Seminal plasma HIV-1 RNA concentration is strongly associated with altered levels of seminal plasma interferon-γ, interleukin-17, and interleukin-5.

Seminal plasma HIV-1 RNA level is an important determinant of the risk of HIV-1 sexual transmission. We investigated potential associations between seminal plasma cytokine levels and viral concentration in the seminal plasma of HIV-1-infected men. This was a prospective, observational study of paired blood and semen samples from 18 HIV-1 chronically infected men off antiretroviral therapy. HIV-1 RNA levels and cytokine levels in seminal plasma and blood plasma were measured and analyzed using simple linear regressions to screen for associations between cytokines and seminal plasma HIV-1 levels. Forward stepwise regression was performed to construct the final multivariate model. The median HIV-1 RNA concentrations were 4.42 log10 copies/ml (IQR 2.98, 4.70) and 2.96 log10 copies/ml (IQR 2, 4.18) in blood and seminal plasma, respectively. In stepwise multivariate linear regression analysis, blood HIV-1 RNA level (p<0.0001) was most strongly associated with seminal plasma HIV-1 RNA level. After controlling for blood HIV-1 RNA level, seminal plasma HIV-1 RNA level was positively associated with interferon (IFN)-γ (p=0.03) and interleukin (IL)-17 (p=0.03) and negatively associated with IL-5 (p=0.0007) in seminal plasma. In addition to blood HIV-1 RNA level, cytokine profiles in the male genital tract are associated with HIV-1 RNA levels in semen. The Th1 and Th17 cytokines IFN-γ and IL-17 are associated with increased seminal plasma HIV-1 RNA, while the Th2 cytokine IL-5 is associated with decreased seminal plasma HIV-1 RNA. These results support the importance of genital tract immunomodulation in HIV-1 transmission.

Pre-clinical evaluation of a 213Bi-labeled 2556 antibody to HIV-1 gp41 glycoprotein in HIV-1 mouse models as a reagent for HIV eradication.

BACKGROUND: Any strategy for curing HIV infection must include a method to eliminate viral-infected cells. Based on our earlier proof-of-principle results targeting HIV-1 infected cells with radiolabeled antibody (mAb) to gp41 viral antigen, we embarked on identifying a suitable candidate mAb for preclinical development. METHODOLOGY/PRINCIPAL FINDINGS: Among the several human mAbs to gp41 tested, mAb 2556 was found to have high affinity, reactivity with multimeric forms of gp41 present on both the surface of virus particles and cells expressing HIV-1 Env, and recognition of a highly conserved epitope of gp41 shared by all HIV-1 subtypes. Also, mAb 2556 was the best in competition with HIV-1+ serum antibodies, which is an extremely important consideration for efficacy in the treatment of HIV patients. When radiolabeled with alpha-emitting radionuclide 213-Bismuth ((213)Bi) - (213)Bi-2556 efficiently and specifically killed ACH-2 human lymphocytes chronically infected with HIV-1, and HIV-1 infected human peripheral blood mononuclear cells (hPBMCs). The number of binding sites for (213)Bi-2556 on the surface of the infected cells was >10(6). The in vivo experiments were performed in two HIV-1 mouse models--splenic and intraperitoneal. In both models, the decrease in HIV-1 infected hPBMCs from the spleens and peritoneum, respectively, was dose-dependent with the most pronounced killing of hPBMCs observed in the 100 µCi (213)Bi-2556 group (P = 0.01). Measurement of the blood platelet counts and gross pathology of the treated mice demonstrated the lack of toxicity for (213)Bi-2556. CONCLUSIONS/SIGNIFICANCE: We describe the preclinical development of a novel radiolabeled mAb reagent that could potentially be part of an HIV eradication strategy that is ready for translation into the clinic as the next step in its development. As viral antigens are very different from "self" human antigens - this approach promises high selectivity, increased efficacy and low toxicity, especially in comparison to immunotoxins.

Repeated lipopolysaccharide (LPS) exposure inhibits HIV replication in primary human macrophages.

Repetitive exposure of macrophages to microbial antigen is known to tolerize them to further stimulation and to inhibit proinflammatory cytokine release. Using transgenic (Tg) mice that incorporate the entire HIV-1 genome we have previously shown that toll like receptor (TLR)-2, -4, and -9 ligands induced tolerance as assessed by decreased proinflammatory cytokine secretion and nuclear factor-kappa beta activation. Yet, despite cytokine modulation, HIV-1 p24 production was enhanced in tolerized cells in vitro and in vivo. Since mice are not natural hosts for HIV infection, in the following report we examined whether TLR2 and TLR4 ligands induced tolerance in human monocytic cell lines stably expressing the HIV-long terminal repeat (LTR) luciferase construct (THP-LTR-Luc) as well as in primary macrophages that had been infected with HIV(BAL)in vitro. In THP-LTR-luc, TLR2 and TLR4 tolerization suppressed tumor necrosis factor (TNF)-alpha release and HIV-LTR transactivation. In HIV(BAL) infected macrophages, repeated LPS exposure inhibited HIV replication as assessed by decreased genetic expression and protein production of HIV-1 p24, although TNF-alpha release was not inhibited. These observations may have important clinical implications in understanding the role of macrophages as HIV reservoirs at anatomical sites where there is repeated exposure to microbial antigens.

