In the human genome the APOBEC3 gene has expanded into a tandem array of genes termed APOBEC3A-G. Two members of this family, APOBEC3G and APOBEC3F, have been found to have potent activity against vif-minus HIV-1. These enzymes become encapsidated in vif-minus HIV-1 virions and in the next round of infection, deaminate the newly synthesized reverse transcripts. The lentiviral Vif protein prevents the deamination by inducing the degradation of APOBEC3G and APOBEC3F. Our study showed that two additional APOBEC3 family members, APOBEC3B and APOBEC3C, have potent antiviral activity against SIV but not HIV-1. Both enzymes were encapsidated in HIV-1 and SIV virions and were active against vif-deleted SIVmac and SIVagm. SIV Vif neutralized the antiviral activity of APOBEC3C but not APOBEC3B. APOBEC3B induced abundant G-to-A mutations in both wild-type and vif-minus SIV reverse transcripts. APOBEC3C induced substantially fewer mutations. These findings raise the possibility that the different APOBEC3 family members function to neutralize specific lentiviruses.

Background: HumanAPOBEC3G (A3G),a deoxycytidine deaminase, is abroadly-acting antiretroviral factor. Given the antiviral effects ofA3G, cytokine-stimulatedT cells may upregulateA3G as a means to counter viral infection. Conversely, various cytokines such as IL-2, IL-4, IL-7, and IL-15 have been reported to convey permissivity to HIV infection in resting CD4 T cells. A3G can exist in two different forms, specifically an enzymatically-active low molecular mass form and an enzymatically inactive, high molecular mass A3G complex containing one or more RNAs. LMMA3G ischaracteristically found in resting CD4T cells where it functions as a potent post-entry restriction factor, while HMM A3G complexes are found in mitogen-activated CD4 T cells (see Chiu et al., Nature 435: 108, 2005).We have surveyed a range of cytokines to determine their effect on both A3G expression and A3G complex assembly.

Methods: Purified CD14- peripheral blood lymphocytes were stimulated individually with IL-2, IL-4, IL-6, IL-7, IL-9, IL15, TNFalpha, and IFNalpha, beta, and gamma; and the potential induction of A3G mRNA and protein was assessed. Interferonalpha-treated macrophages and mature versus immature dendritic cells were also assessed for potential changes in the expression of A3G. In parallel, the potential involvement of select signaling pathways in the observed response was tested by adding inhibitors specific for the Jak/Stat and MAP kinase pathways. Finally, using fast protein liquid chromatography, we assessed whetherA3G was present in the LMM or HMM form after stimulation of purified CD4+ T cells with these cytokines.

Results: A3G expression at the protein level in PBLs was induced by IL-2 and IL-15, and, to a lesser extent, IL-7. No response was observed with IL-4, IL-6, IL-9, TNFalpha or the alpha, beta, or gamma isoforms of IFN. This increase in A3G protein was mirrored by upregulation of A3G mRNA detected by quantitative real time PCR. Inhibition of JAK or MAPK activation sharply impaired the induction of A3G in response to IL-2, IL-7, and IL-15. Dendritic cell maturation and interferon-treatment of macrophages was also associated with a marked increase in the expression of A3G. When A3G complex formation was assessed, IL-2, IL-7 and IL-15 each promoted HMM A3G complex assembly in purified CD4 T cells.

Conclusion: We conclude that IL-2, IL-7, or IL-15 stimulation of resting CD4 T cells leads to transcriptional activation of theA3G gene resulting in increasedA3G mRNA and protein ex-pression. However, the potent post entry restricting activity of the LMM form of A3G is forfeited because the induced A3G protein is recruited into inactive HMM A3G RNA-protein complexes. These effects may explain why these cytokines cause resting CD4 T cells to become permissive to HIV-1 infection.

Background: Many treatment-experienced patients with limited therapeutic options for complete viral suppression remain on a partially suppressive regimen pending the availability of at least 2 fully effective agents. The major risk of this approach is ongoing viral evolution and the loss of future drug options. The rate at which drug options are lost in such patients has not been carefully defined.

Methods: Antiretroviral-treated patients with drug resistance were sampled from a clinic-based cohort of chronically HIV-infected patients. Subjects were included in this analysis if they had: stable antiretroviral regimen for 120 days, plasma HIV RNA level >1000 copies/mL, at least 1 genotypic resistance mutation, and at least 1 follow-up visit. Phenotypic and genotypic resistance testing was performed every 4 months and observations were censored at the time of any treatment modification. The primary endpoints were time to loss of phenotypic susceptibility to at least 1 fully effective drug (or 2 partially effective drugs), as well as time to development of 1 new nucleoside analogue mutation (NAM) or 1 new major protease mutation (IAS-USA guidelines).

Results: A total of 106 patients were eligible; the median duration of observation was 48 weeks (IQR 32-90).The median baseline CD4 cell count and plasma HIV RNA levels were 292 cells/mm3 and 3. 74 log copies/mL, respectively. Subjects had previously received a median 8 prior drugs. Using a Kaplan-Meier analysis, the estimated risk of losing 1 fully suppressive drug (or 2 partially suppressive drugs) was 32% at 1 year (95% CI 22-45).The risk of developing a new NAM at 1 year was 23% (CI 12-40), and of developing a new major protease mutation was 17% (CI 8-33).We also assessed the risk of deferring the use of a single new drug (tipranavir). Only an estimated 5% (CI 216) of protease inhibitor-treated patients experienced genotypic loss of tipranavir susceptibility. Viral load, CD4+ T cell counts, phenotypic susceptibility score, replicative capacity, number of previous drugs, and number of missed doses in last 30 days were not consistently predictive of rate of viral evolution.

Conclusion: In a cohort of heavily pretreated HIV patients with incomplete viral suppression, the risk of losing future drugs options appeared to be moderate. This risk should be considered when deciding to maintain patients on a partially suppressive regimen.

Background: This study examined how people with HIV/AIDS (PWHIVs) experience HIV-related stigma; how stigma experiences are associated with indicators of social, psychological, and physical well-being; and, how other variables affect that association. Stigma is conceptualized as having three key components for PWHIVs: (1.) experiences with enacted stigma (being the direct target of interpersonal rejection, discrimination, and other types of prejudicial behavior based on one's HIV status),(2.) felt stigma (beliefs about the nature and extent of enacted stigma), and (3) internalized stigma (shame, self-blame, and other negative feelings toward the self as a result of having HIV).Previous research on the social psychology of stigma suggests that felt stigma is an especially important construct because expectations of discrimination and ostracism often lead people with a stigmatized condition to avoid enactments of stigma by altering their behavior, often in ways that have deleterious consequences.

Methods: Self-administered questionnaires were completed by 200 PWHIVs recruited through the East Bay AIDS Research Institute (EBARI) at theEast BayAIDS Center (Berkeley) and the Alameda County Medical Center (Oakland).The sample included equal numbers of men and women (with two transgender respondents);was 72% African American, 18% White, and 7% Hispanic; was 39% gay/bisexual and 57% heterosexual;and had a mean age of 45 years and a median income of $5, 000 - $15, 000.

Results: Based on factor- and item-analyses of questionnaire responses, brief measures of enacted stigma, felt stigma, and internalized stigma were constructed (coefficient alpha > .80 for all scales). Correlational analyses revealed that all three forms of stigma were significantly associated with psychological distress, impaired health status and other variables related to wellbeing. Multivariate analyses indicated that the association between stigma and psychological/physical distress was due mainly to the strong connection between felt stigma and the outcome variables. Preliminary analyses suggest that this linkage is partially mediated by the coping strategies adopted by individuals with high levels of felt stigma. These strategies involve avoidance of others and active hiding of one's HIV status.

Conclusion: The experience of stigma is significantly associated with physical and psychological well-being in people with HIV/AIDS.

Background: For the past 25 years, the HIV epidemic has disproportionately affected men who have sex with men (MSM) in the United States. For African American and Latino MSM, the HIV incidence and prevalence has been especially pronounced. Further, African American and Latino MSM with histories of child sexual abuse (CSA), an important predictor of high-risk sexual behaviors, and who are already infected with HIV, are particularly vulnerable populations for high-risk sexual behaviors and negative psychological sequelae such as depression. As HIV continues to spread among this population, it is important to develop interventions to reduce HIV transmission.

Methods: The Enhanced Sexual Health Intervention for Men (ES-HIM), guided by cognitive-behavioral approaches, the Social Learning Model, and the efficacious Women's Health Project, was designed for HIV positive gay and non-gay identifying African American and Latino MSM with histories of CSA. A randomized controlled trial examining depressive symptoms, number of sex partners, and condom use intentions was compared among intervention and control groups with pre-post testing from 1999-2005. The trial compared two 6-week, 120-minute sessions, a sexual risk reduction intervention (ES-HIM) and a comparison standard health promotion condition (HP), implemented by ethnically matched male health educators.

Results: The study included 120 African American and Latino MSM with histories of CSA, ES-HIM (n=60) versus HP (n=60).The sample was marginalized with no significant differences on mean age (ES-HIM = 44. 1 years vs. HP = 43. 0 years) or on mean years of education (ES-HIM = 13. 0 vs. HP = 13. 6 years), and both groups were predominantly unemployed (ESHIM = 88. 9% vs. HP = 86. 7%) with mean monthly incomes of $676. Using the CES-D, the ES-HIM Intervention was efficacious in significantly decreasing depressive symptoms (P<0. 01) at post testing. While both groups decreased their mean number of partners in the previous 30 days, only the Intervention had significant pre-post change in number of partners from 3. 02 to 2. 03 (p<0. 001). Regarding condom use intention, only the ES-HIM Intervention showed significant pre-post change (p<0. 04).

Conclusion: This unique gender and culturally specific intervention focused on perceived male roles and culture, the effects of coercion in relationships, and the importance of condom use in personal protection and protection of partners. Emphasis on these topics may have contributed to it being efficacious at decreasing depressive symptoms and number of sex partners, while increasing condom use intentions among theseAfricanAmerican and Latino MSM who commonly lack such discussions. Future research must assess if these changes are sustained at 3- and 6-month post.

Background: Homeless youth suffer from disproportionate rates of STIs and HIV. The highest risk youth are not found in clinics or shelters, but on the street. Urine-based methods for gonorrhea and chlamydia screening and rapid tests for HIV have made it possible to deliver STI/HIV screening services to youth in non-clinic settings. The Street START Project is a collaboration of non-profit, governmental, and academic partners to create a model program for street-based STI and HIV screening, treatment and linkage to care of homeless youth in San Francisco by Larkin Street Youth Services (LSYS) outreach staff.

Objectives: Our specific aims are: 1) To determine the acceptability and feasibility of outreach-based testing for chlamydia and gonorrhea and to dispense field-based therapy, and 2) To determine the acceptability and feasibility of outreach-based rapid HIV testing. We hypothesize that outreach-based testing will reach a high-risk population of youth, that outreach-based STI treatment will be feasible and acceptable, and that outreach-based HIV testing will reach a higher percentage of youth testing for the first time than clinic-based testing.

Methods: Phase 1: Qualitative assessment of the feasibility of outreach-based STI and HIV testing using focus groups and key informant interviews with street youth, outreach workers and providers. Phase 2: Implementation of joint LSYS-UCSF outreach-based STI testing. Phase 3: Implementation of joint LSYS-UCSF outreach-based STI and rapid HIV testing Phase 4: LSYS outreach-based STI and rapid HIV testing with UCSF consultation.

Results: Phases 1 and 2 are completed. Issues elicited during the focus groups and interviews included confidentiality in the outreach setting, the importance of recognizing neighborhood-specific youth needs, the impact of the study on outreach activities, and obstacles to obtaining necessary contact information for treatment of STI-positives and referral to care of youth testing preliminarily positive for HIV. Since May 2005, 86 street-based youth and a comparison group of 51 clinic-based youth have been recruited. Street-based youth were more likely to be male (74% vs. 45%, p<.01), White (64% vs. 41%, p<.01) and older (mean age 22. 6 vs. 20. 3, p<.01).Street-recruited youth were more likely to engage in HIV-risk behaviors, such as having a higher mean number of sex partners in the past 3 months (5. 2 vs. 2. 2, p<.05), not using a condom at prior vaginal sex (61% vs. 40%, p<.05), having had a sex partner who was an injection drug user (43% vs. 10%,p<.01) or who was perceived to be HIV-positive (30% vs. 10%,p<.01), ever engaging in survival sex (47% vs. 20%, p<.01), or ever injecting drugs (48% vs. 4%, p<.01). No significant differences were found in prior STI testing history between the two groups (96% vs. 89%, p=.11). Street-based youth were more likely to report having ever been tested for Hepatitis C and HIV (78% vs. 46% and 90% vs. 67%,respectively;both p<.01).If tested, street-based youth were more likely to report testing positive for Hep C (32% vs. 0%, p<.01).There was no statistical difference in STI rates in the two groups. All STI-infected street-based youth have been treated.

Preliminary Conclusions: Our preliminary results suggest that street-based STI testing is accessing a higher-risk subgroup of youth, but, unexpectedly, a group with higher rates of prior HIV testing relative to clinic-based youth. The implementation of outreach-based HIV testing will enable us to assess whether street-based rapid HIV testing attracts street-based youth who would be testing for the first time.

Background: Accurate assessment of cognitive impairment due to HIV in the CNS is critical for determining an individual s neurological prognosis and possible response to highly active anti-retroviral therapy (HAART). Neuronal cell damage as well as reversible alterations in neuronal cell function underlies poor neuropsychological performance (NP). Non-invasive brain imaging provides an objective and quantitative assessment of brain integrity and function.

Methods: Arterial Spin Labeled Magnetic Resonance Imaging (ASL-MRI), was used to determine baseline global and regional cerebral blood flow (CBF) changes in 44 HIV associated neurocognitive impairment (HNCI) patients (24 mild cognitive dementia (MCMD) and 20 HIV associated dementia (HAD)) and 10 seronegative controls.

Results: Baseline global CBF values were similar for all groups. However, baseline CBF within areas known to be affected by HIV, the caudate and globus pallidus, was significantly affected in HNCI patients compared to seronegative controls.

Conclusions: These results suggest that HIV downregulates direct subcortical pathways involved in directed movements. Targeted regional ASL-MRI analysis of CBF within subcortical areas of the brain commonly damaged by HIV may predict individuals at risk for development of cognitive impairment and aid in determining responses to HAART or other neuroprotective therapies.

Background: The gamma-herpes viruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are associated with a number of human malignancies, particularly in patients with AIDS. Transmission of these viruses to B-lymphocytes, the primary cell type in which EBV and KSHV establish latency, is thought to be dependent upon production and release of infectious virus particles, or virions, culminating the lytic phase. Murine herpesvirus-68 (MHV-68) is a robust in vitro and in vivo model for gamma-herpesvirus infection. Unlike EBV and KSHV, MHV-68 produces high titers of infectious virions in tissue culture and is capable of establishing both acute and latent infections in laboratory mice. We are studying the structure and protein composition of the MHV-68 virion and have identified two gamma-herpesvirus specific proteins, ORF45 and ORF52, that are critical mediators of virion infectivity. These proteins are found in the virion tegument, a poorly understood structure encasing the nucleocapsid and underlying the virion envelope.

Methods: Virions and infected cell ultrastructures were studied by cryo-electron microscopy, cryo-electron tomography, and transmission electron microscopy (TEM). Virion proteins were analyzed by immunoblotting, mass spectrometry (LC/ MS-MS), and molecular cloning into FLAG-tagged, enhanced green fluorescent protein (EGFP), or dsRed fluorescent protein expression plasmids. Site-specific nonsense mutations in genes encoding tegument proteins were introduced into an MHV-68 genome cloned as a bacterial artificial chromosome (BAC) by homologous recombination with a shuttle plasmid in E. coli. Mutants were analyzed by virological assays and complementation in mammalian cells.

Results: The ORF52 protein is more tightly bound to the nucleocapsidthan the ORF45 protein, suggestingthat ORF52 localizes to a semi-ordered inner layer, and ORF45 to a more labile outer tegument layer. Cryo-electron tomographic reconstructions of MHV-68 virions support the existence of two distinct tegument layers. While the ORF45 protein displays predominantly nuclear localization, ORF52 protein localizes to a cytoplasmic compartment partly overlapping with the trans-Golgi network and actin cytoskeleton. Mutagenesis of ORF45 and ORF52 in an MHV-68/BAC clone revealed that both proteins are required to the complete the lytic phase of infection. Viral lytic gene expression and DNA replication is severely attenuated by loss of ORF45. In the absence of ORF52 protein, the lytic phase arrests after replication of viral DNA, expression of late lytic genes, and assembly and packaging of capsids but prior to tegumentation and envelopment of nascent nucleopcapsids in the cytoplasm.

