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Human beta-defensins (hBDs) are broad-spectrum antimicrobial peptides, secreted by epithelial cells of the skin and mucosae, and astrocytes, which we and others have shown to inhibit HIV-1 in primary CD4+T cells. Although loss of CD4+T cells contributes to mucosal immune dysfunction, macrophages are a major source of persistence and spread of HIV and also contribute to the development of various HIV-associated complications. We hypothesized that, besides T cells, hBDs could protect macrophages from HIV. Our data in primary human monocyte-derived macrophages (MDM)in vitroshow that hBD2 and hBD3 inhibit HIV replication in a dose-dependent manner. We determined that hBD2 neither alters surface expression of HIV receptors nor induces expression of anti-HIV cytokines or beta-chemokines in MDM. Studies using a G-protein signaling antagonist in a single-cycle reporter virus system showed that hBD2 suppresses HIV at an early post-entry stageviaG-protein coupled receptor (GPCR)-mediated signaling. We find that MDM express the shared chemokine-hBD receptors CCR2 and CCR6, albeit at variable levels among donors. However, cell surface expression analyses show that neither of these receptors is necessary for hBD2-mediated HIV inhibition, suggesting that hBD2 can signalviaadditional receptor(s). Our data also illustrate that hBD2 treatment was associated with increased expression of APOBEC3A and 3G antiretroviral restriction factors in MDM. These findings suggest that hBD2 inhibits HIV in MDMviamore than one CCR thus adding to the potential of using β-defensins in preventive and therapeutic approaches.

Highlights • The G-quadruplex-forming aptamer AS1411 strongly inhibits HIV-1 infection. • AS1411 is non-toxic to the host cell at antiviral concentrations. • AS1411 blocks viral attachment to the host cell. • AS1411 binds cell-surface-expressed nucleolin, a putative HIV-1 co-receptor., AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HIV-1) anchorage on target cells. Here we assessed the anti-HIV-1 properties and underlying mechanism of action of AS1411. The antiviral activity of AS1411 was determined towards different HIV-1 strains, host cells and at various times post-infection. Acutely, persistently and latently infected cells were tested, including HIV-1-infected peripheral blood mononuclear cells from a healthy donor. Mechanistic studies to exclude modes of action other than virus binding via NCL were performed. AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell. The aptamer displayed antiviral activity in the absence of cytotoxicity at the tested doses, therefore displaying a wide therapeutic window and favourable selectivity indexes. These findings, besides validating cell-surface-expressed NCL as an antiviral target, open the way for the possible use of AS1411 as a new potent and promisingly safe anti-HIV-1 agent.

Chemokine receptor type 6 (CCR6)⁺CD4⁺ T cells are preferentially infected and depleted during HIV disease progression, but are preserved in non-progressors. CCR6 is expressed on a heterogeneous population of memory CD4⁺ T cells that are critical to mucosal immunity. Preferential infection of these cells is associated, in part, with high surface expression of CCR5, CXCR4, and α4β7. In addition, CCR6⁺CD4⁺ T cells harbor elevated levels of integrated viral DNA and high levels of proliferation markers. We have previously shown that the CCR6 ligands MIP-3α and human beta defensins inhibit HIV replication. The inhibition required CCR6 and the induction of APOBEC3G. Here, we further characterize the induction of apolipoprotein B mRNA editing enzyme (APOBEC3G) by human beta defensin 2. Human beta defensin 2 rapidly induces transcriptional induction of APOBEC3G that involves extracellular signal-regulated kinases 1/2 (ERK1/2) activation and the transcription factors NFATc2, NFATc1, and IRF4. We demonstrate that human beta defensin 2 selectively protects primary CCR6⁺CD4⁺ T cells infected with HIV-1. The selective protection of CCR6⁺CD4⁺ T cell subsets may be critical in maintaining mucosal immune function and preventing disease progression.

Increased levels of the proinflammatory cytokine IL-8 are detected in the sputum of patients with chronic obstructive pulmonary disease (COPD) and during the pathological pulmonary manifestations of HIV infection : To explore a potential interrelationship between smoking, highly active antiretroviral therapy (HAART) and HIV immune status, we collected sputum samples, along with complete pulmonary function tests from groups of HIV-infected women smokers who were either on or off HAART. Analysis of the patient's sputum for cell count along with quantitative measures of IL-8 was performed and correlated with concurrent assessment of pulmonary function test (PFT). We found that HIV-positive smokers had decreased measurements on PFT of the diffusing capacity of the lung for carbon monoxide (D(LCO)) compared to standard reference values that did not differ with HAART usage. HAART, when controlled for CD4, showed a suppressive effect on the levels of pro inflammatory cytokine IL-8 in sputum. We conclude that in the era of HAART, HIV along with concurrent tobacco smoking is associated with declines in PFT in HIV-infected women. The use of HAART in patients appears to mitigate the increases in IL-8 levels in relation to immune status based on CD4 count.

