Levels of murine, but not human, CXCL13 are greatly elevated in NOD-SCID mice bearing the AIDS-associated Burkitt lymphoma cell line, 2F7.

Currently, few rodent models of AIDS-associated non-Hodgkin's lymphoma (AIDS-NHL) exist. In these studies, a novel mouse/human xenograft model of AIDS-associated Burkitt lymphoma (AIDS-BL) was created by injecting cells of the human AIDS-BL cell line, 2F7, intraperitoneally into NOD-SCID mice. Mice developed tumors in the peritoneal cavity, with metastases to the spleen, thymus, and mesenteric lymph nodes. Expression of the chemokine receptor, CXCR5, was greatly elevated in vivo on BL tumor cells in this model, as shown by flow cytometry. CXCL13 is the ligand for CXCR5, and serum and ascites levels of murine, but not human, CXCL13 showed a striking elevation in tumor-bearing mice, with levels as high as 200,000 pg/ml in ascites, as measured by ELISA. As shown by immunohistochemistry, murine CXCL13 was associated with macrophage-like tumor-infiltrating cells that appeared to be histiocytes. Blocking CXCR5 on 2F7 cells with neutralizing antibodies prior to injection into the mice substantially delayed tumor formation. The marked elevations in tumor cell CXCR5 expression and in murine CXCL13 levels seen in the model may potentially identify an important link between tumor-interacting histiocytes and tumor cells in AIDS-BL. These results also identify CXCL13 as a potential biomarker for this disease, which is consistent with previous studies showing that serum levels of CXCL13 were elevated in human subjects who developed AIDS-lymphoma. This mouse model may be useful for future studies on the interactions of the innate immune system and AIDS-BL tumor cells, as well as for the assessment of potential tumor biomarkers for this disease.

Baseline immune phenotypes and CD4+ T lymphocyte responses to antiretroviral therapy in younger versus older HIV-infected individuals.

OBJECTIVE: The purpose of the study was to determine associations between pre-antiretroviral therapy (ART) senescent CD8+ T lymphocytes and naïve versus non-naive CD8+ and CD4+ T lymphocyte subpopulations and CD4+ responses after initiation of ART in younger versus older individuals. METHODS: Retrospective analysis of 100 subjects with pre-ART cryopreserved peripheral blood mononuclear cells samples was performed with flow cytometry. Subjects were divided into four groups by age (30-50 years or > 50 years) and 96-week CD4+ response (<100 or >200 cells/mm(3)). All subjects had 96-week viral suppression to <50 copies/mm(3). Regression was utilized to investigate associations between pre-ART CD8+ and CD4+ T cell phenotypes with age and CD4+ response categories. RESULTS: Individuals <50 years had a lower frequency of senescent CD8+ T lymphocytes of the CD56 + 57+, CD56+, and CD28- phenotypes (95%CI -3.6 to -0.02; 95%CI -4.2 to -0.03; 95%CI -12.5 to -1.4, respectively) and a higher frequency of naïve (CD45RA + CD28+) CD8+ T lymphocytes (95%CI 2.6 to 10.9). Younger age and good CD4+ response were associated with a higher frequency of pre-ART naïve CD4+ T cells (95%CI 2.0 to 16.4 and 95%CI 1.5 to 15.6, respectively). CONCLUSIONS: Prior to ART, younger HIV-infected individuals have a higher frequency of naïve CD4+ and CD8+ T cells and lower frequency of senescent CD8+ T cell phenotypes.

Single mutations in HIV integrase confer high-level resistance to raltegravir in primary human macrophages.

