differences in baseline characteristics and virologic outcomes across dosing groups were analyzed via one-way analysis of variance. Results. One hundred and thirty-five patients were included in the ITT analysis. Most patients had no known DRV RAMs at baseline. Virologic suppression rate was not different among treatment groups: 53.6% of patients on 1,200 mg daily, 52.3% on 600 mg twice daily, and 42.9% on 800 mg daily (P = 0.568). Conclusions. Darunavir 1,200 mg daily should be investigated for use in protease inhibitor-experienced patients.

Autobiographical memory specificity and the persistence of depressive symptoms in HIV-positive patients: rumination and social problem-solving skills as mediators.

Individuals infected with human immunodeficiency virus (HIV) are at elevated risk for depressive conditions, which in turn can negatively impact health-related behaviours and the course of illness. The present study tested the role of autobiographical memory specificity and its interaction with perceived stress in the persistence of depressive symptoms among dysphoric HIV-positive individuals. Additionally, we examined whether rumination and social problem solving mediated these effects. Results indicated that memory specificity moderated the impact of perceived stress, such that perceived stress was more strongly associated with follow-up depressive symptoms among those with greater memory specificity. Rumination, but not social problem solving, mediated this effect. Implications of these findings are discussed.

Similar efficacy of raltegravir when used with or without a protease inhibitor in treatment-experienced patients.

BACKGROUND: Patients with multiclass-resistant HIV-1 have limited treatment options. Raltegravir, an inhibitor of integrase, has shown excellent efficacy when used with protease inhibitors (Pis) in patients with drug-resistant HIV-1. Limited data are available however about the outcomes when using raltegravir without Pis in this population. METHODS: Medical records of subjects who received raltegravir as part of the Merck EAP study 0518 were reviewed and abstracted at participating sites. Eligibility criteria included HIV positivity, age ≥ 16 years, limited or no treatment options due to resistance or intolerance to multiple antiretroviral regimens, detectable viremia on current treatment regimen, and documented resistance to at least one drug in each antiretroviral class (PI, NNRTI, and nucleoside analogue). Demographic, clinical, and laboratory data were collected locally using a standardized collection form. Genotypic susceptibility scores (GSS) were determined from the most recent genotypic resistance test available prior to the initiation of raltegravir. The main objective was to compare virologic results in patients who received raltegravir with a PI versus those who received raltegravir without a PI. RESULTS: Four hundred forty-two subjects were evaluated from the respective sites in the EAP trial, of whom 340 were evaluable. The baseline mean HIV RNA was 4.6 log copies/ mL, and the mean CD4 cell count was 159 cells/µL. The median number of total and new antiretroviral agents in the background regimen was 4 and 2, respectively. Among the 254 patients who received a PI, the most common PI used was darunavir (89%). Etravirine was commonly used in both groups: 39% of the PI group and 67% of the non-PI group. At week 12, 67% of PI patients and 64% of non-PI patients achieved HIV RNA <75 copies/mL and 85% and 86%, respectively, achieved HIV RNA <400 copies/mL GSS, which was similar in both groups at baseline, predicted achieving an HIV RNA of <400 and 75 copies/mL at week 12 (P < .05). CONCLUSIONS: In treatment-experienced patients, the combination of raltegravir with a regimen not containing a PI (used with etravirine in two-thirds of patients) had similar virologic activity when compared to more standard regimens using raltegravir with a PI. The main determinant of efficacy was the number of active drugs as measured by GSS. These data expand the potential utility of raltegravir in patients with multidrug-resistant HIV.

A longitudinal investigation of the impact of life stress on HIV treatment adherence.

Suboptimal antiretroviral adherence is associated with poorer HIV outcomes. Psychosocial factors, including life stress, depression and coping, may influence adherence behavior. This prospective investigation sought to examine the impact of life stress (acute life events, chronic stress, and perceived stress), depression, and coping style on adherence to HIV treatment regimes over time. Participants were 87 treatment-seeking HIV-infected individuals recruited from an urban HIV clinic. They completed clinician-administered interviews and self-report questionnaires at baseline and 3-month follow-up. Acute life events and chronic stress prospectively predicted decreases in treatment adherence more strongly among individuals in a major depressive episode (n = 21) compared to non-depressed individuals (n = 66). Coping style did not appear to be the mechanism by which life stress influenced adherence among depressed HIV-infected individuals. These findings demonstrate that life stress has toxic effects for depressed individuals and suggest that treatment adherence interventions with depressed individuals could be enhanced via development of stress management skills.

Role of chemokine and cytokine polymorphisms in the progression of HIV-1 disease.

Allelic variants of the genes for chemokine receptors and their natural ligands, the chemokines, and cytokines can affect HIV-1 disease progression. This study investigates the level of expression of the CCR5-Delta32, CCR2b-641, RANTES In1.1C, SDF-1 3'A, IL-10-5'-592A and IL-4-589T alleles in two unique HIV-1 infected patient cohorts that represent the two distinct stages of disease progression, namely rapid progressors (RPs) and long term non-progressors (LTNPs) (n=12/group) were recruited. Quantitation of the gene expression of CCR5-Delta32, CCR2b-641, RANTES In1.1C, SDF-1 3'A, IL-10-5'-592A and IL-4-589T in peripheral blood mononuclear leukocytes (PBML) isolated from patients was performed by real time, quantitative (Q)-PCR using DNA was isolated from PBML. We observed that expression of these HIV-protective alleles was generally greater in the LTNP cohort than the RP cohort. LTNPs expressed more of the protective chemokine, SDF-1alpha than RPs, and no statistically significant difference was observed in RANTES production between the LTNPs and RPs. The LTNPs expressed significantly less amounts of cytokines IL-10 and IL-4 as compared to the RPs. Our results demonstrate that gene polymorphisms for CCR5-Delta32, CCR2b-641, RANTES In1.1C, SDF-1 3'A, IL-10-5'-592A and IL-4-589T may be used as clinical markers to predict progression of HIV-1 infections.

