The Potential Role for Group Psychotherapy in the Treatment of Internalized Homophobia in Gay Men.

In order to develop a healthy sense of self, gay males must developmentally move beyond feelings of self-hatred concerning their homosexuality and conquer feelings of internalized homophobia. The struggle with internalized homophobia occurs throughout the lifespan and is detrimental to the physical and mental health of gay men. In order to spur the creation of interventions, a literature review was conducted to show how group psychotherapy could be of service in ameliorating the root causes of shame central to internalized homophobia. While the literature focused on the importance to gay men of feeling a sense of belonging within the gay community-a concept similar to group cohesion-as central to creating affective interventions, group psychotherapy has yet to be sufficiently explored quantitatively in the treatment of internalized homophobia.

Novel delivery system enhances efficacy of antiretroviral therapy in animal model for HIV-1 encephalitis.

Most potent antiretroviral drugs (e.g., HIV-1 protease inhibitors) poorly penetrate the blood-brain barrier. Brain distribution can be limited by the efflux transporter, P-glycoprotein (P-gp). The ability of a novel drug delivery system (block co-polymer P85) that inhibits P-gp, to increase the efficacy of antiretroviral drugs in brain was examined using a severe combined immunodeficiency (SCID) mouse model of HIV-1 encephalitis (HIVE). Severe combined immunodeficiency mice inoculated with HIV-1 infected human monocyte-derived macrophages (MDM) into the basal ganglia were treated with P85, antiretroviral therapy (ART) (zidovudine, lamivudine and nelfinavir (NEL)), or P85 and ART. Mice were killed on days 7 and 14, and brains were evaluated for levels of viral infection. Antiviral effects of NEL, P85, or their combination were evaluated in vitro using HIV-1 infected MDM and showed antiretroviral effects of P85 alone. In SCID mice injected with virus-infected MDM, the combination of ART-P85 and ART alone showed a significant decrease of HIV-1 p24 expressing MDM (25% and 33% of controls, respectively) at day 7 while P85 alone group was not different from control. At day 14, all treatment groups showed a significant decrease in percentage of HIV-1 infected MDM as compared with control. P85 alone and combined ART-P85 groups showed the most significant reduction in percentage of HIV-1 p24 expressing MDM (8% to 22% of control) that were superior to the ART alone group (38% of control). Our findings indicate major antiretroviral effects of P85 and enhanced in vivo efficacy of antiretroviral drugs when combined with P85 in a SCID mouse model of HIVE.

Development of a rapid autopsy program for studies of brain immunity.

Human glia are essential cellular models used for studies of neurodegenerative diseases. Fetal neuroglia are commonly used, as they can be recovered in large quantities and sustained for long periods in culture. However, fetal neuroglia may have limitations in reflecting adult diseases and additionally can pose ethical issues in translating products of abortion for research use. To address these concerns, we developed a rapid autopsy program to procure age- and disease-specific neuroglia from adult brain tissues within hours of death. The challenges in developing this initiative, reflecting experiences from 69 autopsies over 4 years, are presented.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) suppresses Rho GTPases in human brain microvascular endothelial cells and inhibits adhesion and transendothelial migration of HIV-1 infected monocytes.

Under inflammatory conditions (including HIV-1 encephalitis and multiple sclerosis), activated brain endothelium enhances the adhesion and transmigration of monocytes across the blood-brain barrier (BBB). Synthetic ligands that activate the peroxisome proliferator-activated receptors (PPARs) have anti-inflammatory properties, and PPAR stimulation prevents the interaction of leukocytes with cytokine stimulated-endothelium. However, the mechanism underlying these effects of PPAR ligands and their ability to intervene with leukocyte adhesion and migration across brain endothelial cells has yet to be explored. For the first time, using primary human brain endothelial cells (BMVEC), we demonstrated that monocyte adhesion and transendothelial migration across inflamed endothelium were markedly reduced by PPARgamma activation. In contrast to non-brain-derived endothelial cells, PPARalpha activation in the BMVEC had no significant effect on monocyte-endothelial interaction. Previously, our work indicated a critical role of Rho GTPases (like RhoA) in BMVEC to control migration of HIV-1 infected monocytes across BBB. In this study, we show that in the BMVEC PPARgamma stimulation prevented activation of two GTPases, Rac1 and RhoA, which correlated with decreased monocyte adhesion to and migration across brain endothelium. Relevant to HIV-1 neuropathogenesis, enhanced adhesion and migration of HIV-1 infected monocytes across the BBB were significantly reduced when BMVEC were treated with PPARgamma agonist. These findings indicate that Rac1 and RhoA inhibition by PPARgamma agonists could be a new approach for treatment of neuroinflammation by preventing monocyte migration across the BBB.

Peroxisome proliferator-activated receptor-gamma activation suppresses HIV-1 replication in an animal model of encephalitis.