CD4(+) T cells and macrophages are the primary target cells for HIV in vivo, and antiretroviral drugs can vary in their ability to inhibit the infection of these different cell types. Resistance pathways to the HIV integrase inhibitor raltegravir have previously been investigated in T cells. Primary raltegravir resistance mutations, most often at integrase amino acid position 148 or 155, afford some resistance to the drug. The acquisition of pathway-specific secondary mutations then provides higher-level resistance to viruses infecting T cells. We show here that during macrophage infection, the presence of a single primary raltegravir resistance mutation (Q148H, Q148R, N155H, or N155S) is sufficient to provide resistance to raltegravir comparable to that seen in viruses expressing both primary and secondary mutations in costimulated CD4(+) T cells. These data implicate macrophages as a potential in vivo reservoir that may facilitate the development of resistance to raltegravir. Notably, the newer integrase inhibitor MK-2048 effectively suppressed the infection of all raltegravir-resistant viruses in both T cells and macrophages, indicating that more recently developed integrase inhibitors are capable of inhibiting infection in both major HIV cellular reservoirs, even in patients harboring raltegravir-resistant viruses.

Bone density and hyperlipidemia: the T-lymphocyte connection.

Osteoporosis, which contributes to morbidity and mortality, often coexists with cardiovascular disease, especially atherosclerosis. We have reported recently that in vitro exposure of human T-lymphocytes to oxidized lipids induced expression of a key osteoclastogenic cytokine, receptor activator of NF-κB ligand (RANKL). Our previous studies have shown that mice fed an atherogenic high-fat diet developed osteopenia and that bone marrow preosteoclasts from these hyperlipidemic mice have increased osteoclastic potential. To investigate the role of T-lymphocytes in the diet-induced bone loss, C57BL/6 mice were fed either chow or a high-fat diet, and bone parameters and T-lymphocyte activation were assessed at 6 and 11 months. Consistent with our previous findings, peripheral quantitative computed tomographic (pQCT) analysis showed that mice in the high-fat group had lower bone mineral content than mice in the chow group. Furthermore, histomorphometric analysis showed decreased structural parameters in the high-fat group. Coculture studies showed that bone marrow cells isolated from the high-fat group, which contained increased levels of activated memory T-lymphocytes compared with bone marrow cells from the chow mice, supported osteoclastic differentiation of RAW 264.7 cells. Additionally, RANKL expression was upregulated significantly in the T-lymphocytes isolated from the bone marrow of the high-fat group. Splenic T-lymphocytes isolated from the high-fat group also had increased expression of transcripts for the receptor for oxidized lipids (LOX-1) as well as for inflammatory and osteoclastogenic cytokines, including RANKL, interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), IL-1ß, and interferon γ (IFN-γ). Together these findings suggest that T-lymphocytes play a key role in the osteoclastogenesis induced by a high-fat diet and may contribute to the bone loss associated with diet-induced osteopenia.

Expression and Function of the Chemokine, CXCL13, and Its Receptor, CXCR5, in Aids-Associated Non-Hodgkin's Lymphoma.

Background. The homeostatic chemokine, CXCL13 (BLC, BCA-1), helps direct the recirculation of mature, resting B cells, which express its receptor, CXCR5. CXCL13/CXCR5 are expressed, and may play a role, in some non-AIDS-associated B cell tumors. Objective. To determine if CXCL13/CXCR5 are associated with AIDS-related non-Hodgkin's lymphoma (AIDS-NHL). Methods. Serum CXCL13 levels were measured by ELISA in 46 subjects who developed AIDS-NHL in the Multicenter AIDS Cohort Study and in controls. The expression or function of CXCL13 and CXCR5 was examined on primary AIDS-NHL specimens or AIDS-NHL cell lines. Results. Serum CXCL13 levels were significantly elevated in the AIDS-NHL group compared to controls. All primary AIDS-NHL specimens showed CXCR5 expression and most also showed CXCL13 expression. AIDS-NHL cell lines expressed CXCR5 and showed chemotaxis towards CXCL13. Conclusions. CXCL13/CXCR5 are expressed in AIDS-NHL and could potentially be involved in its biology. CXCL13 may have potential as a biomarker for AIDS-NHL.

Oxidized lipids enhance RANKL production by T lymphocytes: implications for lipid-induced bone loss.

