Exosomal Tat protein activates latent HIV-1 in primary, resting CD4+ T lymphocytes.

Replication competent HIV-1 persists in a subpopulation of CD4+ T lymphocytes despite prolonged antiretroviral treatment. This residual reservoir of infected cells harbors transcriptionally silent provirus capable of reigniting productive infection upon discontinuation of antiretroviral therapy. Certain classes of drugs can activate latent virus but not at levels that lead to reductions in HIV-1 reservoir size in vivo. Here, we show the utility of CD4+ receptor targeting exosomes as an HIV-1 latency reversal agent (LRA). We engineered human cellular exosomes to express HIV-1 Tat, a protein that is a potent transactivator of viral transcription. Preparations of exosomal Tat-activated HIV-1 in primary, resting CD4+ T lymphocytes isolated from antiretroviral-treated individuals with prolonged periods of viral suppression and led to the production of replication competent HIV-1. Furthermore, exosomal Tat increased the potency of selected LRA by over 30-fold in terms of HIV-1 mRNA expression, thereby establishing it as a potentially new class of biologic product with possible combinatorial utility in targeting latent HIV-1.

Reduced levels of genital tract immune biomarkers in postmenopausal women: implications for HIV acquisition.

BACKGROUND: Rates of HIV infections are increasing in older adults. Although it is known that the HIV/AIDS epidemics affects women disproportionately, little is known regarding immune functions in the genital tract of postmenopausal women, as relevant to HIV susceptibility. OBJECTIVE: The objective of the study was to compare levels of female reproductive tract immune mediators that are important for HIV-associated immune responses as well as intrinsic anti-HIV activity in the cervical vaginal lavages collected from HIV-negative pre- and postmenopausal women. STUDY DESIGN: Cervical vaginal lavage from 20 premenopausal and 20 postmenopausal women were assayed for interleukin-6, interleukin-8, tumor necrosis factor-α, secretory leukocyte protease inhibitor, elafin, human ß-defensin-2, and macrophage inflammatory protein-3α using standard enzyme-linked immunosorbent assays. Anti-HIV activity of cervical-vaginal lavage was measured using TZM-bl indicator cells against HIV-1 IIIB and BaL. Whereas each postmenopausal woman provided only 1 sample, each premenopausal woman provided 3 samples, during proliferative, ovulatory, and secretory stages, based on menstrual dates. RESULTS: We observed significantly lower levels of tumor necrosis factor-α, MIP-3α, secretory leukocyte protease inhibitor, elafin, and human ß-defensin-2 in cervical vaginal lavage from postmenopausal women compared with premenopausal women. Inhibition of HIV-1 infection was observed for both pre- and postmenopausal women, but cervical vaginal lavage from postmenopausal women showed significantly higher inhibition against HIV-1 BaL after adjusting for total protein concentration, genital pH, and reproductive tract infections. No change in mediators or HIV inhibition was observed through the stages of menstrual cycle. In addition, we observed that postmenopausal women with reproductive tract infections had significantly higher levels of tumor necrosis factor-α and significantly lower levels of interleukin-8, which were not observed in premenopausal women. CONCLUSION: Our findings suggest that female reproductive tract immune microenvironment is distinct in HIV-negative postmenopausal women. Further studies are needed to assess the risk of HIV acquisition/transmission in this population.

The spectrum of undiagnosed hepatitis C virus infection in a US HIV clinic.