Conclusions: Thus, while ORF45 protein is required in the immediate-early stage of infection, ORF52 protein functions to facilitate virion morphogenesis and egress. The protein-protein interactions through which ORF45 and ORF52 function and are packaged into the virion tegument are under investigation, as well as indications that the proteins interact with each other. Elucidating the structure-function relationships making up the gamma-herpesvirus virion tegument develops a potential target for therapeutic interventions aimed at preventing the release of infectious virus from the cell.

During the initial stages of an infection with Toxoplasma gondii, 'tachyzoites'(the rapidly dividing form of the parasite) disseminate throughout the host tissues, including the brain. A robust immune responseduring thisacute phase eliminates mosttachyzoites;however, some parasites convert to encysted 'bradyzoites' that may persist for the life of the host and are mainly located in the brain. Toxoplasma infection is a constant threat for immunocompromised individuals, such asAIDS patients, because reactivation leads to eruption of the cysts in the brain leading to life-threatening Toxoplasmic encephalitis. An improved understanding of how Toxoplasma establishes a persistent infection in healthy individuals and reactivates in AIDS will help to contribute to the development of effective therapeutic strategies.

My research focuses on determining how Toxoplasma manipulates its host enabling it to establish and maintain a life-long persistent infection and then later reactivate and cause problems in AIDS patients. I am using Toxoplasma strains that differ in their ability to establish a persistent infection to investigate this question. Microarray analysis of host gene expression of human cells infected with different Toxoplasma strains shows dramatic differences in the ability of different strains to modulate subsets of host genes. Using similar analyses of cells infected with progeny derived from a cross between type II and type III strains, we have genetically mapped the parasite loci responsible for these differences. Surprisingly, the data indicate a single Mendelian locus is responsible for each of the differences in phenotype. Candidate genes from these regions are currently being investigated and will be discussed. In addition, bioinformatic analysis of the host genes that are coordinately regulated by infection with the different parasite strains indicates that many appear to be regulated by the same transcription factors. Immunofluorescence assays and Western blots have been used to confirm the role of these molecules. Hence, we now have a handle on both the host and parasite molecules that mediate strain-specific differences in host response to infection. The next step will be to investigate the role of these molecules in helping Toxoplasma to establish a persistent infection.

Anecdotal reports indicate that non-gay identified (NGI) Latino men who have sex with men (MSM) are isolated and secretive about their sexual behavior, and are not being reached by existing HIV prevention programs targeting Latino MSM. Thus, this population may be at increased risk for HIV and other sexually transmitted infections (STI).Vista Community Clinic (VCC) seeks to access NGI Latino MSM by providing a free male health exam that assesses general physical health regardless of sexual practices, and includes HIV and STI testing as well as non-judgmental individual risk reduction counseling and referrals.

The purpose of this project is to test the effectiveness of a social marketing campaign to promote condom use, HIV testing, and participation in VCC's risk reduction activities, including the male health exam, among NGI Latino MSM in North San Diego County. Completed formative research activities include (a) 24 one-on-one key informant interviews, including 12 with NGI Latino MSM, 6 with employees of venues frequented by NGI Latino MSM, and 6 with Latina women who are family members, friends, or current or former significant others of NGIs.; (b) two focus groups, one conducted with key gay-identified Latinos who prefer NGI MSM as sexual partners, and the other conducted with VCC staff who have NGI Latino MSM clients; and (c) field visits, direct observations, and head counts conducted at potential intervention sites.

Analyses of qualitative data from key informant interviews and focus groups are currently under way. Significant preliminary themes that have emerged include cultural issues such as the importance of family, religion, and masculinity; the association of condom use with distance between partners, promiscuity, and infidelity; lack of condom use on the basis of a clean or healthy looking partner;and the use of alcohol as a justification for sexual encounters with other men. These and other results will inform the design of preventative materials and activities for the social marketing campaign. In addition, observations and head counts have helped us identify 12 sites (5 high risk and 7 low risk venues) in which we plan to implement campaign activities and conduct surveys to evaluate its effectiveness.

Beginning in December 2005, bi-monthly baseline intercept surveys with independent samples were implemented at both high-risk and low risk venues (N = 230 per survey wave) to assess baseline target behaviors, attitudes, and use of clinic services.

Future plans include the implementation of the social marketing campaign in April 2006, accompanied by continued bi-monthly intercept surveys to assess exposure to the campaign and changes in target behaviors, attitudes, and use of clinic services. The campaign willremain in place through November 2006, at which time all campaign activities will cease. Bi-monthly intercept surveys will continue to be conducted through May 2007.

The results of this project will inform the effectiveness of our social marketing intervention to promote safe sex behavior and enhance health care utilization among Latino males in general and NGI Latino MSM in particular. This study will also explore whether these materials and activities contribute to reduced social stigma, increased social acceptance of individuals who engage in same-sex practices, and community mobilization to promote and reinforce preventive behaviors. Study findings may inform future campaigns to reduce HIV risk among the Latino population in California.

Candida albicans is the most commonly isolated human fungal pathogen and is responsible for both disseminated and superficial infections. Immuno-compromised individuals, including patients undergoing chemotherapy and those suffering from AIDS, are particularly susceptible to Candida infections. In fact, over 90% of AIDS patients suffer from oral candidiasis over the course of the disease. Due to the increasing resistance of C. albicans to the commonly used anti-fungal drugs fluconazole and amphotericin-B, the necessity to better understand the virulence properties of C. albicans is of much greater importance. One virulence property of C. albicans has been attributed to its unique ability to undergo a morphogenic switch from a yeast growth form to a hyphal growth form. A key regulator of this switch is the transcription factor Efg1. Efg1 is essential for the morphogenic switch as the deletion of this gene from C. albicans results in a strain that cannot under go the yeast to hyphal transition in most growth conditions. It has been observed that Efg1 has both transcriptional activation and repression activities. How these activities of Efg1 are regulated is not known. Our preliminary data suggest that the ability of Efg1 to act as a repressor is regulated by CDK. The objective of this research is to determine the molecular mechanisms of the Efg1-mediated transcriptional repression and to ascertain the effect this activity has on morphogenesis.

After cell entry, the RNA genome of retroviruses is converted to a cDNA copy by reverse transcriptase (RT).This viral cDNA, incorporated into a nucleoprotein complex termed the preintegration complex, enters the nucleus and is integrated into the host cell chromosome. Retroviral integrase (IN) catalyzes the integration of viral DNA. Both reverse transcription and integration are essential steps in the viral life cycle. The function of IN is not restricted to integration. For human immunodeficiency virus type 1 (HIV-1) certain mutations of IN, such as substituting the Cys residue at position 130 of IN to Ser (NL-C130S), can cause a specific defect in reverse transcription, but the underlying mechanism for this defect is poorly understood. In vitro, IN and RT can physically interact, and we have determined previously by co-immunoprecipitation that HIV-1 RT interacts with the C-terminal domain of IN. We hypothesize that the IN-RT interaction is functional and critical for reverse transcription. To confirm that the lack of the reverse transcription is caused by the IN mutation, wild type (WT) IN was packaged into the NL-C130S mutant virions by the in-trans method. Incorporation of IN restored reverse transcription to 25% of the WT virus. To understand the mechanism by which IN stimulates reverse transcription, we employed cell-free reverse transcription assays to examine the effects of IN on the RNA- and DNA-dependent polymerase activity of RT. Our preliminary results showed thatIN can stimulate both the initiation and elongation mode of reverse transcription. In the presence of 10 molar excess IN, initiation and elongation products increased nearly 3-and 2-fold, respectively. In addition to initiation and elongation, IN may help stimulate the early steps of reverse transcription by promoting annealing of the tRNA primer to the template, stabilizing the RT-templateprimer ternary complex, or destabilizing secondary structures of the RNA primer and template, all of which could lead to an increase in initiation and extension products. We are currently investigating such possibilities with aims to identify the critical step that IN is required during early events of reverse transcription. The experiments will shed light on the interaction between two key retroviral enzymes and the effect of such an interaction on the essential step of reverse transcription. Characterizing the RT-IN interaction and determining its biological significance may reveal new functional roles for IN as well as potential targets for devising new approaches to inhibit viral replication.

Background: The primary objective of the study is to describe perceptions of maternal uncertainty, stress and distress in HIV-infected women in association with the unknown HIV status of their infants and to document changes in these variables over time. Secondary objectives include determining relationships between maternal social support and perceptions of stress and distress; and between levels of maternal stress and distress and adherence to antiretroviral medications in the mothers and their infants. In California, the majority of women with HIV infection are in their childbearing years. HIV-infected women who become pregnant receive highly active antiretroviral therapy (HAART) both to reduce perinatal HIV transmission as well as to delay maternal disease progression. The use of HAART significantly reduces the risk of perinatal transmission from 25% to less than 2%.As a result, the vast majority of HIV-infected pregnant women are expected to give birth to uninfected, healthy infants. However, the HIV status of these infants is not immediately known and requires several HIV-specific viral laboratory tests during the first few months of life to determine their infection status. Ninety-nine percent of infected infants are diagnosed during the first four months of life and negative HIV viral test results during this time can be reassuring to parents. However, maternal perceptions of uncertainty about an infant's HIV infection status during the testing period can contribute to psychological and physiological distress. Perceptions of stress and distress in HIV-infected individuals have been associated with erratic adherence to antiretroviral medications, alterations in immune function and increased mortality rates. In addition, inconsistent maternal adherence to HIV medications has been linked to sub-optimal administration of HIV prophylactic medications to perinatally-exposed infants, thereby increasing the risk of infection in these infants.

Methods: This is a prospective, repeated measures study of HIV-infected mothers and their infants designed to describe the effects of unknown infantHIV statuson:(1.)maternalperceptions of uncertainty; (2.) maternal perceptions of stress; (3.) maternal perceptions of psychological distress;(4.) maternal social support; and (5.) adherence to maternal and infant HIV medications. Study variables are measured in women at six time points (once during the thirdtrimesterof pregnancy andfive times afterdelivery).Data are collected using standardized questionnaires and open-ended questions. Maternal and infant medical records are abstracted to confirm their health status. Data analyses include repeated measures analysis of variance for quantitative responses and content analysis for qualitative responses.

Results/Expected Results: Demographic data for the 12 enrolled maternal subjects are as follows: mean age = 31. 9 (range = 21-43); ethnicity/race = 41% Black (non-Hispanic), 41% White (non-Hispanic), 9% Hispanic, 9% Native American/ Hispanic; HIV/AIDS stage: 33% with an AIDS diagnosis prior to the current pregnancy. Five maternal subjects have completed data collection, and all of their infants are uninfected by HIV DNA PCR testing.

Conclusions: This study has been designed to investigate aspects of maternal stress, distress, coping and medication adherence that have not been addressed previously in HIV-infected childbearing women. Information gained from this study will provide a foundation for developing interventions that may improve maternal and infant health outcomes during this potentially stressful period.

Background: CD40 ligand (CD40L, CD154),a member of the TNF superfamily (TNFSF), is the major endogenous activator of dendritic cells, enabling them to "license" CD8+ T cells for long-term memory. CD40L is a trimeric, membrane molecule that has been very difficult to apply to vaccination. However, we have constructed new forms of CD40L that are soluble proteins with 1-, 2-, or 4-trimers. Previous work indicated that the immunostimulatory activity of the soluble trimers is related to the valency of the trimers (1 < 2 < 4). In addition, a 4-trimer form of GITRL, another TNFSF molecule, was prepared for study. GITRL is of interest because it reverses the suppressive effects of CD4+CD25+ regulatory T cells (Tregs) and co-stimulates CD4+CD25- effector T cells. We wished to determine the effect of soluble, multimeric CD40L and GITRL on long term immunological memory, either 14 or 90 days after vaccination with a secreted Gag DNA vaccine.

Methods: Antigen plasmidwaspScGag, expressing asecreted form of the Gag protein from a CMV promoter. The following TNFSF plasmids were tested: pMemCD40L (natural membrane CD40L); pAcrp30-CD40L (2-trimer sCD40L produced as a fusion protein with Acrp30); pSP-D-CD40L (4-trimer sCD40L produced as a fusion protein with surfactant protein D); and pSP-D-GITRL (4-trimer sGITRL). BALB/c mice were vaccinated i. m. with 80 ug of antigen plasmid plus 20 ug of adjuvant plasmid every two weeks X 3, and spleen cells were harvested either 14 or 90 days later.

Results: Plasmids for the natural membrane form of CD40L (pMemCD40L) had no adjuvant effects on the formation of tetramer positive CD8+ cells. However, 4-trimer sCD40L (pSP-DCD40L) increased the level of tetramer positive CD8+ cells both 14 and 90 days post vaccination. At 90 days post vaccination, this adjuvant effect was seen for both CD62L low (effector memory) and CD62L high (central memory) pools. pSP-D-GITRL also augmented the level of tetramer positive CD8+ cells, but led to a predominance of CD62L high central memory cells. Conclusions: The addition of pSP-D-CD40L to secreted Gag DNA vaccines produced CD8+ T cell memory responses that survive for more than 90 days after vaccination. A pSP-D-GITRL adjuvant also produced tetramer positive CD8+ T cell responses, and favor the generation of CD62L high central memory cells. Taken together, these soluble multimericTNFSF ligands have significant potential as DNA vaccine adjuvants.

Background: Human cytomegalovirus (HCMV) retinitis is an important etiology of blindness inAIDS patients. The mechanism of HCMV pathogenesis in the retina is unknown; however, the retinal pigment epithelium (RPE) cells play a key role in AIDS retinitis and are the target of HCMV infection in vivo. The ability of HCMV to infect and replicate in RPE cells is responsible for the development of viral-associated retinitis. Similar to that in vivo, RPE cells are fully permissive to HCMV infection in vitro.

Method: Tounderstand HCMVreplication in RPE cells, the HCMV genome was cloned as a bacterial artificial chromosome (BAC) to generate a deletion mutant library. We systematically investigated the necessity of each ORF for replication in RPE cell through phenotypic screening of growth properties in RPE cells. A revertant virus has been constructed to confirm whether the enhanced phenotype is specifically due to deletion of US15. For further unveiling the molecular mechanism of this suppression, Western analysis and microarray study were being carried through.

Results: Compared to wild type BAC, US15 mutant displayed an enhanced growth phenotype. In contrast, the growth of US15 mutant in human foreskin fibroblasts was not significantly different from that of the parental HCMV-BAC. The revertant virus confirmed that the enhanced phenotype is specifically due to deletion of US15. Western analysis showed that since alpha phase of viral life cycle the viral protein expression level of US15 mutant was higher than the parental HCMV-BAC. Further microarray study showed that the US15 mutant had higher viral transcription level from the immediate early stage of viral life cycle comparing to the parental HCMV-BAC.

Conclusion: Through the above study it is clear that the suppression effect of US15 happens during viral life cycle alpha phase and is due to the suppression of viral immediate early genes transcript. This useful information will help us to develop innovative anti-viral strategies for HCMV retinitis.

Background: Gene therapy strategies using genetically modified T cells or hematopoietic stem cells (HSCs) have been proposed as an adjuvant to chemotherapy for treating HIV infection. The discovery that exogenously delivered siRNA can trigger RNAi in mammalian cells raises the possibility of harnessing RNA interference (RNAi) technology as a therapeutic tool against HIV infection. This is confirmed by stable expression of short hairpin RNAs (shRNAs) via HIV vector-mediated gene transfer in T cells, rendering cells resistant to HIV replication. However, HIV resistant strains with mutations in the siRNA target site emerge quickly. Thus, expression of siRNAs against multiple targets in the HIV genome may be necessary to effectively suppress the emergence of resistant strains. Using multiple pol II promoters in a HIV vector to drive siRNA expression is technically difficult. Using multiple pol III promoters is feasible but constitutive siRNA expression from strong pol III promoters will most likely generate undesirable "off-target" effect in the target cell. In contrast to siRNA, multiple microRNAs (miRNAs) can be expressed as a single transcript from a pol II promoter and can be processed efficiently by Drosha based on their unique stem-loop structures.

Methods: We propose to evaluate whether multiple siR-NAs with each siRNA flanked by the stem-loop structure of pri-miR-30a can be expressed and processed properly from a pol II promoter-derived polycistronic transcript. We will determine whether inducible pol II promoters can be used to drive the expression of multiple siRNAs against HIV in the target cells.