Cocaine and sigma-1 receptors modulate HIV infection, chemokine receptors, and the HPA axis in the huPBL-SCID model.

Cocaine is associated with an increased risk for, and progression of, clinical disease associated with human immunodeficiency virus (HIV) infection. A human xenograft model, in which human peripheral blood mononuclear cells were implanted into severe combined immunodeficiency mice (huPBL-SCID) and infected with a HIV reporter virus, was used to investigate the biological interactions between cocaine and HIV infection. Systemic administration of cocaine (5 mg/kg/d) significantly increased the percentage of HIV-infected PBL (two- to threefold) and viral load (100- to 300-fold) in huPBL-SCID mice. Despite the capacity for cocaine to increase corticosterone and adrenocorticotropic hormone levels in control mice, the hypothalamic-pituitary-adrenal axis was suppressed in HIV-infected animals, and corticosterone levels were further decreased when animals were exposed to HIV and cocaine. Activating huPBL in vitro in the presence of 10(-8) M cocaine increased expression of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) coreceptors. Expression of CCR5 was also increased at early time-points in the huPBL-SCID model following systemic exposure to cocaine (54.1+/-9.4% increase over control, P<0.01). This effect preceded the boost in viral infection and waned as HIV infection progressed. Cocaine has been shown to mediate immunosuppressive effects by activating sigma-1 receptors in immune cells in vitro and in vivo. Consistent with these reports, a selective sigma-1 antagonist, BD1047, blocked the effects of cocaine on HIV replication in the huPBL-SCID mouse. Our results suggest that systemic exposure to cocaine can enhance HIV infection in vivo by activating sigma-1 receptors and by modulating the expression of HIV coreceptors.

Tetrahydrocannabinol suppresses immune function and enhances HIV replication in the huPBL-SCID mouse.

Epidemiologic studies identify marijuana as a potential cofactor in the development and progression of HIV infection. To evaluate this interaction we employed a hybrid model in which human peripheral blood leukocytes (PBL) were implanted into severe combined immunodeficient mice (huPBL-SCID) and infected with an HIV reporter construct in the presence or absence of tetrahydrocannabinol (THC) exposure. Administration of THC alone, in the absence of HIV, decreased CD4 counts and the CD4:CD8 ratio. Co-administration of THC and HIV did not reduce CD4 counts further, but significantly increased the percentage of HIV-infected PBL when compared to saline-treated animals (17+/-4.6% vs. 7+/-1.4%). Quantitative PCR confirmed a 50-fold increase in systemic viral load in THC-treated animals. The CCR5 and CXCR4 chemokine receptors function as coreceptors essential for HIV infection. Administration of THC for 5 days increased the percentage of PBL expressing CCR5 and, to a lesser extent, CXCR4. This effect was lost after 10 days of THC administration, but the number of HIV-infected cells had significantly increased by that time suggesting a role for early upregulation of these coreceptors in the pathogenic effect of THC. Finally, the impact of treatment on the number of human interferon-gamma (IFN-gamma) producing cells was determined by ELISPOT. Both THC and HIV infection independently decreased the number of IFN-gamma producing cells and co-administration produced additive effects. These results suggest that exposure to THC in vivo can suppress immune function, increase HIV coreceptor expression, and act as a cofactor to significantly enhance HIV replication.

Amelioration of graft versus host disease by galectin-1.

Graft versus host disease is a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Galectin-1, a mammalian lectin that modulates T cell function and apoptosis, has been shown to be immunomodulatory in animal models of autoimmune disease. We investigated the efficacy of galectin-1 in a murine model of graft versus host disease and found that 68% of galectin-1-treated mice survived, compared to 3% of vehicle-treated mice. Galectin-1-treated animals also had reduced inflammatory infiltrates in tissues compared to animals treated with vehicle alone. Galectin-1 did not affect engraftment of donor hematopoietic cells. However, galectin-1-treated animals demonstrated increased cellularity in bone marrow and spleen with increased numbers of splenic B cells and CD4 T cells compared to those animals treated with vehicle alone. Galectin-1 treatment also significantly improved reconstitution of normal splenic architecture following transplant. Production of type I cytokines interleukin-2 (IL-2) and interferon-gamma was reduced in splenocytes derived from galectin-1-treated transplanted mice when compared to animals treated with vehicle alone, while production of the type II cytokines, IL-4 and IL-10, was similar between the two groups of animals. Although splenocytes from galectin-1-treated transplanted animals responded to both third party antigens and leukemic challenge, host alloreactivity was significantly reduced when compared to cells from vehicle-treated animals. These results demonstrate that galectin-1 therapy is capable of increasing survival and suppressing the graft versus host immune response without compromising engraftment or immune reconstitution following allogeneic hematopoietic stem cell transplant.