Lymphangiogenesis and concurrent angiogenesis are essential in supporting proliferation and survival of AIDS-related lymphomas, which are often metastatic. In vitro studies suggest a candidate angiogienic and lymphangiogenic factor encoded by HIV: the matrix protein p17. p17 accumulates in lymph nodes of patients even when they are undergoing highly active antiretroviral therapy. p17 has been found to affect immune cells, and recent data showed that a variant p17, called S75X, induces cell growth by triggering MAPK/ERK and PI3K/AKT pathways. We tested the in vivo angiogenic activity of p17 by injecting it in Matrigel plugs in nude mice. Plugs were retrieved 7 days after injection, and assessed macroscopically, and by light and confocal microscopy. Our data revealed that both reference and S75X variant p17 promote angiogenesis and lymphangiogenesis in vivo. Our results suggest that the induction of angiogenesis and lymphangiogenesis by HIV-1 p17 may generate a favorable microenvironment that could trigger tumor growth and maintenance. Moreover, the presence of adipocytes infiltration observed at the histological level suggests a possible interplay between angiogenesis, lymphangiogenesis and adipogenesis. These findings offer new opportunities for the development of treatment strategies to combat HIV-related cancers.

Accelerated bone loss leading to osteopenia, osteoporosis, and bone fracture is a major health problem that is increasingly common in human immunodeficiency virus (HIV) infected patients. The underlying pathogenesis is unclear but occurs in both treatment naïve and individuals receiving antiretroviral therapies. We developed an HIV-1 transgenic rat that exhibits many key features of HIV disease including HIV-1 induced changes in bone mineral density (BMD). A key determinant in the rate of bone loss is the differentiation of osteoclasts, the cells responsible for bone resorption. We found HIV-1 transgenic osteoclast precursors (OCP) express higher levels of suppressor of cytokine signaling-1 (SOCS-1) and TNF receptor associated factor 6 (TRAF6) and are resistant to interferon-gamma (IFN-γ) mediated suppression of osteoclast differentiation. Our data suggest that dysregulated SOCS-1 expression by HIV-1 transgenic OCP promotes osteoclastogenesis leading to the accelerated bone loss observed in this animal model. We propose that elevated SOCS-1 expression in OCP antagonizes the inhibitory effects of IFN-γ and enhances receptor activator of NF-kB ligand (RANKL) signaling which drives osteoclast differentiation and activation. Understanding the molecular mechanisms of HIV-associated BMD changes has the potential to detect and treat bone metabolism disturbances early and improve the quality of life in patients.

Background Human Immunodeficiency Virus Type I (HIV-1) infection is associated with a high incidence of B-cell lymphomas. The role of HIV in these lymphomas is unclear and currently there are no valid in vivo models for better understanding HIV-related lymphomagenesis. Transgenic (Tg) 26 mice have a 7.4-kb pNL4-3 HIV-1 provirus lacking a 3.1-kb sequence encompassing parts of the gag-pol region. Approximately 15% of these HIV Tg mice spontaneously develop lymphoma with hallmark pre-diagnostic markers including skin lesions, diffuse lymphadenopathy and an increase in pro-inflammatory serum cytokines. Here we describe the phenotypic and molecular characteristics of the B cell leukemia/lymphoma in the Tg mice. Results The transformed B cell population consists of CD19+pre-BCR+CD127+CD43+CD93+ precursor B cells. The tumor cells are clonal and characterized by an increased expression of several cellular oncogenes. Expression of B cell-stimulatory cytokines IL-1β, IL-6, IL-10, IL-12p40, IL-13 and TNFα and HIV proteins p17, gp120 and nef were elevated in the Tg mice with lymphoma. Conclusions Increased expression of HIV proteins and the B-cell stimulatory factors is consistent with the interpretation that one or more of these factors play a role in lymphoma development. The lymphomas share many similarities with those occurring in HIV/AIDS+ patients and may provide a valuable model for understanding AIDS-related lymphomagenesis and elucidating the role played by HIV-1.