Staphylococcus aureus antimicrobial susceptibility of abscess samples from adults and children from the Kaleida Health System in western New York State, 2003 to 2006.

Staphylococcus aureus is the most common etiologic agent of skin abscesses. The regional rate of methicillin-resistant S. aureus (MRSA) abscesses may reflect the prevalence of local community-acquired MRSA (CAMRSA). A retrospective study was conducted to compare the antimicrobial susceptibility patterns of S. aureus isolates recovered from abscesses from 2003 to 2006 from patients at hospitals of the Kaleida Health System in western New York. S. aureus susceptibility information was obtained from a Vitek Legacy system, and the location and source of each isolate were identified. EpiInfo software was used to analyze the antimicrobial susceptibilities of all isolates and the trends in the rates of MRSA. A total of 2,848 S. aureus abscesses were identified by the Kaleida Health Clinical Microbiology Laboratory. Of those, 978 S. aureus abscess events occurred in four hospitals, including three adult facilities (547 episodes with 62 cases of bacteremia) and one children's facility (431 episodes with 2 cases of bacteremia). The MRSA rates in adults increased from 56% (2003) to 71% (2006), and that in children increased from 26% (2003) to 64% (2006). Of the MRSA isolates in the children's samples, more than 92% were susceptible to clindamycin. Of the MRSA isolates in the adult samples, 50% were susceptible to clindamycin in 2003 and 2004, whereas greater than 75% were susceptible in 2005 and 2006. The increased rates of MRSA abscesses with susceptibility to clindamycin may reflect the high prevalence level of CAMRSA in the western New York community. The variations in S. aureus susceptibilities could serve as an indicator of the changing resistance patterns within a broad urban community.

Modulation of the proteome of peripheral blood mononuclear cells from HIV-1-infected patients by drugs of abuse.

INTRODUCTION: We used proteomic analyses to assess how drug abuse modulates immunologic responses to infections with the human immunodeficiency virus type 1 (HIV-1). METHODS: Two-dimensional difference gel electrophoresis was utilized to determine changes in the proteome of peripheral blood mononuclear cells (PBMC) isolated from HIV-1-positive donors that occurred after treatment with cocaine or methamphetamine. Both drugs differentially regulated the expression of several functional classes of proteins. We further isolated specific subpopulations of PBMC to determine which subpopulations were selectively affected by treatment with drugs of abuse. Monocytes, B cells, and T cells were positively or negatively selected from PBMC isolated from HIV-1-positive donors. RESULTS: Our results demonstrate that cocaine and methamphetamine modulate gene expression primarily in monocytes and T cells, the primary targets of HIV-1 infection. Proteomic data were validated with quantitative, real-time polymerase chain reaction. These studies elucidate the molecular mechanisms underlying the effects of drugs of abuse on HIV-1 infections. Several functionally relevant classes of proteins were identified as potential mediators of HIV-1 pathogenesis and disease progression associated with drug abuse.

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products.

BACKGROUND: While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). RESULTS: Inspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes. CONCLUSION: HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are encoded in a region of the LTR that has already been shown to be deleted in some HIV-infected long-term survivors and represent new potential targets for vaccine development.

Cocaine modulates dendritic cell-specific C type intercellular adhesion molecule-3-grabbing nonintegrin expression by dendritic cells in HIV-1 patients.

We report that cocaine may act as cofactor in HIV pathogenesis by increasing dendritic cell-specific C type ICAM-3-grabbing nonintegrin (DC-SIGN) expression on dendritic cells (DC). Our results show that cocaine-using, long-term nonprogressors and normal progressors of HIV infection manifest significantly higher levels of DC-SIGN compared with cocaine-nonusing long-term nonprogressors and normal progressors, respectively. Furthermore, in vitro HIV infection of MDC from normal subjects cultured with cocaine and/or HIV peptides up-regulated DC-SIGN, confirming our in vivo finding. Cocaine, in synergy with HIV peptides, also up-regulates DC-SIGN gene expression by MDC. Furthermore, the cocaine-induced effects were reversed by a D1 receptor antagonist demonstrating the specificity of the reaction. Our results indicate that cocaine exacerbates HIV infection by up-regulating DC-SIGN on DC and these effects are mediated via dysregulation of MAPKs. These data are the first evidence that cocaine up-regulates the expression of DC-SIGN on DC. A better understanding of the role of DC-SIGN in HIV infection may help to design novel therapeutic strategies against the progression of HIV disease in the drug-using population.

Linezolid-resistant Enterococcus faecalis isolated from a cord blood transplant recipient.

We report a linezolid-resistant Enterococcus faecalis infection in a cord blood stem cell transplant recipient previously treated with linezolid for bloodstream infections by vancomycin-resistant enterococci. Sequencing showed a G2576U mutation in the 23S rRNA gene. Because of the important niche of linezolid in cancer treatment, linezolid-resistant E. faecalis is noteworthy.