Results or expected results: We have so far inserted an shRNA gene against a common exon for the tat and rev genes into the stem-loop structure of pri-miR-30a. A constitutive pol II promoter and the GFP gene were placed upstream from the shRNA gene and the whole cassette was then inserted into a HIV vector. H9 cells were transduced with this vector and challenged with HIV-1. We will determine whether the siRNA is properly processed and whether it can render the cell resistance to HIV-1 replication. If this design allows efficient expression and proper processing of this siRNA, we will evaluate whether inserting two shRNA genes targeted to different regions of the HIV genome can lead to a similar efficiency of expression and processing. We will then determine whether the well-characterized human interleukin 2 (IL-2) promoter can drive T cell-specific and inducible expression of the siRNA genes. We will also evaluate whether HIV long terminal repeat (LTR) which responses strongly to the HIV Tat protein can also serve as an inducible promoter for siRNA expression.

Background: Since 1997, the United States Public Health Service has recommended that injection drug users who cannot or will not stop injecting obtain and use a new needle for every injection. San Francisco has approximately 5,000 injecting drug users aged under 30. Among this high-risk population, HIV seroprevalence is 6% and hepatitis C (HCV) seroprevalence is 50%. Most young injectors in San Francisco acquire at least some of their needle needs through City-funded needle exchanges, however many still share and re-use needles, implying that there are other barriers to the acquisition and use of sufficient needles to prevent HIV and HCV transmission. The existing literature on barriers to the use of needle exchange has identified both operational issues, such as inconvenient or insufficient locations of operation, and structural issues, such as fear of identification or harassment by police, as potential barriers. However, there is little or no research on the ways an individual's exposure to the criminal justice system might impact on their willingness or ability to utilize HIV prevention programs such as needle exchange. Likewise, there is a long tradition of locating HIV prevention services in the communities in which high- risk populations are assumed to reside, however among this highly mobile and largely homeless population, notions of "neighborhood of residence" become largely meaningless. Young injectors move through (or avoid) multiple neighborhoods in the course of their day; understanding how and why they might see these areas as safe or unsafe; as easy to get to or as 'far away'; as places to visit briefly or as places to spend considerable parts of their day in, and understanding how these perceptions might change under the influence of factors such as their interactions with the criminal justice system, are crucial to understanding how and why they utilize, or under-utilize, or fail to utilize, core HIV prevention interventions such as needle exchange.

Goals: The two goals of this project are to quantitatively analyze relationships between judicial status and the closeness of compliance to standard of one needle per injection; and to qualitatively investigate understandings of social space held by young injecting drug users in San Francisco, CA, as they relate to locations of HIV prevention programs such as needle exchange.

Relevance to HIV/AIDS: Acquisition and use of sufficient needles is the single most effective HIV prevention intervention for those who cannot or will not stop injecting drugs. California has led the US in funding needle exchanges; understanding the barriers to the appropriate use of these programs is critical to ensuring the effectiveness of these programs for preventing the spread of HIV in California.

Methods: This study builds on an existing NIDA-funded prospective study of hepatitis C seroconversion being conducted by the University of California, San Francisco. By extending the quantitative data collection conducted as part of the above study, and by conducting a series of 30-40 qualitative interviews with participants of the above study, this project will examine the relationships between utilization of crucial HIV prevention intervention, the involvement of young injectors with the criminal justice system, and the relationships young injectors have with the physical locations in which needle exchanges are located.

HIV is a potent risk factor for tuberculosis (TB). It increases the rate of latent TB reactivation, the rate of progression of the active disease and the risk of new infections by an order of magnitude. Active TB disease also affects HIV, accelerating progression of the HIV infection, increasing infectivity and reducing HIV treatment efficacy. About 13 million people are co-infected with HIV and TB worldwide, and mortality rates during treatment can be as high as 20% compared to the 5% treatment death rates that are more typical of TB cases without HIV co-infection.

In San Diego County, where the TB case rate is more than twice as high as the rest of the U.S., the annual incidence of HIV/TB co-infection has remained at about 10% of all TB cases despite a decline of both TB and HIV cases in the overall population. There is a concern that the preventive measures that have proved effective in the general community are not reaching the HIV/TB patient population. Preliminary analysis of the HIV/TB cohort reported to San Diego County over the last five years indicates that this population is comprised mostly of male immigrants (>70% Hispanic) and that over 15% of them die before TB treatment is completed--on average within 2.5 months of their TB diagnosis. Furthermore, there are grave concerns that if left unchecked, San Diego's largely immigrant HIV/TB population, which currently has a very low prevalence of Multi Drug Resistant (MDR) TB, will become infected with more strains of MDR-TB or the Extensively Drug Resistant (XDR) TB strains that have recently been discovered in California.

The synergy of HIV and TB co-infection and the unique risk environment of the patients that acquire these dual infections will require innovative and holistic approaches for management that are based on an intimate understanding of the epidemiology of both HIV and TB. The overall goal of this proposed project is to characterize the epidemiology of HIV and TB co-infection in San Diego and to develop an HIV/TB co-infection surveillance tool for long-term use. We have four specific aims: 1) To characterize risk factors for HIV infection in HIV/TB Cases in San Diego County using a two year prospective survey, 2) To determine the etiology of TB in HIV/TB Cases in San Diego County using genotype cluster analysis of TB isolates, 3) To characterize risk factors for TB infection among HIV/TB Cases in San Diego County using a 15 year retrospective analysis and lastly, 4) To identify risk factors associated with mortality during TB treatment among HIV/TB co-infected individuals, using a 15 year retrospective analysis and a two year prospective survey.

Increasingly specialized HIV and TB treatments and complex socio-economic barriers are widening the gap between the control and care of HIV and the control and care TB at a time when it is becoming clear that the best prevention and care is delivered when these dual infections are managed together. The proposed study will provide the first comprehensive characterization of both HIV and TB risk factors unique to San Diego County, as well as a clear understanding of the factors driving the high treatment mortality seen in this extraordinarily vulnerable patient group. Armed with insights from our project, we expect to be able to identify critical points of intervention for increasing the effectiveness of future preventive strategies and improving treatment outcomes in this population.

Background: Non-Hodgkin's lymphoma (NHL) is the second most common cancer in AIDS patients, and is thought to result due to errors in class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. Both processes are tightly regulated, occur in the germinal center (GC), and require the expression of the activation induced cytidine deaminase (AID) gene. Normally, AID is expressed only in GC B cells in secondary lymphoid organs, which is the site of CSR and SHM. The aberrant expression of AID (e. g., elevated and/or extranodal expression) could potentially lead to mistakes in the CSR and SHM processes, resulting in the mutation/translocation of protooncogenes, leading to the development of NHL. Since NHL is a common cancer in HIV infection and aberrant expression of AID could potentially lead to the development of lymphoma, we hypothesized that AID expression might be detectable in circulating cells in HIV infection, and preferentially elevated in those HIV+ subjects who went on to develop lymphoma.

Methods: In order to determine if AID was elevated in the peripheral circulation of HIV+ subjects who went on to develop lymphoma, AID expression was measured by quantitative real time PCR in peripheral blood mononuclear cells (PBMC) collected prior to lymphoma diagnosis; PBMC from HIV+ controls with AIDSbut nolymphoma, and HIV-negativecontrolsatcomparable time points were also evaluated. This study was done using archival viably frozen PBMC from the Multicenter AIDS Cohort Study (MACS) at UCLA. The MACS is a study of the natural history of AIDS that was initiated more than 20 years ago. Subjects in the MACS are seen at two study visits per year, during which serum, plasma, and PBMC are collected and stored. Additionally, extensive clinical and epidemiological information is collected on these study subjects. PBMC, collected longitudinally over an extended period of time pre-NHL diagnosis, were tested from a total of 16 HIV+ lymphoma subjects at a study visit within 1 year prior to lymphoma diagnosis (n=14), between 1 to 5 years pre-lymphoma diagnosis (n=11), and more than 5 years prior to lymphoma diagnosis (n=11).

Results: It was seen that PBMC AID expression was undetectable in virtually all HIV-negative controls, but detectable at low to moderate levels in some of the HIV+/AIDS controls. Mean AID expression was significantly elevated at all three time points in those HIV+ subjects who developed lymphoma, with mean levels 100-1000 times higher compared to AIDS controls. When stratified by lymphoma subtype, it was seen that PBMC from those who developed central nervous system (CNS) lym-phomas had little or no detectable AID expression, while moderate to high levels of AID expression were seen in the majority of those who developed non-CNS lymphomas (Burkitt's lymphoma and large cell lymphoma). Purified B cells from one lymphoma subject showed a 25-fold increase in AID expression compared to PBMC.

Conclusions: These results suggest that elevated levels of AID expression in circulating B cells precede lymphoma in HIV-infected persons, and support the hypothesis that aberrant AID expression may play a significant role in the development of AIDS-associated NHL.

The HIV-1 Tat protein is a transcriptional activator of the HIV promoter and is also thought to be involved in the activation of immune T cells. We have investigated the mechanism of Tat action by examining the expression of 21, 000 human genes using oligonucleotide microarray analysis in response to endogenous Tat expression. We compared two forms of Tat, Tat72 and Tat101 with control cells. Among the genes identified is the chemokine receptor, CXCR4. We found that both Tat72 and Tat101 induce CXCR4 at the cell surface. Furthermore, this increase in CXCR4 expression is paralleled by an increase in the chemotaxis of cells that express Tat to the ligand of CXCR4, SDF-1. Both Tat72 andTat101 also activate the CXCR4 promoter. The results may implicate Tat in altering cellular homing to sites of infection as well as signaling through chemokine receptors.

Background: B cell hyperactivity, including hypergammaglobulinemia, is very common in HIV infected subjects. Interestingly, higher serum immunoglobulin titers are associated with rapid loss of CD4 T cells; this is believed to be due to increased formation of autoimmune complexes, which often contain the gp120 antigen and specifically target CD4 T cells for apoptosis. We hypothesize that regulatory T cells (Treg, NKT cells) directly suppress activation, proliferation, and immunoglobulin secretion by B cell populations. We propose that B cell aberrancies, including hypergammaglobulinemia and the excess of gp120-specific antibodies present in many persons infected with HIV, are a direct result of regulatory T cell dysfunction in these patients. It may be possible to abrogate the B cell dysfunctions present in many HIV-infected patients through enhancement of the suppressive mechanisms of regulatory T cells; this could lead to the generation of novel therapies that alter the frequencies and /or effector functions of regulatoryT cells to reduce and/or prevent both CD4 T cell loss (induced by antibody complexes) and B cell diseases, including autoimmune disorders and lymphomas, that are present in afflicted individuals.

Methods: The frequencies of ASCs will be enumerated using IgG, IgM, and IgA Elispot assays. These assays can measure the total number of Ig secreting cells, as well as the frequencies of ASC secreting antibodies for specific viral or protein antigens. Total plasma titers for Ig isotypes will be measured by ELISA. The frequencies and functions of Treg and NKT populations will be evaluated by Flow Cytometry and Elispot assays.

Results/ Expected Results: Preliminary data suggestTreg cells are capable of directly inhibiting immunoglobulin secretion from ASCs in vitro. This novel 'B regulatory' effector function of Treg cells will be evaluated from PBMC of acute and chronic HIV-infected individuals to determine if there is a correlation between the ex vivoeffector functions of regulatoryT cell populations and the presence of B cell disorders, including lymphomas, found in many HIV-infected individuals. A subset of these infected patients will be studied longitudinally to determine if ex vivo Treg / NKT cell frequencies/functions are related to the rate of CD4 T cell loss and progression to AIDS.

Background: In order to balance their budgets, many states are considering reducing eligibility for Medicaid, one of the fastest growing state expenditure categories. Using variation in state policies, this paper models the effect of more stringent eligibility criteria for Medicaid on the insurance status and the use of antiretroviral therapy (HAART) for people living with HIV (PLWH). The paper also investigates whether there are differential effects of the state policies for those who are disabled among the PLWH.

Data: The data is from the HIV Cost and Services Utilization Study (HCSUS),a nationally representative probability sample of adults receiving care for HIV/AIDS in 1996 in the contiguous United States. The sample used in this paper includes all 2, 864 respondents, sampled with known probability at each stage. For this analysis, the HCSUS data was merged with a separate database on states'Medicaid eligibility rules and other state-level covariates like unemployment and uninsurance rates.

Methods: Multinomial logistic regressions were used to derive the predicted probabilities of various types of insurance and the marginal effects of relaxing states' Medicaid eligibility rules on the insurance status of PLWH. For the second analysis involving HAART use, reduced form logit regressions were estimated. Both analyses were carried out for the full sample, as well as for various subsets of the sample, using disability and work status to differentiate among patients.

Results: The results suggest that restricting eligibility increases uninsurance and has no effect on private coverage for PLWH. Further, more stringent eligibility criteria have a larger effect on the uninsurance rates of the disabled and an even larger effect for those who are both disabled and unemployed. The same holds true for the analysis involving HAART use.

Conclusion: Lower eligibility thresholds are likely to raise uninsurance rates and reduce the use of antiretroviral therapy among PLWH - more so, for those who are disabled and unemployed. There is no evidence of a "crowding out" effect of public insurance on private coverage for PLWH.

The maintenance of human immunodeficiency virus (HIV) life cycle in infected host human cells requires host proteins. A class of transcription factors known as nuclear factor kappaB (NF-kappaB) is among the host proteins that HIV uses for its gene expression. The NF-kappaB family of dimeric transcription factors regulates the expression of genes that are key to a variety of biological responses. NF-kappaB dimers are formed by the combinatorial association of five members, p50, p52, RelA, c-Rel and RelB. It is known that these dimers bind to a class of DNA sequences (kappaB sites) located within the promoter/enhancer region of a large number of response genes. The HIV-LTR contains such kappaB sites, which have been shown to utilize the NF-kappaB/Rel proteins to regulate transcription through binding to these kB sites. One of the HIV isolates contains two such kappaB sequences arranged in tandem. Binding of two NFkappaB dimers to these tandem sites is essential for synergistic transcriptional activation from HIV-LTR. Surprisingly, our biochemical experiments have shown that NF-kappaB dimers fail to bind to specific sequences arranged in tandem with high affinity. I will investigate if the transcriptional coactivator (CBP) binds directly and simultaneously to both NF-kappaB molecules and thereby promoting stable association of CBP and NF-kappaB on the HIV-LTR. This is thought to play a role in relieving the anti-cooperative effects of NF-kappaB bound to tandem kappaB sites. Different biochemical techniques such as electrophoretic mobility shift assay (EMSA), fluorescence anisotropy assay (FAA) and affinity pull down assays will be utilized to demonstrate the assembly of CBP and NF-kappaB on different DNA fragments taken from various HIV strains.
Recent studies have shown that two different NF-kappaB dimers are responsible for the regulation of HIV gene expression in different cells;NF-kappaB p50/p65 primarily acts in CD4 T cells and NF-kappaB p52/RelB heterodimer acts in monocyte and macrophage cells. Whereas the molecular structure of HIVDNA bound p50/p65 hetrodimer is known, no such structure is known for the other dimer bound to HIV-DNA. Large crystals of p52/RelB complexed to DNA have been obtained. The crystals have diffracted to 3Å and the X-ray diffraction data has been collected and processed. During the process of improving the crystals I determined that p52/RelB binds to DNA in a different manner than other NF-kappaB family members. Therefore determining the structure will be essential before I can proceed with the cooperative effects of tandem kappaB sites.

Background: Gay and bisexual males who use methamphetamine are at extremely high risk for HIV infection. Methamphetamine use is highly integrated into gay male social contexts, such as circuit parties, sex clubs, andbathhouses. Methamphetamine use in this population, is highly associated with risky sexual behavior, with users of the drug reporting an increased number of sexual partners, decreased use of condoms, and an increased likelihood of being HIV-infected or having a sexually transmitted disease. By contrast, interventions that reduce methamphetamine use among methamphetamine dependent, treatment-seeking gay and bisexual men result in concomitant decreases in HIV-related sexual transmission behaviors that can be measured up to one year following treatment entry. These interventions include contingency management, i. e., provision of increasingly valuable reinforcements for consecutive urine samples that document abstinence from methamphetamine and cognitive behavioral therapy, i. e., a psychological and educational intervention that teaches participants how to initiate abstinence and to prevent relapse. The proposed study will (1) adapt an evidenced-based, gay-specific cognitive behavioral therapy (GCBT) intervention for methamphetamine-abusing gay and bisexual males and transfer its use in a community-based HIV prevention setting; (2) incorporate this GCBT intervention with a contingency management (CM) intervention to yield an optimal behavioral intervention for producing sustained HIV sexual and drug risk reductions; and (3) develop an intervention to provide continuing care to support participants after completion of the intensive, synthetic GCBT+CM intervention with the objective being maintenance of longer-term behavior changes.