Clinical and experimental observations suggest that human herpesvirus-6 (HHV-6), a T-lymphotropic herpesvirus, may act as a cofactor in the acquired immunodeficiency syndrome (AIDS). Moreover, a possible role of HHV-6 in the increased incidence and severity of cervical carcinoma in human immunodeficiency virus (HIV)-infected women was suggested by the recent observation that HHV-6 can infect cervical carcinoma cells, accelerating their tumorigenicity in vivo. Therefore, the ability of four HHV-6 genomic clones derived from HHV-6 to transactivate the long terminal repeat (LTR) of HIV-1 in two cervical carcinoma cell lines and in a T-lymphoid cell line was tested. Two HHV-6 clones, pZVH-14 and pZVB-70, which were previously shown to increase the expression of human papillomavirus (HPV)-transforming genes, were, per se, weak transactivators of the HIV-1 LTR. However, an increased effect occurred when these clones were combined with the HIV-1 transactivator TAT-1. No such effect was seen with two other HHV-6 clones used as controls. Analysis with HIV-1 LTR deletion mutants indicated that this enhancing effect requires the presence of elements contained in both the enhancer region and the TAT activation region (TAR) of HIV-1. This data may have implications for the potential role of HHV-6 in AIDS and AIDS-related cervical carcinoma.

s-defensins are antimicrobial peptides secreted by epithelial cells that can bind to cellular receptors. CCR2 and CCR6 are the two cellular receptors known to bind human s-defensin (hBD) 2 and -3. Both of these receptors are of crucial relevance in HIV infection. CCR6 is expressed, often in concert with CCR5, on cells that are highly susceptible to HIV infection: memory T cells, Th17 cells, α4s7+ cells, and defects in CD4+CCR6+ cells have been associated with faster AIDS progression. CCR2 is expressed on monocytes and macrophages, cells that are reservoirs of HIV infection and that are known to mediate central nervous system damage. Our studies show that hBD2, hBD3, and CCR6 ligand MIP-3α/CCL20 inhibit HIV infection via CCR6 by increasing expression of the antiviral protein APOBEC3G. This increase is due to a transcriptional mechanism mediated by intracellular signaling. hBD2 also inhibits HIV replication in macrophages that express CCR2. Our findings suggest novel therapeutic and preventive approaches that exploit CCR6 and CCR2-mediated intracellular signaling to inhibit HIV infection in highly susceptible cells.

The Th17/interleukin (IL)-17 axis controls inflammation and might be important in the pathogenesis of experimental autoimmune myocarditis (EAM) and other autoimmune diseases. However, the mechanism underlying the increased Th17 cell response in coxsackievirus-induced myocarditis remains unclear. This study aimed to elucidate the regulatory mechanisms affected by blocking IL-17A responses in acute virus-induced myocarditis (AVMC) mice. The results showed that IL-17A and COX-2 proteins were significantly increased in the cardiac tissue of acute myocarditis, as were Th17 cells in the spleen. Using anti-mouse IL-17Ab to block IL-17A on day 7 of the viral myocarditis led to decreased expressions of cardiac tumor-necrosis factor alpha, IL-17A and transforming growth factor beta in AVMC mice compared to isotype control mice. COX-2 and prostaglandin E2 proteins were dramatically elevated, followed by marked reductions in CVB3 replication and myocardial injury. These results hint that the Th17/IL-17 axis is intimately associated with viral replication in acute myocarditis via induction of COX-2 and prostaglandin E2.

Neurotrophins control cell survival. Therefore, we examined whether HIV-1 reduces neurotrophin levels. Serum of HIV-positive individuals exhibited lower concentrations of brain-derived neurotrophic factor (BDNF), but not of other neurotrophins, than HIV-negative individuals. In addition, R5 and X4 strains of HIV-1 decreased BDNF expression in T cells. Our results support the hypothesis that reduced levels of BDNF may be a risk factor for T-cell apoptosis and for neurological complications associated with HIV-1 infection.

Relative resistance to HIV infection has been associated with genetic polymorphisms. Both single nucleotide polymorphisms and copy number variations (CNVs) have been documented for defensins, which are natural inhibitors of HIV infection. We tested the hypothesis that these CNVs may be related to susceptibility to HIV infection and vertical transmission by evaluating the CNVs of 13 defensin genes in Brazilian HIV-infected children. Study groups included seronegative controls, HIV-infected subjects, and subjects who did not contract HIV despite exposure at the time of birth from HIV-infected mothers. We observed that the copy number of one of these genes, DEFB104, was significantly lower in HIV-positive subjects than in HIV-exposed uninfected children, suggesting DEFB104 as a candidate HIV-protective gene.

The oral cavity is a primary target for opportunistic infections, particularly oral candidiasis caused by the opportunistic pathogen Candida albicans. HIV+ individuals constitute a population highly susceptible to oral candidiasis possibly due to a change in the environment of the oral cavity as the result of salivary gland dysfunction. Histatins are a family of salivary antimicrobial peptides which under normal circumstances have a protective function on the oral mucosa. This study aimed to compare salivary histatin concentrations and oral fungal colonisation in an HIV+ and HIV- control populations. Oral samples for fungal cultures and parotid saliva were collected from all subjects. Fungal identification was determined using standard mycological procedures. In order to determine salivary histatin levels a semi-quantitative ELISA was designed using a specific polyclonal antibody and extensive statistical analysis was performed. Forty-seven percent of HIV+ and 17% of control subjects had positive fungal cultures. Mean histatin levels were 7.32 microg ml(-1) for the HIV+ group and 9.17 microg ml(-1) for control group (P = 0.003). The data from this study demonstrate that the level of fungal colonisation is significantly higher in HIV+ individuals whereas histatin-5 concentrations are significantly lower, likely contributing to the enhanced predisposition of this population to oral candidiasis.