OBJECTIVE: Poor penetration of antiretroviral therapy across the blood-brain barrier poses an impediment on control of HIV-1 infection in brain macrophages. Peroxisome proliferator-activated receptor (PPAR)-gamma, a member of the nuclear receptors family, regulates important physiological functions (including anti-inflammatory effects) in response to ligand-mediated activation. As PPARgamma agonists are rapidly absorbed by oral administration and efficiently permeate the blood-brain barrier, we hypothesized that PPARgamma stimulation may suppress HIV-1 replication. DESIGN AND METHODS: We investigated the effect of PPARgamma ligand (rosiglitazone) on HIV-1 replication in human monocyte-derived macrophages and in vivo using a murine model (immunodeficient mice reconstituted with human lymphocytes and intracerebrally inoculated with HIV-1 infected macrophages) of HIV-1 encephalitis. RESULTS: Treatment with rosiglitazone caused a significant decrease of virus infection in macrophages. PPARgamma stimulation inhibited virus replication by modulating NF-kappaB activation in a receptor-dependent manner, leading to downregulation of HIV-1 long terminal repeat (LTR) promoter activity and suppression of HIV-1 replication. These effects were PPARgamma specific as PPARgamma silencing or addition of PPARgamma antagonist abolished effects of PPARgamma stimulation on HIV-1 LTR and virus replication. Using a murine model for HIV-1 encephalitis, we demonstrated that PPARgamma ligand suppressed HIV-1 replication in macrophages in brain tissue and reduced viremia by 50%. CONCLUSION: In vitro data delineated the novel mechanism by which PPARgamma activation suppresses HIV-1 replication, and in vivo findings underscored the ability of PPARgamma agonists to reduce HIV-1 replication in lymphocytes and brain macrophages, thus offering a new therapeutic intervention in brain and systemic infection.

Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE).

The blood-brain barrier (BBB) is compromised during progressive HIV-1 infection, but how this occurs is incompletely understood. We studied the integrity of tight junctions (TJs) of brain microvascular endothelial cells (BMVECs) in an in vitro BBB system and in human brain tissues with HIV-1 encephalitis (HIVE). A downregulation of TJ proteins, claudin-5 and occludin, paralleled monocyte migration into the brain during HIVE. Because small G proteins (such as Rho) can play a role in BMVEC TJ assembly, an artificial BBB system explored the relationship among TJs, Rho/Rho kinase (RhoK) activation, and transendothelial monocyte migration. Coculture of monocytes with endothelial cells led to Rho activation and phosphorylation of TJ proteins. Rho and RhoK inhibitors blocked migration of infected and uninfected monocytes. The RhoK inhibitor protected BBB integrity and reversed occludin/claudin-5 phosphorylation associated with monocyte migration. BMVEC transfection with a constitutively active mutant of RhoK led to dislocation of occludin from the membrane and loss of BMVEC cell contacts. When dominant-negative RhoK-transfected BMVECs were used in BBB constructs, monocyte migration was reduced by 84%. Thus, loss of TJ integrity was associated with Rho activation caused by monocyte brain migration, suggesting that Rho/RhoK activation in BMVECs could be an underlying cause of BBB impairment during HIVE.

Alcohol abuse enhances neuroinflammation and impairs immune responses in an animal model of human immunodeficiency virus-1 encephalitis.

Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1(+) MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8(+)/interferon-gamma(+) T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8(+) T-lymphocyte infiltration in brain areas containing HIV-1(+) MDMs, ethanol-fed mice featured double the amounts of HIV-1(+) MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1(+) MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain.

Second virial coefficient determination of a therapeutic peptide by self-interaction chromatography.

Self-interaction of macromolecules has been shown to play an important role in a number of physical processes, including crystallization, solubility, viscosity, and aggregation. Peptide self-interaction is not as well studied as for larger proteins, but should play an equally important role. The osmotic second virial coefficient, B, can be used to quantify peptide and protein self-interaction. B values are typically measured using static light scattering (SLS). Peptides, however, do not scatter enough light to allow such measurements. This study describes the first use of self-interaction chromatography (SIC) for the measurement of peptide B values because SIC does not have the molecular size limitations of SLS. In the present work, SIC was used to measure B for enfuvirtide, a 36-amino acid therapeutic peptide, as a function of salt concentration, salt type, and pH. B was found to correlate strongly with solubility and apparent molecular weight. In general, the solubility of enfuvirtide increases with pH from 6 to 10 and decreases as the salt concentration increases from 0 to 0.5M for three different salts. The effect of peptide concentration on B was also investigated and shown to have a significant effect, but only at high concentrations (>80 mg/mL).

Ethanol-induced activation of myosin light chain kinase leads to dysfunction of tight junctions and blood-brain barrier compromise.