Osteoporosis is a systemic disease that is associated with increased morbidity, mortality and health care costs. Whereas osteoclasts and osteoblasts are the main regulators of bone homeostasis, recent studies underscore a key role for the immune system, particularly via activation-induced T lymphocyte production of receptor activator of NFkappaB ligand (RANKL). Well-documented as a mediator of T lymphocyte/dendritic cell interactions, RANKL also stimulates the maturation and activation of bone-resorbing osteoclasts. Given that lipid oxidation products mediate inflammatory and metabolic disorders such as osteoporosis and atherosclerosis, and since oxidized lipids affect several T lymphocyte functions, we hypothesized that RANKL production might also be subject to modulation by oxidized lipids. Here, we show that short term exposure of both unstimulated and activated human T lymphocytes to minimally oxidized low density lipoprotein (LDL), but not native LDL, significantly enhances RANKL production and promotes expression of the lectin-like oxidized LDL receptor-1 (LOX-1). The effect, which is also observed with 8-iso-Prostaglandin E2, an inflammatory isoprostane produced by lipid peroxidation, is mediated via the NFkappaB pathway, and involves increased RANKL mRNA expression. The link between oxidized lipids and T lymphocytes is further reinforced by analysis of hyperlipidemic mice, in which bone loss is associated with increased RANKL mRNA in T lymphocytes and elevated RANKL serum levels. Our results suggest a novel pathway by which T lymphocytes contribute to bone changes, namely, via oxidized lipid enhancement of RANKL production. These findings may help elucidate clinical associations between cardiovascular disease and decreased bone mass, and may also lead to new immune-based approaches to osteoporosis.

Predicting adherence to treatment for methamphetamine dependence from neuropsychological and drug use variables.

Although some individuals who abuse methamphetamine have considerable cognitive deficits, no prior studies have examined whether neurocognitive functioning is associated with outcome of treatment for methamphetamine dependence. In an outpatient clinical trial of bupropion combined with cognitive behavioral therapy and contingency management (Shoptaw, S., Heinzerling, K.G., Rotheram-Fuller, E., Steward, T., Wang, J., Swanson, A.N., De La Garza, R., Newton, T., Ling, W., 2008. Randomized, placebo-controlled trial of bupropion for the treatment of methamphetamine dependence. Drug Alcohol Depend 96, 222-232.), 60 methamphetamine-dependent adults completed three tests of reaction time and working memory at baseline. Other variables that were collected at baseline included measures of drug use, mood/psychiatric functioning, employment, social context, legal status, and medical status. We evaluated the relative predictive value of all baseline measures for treatment outcome using Classification and Regression Trees (CART; Breiman, L., Friedman, J.H., Olshen, R.A., Stone, C.J., 1984. Classification and Regression Trees. Wadsworth, Belmont, CA.), a nonparametric statistical technique that produces easily interpretable decision rules for classifying subjects that are particularly useful in clinical settings. Outcome measures were whether or not a participant completed the trial and whether or not most urine tests showed abstinence from methamphetamine abuse. Urine-verified methamphetamine abuse at the beginning of the study was the strongest predictor of treatment outcome; two psychosocial measures (e.g., nicotine dependence and Global Assessment of Functioning) also offered some predictive value. A few reaction time and working memory variables were related to treatment outcome, but these cognitive measures did not significantly aid prediction after adjusting for methamphetamine usage at the beginning of the study. On the basis of these findings, we recommend that research groups seeking to identify new predictors of treatment outcome compare the predictors to methamphetamine usage variables to assure that unique predictive power is attained.

Primary human immunodeficiency virus type 1 (HIV-1) infection during HIV-1 Gag vaccination.