Methods: The formative stage of the study included tailoring the evidenced-based GCBT and CM interventions, which received both a scientific and community review. Enrollment began in November 2005. The study will enroll 210 gay or bisexual males who meet criteria for methamphetamine abuse into the 16-week behavioral intervention consisting of GCBT+CM during weeks 1 through 8, and Continuing Care+CM during weeks 9 through 16. Assessments will be conducted at baseline, 8-week, 16-week, and 26-week post admission.

Background: CARE-HIPP (Health Insurance Purchase Program) is a California State program that purchases private, continuation health insurance for persons living with HIV (PLH) in lieu of directly providing them antiretroviral medication through the AIDS Drug Assistance Program (ADAP).

Methods: This project examines both the cost-effectiveness of the current program and the potential to expand it. This research involves a number of steps: (1.) estimating the numbers and characteristics of current CARE-HIPP recipients; (2.) calculating the full cost of providing coverage for antiretroviral medication to CARE-HIPP enrollees through the premium purchase program and through ADAP; (3.) determining what the cost of providing antiretroviral medication to this population through ADAP coverage uniquely would be in the absence of private insurance purchase; (4.) calculating the cost-effectiveness ratio by comparing incremental costs and benefits; and (5.) making comparable calculations for populations of PLH who may reasonably be expected to have the option of continuing their private health insurance policies, namely those PLH with private insurance other than CARE-HIPP who currently also receive some ADAP assistance.

Results: The current CARE-HIPP program is highly cost-effective. It not only is less costly to provide antiretroviral medication through CARE-HIPP than directly through ADAP, but CARE-HIPP also provides coverage for physician and hospital services. The program would also be cost-effective for likely expansion populations.
Conclusions: The CARE-HIPP program should be continued and expanded.

Background and Methods: The HIV epidemic among men who have sex with men (MSM) is of substantial concern. Recent findings indicate HIV prevalence among MSM in San Francisco has increased from 20% in 1996 to approximately 28% in 2003. The proposed work examines unexplored population level processes influencing HIV risk behaviors and HIV distributions among MSM (sexual mixing, demographic changes, migration). In this phase of the work, we investigated the relationship between indices of MSM migration and HIV risk and prevention practices in survey probability sampled based research conducted at the City (Urban Men's Health Study III, 2002-03, N = 850) and State levels [California Health Interview Survey-MSM Survey, 2002-03; N = 395).

Results: Approximately 75% of adult MSM residents of California migrated into the state at some earlier point in their adult life. In San Francisco, approximately 80% of MSM are in-migrants from other locales. Preliminary findings at the state level indicate that more recent MSM in-migrants have higher levels of risk behavior, greater use of sex venues, and are less likely to have been tested for HIV;the age of in-migration, the number of moves made since age 18, and the number of MSM-urban centers lived in are related to HIV risk indices. In San Francisco (SF), MSM not born in SF are more likely to attend sex venues, have more sex partners, greater HIV risk, and greater sexual mixing. In addition, migrants from outside the US vs. US residents had higher levels of risk behavior, use of sex venues, less HIV testing, more sex partners, and less condom use. Recent in-migrants to SF had higher levels of risk behavior, less condom use and serosorting, more use of sex venues, more sex partners, and great sexual mixing than older in-migrants.

Conclusion: The findings suggest that new migrants/inmigrants to California and large urban MSM communities, are at greater risk for HIV infection; while native born MSM are at lower risk. This suggests that community norms for safe sex are strongest for native born MSM and weakest for in-migrants; implications for HIV prevention will be discussed.

Background & Rationale: Early in the HIV epidemic in the USA, HIV/AIDS was a predominately "gay white male" disease. Recently, the prevalence of HIV in women has increased world-wide. Females now make up about half of all persons infected with HIV globally. Historically women have been under-represented in HIV clinical trials. This disparity is most evident in early phase studies of newer agents. Consequently, current knowledge about the pharmacokinetics (PK),efficacy and toxicity of anti-retroviral drugs is derived from studies of predominately male subjects. Data from recent studies focusing on HIV-infected female subjects suggest the existence of sex-related PK differences. While the clinical significance of some of these differences is not fully apparent, several studies suggest that women achieve higher drug levels than men even after controlling for weight. Much of the existing data have important limitations. Some studies were retrospective reviews of therapeutic drug monitoring databases in which the indication for monitoring was not controlled. Other studies were conducted in HIV-uninfected subjects, included random drug levels and often enrolled too few women. Ours is the first large prospective formal PK study of sex differences in Kaletra pharmacokinetics. Kaletra is a co-formulation of lopinavir (LPV) -a potent inhibitor of HIV-1 protease and ritonavir (RTV) whichinhibitsCYP3A-mediatedmetabolismof lopinavir. Kaletra (LPV/r) is widely used to treat HIV infection.

Patients and Methods: Population: HIV-infected men and women, who were aged 18 and older on LPV/r in combination with one or more antiretroviral agents for at least 2 weeks prior to screening, were eligible for this study. Subjects on medications interacting with LPV/r, pregnant females and subjectstreated with dual HIV protease-inhibitors were excluded.

Study Design: This is a multi-center stratified prospective non-randomized PK study. Females are hypothesized to have a LPV area-under the concentration-time curve (AUC) 30% higher than their male counterparts. A total of 78 subjects were divided into two groups by sex. Each group was further divided into 4 approximately equal strata by race/ethnicity. Three study visits were required; a screening, pre-entry and a PK visit. LPV PK was assessed after subjects kept a Kaletra medication diary for a 48-hour period. One hundred percent compliance with medications during this period was required to enter the study. On the PK day, blood samples were drawn at time 0 and at again at 1, 2, 3, 4,5, 6,8, 10 and 12 hours after subjects took the morning dose of LPV/r. All subjects received a standardized breakfast.

Statistical Analysis Plan:
Primary Endpoint: Lopinavir AUC 0-12h

Secondary Endpoints: LPV PK parameters maximum concentration (Cmax), concentration at 12 hours (C12h) and apparent oral clearance (CL/F)

Assumptions: AUC of LPV/r follows a normal distribution. The variance of LPV PK parameters between HIV-infected men and women is equal. A Student's T-test will be used to compare the mean LPV AUC as well as the other PK parameters in the 2 groups. A multiple regression model fitted with stepwise model selection will be used to evaluate the relationship of the covariates age, weight, use of tenofovir and body mass index on the PK of LPV. A non-parametric Kruskall-Wallis test will be performed to evaluate the differences in LPV PK parameters among the racial groups.

Results: This study opened to accrual nationwide on 10/06/ 05 and rapidly accrued by 11/30/05. Final results are pending.

Background: Disparities in HIV/AIDS preventive and clinical care due to linguistic and cultural barriers are known and exist among California's large number of Latinos with limited English proficiency (LEP). Clinicians who serve LEP patients may not be prepared to discuss sensitive issues necessary to screen for behavioral risk factors among patients who are asymptomatic for HIV/AIDS. A prevention study is proposed to examine if health centers whose staff receive cultural competency training focused on HIV/AIDS for LEP Latinos increase their overall rate of screening of HIV behavioral risk factors and HIV testing among asymptomatic or unknown HIV status, LEP Latinos. The presentation will describe the development and pilot testing of the training to help improve screening and education of LEP Latinos for HIV/AIDS.
Methods: The development of the training was built on: (1.) review of similar trainings with published results, (2.) identification of recommended clinical guidelines and evidence-based practices for clinician-led HIV/AID prevention, (3.) cultural competency and diversity training,(4.) literature on sexual beliefs and practices among Latin Americans, with a focus on Mexicans, (5.) pilot data from clinician HIV/AIDS clinical practices, and (6.) consultation with HIV/AIDS clinician training experts in the USA and Mexico. Content and procedures of the training were presented and critiqued by research and clinical staff prior to formal implementation.

Results: Most clinician training materials and content identified for the development of this study's training was focused on providers who care for patients with HIV/AIDS (rather than prevention).Limited items were found to explicitly address health disparities and cultural and linguistic factors that may be address to facilitate clinician HIV/AIDS screening and education among LEP Latinos. Review of similar cultural competency trainings (found for non-HIV/AIDS issues) and conversations with research and clinical experts led to the development of role plays and interactive exercises intended to increase practice and retention of skills among clinicians and to increase comfort in discussing sexual health with LEP Latinos, with and without an interpreter. Trainings will include a formal four-hour workshop, booster sessions and case study presentations at monthly meetings.

Discussion: Given California's large and growing Latino population it is critical to address cultural and linguistic barriers to clinician screening and education of HIV/AIDS among Latino patients. This study will examine the development of a training to improve such screening and education. Sexual health screening and education can be adapted to better attend to customs, beliefs and values in Latin American culture. Ethical issues related to sexual health screening will be considered, especially when using bilingual staff and family for interpreting. The study will examine challenges and opportunities for intervention at the provider and organizational levels that may lead to more culturally and linguistically appropriate care for HIV/AIDS among diverse populations.

Racial/ethnic disparities in the U.S. HIV/AIDS epidemic have been most apparent among women. Since the early 1980s, African American women have accounted for at least six of every ten female AIDS cases, while composing just 13% of the population. National HIV/AIDS rates among Black women are currently 24 and 4.1 times those of White and Latina women. The majority of these HIV/AIDS cases have been attributed to heterosexual sex, often with a male of unknown HIV risk who may have had sex with other men or injected drugs (IDUs). The risk of transmission from bisexual men is particularly relevant in California, where men who have sex with men compose more than 60% of all HIV/AIDS cases and heterosexual transmission from IDUs plays a relatively minor role in the epidemic. Sociocultural factors that uniquely impact African American heterosexual partnerships call for HIV risk-reduction interventions that are specifically designed for African American women with at-risk male partners (i.e., behaviorally bisexual or drug-using partners, those with unknown HIV risk histories, etc.). We propose to develop and evaluate the Females of African American Legacy Empowering Self (FemAALES) Project, a novel and culturally congruent intervention guided by the Theory of Reasoned Action and Planned Behavior, the Critical Thinking and Cultural Affirmation (CTCA) Model, and the Empowerment Theory. This small-group intervention involves six two-hour group sessions lead by an ethnically and gender-matched facilitator and uses online technologies to reinforce concepts, foster social support, and provide low-cost opportunities for sustained intervention exposure. The primary aims are to: (1) develop and test the FemAALES intervention based on formative research and review of local data and published literature and to (2) reduce HIV-related sexual risk behaviors and (3) increase sexual negotiation skills and racial/ethnic and gender pride among African American women with at-risk male partners. In addition, we will explore the intervention’s impact on the frequency of sex under the influence of drugs. FemAALES will be fully developed and implemented in three phases. Phase 1 will involve formative research to guide and develop the intervention curriculum, including conducting and analyzing three focus groups with the target population and one with community service providers. Phase 2 will involve a pilot test of the intervention with two groups of FemAALES participants and one group of waitlisted controls. In the full trial, controls will be waitlisted to later receive Healthy Alternatives for Reducing the Risk for HIV Infection Project (HARRP) -- an untested and non-risk, gender, or ethnic group-specific intervention currently being used by the applicant organization. Phase 3 will involve a preliminary test of both interventions with a total of 120 African American women evenly randomized to the FemAALES and waitlisted HARRP conditions. Participants will be assessed at baseline, post- and three months following intervention completion. Bivariate and multiple regression analyses will be used to examine changes in the outcomes of interest among the intervention conditions (FeMAALES, waitlist, and HARRP), mediating effects, and dose- response associations. Study findings will be used to seek funding for a large-scale intervention trial and will be disseminated throughout California for use by policy makers, researchers, and other community organizations.

The decrease in risky sexual and injection drug use behaviors has been the primary focus of prevention efforts against the spread of theHIV. Foropioidaddicts, abundant datahasshownthereduction in risky behavior when treatment is offered with opioid replacement therapy. For more than three decades Methadone, administered in a structured strictly prescribed program, was the only treatment available until the release of Buprenorphine. California laws have restricted opioid replacement therapy options in many communities, and this has made it virtually impossible for jails to begin opioid replacement therapy on incarcerated opioid addicts. Physicians can now prescribe effective opioid replacement therapy for opioid dependent persons using Buprenorphine, including persons who are incarcerated. In San Francisco, leadership in public health and in the sheriff 's department has been supportive of providing opioid replacement therapy to HIV+ and HIV-addicts, but the program in the jail has been slow to be implemented.

Buprenorphine is viewed as an effective, economical and viable alternative to methadone, especially in a jail setting where the complex rules of methadone licensing may be difficult to implement. But, Buprenorphine has yet to be fully utilized in a jail-based setting as opiate replacement. Initially, the San Francisco Jail Health Services program for treating opiate dependent incarcerated persons was administered to opiate dependent individuals undergoing opioid withdrawal in jail. Upon release, each individual was referred to the San Francisco Department of Public Health's Induction Clinic (OBIC), located in the Mission District of San Francisco. However, a simple referral to OBIC has not been effective for linking released inmates to ongoing Buprenorphine care.

Medication adherence and treatment outcomes are optimized when linked with substance abuse treatment. The largest challenge is to stabilize peoples'lives so that they can consistently access care and to provide comprehensive, quality care to those whose lives remain chaotic. As Ryan White-funded primary care providers, the PHP clinic provides substance abuse counseling and has access to substance abuse treatment for its patients through multiple community linkages and collaborations. Substance abuse services are coordinated by five full-time social workers. Services include on-site substance abuse counseling, referrals to inpatient and outpatient substance abuse treatment programs, and a strong linkage to OTOP on the hospital campus.

Concurrent studies of this population in San Francisco indicate that a lack of ongoing effective opiate replacement therapy carries a direct correlation with high rates of progression from HIV to AIDS diagnosis and death, recidivism, risky behavior and subsequent spread of infectious diseases such as HIV and HCV. This study is designed to evaluate linkages between opiate replacement therapy in jails and community healthcare. Our research goals for this pilot are: (1.) to document the implementation of a program to induce and maintain HIV positive and negative inmates on Buprenorphine while in jail, (2.) to implement a transition program for jail inmates on Buprenorphine into the community, and (3.) to describe the characteristics ofHIV positive persons, including changes in reported HIV risk behavior, who continue on Buprenorphine after release from jail.

Toxoplasma gondii is a protozoan parasite that is estimated to chronically infect approximately 25% of the global human population. Following acute infection of immunocompetent individuals, this parasite differentiates into a semi-quiescent, encysted state, primarily in the brain and muscle tissues, persisting for the life of the host. Reactivation of this latent infection, as a result of immunosuppression in the host, can lead to fatal disease, particularly in HIV/AIDS patients. Opportunistic infection of HIV/AIDS patients by Toxoplasma is a serious concern, as nearly a third of these patients are at risk of developing toxoplasmosis encephalitis.

Central to the prevalence and persistence of Toxoplasma infections in humans is the ability of this parasite to efficiently invade nearly any human cell. This obligate intracellular parasite actively invades target cells and generates a vacuole by forcing invagination of the host cell plasma membrane. A distinctive feature of this process is the formation of a close apposition between the parasite and host cell plasma membranes that migrates down the length of the parasite as invasion proceeds. In forming the vacuolar membrane, this moving junction (MJ) somehow selectively excludes certain host cell membrane proteins, thereby preventing recognition by host degradative pathways and creating an intracellular niche for parasite development. Thus, invasion is not only an essential step in initiating the intracellular life-cycle of the parasite, but it is also critical to the parasite's ability to evade host immune defenses. Despite its absolutely central role in parasite pathogenesis, very little is known about how the MJ forms and operates. To better understand the molecular mechanisms of Toxoplasma invasion, it is imperative to define the MJ composition and, in particular, identify its association with both the host and parasite plasma membranes. Recent work from our laboratory determined that the Toxoplasma MJ is a secreted, heteromultimeric complex consisting of at least four proteins. It is not clear if this protein complex alone facilitates the functions attributed to the MJ or if these proteins recruit additional factors to aid in the invasion process. One of these secreted proteins, RON2, has putative transmembrane domains, suggesting it may act as an anchor for the MJ in one of the two membranes that the complex spans. Given that Toxoplasma can invade virtually any nucleated cell, we hypothesize that, rather than using a host cell receptor, Toxoplasma inserts its own receptor into host cells and that RON2 functions in this role. The experiments outlined in this proposal will define the role of RON2 in host cell invasion and specifically test this hypothesis. In addition, to define the structure and composition of the Toxoplasma MJ, we propose the use biochemical methods to identify proteins, both parasite and host cell in origin, that associate with the MJ, and in this way, contribute to the operation of this unique structure.