United States guidelines endorse one-time HCV antibody screening at HIV diagnosis. Rescreening HCV-seronegative patients on a regular basis is still not policy, although HIV-infected persons have reasonably substantial HCV incidence. We evaluated routine risk factor-independent HCV antibody re-testing in a Rhode Island HIV clinic. We instituted annual HCV antibody testing for HCV-seronegative patients who had not been rescreened in a year or more. Testing based on clinical suspicion continued. We conducted a chart review of new antibody-positive cases in the first year of rescreening, July 2006 to June 2007. Of 245 rescreened patients, 11 (4.5%) seroconverted. Five (45%) were female. Median time between last negative and first positive result was 32 months (range 8-98 months). Six (55%) had documented risk factors and 6 (55%) elevated ALT (> 45 IU/L) between antibody tests; none prompted re-testing. One seroconverter died of hepatocellular carcinoma 3.7 years after HCV diagnosis. A twelfth was rescreened for suspected acute HCV based on ALT of 515 IU/L. He had newly detectable HCV RNA then seroconversion, and achieved SVR following 6 months of treatment in the acute phase for genotype 1 infection. Incident HCV is not uncommon among HIV-infected patients in care. Rescreening identified undiagnosed HCV in this population. HCV RNA should be checked promptly in HCV-seronegative persons with ALT elevation. We observed consequences of late diagnosis (hepatocellular carcinoma) and benefits of early diagnosis (cure with treatment of acute HCV). Adding annual rescreening to the Ryan White Program would facilitate earlier identification of undiagnosed HCV and create an instant widespread surveillance system, providing HCV incidence data.

Hepatocellular carcinoma in HIV-infected women: two case reports.

With the widespread availability of highly active antiretroviral therapy (HAART), and increased life expectancy among HIV-infected individuals, liver-related mortality has emerged as the leading cause of non-AIDS-related death. The incidence of hepatocellular carcinoma (HCC), a sequela of chronic liver disease, is rising among HIV-infected individuals. While women are increasingly and disproportionately affected by HIV, little is known about HCC in HIV-infected women given HCC's predilection for men. In 2007, 2 out of 398 HIV-infected women seen at a Rhode Island HIV clinic were diagnosed with HCC. Of 351 HIV-infected individuals with HCC described in the published literature, 12 (3.4%) were women. These 2 cases add to the existing literature on this topic.

Persistent genital tract HIV-1 RNA shedding after change in treatment regimens in antiretroviral-experienced women with detectable plasma viral load.

OBJECTIVE: To longitudinally assess the association between plasma viral load (PVL) and genital tract human immunodeficiency virus (GT HIV) RNA among HIV-1 infected women changing highly active antiretroviral therapy (HAART) because of detectable PVL on current treatment. METHODS: Women were eligible for the study if they had detectable PVL (defined as two consecutive samples with PVL>1000 copies/mL) and intended to change their current HAART regimen at the time of enrollment. Paired plasma and GT HIV-1 RNA were measured prospectively over 3 years. Longitudinal analyses examined rates of GT HIV-1 RNA shedding and the association with PVL. RESULTS: Sixteen women were followed for a median of 11 visits contributing a total of 205 study visits. At study enrollment, all had detectable PVL and 69% had detectable GT HIV-1 RNA. Half of the women changed to a new HAART regimen with ≥3 active antiretroviral drugs. The probability of having detectable PVL ≥30 days after changing HAART was 0.56 (95% CI: 0.37 to 0.74). Fourteen women (88%) had detectable PVL on a follow-up visit ≥30 or 60 days after changing HAART; and 12 women (75%) had detectable GT HIV-1 RNA on a follow-up visit ≥30 or 60 days after changing HAART. When PVL was undetectable, GT shedding occurred at 11% of visits, and when PVL was detectable, GT shedding occurred at 47% of visits. CONCLUSIONS: Some treatment-experienced HIV-infected women continue to have detectable virus in both the plasma and GT following a change in HAART, highlighting the difficulty of viral suppression in this patient population.

Treatment for hepatitis C virus genotype 1 infection in HIV-infected individuals on methadone maintenance therapy.