Vaccination for human immunodeficiency virus type 1 (HIV-1) remains an elusive goal. Whether an unsuccessful vaccine might not only fail to provoke detectable immune responses but also could actually interfere with subsequent natural immunity upon HIV-1 infection is unknown. We performed detailed assessment of an HIV-1 gag DNA vaccine recipient (subject 00015) who was previously uninfected but sustained HIV-1 infection before completing a vaccination trial and another contemporaneously acutely infected individual (subject 00016) with the same strain of HIV-1. Subject 00015 received the vaccine at weeks 0, 4, and 8 and was found to have been acutely HIV-1 infected around the time of the third vaccination. Subject 00016 was a previously HIV-1-seronegative sexual contact who had symptoms of acute HIV-1 infection approximately 2 weeks earlier than subject 00015 and demonstrated subsequent seroconversion. Both individuals reached an unusually low level of chronic viremia (<1,000 copies/ml) without treatment. Subject 00015 had no detectable HIV-1-specific cytotoxic T-lymphocyte (CTL) responses until a borderline response was noted at the time of the third vaccination. The magnitude and breadth of Gag-specific CTL responses in subject 00015 were similar to those of subject 00016 during early chronic infection. Viral sequences from gag, pol, and nef confirmed the common source of HIV-1 between these individuals. The diversity and divergence of sequences in subjects 00015 and 00016 were similar, indicating similar immune pressure on these proteins (including Gag). As a whole, the data suggested that while the gag DNA vaccine did not prime detectable early CTL responses in subject 00015, vaccination did not appreciably impair his ability to contain viremia at levels similar to those in subject 00016.

Targeting c-Mpl for revival of human immunodeficiency virus type 1-induced hematopoietic inhibition when CD34+ progenitor cells are re-engrafted into a fresh stromal microenvironment in vivo.

The inhibition of multilineage hematopoiesis which occurs in the severe combined immunodeficiency mouse with transplanted human fetal thymus and liver tissues (SCID-hu Thy/Liv) due to human immunodeficiency virus type 1 (HIV-1) infection is also accompanied by a severe loss of c-Mpl expression on these progenitor cells. Inhibition of colony-forming activity (CFA) of the CD34(+) progenitor cells is partially revived to about 40% of mock-infected Thy/Liv implants, following reconstitution of the CD34(+) cells that were exposed to HIV-1 infection, in a new Thy/Liv stromal microenvironment of irradiated secondary SCID-hu recipients at 3 weeks post-re-engraftment. In addition, in these reconstituted animals, the proportion of c-Mpl(+) CD34(+) cells relative to c-Mpl(-) CD34(+) cells increased by about 25%, to 35% of mock-infected implants, suggesting a reacquirement of c-Mpl phenotype by the c-Mpl(-) CD34(+) cells. These results suggest a correlation between c-Mpl expression and multilineage CFA of the human CD34(+) progenitor cells that have experienced the effects of HIV-1 infection. Treatment of the secondary-recipient animals with the c-Mpl ligand, thrombopoietin (Tpo), further increased c-Mpl expression and CFA of re-engrafted CD34(+) cells previously exposed to virus in the primary implants to about 50 to 70% over that of those re-engrafted CD34(+) cells derived from implants of untreated animals. Blocking of c-Mpl with anti-c-Mpl monoclonal antibody in vivo by injecting the SCID-hu animals resulted in the reduction or loss of CFA. Thus, inhibition, absence, or loss of c-Mpl expression as in the c-Mpl(-) CD34(+) subset of cells is the likely cause of CFA inhibition. Further, CFA of the CD34(+) cells segregates with their c-Mpl expression. Therefore, c-Mpl may play a role in hematopoietic inhibition during HIV-1 infection, and control of its expression levels may aid in hematopoietic recovery and thereby reduce the incidence of cytopenias occurring in infected individuals.

CD4 on CD8(+) T cells directly enhances effector function and is a target for HIV infection.

Costimulation of purified CD8(+) T lymphocytes induces de novo expression of CD4, suggesting a previously unrecognized function for this molecule in the immune response. Here, we report that the CD4 molecule plays a direct role in CD8(+) T cell function by modulating expression of IFN-gamma and Fas ligand, two important CD8(+) T cell effector molecules. CD4 expression also allows infection of CD8 cells by HIV, which results in down-regulation of the CD4 molecule and impairs the induction of IFN-gamma, Fas ligand, and the cytotoxic responses of activated CD8(+) T cells. Thus, the CD4 molecule plays a direct role in CD8 T cell function, and infection of these cells by HIV provides an additional reservoir for the virus and also may contribute to the immunodeficiency seen in HIV disease.