Given that host cell invasion is a critical step in the establishment as well as the progression of Toxoplasma infection, identification of the factors that facilitate this process will provide important information for the development of therapeutics, such as vaccines, to prevent disease caused by this parasite. The development of such therapeutics has direct implications for preventing morbidity and mortality among those who suffer from HIV/AIDS.

CD28 provides an important costimulatory signal for T cell activation that regulates multiple cellular processes including proliferation, expansion and survival. However, the mechanism by which CD28 regulates these different processes is still not clear. Using MCC-reactive AND transgenic T cells, we demonstrate that after the recognition of antigen, T cells lacking CD28 have reduced expression and activity of Aurora B (AIM1, Aurora-1), a serine/threonine kinase, described as a chromosome passenger involvedincytokinesisandchromosome architecture. It isthought thatAurora B kinases are responsible for chromatin modifications, including phosphorylating histone H3, however, there is no information on the role of Aurora B in T cells. To understand its function, we used retroviral transduction of Aurora B constructs. In vitro, dominant negative Aurora B expressed in activated wild type T cells decreased cell expansion at an early stage. Conversely wild type Aurora B transduced into responding CD28-deficient T cells enhanced early cell expansion during the phase of active division, but did not provide a long-term survival advantage. Interesting, wild type Aurora B co-introduced with Bcl-xL in responding CD28-deficient T cells enhanced cell expansion during the phase of active division, and provided a long-term survival advantage. These results indicate thatAurora B is induced by CD28 cosignaling and allows T cells to maintain cell division while synergizing with Bcl-xL to control T cell survival.

Background: In ongoing ethnographic research with homeless sexualminorityyoung people (15-28),Iam considering how their stigmatized and illegal behaviors are often concealed by not only themselves, but also by cultural and structural forces within the local gay and larger communities, local and national governments, and scientific practice itself. While identifying these forces (discursive, violent, political, legal, economic, symbolic, epistemological) I came to see the increasing importance of quantitative behavioral survey research since the advent of the AIDS Pandemic to enumerate--and thereby reveal--stigmatized and illegal behaviors. For example, when exploring my informants' participation in survival sex, explicit speech about it was constrained. But contrary to the theorizing of anthropologists on the inexpressibility of suffering and pain in language, I learned that it was not that the youth cannot not speak about their participation in survival sex, but rather that their speech and actions about it are not heard. My ability to label or translate such speech and actions came in part from the work of behavioral epidemiologists to assemble a variety of behaviors into the category of 'survival sex'. As a result I bore a kind of knowledge that many I observed lacked, a sexual knowledge generated from the analysis of data abstracted from others 'like' them in the past. While the quantitative social sciences have been critiqued for concealing structural oppression and the politics of research, the research on survival sex provides a counterexample of how they can also be used in attempts at the opposite. This led me to consider how numbers and enumeration can be used to reveal behavior which other cultural forces serve to conceal or elide, and thereby expanded my dissertation project to consider both the human researchers and researched humans in the production of sexual and drug-using knowledge.

Methods: The majority of my data is constituted by my detailed field notes and interviews with homeless (25) and scientific (15) informants. I have been accompanying my homeless youth and young adult informants in many activities of their daily lives and my scientific informants in their professional practices, including project planning, funding activities, instrument design, writing up and public presentation of findings, and recruitment, outreach and surveying of research subjects.

Results/Conclusion/Relevance: This project seeks to understand how epidemiological categories of sexual behavior and 'at-risk' populations are constituted; a critical feature of this analysis examines how behavioral epidemiologists and the humans they study interact to produce scientific sexual knowledge. This project therefore links an anthropology of science attentive to knowledge practices with an urban anthropology rooted in gay and lesbian studies that foregrounds the subjectivity of stigmatized groups. To understand the relation between the lived experience and social worlds of persons and the scientific practices by and in which they are transformed into a sexual category of risk, I am examining the everyday practice both of behavioral epidemiologists and their human objects of study.

Probiotics are live microorganisms that enhance health and wellbeing of man or animals by exerting beneficial effects on host mucosal surfaces of the gastrointestinal tract, the upper respiratory tract, or the urogenital tract. Some Lactobacillus species play a predominant role as advantageous components of the microflora, however, little is known about the molecular mechanisms these "good bacteria" use to interfere with pathogenic microorganisms. In order to elucidate how probiotic Lactobacillus species are able to directly suppress the growth of pathogens, we are using Candida albicans as a sensor organism. While commensal in healthy individuals, C. albicans has become the most important opportunistic fungal pathogen in the immunosuppressed causing severe mucosal or disseminated infections. DNA microarrays for C. albicans enable us to utilize genome-wide expression analysis to characterize the transcriptional response of the fungi to the presence of probiotic lactobacilli or their antimicrobial products. We have found that lactic acid production by the lactobacilli is a major, but not the only component inhibitory for fungal growth. Lactobacilli can generate oxidative stress responses in the fungi and appear in fact able to kill C. albicans cells. Consequently, they are also capable of interfering with formation of C. albicans biofilms. We also have gathered evidence that lactobacilli react to the presence of fungal cells and might actively induce components of their armamentariumto attack C. albicans. Our genomicapproach will help to understand mechanisms of probiotic interference and results will provide a basis for further studies on the development of therapeutic regimens for opportunistic infections using pro-biotic bacteria. Additionally, the bacteria may also be employed for preventive measures, which could ultimately result in reduced transmission of HIV due to a healthy and robust mucosal flora.

Candida albicans is a commensal inhabitant of mucosal surfaces in immunocompetent individuals, but in patients with impaired immunity, this opportunistic fungus can invade tissues and cause life-threatening disease. We show that C. albicans cultured in the presence of the essential omega-3 fatty acid eicosapentaenoic acid (EPA) synthesizes several oxygenated products of EPA including resolvin E1 (RvE1), a lipid mediator of inflammation produced in humans by acetylated cyclooxygenase-2 (COX-2) or cytochrome 450 monoxygenase (CYP450) and 5-lipoxygenase (5LO).Importantly, RvE1 promotes the resolution of inflammation in rodent models of dermal inflammation, peritonitis, and colitis. Although there is no known 5-LO equivalent in Candida, specific inhibitors of 5-LO markedly reduced fungal RvE1 synthesis without affecting growth. Human neutrophils, cells important for innate defense against pathogens, responded to nanomolar levels of RvE1 by exhibiting reduced IL8-stimulated chemotaxis, but enhanced phagocytosis and killing of Candida. Our results demonstrate that by producing a human immunomodulatory lipid, C. albicans can alter the host immune response, thereby potentially affecting fungal persistence in the host. Our finding that C. albicans can synthesize oxygenated lipids involved in the resolution of inflammation suggests that lipid-derived signaling molecules represent a new class of mediator that can function in the host-pathogen dialogue.

Background: Behavioral interventions promoting safer sex among people living with HIV may need to be tailored to the sexual behaviors and relationships of individual patients to be most effective. This study examines correlates of unprotected sex among specific subgroups of people living with HIV to determine whether easily assessed characteristics can be used as a basis for tailoring safer sex counseling in the clinic setting. Preliminary results are available for a subgroup of male heterosexual patients.

Methods: 121 adult HIV-positive heterosexual men who were attending one of six clinics in California and were sexually active with one partner in the prior 3 months were included. Potential correlates of self-reported unprotected oral (receptive) and vaginal sex included participant demographics (e. g., age, ethnicity), disease status (CD4 counts, viral load, years since diagnosis), safer sex beliefs (e. g., condom attitudes, safer sex self-efficacy), substance use, psychological characteristics (depressive symptoms, dispositional optimism and pessimism), and sex partner characteristics (main/casual partner, status, and duration of relationship).A series of logistic regression analyses were used to determine significant relationships.

Results: Correlates of reported levels of unsafe oral (24%) and vaginal (21%) sex were not associated with the type of relationship (main or casual) or perceived HIV infection status of the partner (positive, negative, or unknown). Preliminary results indicate that unsafe oral sex was positively associated with age and CD4 counts and inversely associated with optimism and positive condom attitudes (all p's<.05). Unsafe vaginal sex was inversely associated with positive condom attitudes.

Conclusion: Prevention efforts among sexually active adult heterosexual men living with HIV may benefit from focusing on improving attitudes towards condom use regardless of partner relationship status.

The rapid appearance of resistant HIV-variants, adverse effects on contemporary drugs, recent indications for HIV "superinfections" and disappointing results with experimental vaccines, all necessitate the continuous development of independent therapeutic strategies to combat HIV infection. The goals of the proposed project are to design, synthesize and evaluate novel nucleotideguanidinoglycoside conjugates as potential anti HIV-1 agents.

The underlying hypothesis guiding the proposed strategy is that the anti HIV efficacy of nucleoside-based ReverseTranscriptase inhibitors (NRTIs) can be enhanced by covalently conjugating their corresponding monophosphates to guanidinoglycosides, a novel family of cellular uptake vehicles that also exhibit high affinity to viral RNA sequences. Two objectives are therefore concomitantly met: (1.) RT inhibitors are actively transported into the cell and then released in a semi-activated form, hence the necessary monophosphorylation step is circumvented, and (2.) essential regulatory events involving viral-specific protein-RNA interactions (e. g., Rev-RRE) are inhibited.

The unique fundamental as well as practical features offered by this approach include: (1.) two distinct stages in the lifecycle of the virus are targeted with one anti-HIV agent, and (2.) facilitated import of semi-activated NRTI's may enhance the therapeutic factor of clinically proven agents (since fewer metabolic activation steps are needed and the released negatively charged nucleotide may reside longer in the cell), and possibly revive NRTI's that have clinically never materialized due to lack of appropriate metabolic activation.

The overarching theme for the San Mateo County, San Francisco Peninsula HIV/AIDS Research Center is the innovative application of technologies to public health interventions that advance the surveillance, prevention, care and treatment of HIV/AIDS. The partnerships of the San Mateo County, Health Department and Medical Center's Clinical Trials and Research Unit, Stanford's University School of Medicine's Center for AIDS Research, and the San Francisco Department of Public Health's STD Program will promote this theme through interrelated research studies which address problems important to advances in the prevention transmission, pathogenesis, care and treatment of HIV/AIDS. Specific research efforts will focus on (1) evaluation of the detection of early and acute HIV infection as a public health strategy through the development and validation of assays of saliva and oral secretions; (2) providing the foundation for a cohort study to evaluate shedding of HIV in various biological compartments. Informatics and experimental methods in molecular virology will provide new information about the role of drug resistance, fi tness and envelope tropism in transmission and pathogenesis. The aim is to understand and improve antiretroviral treatment and secondary prevention strategies in vulnerable patient populations.

Infrastructure components designed to support the Center's theme will build the individual and shared capacity of each partner. This includes strategies to support an emerging community based HIV/ AIDS research center on the San Francisco Peninsula focused on underrepresented populations; create joint mentoring and training infrastructure, to support development of young investigators committed to public health careers in HIV / AIDS research; create a multi-jurisdictional information system, to serve the clinical and research needs of HIV/AIDS patients, providers and researchers; implement community resource strategies, to enroll and retain women and people of color on ART and in clinical trials that are based on principles of participatory community research; award pilot study funding to promising young investigators for projects that advance the Center's research theme and promote the transfer of knowledge, skills and technology.

The Center presents a unique opportunity in California to form a three-way partnership involving two neighboring health jurisdictions. The San Mateo Clinical Research and Trials Unit (CTRU), an emerging research entity with a strong communitybased research track-record, will be the coordinating center for this highly innovative partnership that will bring together advanced methods in public health and laboratory science to serve a research agenda that is responsive to the needs of historically understudied populations.

Anti-retroviral therapy has greatly curbed HIV proliferation; however cognitive deficits continue to have a high prevalence among the infected. Converging evidence from neuropsychological, neurophysiological, and autopsy studies implicate dysfunction of the dopaminergic fronto-striatal system of the brain as the basis for these deficits. However, neuropathological changes do not consistently correlate with impairment, as some individuals are more vulnerable to developing neurocognitive disorders and deficits than others. This suggests that as of yet unknown variables mediate this relationship. In the current application, we propose to examine the contribution of genetic polymorphisms (GPs) that affect the metabolism and activity of dopamine (DA), a key neurotransmitter in the fronto-striatal. Consistent with current theories of Gene X Environmental interactions in development of psychiatric illness, we will consider both endogenous (GPs) and exogenous (HIV and stimulant use) factors in the development of neurocognitive disorders and/or deficits in HIV.

It is well established that both HIV and stimulant use are exogenous stressors that adversely affect the subcortical dopaminergic pathways of the brain. More recently, studies have shown that certain GPs that affect DA metabolism are risk factors for neuropsychiatric disease, and that they also explain significant variation in cognitive functioning across both healthy and clinical populations. Such findings are relevant to NeuroAIDS research, as information about host genotype may add a new tier of explanatory power to the current model of neuropathogenesis in HIV-related neurocognitive disorders. Our proposed study may help explain why some individuals with HIV, despite having stable immune functioning, experience cognitive decline while others do not.

The potential value of elucidating the contribution of genotype to neurocognitive vulnerability in HIV is great; such understanding could lead to more empirically-based preventative strategies and target-specific pharmaceutical interventions. With the new technologies available to quickly and inexpensively obtain patient genotype, the opportunity to determine tailored therapeutic interventions is upon us. In addition, using information about host-genotype in developing preventative strategies has the potential to save lives and health-related resources, as well as reducing the financial burden of HIV-related cognitive disorders on the state of California.

Towards this end, we have assembled a multidisciplinary team composed of investigators with expertise in neuropsychology, neurology, genetics, and virology, all who have extensive research experience in HIV infection and/or genetics of neurocognitive disorders. Using existing tissue and data from the National NeuroAIDS Tissue Bank (NNTC), we will have the ability to examine a number of known GPs in relation to HIV-related neurocognitive disorders and deficits. Using regression modeling, we will be able to examine the degree of risk that various endogenous and exogenous variables confer for neurocognitive disorder and/or deficit. This study, focusing on DA-related GPs, will be an initial probe into this relationship, as in the future we plan to examine additional GPs and their relation to neurocognitive outcomes. We are confident that the results of this study will lead to additional funding opportunities through the National Institutes of Health.

Background: Medicaid is the principal payer of care for Americans living with advanced HIV-infection. Individual state Medicaid programs have promoted managed care plans (MCPs) over traditional Fee-For-Service (FFS) for controlling healthcare costs for individuals with chronic disease, such as persons living with HIV. California has the second largest cohort of HIV-infected Medicaid enrollees in the country and has introduced MCPs on a county-by-county basis. We examine the impact of MCP enrollment on mortality, hospitalization, changes in enrollment and cost among Medicaid beneficiaries with AIDS.

Methods: This study uses a retrospective longitudinal cohort study of 12, 078 Medicaid beneficiaries with AIDS in urban counties of California, enrolled in January 1, 1999 and followed through December 31, 2003. We are analyzing Medi-Cal enrollment data linked to Medi-Cal claims, state hospital discharge abstracts, the state death certificate registry and the state AIDS Registry. The impact of MCP enrollment on mortality, hospitalization and changes in enrollment is estimated using multivariate regression models.

Results: In preliminary results, we identify 12, 078 individuals, with 14. 5% enrolled in MCPs. Characteristics of enrollees were: 83% male, age 41. 5 years old (mean), 4. 7 years since AIDS diagnosis (mean),46% non-LatinoWhite, 28% non-Latino Black, and 24% Latino. One third of the enrollees were hospitalized in 1998. During the study period, 63. 3% of enrollees were hospitalized and 23. 3% died. Rates were similar among MCP and FFS enrollees. However, 24. 3% of MCP enrollees changed to FFS by the end of the period, while only 5. 6% of FFS enrollees changed to a MCP. Multivariate regression models found no significant relationship betweenMCPenrollmentand mortality, but MCP enrollment did appear to be associated with lower rates of hospitalization. MCP enrollment was associated with a greater odds of changing plans (Odds Ratio: 6. 81, 95% Confidence Interval: 5. 71 to 8. 13).

Conclusions: Similar death rates with lower utilization among managed care recipients suggest that MCP enrollment may reduce hospitalization rates. Unmeasured severity may bias these estimates, suggesting benefit where there is none. Greater likelihood of disenrollment from MCPs is suggestive of low patient satisfaction or difficulties with access to care. Before wholesale changes are made in the delivery of care, policy makers should address whether non-clinical measures such as cost, satisfaction and access to care differ substantially between MCP versus FFS Medicaid for HIV-infected and other chronically ill beneficiaries. Future work will focus on revising estimates of the impact of MCP on outcome, comparing the cost of care for current MCP and FFS enrollees and examining physician readiness where mandatory MCP enrollment would be instituted.