BACKGROUND: A minority of HIV/HCV coinfected patients with opiate addiction undergo HCV treatment. HCV therapy for HCV-monoinfected methadone maintenance (MM) recipients is safe and effective. We evaluated treatment efficacy and adherence to pegylated interferon (pegIFN) among HIV/HCV coinfected MM recipients. METHODS: HCV treatment-naïve, HIV-infected persons 18-65 years with chronic HCV genotype 1 on MM were prospectively enrolled in an HCV treatment study at two HIV clinics. At weekly visits pegIFN alfa-2a injections were directly administered. Daily MM recipients had morning ribavirin delivered with methadone at off-site methadone clinics. Weekly take-home MM recipients took ribavirin unsupervised. Target enrollment was 30 participants. RESULTS: During 18 recruitment months, 11 participants were enrolled, 6 of whom received daily methadone. Mean age was 46, 64% were female, 5 were Caucasian, 4 Black and 2 Hispanic. At baseline, 82% had high HCV RNA and 55% had stage 2 fibrosis or greater. The majority (91%) were on HAART, and 82% had undetectable HIV RNA with a median CD4(+) of 508cells/µL. All had polysubstance use history, non-substance-based psychiatric diagnoses and were on psychotropic medications pre-enrollment. Two (18%) participants achieved a Sustained Virologic Response (SVR). Two completed 48 treatment weeks, 5 were withdrawn due to adverse events, 2 dropped out prematurely and 2 had treatment discontinued for virologic non-response. Of on-treatment weeks, adherence to pegIFN was >99%. CONCLUSIONS: SVR rate was comparable to historic controls for coinfected genotype 1 patients, with optimal pegIFN adherence. Adverse effects often prevented therapy completion in this population.

Hepatic steatosis is associated with fibrosis, nucleoside analogue use, and hepatitis C virus genotype 3 infection in HIV-seropositive patients.

BACKGROUND: We conducted a study to determine the prevalence and factors associated with hepatic steatosis in human immunodeficiency virus (HIV)-seropositive patients with hepatitis C and to investigate whether steatosis is associated with liver fibrosis. METHODS: Retrospective chart reviews were conducted in 4 hospitals that serve community-based and incarcerated HIV-infected patients who had undergone a liver biopsy for evaluation of hepatitis C virus (HCV) infection during the period of 2000-2003. Demographic characteristics and medication and laboratory data were collected from the time of the biopsy. A pathologist blinded to all clinical data evaluated the specimens. The primary outcome was presence or absence of steatosis. RESULTS: Of 260 HIV-HCV-coinfected patients, 183 met inclusion criteria and had a biopsy specimen adequate for review. Steatosis was present in 69% of patients (graded as minimal in 31%, mild in 27%, moderate in 18%, and severe in 1%). Factors associated with steatosis included use of dideoxynucleoside analogues, such as didanosine and stavudine (odds ratio [OR], 4.63; 95% confidence interval [CI], 1.55-13.82). There was a trend toward presence of steatosis and use of other nucleoside analogues or infection with HCV genotype 3 (OR, 2.65 [95% CI, 0.95-7.41] and 3.38 [95% CI, 0.86-13.28], respectively). The presence of steatosis was associated with fibrosis (OR, 1.37; 95% CI, 1.03-1.81). CONCLUSIONS: In this multiracial population of HIV-HCV-coinfected patients, steatosis was prevalent and was associated with severity of liver fibrosis. Use of nucleoside analogues (particularly didanosine and stavudine) and HCV genotype 3 infection were associated with hepatic steatosis. The development of steatosis is multifactorial in nature and may play a contributory role in the progression of liver disease in HIV-infected patients.

Intrahepatic cytokine expression is downregulated during HCV/HIV co-infection.

HIV co-infection is associated with reduced HCV treatment response rates and accelerated HCV-related liver disease. Cytokines play an important role in regulating hepatic inflammation and fibrogenesis during chronic HCV infection, yet the roles of HIV and/or its therapies on cytokine expression are unknown. Total RNA was extracted from liver biopsies of 12 HCV mono-infected and 14 HCV/HIV co-infected persons. We used real-time PCR to quantify cytokines that contribute to innate and adaptive immune responses, including IFNalpha, IFNgamma, TNFalpha, TGFbeta(1), IL-2, IL-4, IL-8, IL-10, and IL-12p40. Positive- and negative-strand HCV RNA levels were quantified using a molecular beacon approach. Detection of positive-strand HCV RNA was 100% in both groups; negative-strand HCV RNA was detected in four (33%) HCV mono-infected persons and in nine (64%) HCV/HIV co-infected persons. Median strand-specific HCV RNA levels were not significantly different between the two groups. Detection rates of cytokine mRNAs were lower for the HCV/HIV co-infected group compared to the HCV mono-infected group; the detection rates for TNFalpha, IL-8, and IL-10 were statistically significant. Overall, cytokine mRNA quantities were lower for HCV/HIV co-infected compared to HCV mono-infected persons, with the exception of TGFbeta1. These data suggest that a defect in cytokine activation may occur in HCV/HIV co-infected persons that limits efficient clearance of HCV from the liver.