BACKGROUND: Brain endothelial cells form the blood-brain barrier (BBB) that regulates solute and macromolecule flux in and out of the brain, leukocyte migration, and maintains the homeostasis of the central nervous system. BBB dysfunction is associated with disruption of tight junctions (TJ) in the brain endothelium. We propose that alcohol abuse may impair BBB permeability through TJ modification. METHODS: Primary cultured bovine brain microvascular endothelial cells (BBMEC) were treated with 50 mM ethanol (EtOH), and monolayer tightness was assessed by measurement of transendothelial electrical resistance (TEER). Changes in TEER were correlated with alterations in TJ protein distribution [occludin, zonula occludens-1 (ZO-1), claudin-5] using immunofluorescence (IF). Expression of myosin light chain (MLC) kinase (MLCK), ZO-1, claudin-5, and phosphorylated MLC, occludin and claudin-5 were determined by immunoprecipitation and Western blot. EtOH-induced changes in monocyte migration across in vitro BBB constructs were also examined. RESULTS: EtOH induced a decrease in TEER of BBMEC monolayers that was reversed by EtOH withdrawal. Treatment of BBMEC with EtOH or its metabolite, acetaldehyde, prior to monocyte application resulted in a 2-fold increase in monocyte migration across the BBB. IF demonstrated decrease in claudin-5 staining, occludin translocation from cell borders to cytoplasm and gap formation in EtOH-treated BBMEC monolayer. These changes paralleled significant increase in phosphorylation of MLC, occludin and claudin-5. EtOH-treated BBMEC showed reduction of total occludin and claudin-5 without changes in ZO-1 or MLC. TEER decrease, changes in occludin/claudin staining, increase in MLC, occludin and claudin-5 phosphorylation and enhanced monocyte migration across the BBB were all reversed by inhibition of MLCK. Inhibition of EtOH metabolism in BBMEC also reversed these events. CONCLUSION: These results suggest that EtOH activates MLCK leading to phosphorylation of MLC, occludin and claudin-5. Cytoskeletal alterations (MLC) and TJ changes (occludin and claudin-5 phosphorylation) result in BBB impairment (decrease in TEER). TJ compromise is associated with increased monocyte migration across the BBB.

Inhibition of indoleamine 2,3-dioxygenase (IDO) enhances elimination of virus-infected macrophages in an animal model of HIV-1 encephalitis.

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine pathway of tryptophan metabolism. IDO activity is linked with immunosuppression by its ability to inhibit lymphocyte proliferation, and with neurotoxicity through the generation of quinolinic acid and other toxins. IDO is induced in macrophages by HIV-1 infection, and it is up regulated in macrophages in human brain tissue with HIV-1 encephalitis (HIVE). Using a model of HIVE, we investigated whether IDO inhibitor 1-methyl-d-tryptophan (1-MT) could affect the generation of cytotoxic T lymphocytes (CTLs) and clearance of virus-infected macrophages from the brain. Severe combined immunodeficient mice were reconstituted with human peripheral blood lymphocytes, and encephalitis was induced by intracranial injection of autologous HIV-1-infected monocyte-derived macrophages (MDMs). Animals treated with 1-MT demonstrated increased numbers of human CD3+, CD8+, CD8+/interferon-gamma+ T cells, and HIV-1(gag/pol)-specific CTLs in peripheral blood compared with controls. At week 2 after MDM injection in the basal ganglia, mice treated with 1-MT showed a 2-fold increase in CD8+ T lymphocytes in the areas of the brain containing HIV-1-infected MDMs compared with untreated controls. By week 3, 1-MT-treated mice showed 89% reduction in HIV-infected MDMs in brain as compared with controls. Thus, manipulation of immunosuppressive IDO activity in HIVE may enhance the generation of HIV-1-specific CTLs, leading to elimination of HIV-1-infected macrophages in brain.

Alcohol and HIV decrease proteasome and immunoproteasome function in macrophages: implications for impaired immune function during disease.

Proteasomes (proteinase complexes, PR) and immunoproteasomes (IPR) degrade damaged proteins and affect protein processing required for antigen presentation by mononuclear phagocytes. These critical immune processes are attenuated during progressive HIV-1 infection and are affected by alcohol abuse. To investigate the mechanisms underlying these functional changes, we measured PR and CYP2E1 activities [an ethanol (EtOH) metabolizing enzyme] and reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM) following HIV-1 infection and EtOH treatment. We observed progressive declines of PR activity and PR/IPR contents in HIV-1-infected MDM. PR activity and IPR expression increased after IFN-gamma stimulation but reduced after HIV-1 infection. EtOH inhibited both IFN-gamma-induced PR and IPR. Paradoxically, EtOH attenuated PR catalytic activity in infected MDM and suppressed viral replication. Elevated ROS followed EtOH exposure and paralleled decreased PR activity. The latter was restored by anti-oxidant. The data support the notion that HIV-1 infection and EtOH may work in concert to affect immune function including antigen presentation and thereby affect disease progression.