Background: More than 72, 000 Californians are HIV-infected and an additional 125, 173 Californians have been diagnosed with AIDS. Currently, most HIV prevention programs are based in health clinics and community centers and are not reaching substantial numbers of people at risk for HIV-infection. The current project explores the feasibility of bringing HIV prevention into business settings. At-risk people visit businesses in their community every day, but little research has explored strategies to engage business owners in AIDS prevention.

Methods: Approximately 40 businesses located in Hillcrest, San Diego, will be engaged in distributing condoms and HIV-testing flyers to their customers and employees. Condoms will be wrapped in cute packages with humorous slogans. We are conducting a process evaluation of the feasibility of this approach. Process measures will include number of condoms distributed, types of condom packaging and slogans preferred by different businesses, business participation rate, business owner satisfaction with the program, and customer reactions.

Results: Preliminary pilot testing with 8 randomly selected business owners in Hillcrest, San Diego indicated that about 50 percent would be willing to display condoms or other AIDS prevention materials if given these materials free of charge. Process measures being collected from business owners and customers will provide further data on the viability of engaging business owners in AIDS-prevention initiatives.

Conclusion: This is the first study to address the extent to which businesses of different types are willing to display free condoms and other AIDS prevention materials at their business sites, and to determine satisfaction with this type of program. Lessons learned will guide future HIV-prevention interventions with at-risk groups.

Monocytes are thought to transport human immunodeficiency virus (HIV) and its simian counterpart SIV across the blood-brain barrier (BBB) by a poorly defined mechanism, triggering events that can lead to neurological complications and development of neuro-AIDS. Other neuroinvasive pathogens, such as cytomegalovirus (CMV) and Listeria, similarly infect monocytes, however dissemination into the brain may occur by an alternate route. We hypothesize that SIV infection triggers a unique monocyte migratory program promoting enhanced neuroinvasion and CNS inflammation, distinct from monocyte trafficking characteristics initiated byinfection withCMV or Listeria as well as Salmonella, a non-neuroinvasive pathogen. The specific objectives of the newly funded study are 1) to identify unique phenotypic and functional monocyte trafficking characteristics in SIV infection and 2) to delineate autocrine regulatory mechanism(s) in monocytes, such as production of proinflammatory chemokines, that are likely to contribute to continued monocyte brain recruitment.

To address these specific aims, we will conduct in vitro infection experiments and contrast migratory characteristics of monocytes infected with neurovirulent strains SIV/17E-Fr and SIV DeltaB670 to monocytes infected with rhesus CMV expressing enhanced green fluorescent protein (EGFP), Listeria monocytogenes wildtype strain 10403S or Salmonella typhimurium wildtype derivative IR715 (both transformed with a GFP-expressing construct).We will recapitulate the optimized conditions previously described for HIV infection to enhance susceptibility of rhesus monocytes to SIV infection in vitro. Freshly isolated monocytes will be cultured short-term in the presence of monocyte colony-stimulating factor and SIV-infected, prior to their differentiation and upregulation of macrophage marker CD71. For phenotypic and functional analysis, we established multi-color staining panels to define activation markers, chemokine receptor profiles and intracellular cytokine/chemokine production using flow cytometry. Assays of monocyte function in vitro will be complemented with characterization of infiltrating monocytes in brain tissues derived from acutely SIV-infected rhesus macaques (Clay et al, 2005).

Our anticipated results include expression of distinct chemokine receptors in rhesus monocytes infected with neurovirulent SIV strains, correlating with a neuroinvasive phenotype and with monocytes' suggested role as 'Trojan horse' for viral entry into the brain. This is supported by preliminary findings demonstrating increased CNS infiltration of fluorescein dye+ monocytes in acutely SIV-infected, but not uninfected macaques, following autologous cell transfer of fluorescein dye+ peripheral blood mononuclear cells after a 2-day in vivo migratory period. We also expect production of brain-specific chemokine signals in SIV-infected monocytes, such as fractalkine/CX3CL1 and MCP1/CCL2, that are likely to promote continued monocyte brain recruitment in vivo, potentially shared by other brain-invading pathogens.

Determining unique monocyte migratory parameters triggered upon SIV infection (versus infection with other viral or bacterial pathogens) as proposed under this new award, will help delineate mechanism(s) of monocyte neuroinvasion. This may lead to the development of novel drug targeting options and treatment strategies that inhibit aberrant monocyte neuroinfiltration during HIV infection by targeting specific monocyte subsets and their trafficking signals.

Background: Outbreaks of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) among HIV-infected men who have sex with men (MSM) have been increasingly reported. In many HIV clinics, including ours, the majority of skin and soft tissue infections are now caused by CA-MRSA. Nasal MRSA colonization is known to be a risk factor for MRSA infection in hospitalized patients. However, the role of S. aureus nasal colonization in the pathogenesis of non-hospitalized CA-MRSA remains poorly understood. We hypothesized that nasal colonization with CA-MRSA would exceed that of methicillin-susceptible S. aureus (MSSA), akin to observations that the incidence of clinical disease from CA-MRSA exceeds that of CA-MSSA in our population. To test this hypothesis, we performed a survey of S. aureus nasal colonization among HIV-infected persons.

Methods: We prospectively surveyed HIV-infected MSM followed in the Harbor-UCLA Medical Center HIV clinic. After informed consent was obtained, patients were administered a questionnaire on CA-MRSA risk factors and a nasal swab for S. aureus was performed.

Results: Of 309 enrolled subjects, 78 (25%) had nasal colonization with S. aureus. Of these, 61 (78%) were colonized with MSSA and 17 (22%) with MRSA. MRSA colonized subjectswere more likely than those not colonized with MRSA to report a skin infection in the past six months (53% v 18%, RR 3. 0 [95% CI 1. 8 - 5. 0],p=0. 0004) and to have close contact with a person with a skin infection in the past six months (21% v 5%, RR 4. 3 [95% CI 1. 4 - 13. 2], p=0. 04).There was a non-significant trend towards persons with MRSA colonization having high risk sexual behavior compared to non-colonized patients (18% versus 6%, RR 2. 9 [95% CI 0. 95 - 9. 1], p=0. 09).There was no relationship between MRSA colonization and: gender, MSM, recent hospitalization, residence in a chronic care facility, self-reported drug use, recent incarceration or anonymous sex while using drugs or alcohol. No factors were associated with MSSA colonization except lack of anonymous sex while using drugs or alcohol (RR 0. 22 [95% CI 0. 06 - 0. 88], p=0. 01).

Conclusion: Although most skin and soft tissue infections in our clinic population are caused by MRSA, the majority of
S. aureus nasal colonization is with MSSA. Additionally, MRSA colonization is associated with previous skin infection or contact with persons with skin infections, while MSSA colonization is not associated with these factors. Our findings suggest that the infectivity and transmissibility of nasal CA-MRSA may be higher than nasal CA-MSSA strains. Longitudinal studies are needed to clarify this relationship.

HIV/AIDS researchers are increasingly turning their attention to women, and many recognize the importance of jails as a unique opportunity to reach the many disenfranchised women who move quickly through jails and return to their communities and to address the many conditions that put many women at risk of infection. However, many of the most innovative HIV/AIDS education and prevention programs continue to focus on incarcerated men. This pilot study aims to establish a foundation for a participatory, collaborative research program that will improve the HIV/AIDS education and services currently available to women moving through California's jails. Specific aims include understanding the many ways incarcerated women experience HIV/AIDS risk and infection; identifying the obstacles that incarcerated women confront when trying to implement HIV/AIDS prevention strategies; and determining the feasibility and impact of jailed women, health educators, and university researchers collaborating in the study of HIV/AIDS and education in incarcerated women's lives.

This project combines qualitative and participatory action research (PAR). Jailed HIV+ and HIV- women, HIV/AIDS educators, and university researchers will collaboratively study participants' experiences with HIV/AIDS and HIV/AIDS education. Qualitative interviews and observations will help provide a comprehensive understanding of HIV/AIDS in women's lives; illuminate the culturally specific social conditions that compromise disenfranchised women's ability to negotiate and maintain their sexual and reproductive health; and determine the potential for collaborative research on HIV/AIDS with incarcerated women. PAR will occur in four series of four two-hour training and research workshops. Workshops will include discussions of HIV/AIDS prevention strategies, interviews about obstacles to women acting on these lessons, data analysis in which women examine transcripts from the other unit, and planning and reflection sessions in which collaborators decide the structure and content of the next workshops. Participants will be women and, following the San Francisco County Jails female population, predominantly African American (55%) and Latina (21%) (n=approximately 240). Data analysis will be grounded and allow jailed women to participate as "co-researchers." Once data collection is complete, open coding of the transcripts and fieldnotes will begin. The themes, patterns, and categories identified in this process will lead to later "focused coding" using NVivo, a qualitative data analysis software package. In biweekly meetings, team members will share emerging analyses with women in the participating housing units.

This pilot study is an exploration of the possibilities for researcher, educator, and inmate collaborations in California's jail-based HIV education programs. Much of the insight will come through the women serving time working with educators and researchers to illuminate and eradicate the conditions that leave women vulnerable to HIV/AIDS and incarceration. The comprehensive understanding that results from this collaboration will lead to an expanded collaborative research effort to develop and evaluate jail-based interventions that reflect culturally specific understandings of HIV/AIDS and incarceration in women's lives. This subsequent intervention and evaluation study will provide a model HIV/AIDS curriculum that could be of use throughout California jails.

The assembly and release of retroviruses from human cells is inhibited by a dominant restriction factor. Different retroviruses have evolved mechanisms to counteract this factor, and this enhancement of virus release (EVR) activity is a property of the HIV-1 Vpu protein, the HIV-2 Env protein and the MLV Env protein. Although the EVR activity of Vpu was first described in 1989, the mechanism by which it enhances virus release is still unknown and represents a significant gap in our knowledge of HIV biology.

Previously, we have described a HeLa cell line (HeLa-T17) that has lost the ability to respond to Vpu, while remaining sensitive to stimulation by the HIV-2 Env. Confocal analysis revealed that the distribution of Vpu in these cells is aberrant, with a concentrated perinuclear distribution that colocalized with markers for the TGN and Golgi. To identify the defect in T17 cells we performed microarray analysis on wild-type and T17 HeLa cells, and observed that two different alkaline phosphatase (ALP) genes were highly up-regulated in the T17 cells. In yeast, ALP traffics from the Golgi to the large central vacuole through an interaction with AP-3, suggesting that the phenotype of T17 cells could arise from a defect in the equivalent mammalian pathway. To test this hypothesis, we performed RNAi knockdown of AP-3 in HeLa cells and found that this recapitulated the loss of Vpu activity. We also observed the same aberrant distribution of Vpu. Interestingly, in both T17 and AP-3 knock-down cells, the distribution of the CD63 marker was also altered. CD63 is present in late endosomes and lysosomes and uses AP-3 to traffic to these compartments from the TGN.

Previous studies have mapped the EVR function of Vpu to its membrane-spanning domain (MSD), a region known to have ion channel activity. Although no model has emerged to explain how ion channel activity could enhance HIV release this early observation has tended to focus studies on the MSD alone. We have now also found that the cytoplasmic tail of Vpu is essential for EVR activity, and mapped this to a [DE]XXXLq motif. Such sequences have previously been shown to interact with AP-1, AP-2, and AP-3 subunits. Mutation of residues within this motif both modified the protein's intracellular location and abrogated EVR activity, further suggesting that interaction with the correct cellular trafficking pathway is essential for Vpu's activity.

Latent HIV, a pool of replication-competent virus that "hides" in host cells, is a significant barrier to complete eradication of HIV from a patient; however, the molecular basis of latency remains unknown. Using a retroviral model of the HIV-1 Tat-mediated positive feedback loop, the laboratories of Prof. David Schaffer (mentor, UC Berkeley) and Prof. Adam Arkin (collaborator, Lawrence Livermore) showed that numerous clonal populations of infected Jurkat cells with a single viral integration can either rapidly initiate viral gene expression or exhibit long periods of low gene expression analogous to a latent infection, a phenomenon referred to as phenotypic bifurcation. We hypothesize that long stochastic transcriptional and translational delays inherent in these bifurcating clones contribute to the establishment of HIV latency.

I am using experimental and computational techniques to probe how key transcriptional regulators of the HIV LTR promoter influence this phenotypic bifurcation and latency. My central hypothesis is that fluctuations in transcriptional regulators within single T cells--primarily the regulators NF-kB, Sp1 and p300--lead to the activation or latency decision. This hypothesis will be tested through a set of experiments organized into two specific aims.

In Specific Aim I, I will experimentally perturb the NF-kB, Sp1 and p300 environment in Jurkat cells infected with the HIV-1 retroviral model, and quantitatively measure the effect on transcription, PheB and latency. I will genetically vary the protein environment by overexpressing NF-kB p65, Sp1 and p300 with tetracycline-regulated plasmids delivered with lentiviral vectors, and (separately) inhibit expression using RNA interference. I will also measure HIV transcription directly in single Jurkat clonal cells using tandem fluorescent probes and microscopy. These experimental data will be used to build a computational model of transcription from the LTR to mathematically explain how phenotypic bifurcation (or the choice between activation and latency) is a function of the NF-kB, Sp1 and p300 cellular environment.

In Specific Aim II, I will stimulate infected Jurkat cells with different T cell activators and measure the effect on phenotypic bifurcation. For a subset of activators, I will experimentally measure signaling dynamics, including protein and phosphorylation levels of the transcriptional regulators over time. I will then use these data to develop a mathematical model linking upstream signaling dynamics to nuclear levels of transcriptional regulators, and ultimately, to HIV transcription controlling active infection or latency. We hope that such a model will yield fundamental insights into HIV gene regulation, as well as serve as a platform for designing new signal transduction modulating drugs to treat latency.

Many U. S. adolescents engage in sexual activities that put them at risk for HIV, other STDs, and unintended pregnancies. At least half of new HIV infections are estimated to be among people younger than 25yearsold. Some adolescent HIV prevention programs have shown effectiveness in reducingrisk behaviors, butprogrameffects often do not endure. Research suggests that parents can have a strong influence on their child's behaviors through parenting and communication behaviors that reduce risk.

A promising yet relatively new approach for increasing the effectiveness and duration of such prevention programs is to incorporate parents along with their adolescent children. It can be a challenge, however, to recruit parents and children to participate in a program that deals with such a sensitive and potentially stigmatizing topic. Many parents avoid discussing sex with their children because they feel uninformed and inhibited, or fear that talking about sex will "encourage" sexual activity. There are also many other important health issues that concern parents, such as substance use and obesity. One approach that addresses these points is to provide HIV prevention education in the context of broader health issues. Incorporating additional topics may give a program wider appeal while reducing the stigma of participating in a program about sex and HIV.

Our program helps parents and adolescents become more comfortable and skilled at communicating with each other about sensitive issues, with the goal of creating a fundamental change in the parent-adolescent relationship and a family environment that encourages adolescents to make healthy choices. Our Center previously designed a program to promote healthy adolescent sexual development and risk reduction by helping parents learn skills to communicate effectively with their adolescent children. We are now expanding this program to include adolescents along with their parents and to cover multiple topics. The adapted program will aim to help parents and adolescents learn more effective approaches for interacting with each other while simultaneously imparting knowledge related to sexual health and development, substance use, and obesity so that parents and adolescents will be able to communicate about these sensitive topics in an open and constructive manner.

We are conducting formative research to guide and refine the adaptation of the program as well as a pilot study of the program with middle school students and their parents. The formative research consists of key informant interviews with school personnel and health educators who work with parents and adolescents, and focus groups with parents of middle school students and with middle school boys and girls. The key informant interviews and focus groups will be conducted in March of 2006. The results of this formative research are expected to include information regarding recruitment strategies, logistical issues regarding the implementation ofthe program, andfeedback about program content. These results will be used to refine the program content and structure and tailor the program recruitment and implementation to our population of middle school students and their parents. We will use the pilot study to further refine the program, measure the short-term efficacy of the program, and help determine whether to apply for additional funding to run a randomized controlled trial of the program.

Background: HIV-1 Vif is essential in vivo for it prevents incorporation of the antiviral enzyme APOBEC3G (A3G) into assembling virions. A deoxycytidine deaminase, A3G antiviral activity results in extensive dC ' dU deamination, effectively terminating viral replication. It has previously been demonstrated that cellular A3G is subject to regulation by assembly into a high molecular mass (HMM) complex. RNaseA treatment of HMM A3G generates low molecular mass (LMM) forms. Given that cellular HMMA3G is enzymatically inactive and that LMMA3G is enzymatically active, it was initially assumed that viral A3G, which we know is potently active during reverse transcription, would be incorporated into virions as the enzymatically active LMM form. These studies focus on the form and regulation of virion-packaged A3G.