Identification of T cell-signaling pathways that stimulate latent HIV in primary cells.

Eradication of HIV infection depends on the elimination of a small, but stable population of latently infected T cells. After the discontinuation of therapy, activation of latent virus can rekindle infection. To purge this reservoir, it is necessary to define cellular signaling pathways that lead to activation of latent HIV. We used the SCID-hu (Thy/Liv) mouse model of HIV latency to analyze a broad array of T cell-signaling pathways and show in primary, quiescent cells that viral induction depends on the activation of two primary intracellular signaling pathways, protein kinase C or nuclear factor of activated T cells (NF-AT). In contrast, inhibition or activation of other important T cell stimulatory pathways (such as mitogen-activated protein kinase, calcium flux, or histone deacetylation) do not significantly induce virus expression. We found that the activation of NF-kappaB is critical to viral reactivation; however, all pathways that stimulate NF-kappaBdonot reactivate latent virus. Our studies further show that inhibition of NF-kappaB does not prevent activation of HIV by NF-AT, indicating that these pathways can function independently to activate the HIV LTR. Thus, we define several molecular pathways that trigger HIV reactivation from latency and provide evidence that latent HIV infection is maintained by the functional lack of particular transcription factors in quiescent cells.

Hierarchical phylogenetic models for analyzing multipartite sequence data.

Debate exists over how to incorporate information from multipartite sequence data in phylogenetic analyses. Strict combined-data approaches argue for concatenation of all partitions and estimation of one evolutionary history, maximizing the explanatory power of the data. Consensus/independence approaches endorse a two-step procedure where partitions are analyzed independently and then a consensus is determined from the multiple results. Mixtures across the model space of a strict combined-data approach and a priori independent parameters are popular methods to integrate these methods. We propose an alternative middle ground by constructing a Bayesian hierarchical phylogenetic model. Our hierarchical framework enables researchers to pool information across data partitions to improve estimate precision in individual partitions while permitting estimation and testing of tendencies in across-partition quantities. Such across-partition quantities include the distribution from which individual topologies relating the sequences within a partition are drawn. We propose standard hierarchical priors on continuous evolutionary parameters across partitions, while the structure on topologies varies depending on the research problem. We illustrate our model with three examples. We first explore the evolutionary history of the guinea pig (Cavia porcellus) using alignments of 13 mitochondrial genes. The hierarchical model returns substantially more precise continuous parameter estimates than an independent parameter approach without losing the salient features of the data. Second, we analyze the frequency of horizontal gene transfer using 50 prokaryotic genes. We assume an unknown species-level topology and allow individual gene topologies to differ from this with a small estimable probability. Simultaneously inferring the species and individual gene topologies returns a transfer frequency of 17%. We also examine HIV sequences longitudinally sampled from HIV+ patients. We ask whether posttreatment development of CCR5 coreceptor virus represents concerted evolution from middisease CXCR4 virus or reemergence of initial infecting CCR5 virus. The hierarchical model pools partitions from multiple unrelated patients by assuming that the topology for each patient is drawn from a multinomial distribution with unknown probabilities. Preliminary results suggest evolution and not reemergence.

Molecular characterization, reactivation, and depletion of latent HIV.

Antiretroviral therapy is unable to eliminate HIV infection in a small, long-lived population of latently infected T cells, providing a source for renewed viral replication following cessation of therapy. Analysis of individual latently infected cells generated in the SCID-hu (Thy/Liv) mouse demonstrated no functional viral RNA produced in the latent state. Following reactivation viral expression was dramatically increased, rendering the infected cells susceptible to an anti-HIV immunotoxin. Treatment with the immunotoxin in conjunction with agents that activate virus expression without inducing cell division (IL-7 or the non-tumor-promoting phorbol ester prostratin) depleted the bulk of the latent reservoir and left uninfected cells able to respond to subsequent costimulation. We demonstrate that activation of latent virus is required for targeting by antiviral agents and provide the basis for future therapeutic strategies to eradicate the latent reservoir.

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.