Beta A versus beta B: is it merely a matter of expression?

Activins are members of the transforming growth factor (TGF) beta (beta) superfamily of proteins that function in a wide array of physiological processes. Like other TGFbeta ligands, activins are biologically active as dimers. An activin molecule is comprised of two beta-subunits, of which four isoforms have been identified: betaA, betaB, betaC, and betaE. The most widely studied activins to date are activin A (betaA/betaA), activin B (betaB/betaB), and activin AB (betaA/betaB). Inhibin is a naturally occurring activin antagonist that consists of an alpha-subunit disulfide-linked to one of the activin beta-subunits, producing inhibin A (alpha/betaA), or inhibin B (alpha/betaB). The development of assays distinguishing between different forms of activins and inhibins, along with knock-in and knock-out models, have provided evidence that the betaA- and betaB-subunits have independent and separate roles physiologically. Additionally, evaluation of ligand-receptor interactions indicates significant differences in receptor affinity between activin isoforms, as well as between inhibin isoforms. In this review we explore the differences between activin/inhibin betaA- and betaB-subunits, including expression patterns, binding properties, and the specific structural aspects of each. From the growing pool of knowledge regarding activins and inhibins, the emerging data support the hypothesis that betaA- and betaB-subunits are functionally differently.

Betaglycan localization in the female rat pituitary: implications for the regulation of follicle-stimulating hormone by inhibin.

Activin-stimulated FSH synthesis and secretion from the pituitary gonadotrope is negatively modulated by ovarian inhibin; however, the cellular mechanism of inhibin antagonism is unknown. Inhibin and activin share a common beta-subunit through which inhibin can compete with activin for binding to the activin type II receptor and prevent activin signal transduction. Although the affinity of inhibin for binding to the activin receptor is far lower than that of activin itself, inhibin is capable of inhibiting activin-stimulated FSH synthesis and secretion even at low or equimolar concentrations. It is now known that the TGFbeta type III receptor, betaglycan, acts as an inhibin coreceptor that binds the inhibins and increases their affinity for the activin type II receptor, thereby enhancing the antagonistic effect of inhibin on activin signal transduction. Yet, despite the characterization of betaglycan is an inhibin coreceptor in several cell models in vitro, the role of this protein in the regulation of FSH in vivo has not been demonstrated. In this study we sought to understand more fully the function of betaglycan in the control of FSH release by the gonadotrope by describing betaglycan immunolocalization in the pituitary and assessing its correlation to fluctuations in FSH and inhibin throughout the rat estrous cycle. In general, betaglycan immunoreactivity was present in the anterior pituitary at all estrous cycle time points, but was confined to the membrane of gonadotropes just before and after the primary and secondary FSH surges. Importantly, betaglycan localized to the gonadotrope membrane when inhibin must rapidly reduce FSH to basal levels after the secondary FSH surge. These data indirectly support a role for betaglycan in vivo as a coreceptor that is required for inhibin-modulated FSH release from the pituitary.

A pilot study of treatment of bacterial vaginosis with a buffering vaginal microbicide.