Methods: Virions were produced by transient transfection of 293T cells, clarified and concentrated, and lysed under mild conditions. The virions were then subject to FPLC analysis followed by Western blot using antibody to A3G. Fractions identified as containing A3G were then subjected to A3G immunoprecipitation and the immunoprecipitates subjected to enzymatic analysis. Enzymatic tests assessed deoxycytidine deaminase activity and/or RNaseH activity.

Results: Surprisingly, FPLC analysis of virion lysate revealed that virion-incorporatedA3G is in the HMM form. Furthermore, analysis of other virion components suggests that virion-incorporated A3G is in a complex containing IN and Vpr and viral genomic RNA. RNaseA-treatment of virion lysate results in redistribution of virion A3G into LMM complex forms. Since cellular HMM A3G is enzymatically inactive, we examined the activity of virion-derived HMM A3G. Similar to the activity profile for cellular A3G, immunoprecipitated virion HMM A3G is enzymatically inactive while LMM A3G derived from RNaseA-treated virions is enzymatically active. Furthermore, RNaseA treatment of the immunoprecipitated HMM A3G complex restored deaminase activity, indicating that the RNA component of the complex inhibits A3G activity. Similarly, A3G derived either from whole virion lysate or producer cell lysate is enzymatically inactive unless treated with RNaseA. Thus, we conclude from these observations that virion RNA inhibits A3G enzymatic activity.

Integral to the process of reverse transcription is removal of the RNA genome template by the RNaseH activity of Reverse Transcriptase (RT). A3G exerts its enzymatic antiviral activity during reverse transcription, suggesting that removal of RNA from virion A3G may be required for activation of its antiviral activity. When endogenous reverse transcription was initiated by incubation of lysed virions with dNTPs and Mg2+,A3G activity was indeed induced in the absence of any RNaseA addition. Importantly, addition of the RNaseH inhibitor, Compound I, during the incubation, completely inhibitedA3G activation by the endogenous reverse transcription conditions. If the endogenous reverse transcription reactions were performed with virions that contain a point mutation in RNaseH rendering it inactive, A3G deaminase activity was no longer induced. These data strongly suggest that RNaseH activity is sufficient to activate HMM A3G derived from virions.

Conclusion: We propose that RNaseH is not only required for generation of the single-stranded DNA template that serves as a target for A3G deamination, but also "activates" virion A3G activity by degrading any associated inhibitory RNA from it, allowing it to now act on the ssDNA.

A recent study of Latino male immigrant day labors in Los Angeles County found that engaging in risk behaviors, such as having sex with men, was not associated with HIV testing. Thus some day laborers at high risk may not be accessing HIV testing services. However, increasing HIV testing among Latino day laborers may not solely be a function of offering the HIV test to them. Unique ways of offering HIV testing may be required in order to increase HIV testing. Some Latino men involved in high risk behaviors have been found to be more likely to choose to get tested for HIV when the HIV test is “bundled” with other tests compared to when it is offered by itself. The objective of this study is to increase HIV testing among day laborers at risk for HIV. The specific aims of this study are (1) to determine if a “bundled” HIV testing protocol directed at day laborers results in more immigrants accessing HIV testing compared to an HIV-only testing protocol., and (2) to determine if a “bundled” HIV testing protocol results in more men engaging in HIV prevention services among those involved in high-risk activities compared to when an HIV-only testing protocol is used. The “bundled” HIV testing protocol will include tests or screeners for syphilis, gonorrhea, Chlamydia, alcohol problems, drug dependence, depression and HIV. Individuals in the HIV-bundled protocol will be able to choose to be tested for all conditions or just some of them.

In contrast to previous studies with day laborers, this study will involve a random recruitment of day laborers using a spatial-temporal sampling frame. Two HIV testing protocols, one “bundled” and the other “HIV-only,” will be carried out at day labor sites in Los Angeles County. In the formative stage of our project, we plan to determine which day labor sites contain the largest number of day laborers reporting high-risk activities. We will use the Service Planning Areas (SPAs) of Los Angeles County. We will utilize SPAs 4, 6, 7 and 8. By doing so, we will be focusing on the areas in which 73.9% of all male Latino HIV/AIDS cases were identified between 2004 and 2007. Once all day labor sites in these SPAs are identified, we will visit them at randomly selected times and randomly sample 15% of the individuals who are present when we arrive. These participants will be administered a short screener to assess HIV-related high-risk behaviors. After determining which sites contain the largest number of day laborers reporting high-risk activities, we will develop a sampling unit of “site-day-shift,” where “site” refers to the day labor site, “day” to the day of the week, and “shift” to a 5-hour time period within the day. The sampling plan for the study recruitment for the main part of the study will involve 4 stages: the random assignment of the 4 SPAs to either the HIV-only protocol or the HIV-bundled protocol, the monthly random selection of the day labor sites, the monthly random selection of the sampling units and the random selection of participants at the day labor sites.

A total of 800 participants will be recruited, with one-half being recruited through the HIV-testing only protocol and the other half through the HIV-bundled protocol. Tallies will be kept of the number of people who are approached and asked to participate for each testing protocol and those who accept and decline HIV testing. Rates of HIV testing for the two HIV testing protocols will then be compared.

This study is interested also in which protocol results in greater engagement in HIV prevention services among those reporting high-risk activities. Those reporting HIV-related high risk behaviors will be contacted 2 months later for a brief phone interview to determine if they subsequently utilized the referrals to HIV prevention programs. The two HIV testing protocols will then be compared to determine which resulted in a greater use of HIV prevention services by day laborers.

Information on potential increased HIV testing by day laborers using a “bundled” testing protocol would contribute to advancing HIV prevention science and also provide important information for prevention programs throughout California. Thus, this project is very attentive to the needs of public health and community based organizations in California attempting to counter the present trend of late testing among Latinos.

In HIV-infected patients, virus from a reservoir of latently infected cells quickly repopulates the system after the cessation of antiretroviral treatment. To better understand the molecular mechanisms of HIV latency and eliminate this reservoir, our laboratory has developed J-Lat cells, a model system for the study of HIV latency in vitro. In these cells, the production of virus is reversibly suppressed at the level of transcription and can be measured by GFP fluorescence. As in latently infected CD4+ T cells recovered from patients, in J-Lat cells the majority of virus integrations have occurred within actively transcribed genes.

Few gene products or molecular mechanisms that regulate HIV latency in cells have been described. Here, we propose a novel approach to identify the full spectrum of genes involved in latency. A library of siRNAs will be expressed in J-Lat cells; those cells in which HIV expression is reactivated will be identified by GFP fluorescence. As this is the first reported attempt to identify regulators of HIV latency by phenotype, we expect to identify novel factors in this screen.

Among the genes isolated, we expect three functional groups to be the most prominent: (1) Those that interact directly with the proviral promoter and repress HIV transcription. Chromatin immunoprecipitation will be used to identify proteins that bind the HIV promoter during latency and are modified or dissociate upon virus reactivation. (2) Those that modulate signaling pathways involved in HIV transcription. Modulation of these pathways by these genes can be observed by standard assays of signaling molecules. (3) Those that decrease the transcription of cellular genes proximal to the latent provirus. A reduction in transcription of these genes may decrease interference with provirus transcription, leading to reactivation. Expression of these cellular genes will be determined in cells with reactivated HIV.

The genes identified in this screen can then be utilized in future studies with primary cells recovered from infected patients and in the development of specific inhibitors to reactivate latent virus. Only then, with the depletion of the latent reservoir, can clearance of HIV from infected patients be contemplated.

The viral Tat protein transactivates the HIV-1 promoter through binding to the TAR RNA element. Tat also modulates the expression of cellular genes in a TAR-independent manner. Here, we report that Tat increases the activity of the transcription factor NF-kappa B by interfering with the nicotinamide adenine dinucleotide-dependent deacetylase SIRT1. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate lysine 310 (K310) in recombinant NF-kappa B/p65. Consistent with these findings, we show that Tat expression induces hyperacetylation of cellular p65 in the presence of SIRT1 using antibodies specific for acetylated K310 in p65. In cotransfection experiments using NF-kappa B reporter genes, Tat neutralizes the negative effect of SIRT1 on the transcriptional activity of wildtype p65, but not of mutant p65 in which lysine 310 was changed to arginine (K310R).This neutralizing effect is lost on a mutant SIRT1 protein that no longer binds to Tat. Tat expressed in mouse embryonic fibroblasts (MEFs) isolated from SIRT1-/- mice has no effect on the activity of endogenous NF-kappa B responsive genes as determined by real-time PCR. In contrast, Tat expressed in MEF cells in which SIRT1 expression has been reconstituted after retroviral infection superinduces transcriptional activities of endogenous I kappa B alpha and E-selectin genes in response to TNF alpha. These experiments collectively show that Tat activates NF-kappa B responsive gene expression through SIRT1, a TAR-independent mechanism that can contribute to Tat-mediated superinduction of the HIV promoter as well as cellular promoters in activated CD4+ T cells.

Effective antiretroviral therapy has been available to patients in the U. S. for nearly 10 years and has resulted in remarkable improvements in the longevity and quality of life of people living with HIV. As the virus has become manageable among their own patients, many U. S.-based AIDS researchers and clinicians have begun extending their research interests beyond U. S. borders in an attempt to address the global epidemic. As a result, a growing number of American AIDS researchers are now involved in projects in sub-Saharan Africa, which bears the burden of over 70% of the world's HIV infections.

As these researchers confront the African epidemic, they encounter a landscape of disease that is both eerily reminiscent of places like San Francisco in the 1980s and at the same time is radically different from the epidemic as they know it. This paper is a preliminary exploration of some of the challenges involved in such research, using qualitative data collected through interviewing and observing AIDS researchers in California and Uganda.

One of the challenges is the problem of "translatability." "Translatability" refers to the ways in which knowledge about HIV/AIDS gleaned from the American experience does or does not translate to a Ugandan context. Specifically, there are problems of "biological translation"that arise in relation to measuring biological markers of HIV disease across contexts and problems of "ethical translation" that arise in attempting to apply Western standards of care to a resource-limited setting. Understanding these problems of translation may assist researchers in both the

U. S. and developing countries to negotiate the challenges of collaboration and improve their ability to conduct relevant and accurate HIV research.

Background: Conspiracy theories have accompanied the emergence of plagues from the Middle Ages to the present day. The types of conspiracy theories associated with HIV/AIDS range from theories involving government involvement in the creation of the virus to beliefs that testing and medications themselves can be used as instruments to wipe out "undesirable" populations. HIV/AIDS conspiracy theories place HIV prevention in the context of the historical, economic and cultural context in which people become vulnerable to HIV. Conspiracy theories speak to the structural determinants of HIV and, therefore, suggest structural-level interventions to address disparities in HIV/AIDS, in contrast to the majority of HIV prevention efforts that focus on individual behaviors. In California, AfricanAmericans comprised 7% of the general population yet 21% of all reported HIV cases from 2002 - 2003. In Alameda County, African Americans are similarly overrepresented amongAIDS cases as compared to their proportion of the general population -- African American men and women constitute 15% of the general population, yet 51% of reported AIDS cases in 2002-2003. These trends indicate that current HIV prevention efforts in California are having limited success in decreasing the rates of HIV infection among African American men and women. Research on HIV/AIDS conspiracy theories has consistently found that approximately one quarter of African Americans of all socioeconomic backgrounds hold conspiracy beliefs, and has concluded that they present significant challenges to HIV prevention. HIV/AIDS conspiracy theories have been hypothesized as barriers to HIV testing amongAfrican Americans; as barriers to health education and health care; as barriers to needle exchange; and as barriers to participation in research. Recent research has found stronger conspiracy beliefs among men. This study aims to contribute to the development of HIV prevention measures for African Americans by examining the ways that HIV/AIDS conspiracy theories might present barriers to HIV prevention efforts with African Americans, and by exploring gender differences in these theories.

Methods: This research presents preliminary qualitative work of a larger mixed-method dissertation project on HIV/AIDS conspiracy theories. This research will investigate through in-depth qualitative interviews the types, meaning and implications of HIV/AIDS conspiracy theories among a sample of HIV- and HIV+ African American men and women (n=40), as well as among a sample of 10 HIV prevention providers. Study participants will be recruited using targeted street-based and venue-based convenience sampling. Qualitative interviews will allow this study to generate in-depth narratives regarding if and how HIV/AIDS conspiracy narratives resonate for African American men and women; which conspiracy theories might be particularly meaningful; and a consideration of the implications of these theories on service use and on risk behaviors. The combination of interviews with community members and providers aims to generate a dataset of interviews from two key perspectives to inform prevention practice and future research.

Expected Results: At this time, a Community Meeting has been held with HIV prevention providers to launch the project. This community dialogue generated rich formative data vital to the development of this project. Pilot interviews will be conducted. Preliminary data have been collected from the Community Meeting and from an initial analysis of interviews.

Background: HIV/AIDS rates continue to be disproportionately high among homosexually and bisexually active men, especially those who are African American. Recent attention to the high levels of HIV infection among African American men who have sex with men or both men and women (MSM/W) and the frequent non-gay-identification in this population highlight the importance of understanding how HIV-positive African American MSM/W perceive safer sex, experience living with HIV, and decide when to disclose their HIV status.

Methods: In June 2005, three focus groups were conducted with 30 predominately HIV-positive and non-gay identifying African American MSM/W. Participants were recruited from Los Angeles County through collaborating community-based organizations, posted fliers, and word of mouth. Using a constant comparison methodology we examined responses to focus group questions regarding perceptions of condom use and the effect of being HIV positive on sexual activity and serostatus disclosure.

Results: The major themes regarding condom use included its protective role against disease and pregnancy, condom acceptability including aesthetics factors and concerns about effectiveness, and situational pressures including exchange sex, substance use, and potential questioning from female partners. Themes pertaining to the impact of HIV infection on the sexual lives of these men included isolation, perceived rejection, and decreased partner seeking. Themes regarding disclosure of HIV status included selective disclosure and the responsibility to disclose. These themes influenced some men to disclose and to practice safer sex and others to forego condoms and disclosure or avoid the issue altogether by not seeking out regular sex partners.

Conclusions: Non-gay-identified African American MSM/W must weigh a complicated set of risks and benefits associated with safer sex and HIV disclosure to sexual partners. These concerns, together with condom acceptability issues and situational factors that reduce individual control over condom use, may interfere with a desire to protect others from HIV infection or oneself from other sexually transmitted infections and HIV reinfection. The results of this analysis will inform curriculum development for a UARP-funded HIV prevention intervention for non-gay-identified African American MSM/W.

Background: One of the hallmarks of advanced acquired immune deficiency syndrome (AIDS) is an increased susceptibility to bacterial pathogens, many of which utilize a type III secretion system (TTSS) to deliver toxin proteins (effectors) directly into the cytosol of host cells. Our long-term goal is to understand the steps required for protein transport by the TTSS. Here, we investigate the function of TTSS chaperone proteins. Inside the bacterium, TTSS chaperones bind one (or sometimes two) specific effector proteins. Although chaperones are not translocated into host cells, they are required for the translocation of their cognate effectors, and thus are central to virulence. Two alternative hypotheses have been proposed to describe the role of chaperones in type III secretion. The first hypothesis is that chaperones unfold their bound effectors, thereby maintaining them in a conformation that is competent for secretion. The alternative hypothesis suggests that chaperones mediate effector recognition by templating the formation of a three-dimensional motif that serves to target the effector to a component of the TTSS. Our specific aim is to determine how association of the Yersinia chaperone SycE affects the structure and dynamics of its corresponding effector YopE. If the chaperone werefound to locallyunfold the effector, this would provide direct evidence for the localized unfolding hypothesis. If instead the chaperone were found to locally fold the effector, this would support the hypothesis that structuring of the effector, as induced by the chaperone, may be required for interactions with bacterial components involved in TTSS transport.

Methods: Here, we use Nuclear Magnetic Resonance (NMR) Spectroscopy to compare an intact effector (YopE) in its free and chaperone-bound states, thereby determining how association with SycE affects the structure and dynamics of YopE.
Results: By using NMR spectroscopy we found that the chaperone-binding domain ofYopE is flexible and lacks secondary structure in the "free" form, but becomes well-structured upon association with SycE.

Conclusions: These data demonstrate a "disorder to order" mode of chaperone action, thereby supporting the translocation-targeting hypothesis.