Impairment of antimicrobial activity and nitric oxide production in alveolar macrophages from smokers of marijuana and cocaine.

Human alveolar macrophages (AMs) were recovered from the lungs of healthy nonsmokers (NS) or smokers of tobacco (TS), marijuana (MS), or crack cocaine (CS) and challenged in vitro with Staphylococcus aureus. AMs from NS and TS exhibited potent antibacterial activity that correlated with the production of nitric oxide (NO) and induction of NO synthase without the requirement for priming with exogenous cytokines. In contrast, AMs from MS and CS exhibited minimal antibacterial activity and failed to produce NO unless primed with additional cytokines. These results confirm that NO plays a significant role as an effector molecule used by normal human AMs, but this capacity is suppressed in AMs from MS and CS because of a lack of intrinsic cytokine priming.

Evidence of chronic damage to the pulmonary microcirculation in habitual users of alkaloidal ("crack") cocaine.

STUDY OBJECTIVE: To evaluate BAL cells obtained from habitual users of alkaloidal ("crack") cocaine alone or in combination with tobacco, for evidence of cocaine-associated alveolar injury. DESIGN: Prospective cohort study. PATIENTS: A total of 36 healthy men and women (mean age [SD], 37.5 [7.5] years), including 10 cocaine-only smokers (CS), 6 cocaine-plus-tobacco smokers (CTS), 10 tobacco smokers (TS), and 10 nonsmokers (NS), underwent fiberoptic bronchoscopy and BAL. METHODS: Cytospins were prepared from BAL cells and stained with Wright-Giemsa for cell differentials and Gomori's stain for detection of hemosiderin. Endothelin (ET)-1 levels were determined from lavage fluid by enzyme-linked immunosorbent assay. RESULTS: None of the cocaine users reported episodes of hemoptysis or respiratory distress, and routine spirometry findings were within normal limits in all subjects. While there was little effect on total cell numbers or differential counts, the percentages of hemosiderin-positive alveolar macrophages (AMs) were markedly increased in CS (33.8 +/- 8.7% [SEM]) compared to TS and NS (< 2%; p < 0.05). The percentages of hemosiderin-laden AMs were also numerically increased in CTS (11.8 +/- 7.8%), but this value was not statistically significant from that of TS or NS. ET-1 levels were significantly increased in the fluid recovered from CS (6.2 +/- 0.8 pg/mL) when compared to NS (1.2 +/- 0.4 pg/mL) and TS (1.3 +/- 0.2 pg/mL) [p < 0.05], while ET-1 levels were elevated to a lesser extent in CTS (2.5 +/- 0.6 pg/mL). ET-1 levels correlated with the percentage of hemosiderin-positive AMs when CS were analyzed in conjunction with CTS (r = 0.64; p = 0.0004). CONCLUSION: Clinically inapparent alveolar hemorrhage occurs frequently in otherwise healthy crack cocaine smokers and is associated with elevated levels of ET-1, indicative of cocaine-induced pulmonary microvascular injury.

Cocaine enhances human immunodeficiency virus replication in a model of severe combined immunodeficient mice implanted with human peripheral blood leukocytes.

Epidemiologic studies have identified cocaine as a cofactor for development of acquired immunodeficiency syndrome (AIDS). To evaluate this interaction, human peripheral blood leukocytes (PBL) were implanted into severe combined immunodeficient mice and infected with human immunodeficiency virus (HIV) in both the presence and absence of cocaine. Concurrent administration of cocaine resulted in significantly more PBL becoming infected with HIV in vivo (38.85% vs. 18.5%). The number of CD4(+) cells recovered from HIV-infected, cocaine-treated animals was significantly lower than that from mice infected with HIV in the absence of cocaine (6.5 x 10(4) vs. 19 x 10(4)) and was associated with a lower CD4:CD8 ratio and a dramatic increase in virus load. Exposure to cocaine alone did not affect the implantation of PBL, suggesting a specific interaction between cocaine and HIV. This report describes a model for evaluating HIV cofactors and supports cocaine's role in the development and progression of AIDS.