BACKGROUND: Bacterial vaginosis (BV) is an extremely common problem for women and is associated with adverse pregnancy outcomes and HIV infection. Currently available antibiotic treatments are moderately effective but may need to be repeated frequently because of the recurrent nature of the disease. We undertook a pilot study of a buffering vaginal microbicide in the treatment of BV. METHODS: Women with clinically diagnosed BV were recruited to receive seven applications (5 g per application) of BufferGel trade mark (ReProtect, LLC, Baltimore, MD), a topical vaginal microbicide, and had clinical and gram stain evaluation of response. Subjects were evaluated at 2-3 days after the last application of BufferGel as a test of cure and again at 1 month to assess for relapse. Subjects with BV at test of cure were offered oral metronidazole. RESULTS: Thirty-one women were screened, 16 were offered enrollment, and 10 completed the study. Treatment with BufferGel was clinically effective in 70% of women at 2-3 days after treatment and in 40% of women by 1-month follow-up. CONCLUSIONS: These results suggest that 5 g of BufferGel vaginally once a day appears to be a moderately effective treatment for BV.

Properties of inhibin binding to betaglycan, InhBP/p120 and the activin type II receptors.

Activin-stimulated FSH synthesis and release by the pituitary gonadotrope is antagonized by gonadally derived inhibins. The two isoforms of inhibin, inhibin A and B, bind to the activin type II receptors, though at a lower affinity than the activins, but do not stimulate intracellular signaling. Theoretically, therefore, inhibins can prevent activin signaling through competitive binding if present at higher concentrations than the activins. In reality, the inhibins have been shown to antagonize activin signaling when the two ligand types are present at equimolar concentrations. These observations led to the hypothesis that inhibin binding proteins or co-receptors exist that either increase the affinity of the inhibins for the activin receptors or propagate inhibin-specific intracellular signals. Two candidate inhibin co-receptors, betaglycan and InhBP/p120, interact with activin receptors and augment inhibin antagonism of activin action. Here, we report the effect of betaglycan and InhBP/p120 on both inhibin A and inhibin B binding to the activin receptors ActRIIA and ActRIIB2. InhBP/p120 did not bind inhibin A or B when expressed alone or in combination with activin receptors, requiring a re-examination of the role of this protein in inhibin biology. Both inhibins bound the activin type II receptor, ActRIIB2. Inhibin B had a higher affinity for this receptor than inhibin A but an approximately 10-fold lower affinity than that of activin A. Inhibin A and B bound betaglycan with high affinity; however, only inhibin A binding to ActRIIB2 was significantly enhanced in the presence of betaglycan. Both inhibin isoforms showed slight but significant binding to ActRIIA, yet this binding was potentiated in the presence of betaglycan. Additionally, the complex formed between the inhibins, ActRIIA, and betaglycan was resistant to disruption by activin A, whereas activin A potently competed for inhibin binding to ActRIIB2 and betaglycan. Collectively, these data show that the inhibin isoforms have different affinities for the activin type II receptors but bind betaglycan with high affinity. A recently developed model of inhibin action proposes that inhibins form a high affinity, activin-resistant ternary complex with activin type II receptors and betaglycan, thereby providing a mechanism for inhibin antagonism of activin signaling. Importantly, the results presented here clearly show that this model does not apply equally to both forms of inhibin nor to the different activin type II receptor isoforms. Thus, it appears that the mechanisms of inhibin action may vary depending on the ligand and receptor types involved.

Inhibin binding protein (InhBP/p120), betaglycan, and the continuing search for the inhibin receptor.

Betaglycan (the TGFbeta type III receptor) and InhBP/p120 (a membrane-tethered proteoglycan) were recently identified as putative inhibin receptors. Here, we review the current state of knowledge regarding these two proteins with respect to their potential roles in inhibin biology. Importantly, neither protein appears to satisfy all of the criteria required for classification as a bona fide inhibin receptor. Betaglycan does not appear to be expressed in pituitary gonadotropes, the primary target of circulating inhibins, and InhBP/p120 does not bind inhibins in conventional receptor binding assays. While both proteins appear capable of promoting inhibin-mediated antagonism of activin signaling, neither appears to generate inhibin-specific intracellular signals. Recently, additional inhibin binding proteins were identified in inhibin target tissues, including pituitary and Leydig cells. Characterization of these proteins, coupled with ongoing investigations of betaglycan and InhBP/p120, will lead to a clearer understanding of mechanisms of inhibin action.