Significant disparities in HIV transmission, treatment outcomes and quality of life exist among California's disadvantaged populations. We need a fresh, collaborative approach to developing effective HIV prevention campaigns and treatment programs among these populations.

We believe that the proposed Public Health Consortium for HIV Disparities Research offers that fresh approach. We will promote, conduct and support innovative research on community contexts and social networks that have the potential to reduce HIV-related disparities. This approach holds particular promise for reducing HIV transmission and improving care among communities who often face structural barriers to services and whose cultural beliefs strongly value social, faith-based and friendship networks.

To achieve our goal of stimulating innovative research in HIV disparities, we draw on the unique and complementary strengths of our three partner institutions--the Charles R. Drew University of Medicine and Science, a historically African American medical school; RAND, a public policy and research institute; and the Los Angeles County Division of Public Health, the largest public health department in California. The Consortium will be led by a team with representation from all three institutions, as well as community members impacted by HIV/AIDS.

The Consortium will conduct two original research projects on community contexts and social networks. In the fi rst project, an inter-institutional and multi-disciplinary team will investigate the geographic reach of HIV prevention programs to aid the development of more effective prevention policies and interventions. In the second project, Consortium investigators will evaluate the impact of social support and networks on adherence to treatment in order to develop more effective strategies for keeping African Americans and Latinos with HIV in care.

The Consortium will enhance the research capacity and sustainability of partner institutions to conduct innovative HIV disparities research through strategic planning, project oversight, statistical support, stimulating think tanks, pilot projects, methodological training and support for proposal development.

We believe that our approach has strong potential to invigorate the fi eld through high-quality research and to possibly change the course of HIV/AIDS within disadvantaged and under- researched populations of California.

Background: The Internet allows men who have sex with men (MSM) to find new sex partners and practice sexual behaviors that may be risky for HIV. Outbreaks of sexually transmitted diseases and cases of HIV infections have been traced to sexual networks formed in specific chatrooms. In response, community-based organizations and public health departments have begun using the Internet in their HIV-prevention programs. This study will develop a website to test new ways to prevent HIV transmission among California MSM using the Internet.

Methods: We are currently in the formative research phase of this project, and we have conducted four focus groups of MSM who meet sexual partners online (n=38).We recruited men from Internet venues to attend the in-person groups in LosAngeles, San Jose, Orange County, and Fresno. We asked participants for their reactions toourpreliminary ideas aboutthewebsite'sfeatures, their thoughts about how the Internet can be used to encourage MSM to reduceriskbehaviors, ideas about usingtheInternet topromote community health and resilience, suggestions for the website design, marketing, and branding. We took detailed notes of each focus group and are currently conducting a thematic analysis of the qualitative data. Basedonourpreliminaryfindings(below),weare nowworking with web designers and programmers to develop the website which we will later beta-test, revise, launch, and evaluate.

Results: Several themes emerged that were directly relevant to the design of the website. First, the men in the groups expressed that they wanted opportunities to socialize and communicate with each other about common interests and concerns, including sexual health, in a more in-depth and genuine fashion than occurs in most online venues. Some men felt that the Internet can potentially reduce feelings of isolation, and many expressed the need for websites that, besides opportunities for hook-ups, would offer more ways to interact in fun and interesting ways, including in the offline world. Some of the desired features include events calendars, links to local resources, opportunities to get sexual health information, and instruction on writing safe and effective online profiles. In general, men wanted a site that was cleanly designed, functional, easy to search, non-judgmental, practical, and not preachy. The group discussions often turned to participants'likes and dislikes about current online venues. While no consensus emerged about the particular sites, it was clear that the men wanted a forum where they could share their online experiences and opinions of different websites with each other.

Conclusions: The groups endorsed a site with the following features: (1) Blogs: 3 featured bloggers (with at least one openly HIV+ blogger) will talk about issues of importance to MSM in California, including HIV prevention-related subjects, online cruising tips, resources, and community events. This will include a user comment area where men can have in-depth discussion about the issues presented;(2) Site reviews: An area containing an overview of hook-up sites and space for user reviews and ratings (similar to Cnet. com or Amazon. com); (3) Little Black Book: Application where users can organize information about their sexualencounters andnetworks, onlinebuddies, HIV/STDtesting history, and a personalized calendar to remind them when to get tested based on their actual risk behavior. This will be available online in a password-protected area, as well as downloadable to their own computers and/or handheld devices; (4) Safer sex content: Articles written by health professionals with resource and referral links, and the possibility for users to ask specific questions.

HIV/AIDS constitutes a dire medical and social emergency for MTF transgender women in California. In Los Angeles county, HIV prevalence in the MTF transgender population is reported to be 22%, and in San Francisco, 35%. Recognizing that HIV risk is shaped by synergetic biological, social-structural, and individual factors, this study proposes an anthropological investigation of the social and cultural context of HIV/AIDS risk among transgender women in San Francisco. It has four aims: 1) To identify the significant social, political, economic, and demographic factors that place transgender women at elevated risk for HIV/AIDS; 2) To examine the social support networks of transgender women and assess the impact of these networks on HIV/AIDS risk; 3) To document the views and opinions of transgender women and their healthcare providers towards current HIV/AIDS programs; 4) To identify the HIV/AIDS prevention and healthcare needs of transgender women. The primary research participants include at least 45 transgender women in San Francisco, a community comprised of many immigrant women from Mexico and Southeast Asia. Secondary subjects include at least 25 members of their social networks - partners, friends, family members - and at least 25 healthcare providers. The research design incorporates a multi-sited ethnography, utilizing the recruitment and sampling methods of snowballing, venue-based, and respondent driven recruitment of peers. Field-sites include medical clinics, social support agencies, community and social venues, and fieldwork in a low-income urban neighborhood that is home, worksite, and social center for many immigrant transgender women. To obtain a comprehensive picture of the social and cultural context of HIV risk, the study employs the ethnographic methods of extensive participant-observation, qualitative surveys, in-depth interviews/collection of life histories, and qualitative analysis of data. This study responds to California's HIV/AIDS needs in 3 significant ways. 1) As defined by California's DHS, transgender individuals and immigrant communities constitute two vulnerable populations in California that are disproportionately at risk for HIV/AIDS. 2) As data from Los Angeles and San Francisco report, the MTF transgender population is experiencing some of the highest HIV prevalence rates for a vulnerable community ever recorded in the history of the HIV/AIDS epidemic in the United States. 3) Given San Francisco's unique role as an internationally recognized center for gender and sexual diversity, it is imperative that comprehensive qualitative data is collected describing the diverse HIV/AIDS needs of those who migrate to the City due to gender and sexual identities. Thus, this study will increase knowledge about the life circumstances of individuals who are members of two vulnerable and marginalized communities in California, and provide insight into the international dynamics at play in California's HIV/AIDS crisis, particularly San Francisco's unique position in the global epidemic. These research findings will support the design of culturally appropriate and competent HIV/AIDS services for the diverse population of transgender women who are presently underserved by HIV/AIDS efforts in the State. This study is one component of a broader effort to reduce the burden of HIV/AIDS in a Californian population that is not only disproportionately effected by the epidemic, but misunderstood and stereotyped.

Increasing evidence suggests that acute HIV infection is a main driver of the epidemic, emphasizing the potential impact of early interventions at both patient and population level. Although recent studies have demonstrated the value of acute case detection, those initiatives have so far been limited to publicly funded HIV testing sites. To be truly effective at reducing HIV transmission, acute HIV-centered prevention strategies must move beyond such sites, to doctor's offices, clinics and medical centers where the majority of HIV testing occurs. By extending the highly successful public health model for acute HIV screening currently used at the San Francisco STD clinic, into primary care, urgent care and emergency department settings, this proposal intends to include a more diverse Bay Area population and establish methods suitable for further dissemination statewide.

Working closely with providers, community representatives, University of California-San Francisco (UCSF) and San Francisco Department of Public Health (SFDPH), we will 1) develop and implement integrated models designed to facilitate the accurate screening, results notification, counseling, and both clinical and public health case management of cases of acute HIV infection in our expansion sites; and 2) evaluate the feasibility, acceptability and impact of those acute HIV screening models, and 3) develop specific recommendations for expansion to additional sites with California based on lessons learned.

Over the first 3 months of the award, current SFDPH protocols for acute HIV screening will be adapted to the new expanded testing. Sites to be included are the San Francisco General Hospital Medical Center and affiliated clinics; the San Francisco Department of Public Health community-oriented primary care centers and affiliated clinics, including the Castro-Mission Heath Center, Tom Waddell Health Center and Mission Neighborhood Health Center; and the Edison Clinic in San Mateo County. As with the current SFDPH, appropriate information, disclosure, case management/confirmatory testing and partner services will be implemented. HIV RNA laboratory testing will continue to take place at the SFDPH Laboratory which has been doing such pooled screening since 2003. .

A research plan will be developed to evaluate already routinely collected information on negative and positive HIV test results, notification/partner outcomes and some risk behavior information at each testing site. Outcomes to be measured during the project include the number of patients tested, number of (true/false) positives, patient demographics, risk behaviors, current STDs, CD4+ T-cell counts, time-to-first CD4+ T-cell count, time-to-first HIV care visit, number and characteristics of the partners, adverse events, and acceptability/perceptions of program procedures. To assess the performance and potential impact of proposed models, we will screen about 15,000 patients for acute and chronic HIV infection over a 12-month period.

To increase the generalizability of our findings for California, we will conduct comparative analyses with data obtained from other public health jurisdictions in California (including the San Diego Department of Health). Finally, by incorporating both our own evaluations and possible collaborative analyses in a final summary report, we will develop recommendations for expansion of RNA testing in California.

Background: Results from the VaxGen 004 trial indicated that vaccination with recombinant gp120 (rgp120) was ineffective in preventing sexually transmitted HIV infection. However, vaccine induced antibody titers correlated inversely with HIV infection risk. Since antibody function is often highly dependent on interactions between the Fc part of antibody and Fc gamma receptors (FcgRs) found on monocytes, macrophages, dendritic cells, and natural killer cells, we sought to determine if a functional polymorphisms in an FcgR gene might influence the relationship between antibody titer and infection risk.

Methods: Allele-specific primers were used to determine the FcgRIIa (histidine [H] vs. arginine [R]) genotypes in rgp120vaccinated participants in the VaxGen 004 trial. The sample included 221 of the total of 241 vaccinated participants that became infected during the study and 138 randomly selected uninfected vaccinees. 50% neutralizing antibody titers against HIVMN have been reported previously and were measured by determining the dilution of serum that blocked HIV-1-induced cytopathic effect on MT4 cells.

Results: There was an inverse correlation between HIV risk and HIVMN neutralizing antibody titer among individuals with RR and RH genotypes, but not among those with the HH genotype:

Furthermore, the relative risk of infection for vaccinees with the HH genotype compared to those with the RR genotype increased with increasing antibody titers (relative risks = 0. 34, 0. 74, 0. 90, and 1. 64 for antibody titer quartiles 1, 2, 3, and 4, respectively).

Conclusions: These results suggest that the FcgRIIa polymorphism influences the previously reported inverse correlation between vaccine-induced antibody responses and risk of HIV infection among participants in the VaxGen 004 trial. Specifically, individuals with the HH genotype have a non-statistically significant increased risk of infection with increasing antibody titers, whereas individuals with the RH or RR genotype have a lower risk of infection with higher antibody titers. The HH receptor is known to have higher affinity for IgG2- and IgG3immune complexes than do the RH or RR isoforms. Thus, our findings might be explained by FcgRIIa-mediated antibody-dependent enhancement (ADE) of infection by infectious immune complexes that is most likely to occur in the presence of the higher-affinity receptor isoform. This would in turn suggest that potentially beneficial antibody or innate immune functions that might operate in individuals with RH or RR genotypes are partially overcome by ADE in HH homozygotes. Our data are consistent with the recent observation that infants who are homozygous for the H allele are more likely to acquire HIV perinatally from their infected mothers than infants with other FcgRIIa genotypes.

Over a third of all AIDS cases in California have been reported in Los Angeles County, and young men who have sex with men (YMSM) represent a sizeable portion of those affected. YMSM are an important target for HIV prevention campaigns because they report engaging in high levels of sexual risk behaviors; they are more likely to engage in sexual risk than older MSM are. In this context, the as-yet-unpublished findings of our previously funded UARP grant are particularly striking: in a sample of 170 18- to 24-year-old MSM recruited outside gay- oriented clubs and bars, virtually all were sexually experienced, yet fully 25% reported they had not had sex within the past 6 months. Our data further indicate that the abstainers may be likely to engage in less sexual risk even when they do have sex. Their risk for HIV and other sexually transmitted infections is therefore very low. These findings suggest an important possibility for prevention interventions that is virtually unheard of in the published literature on YMSM: promoting a return to sexual abstinence. Understanding more about YMSM who are no longer sexually active will help us develop interventions to promote a return to abstinence and thereby achieve a great public health impact.

The data from our preliminary study were not designed to explore issues related to sexual abstinence; nevertheless, they do provide some insights into how abstainers may differ from YMSM who are sexually active. The abstainers tended to be men of color, and appear to have what can be described as a stronger sense of self, relating to the importance of race, community and religion in their lives. Based on these preliminary findings, and guided by theories on self- categorization and adaptive identity, we propose carrying out a qualitative study to further explore these issues and others that may be important in return to sexual abstinence. We will conduct 50 in-depth interviews with YMSM who report that they are sexually experienced but have not had sex in the previous 6 months. The specific aims of the project are to answer the following questions:

1. How do these YMSM perceive their own sexuality and sexual behavior? What norms do they hold regarding sexual behavior?
2. For those YMSM who have actively chosen to remain sexually abstinent, what factors did they weigh in making this choice?
3. What groups--sexual, racial ethnic, religious, etc.--do these YMSM identify with, and what characteristics associated with these groups support a return to abstinence?
4. Who are their friends, and to what groups do they belong? What are their friends' norms about sexual behavior?
5. What is the pattern of drug and alcohol use among these YMSM, and how is substance use related to their sexual behavior?
6. What is the club experience like for these YMSM? Why do they go to venues such as clubs, and how or why do they refrain from meeting sexual partners there?

The participants in this study will be 18 to 24 years old, and will be ethnically diverse. The data collected will be transcribed and coded, and then analyzed to identify patterns. Findings will be applied to the development of future research: quantitative studies on larger and more representative samples of YMSM, and intervention studies aimed at promoting a return to abstinence and thus a dramatic reduction in HIV risk.

Background: African American men who have sex with men (MSM) and who do not identify as gay are at high risk for HIV infection. ManyAfricanAmerican MSM do not seek HIV testing, despite engaging in behaviors that place them at risk for infection. Previous strategies have proven to be ineffective in reaching non-gay identified (NGI) African American (MSM) who do not fit into traditionally defined gay or bisexual categories. Many NGI African Americans cannot relate to HIV prevention messages that are designed for the gay community; furthermore, many do not read gay print media, and few participate in gay identified events or patronize gay establishments. This study will inform prevention efforts by exploring the motivations, capacities, and challenges associated with HIV prevention among this hard-toreach population.

Methods: A two-tiered qualitative study design will be used to gather data to examine the perceptions of NGI African American MSM in the San Francisco Bay Area regarding their HIV risk and thesocial, cultural, community, and family influences associated with conducting community outreach, HIV testing, and prevention. During the first phase of the project, 25 in-depth qualitative interviews will be conducted. During the second project phase, 12 individuals from the initial sample will be invited to participate in Photovoice, an innovative participatory action research strategy that uses a photographic technique to promote critical reflection by research participants regarding the phenomenon of interest. This method will explore further the perceived strengths and concerns related to HIV risk of participants.

Expected Results: As of November 2005, we have conducted in-depth interviews with 19 men who fit the inclusion criteria of the study. Preliminary results have revealed a few notable observations that have relevance for culturally relevant HIV prevention efforts. Women have been identified as major supports in the lives of many of the men and may serve as valuable resources for HIV prevention efforts. Despite the fact that many men have characterized the church as opposing same sex behavior, many still consider themselves to be observant Christians. For most, if not all of the men, the concept of harm reduction has not been discussed as an HIV prevention strategy. The Photovoice phase is scheduled to begin in the second year of the study. Most of the 19 men interviewed have expressed interest in participating in the Photovoice component of the project. The outcomes of this participatory action research are expected to inform policies and programs related to HIV prevention, testing, counseling, and outreach to African American NGI-MSM. The collection of visual images may provide data for use in innovative community awareness campaigns. Documenting innovative outreach methods will also assist other researchers in formulating their sampling strategies with this hard-to-reach population.