HIV infection increases the risk of many types of cancer, including lymphoma. Combination antiretroviral therapy (cART) has reduced, but not eliminated, the risk of HIV-associated lymphoma. There has been a substantial shift in the subtypes of lymphoma observed in HIV-infected patients treated with cART. In this review, we will first outline these changes based on epidemiological studies and describe the impact of cART on lymphoma risk and mortality. Then, we will discuss some immunological factors that may contribute to the increased risk of lymphoma persisting after the administration of cART, including immunological non-response to therapy, chronic B-cell activation and dysfunction, T follicular helper cells, natural killer cells and altered lymphopoiesis. A better understanding of the pathophysiologic mechanisms of HIV-associated lymphoma under effective cART will inform future treatment strategies.

of Human Virology Meeting abstracts. A s ingle PDF containing all abstracts in this Supplement is available here http://ww w.biomedcentral. com/content/pdf/ 1742-4690-3-S1-i nfo.pdf

Results The CD4+ T cells produced IFN-y and MIP-1a in response to gp120. Also detected were RANTES and MIP-1a, but not MDC (CCL22). The b-chemokines displayed potent inhibitory activities against R5-tropic HIV-1. After the cells were exposed to BX08 (an R5 subtype B HIV-1 primary isolate), the capacity of these cells to produce IFN-y and MIP-1a in response to gp120 was initially comparable with that of the unexposed control. But after 3–4 days of infection, the numbers of cells capable of producing IFNy and MIP-1a decreased significantly, while the percentage of infected cells reached >80%. Conclusion HIV-specific CD4+ T cells are susceptible to HIV. HIV reduces the capacity of the cells to produce IFN-y and bchemokines. On-going efforts to enhance HIV-specific CD4+ T cell resistance against the virus will be presented. from 2006 International Meeting of The Institute of Human Virology Baltimore, USA. 17–21 November, 2006

Background We have shown that the Type C style of coping with stress (diminished ability to recognize and express stress/ distress/emotions) is related to HIV progression. We recently found in a study of 50 HIV+ patients in Baltimore, that stronger Type C coping is associated with decreased production of beta-chemokines that bind to the HIV coreceptor CCR5. Under the aegis of NIH, we have initiated a longitudinal study with a final N = 200 to evaluate the core hypothesis that lower production of the beta-chemokines MIP-1 α/β mediates the relationship between Type C coping and HIV progression.

Chemokines are key players in the elicitation of immune response, by selectively attracting subpopulations of immune cells to the site of antigen presentation. Therefore, they are natural candidates for modulating immune responses to antigens qualitatively and quantitatively. We have selected chemokines associated with different arms of the immune response, i.e. RANTES/CCL5, B-lymphocyte chemoattractant/CXCL13, and monocyte chemoattractant protein-1/CCL2, and co-injected DNA expression constructs encoding these chemokines with constructs encoding two HIV antigens, gp120 and gp160, in mice. We subsequently measured markers of both cellular and humoral immune responses, and found that these chemokines qualitatively influenced the outcome of immune responses to both antigens, essentially according to their predicted association to Th profiles. These results are relevant towards the engineering of novel vaccine and immune-based therapies, and point to chemokines as candidate adjuvant and immunomodulatory molecules.

Mapping coreceptor epitopes used by the prototypic R5 and X4 strains, HIV-1BaL and HIV-1IIIB, in comparison with epitopes involved in the activation and signaling induced by the natural ligands, RANTES and SDF-1beta.Receptor hybrids between CCR5 and CXCR4 were constructed.Using single-overlap and extension PCR, increasing portions of CCR5 were replaced with corresponding parts of CXCR4. Viral interaction with these constructs was monitored in infection experiments using stably transfected cell lines, and ligand-induced activation of cells transiently expressing the constructs was measured in terms of calcium fluxes.SDF-1beta required an essentially complete CXCR4, whereas RANTES demanded both the N terminus and the first two extracellular loops of CCR5. HIV-1 infection experiments emphasized the importance of the CCR5 N terminus for infection with HIV-1BaL, whereas HIV-1IIIB was less demanding in its use of CXCR4.This study, for the first time monitoring CCR5 and CXCR4 ligand activation and HIV-1 interaction concomitantly, indicates that ligands and virus use different receptor epitopes which, in turn, vary between the two receptors. One particular chimera (FC-4b), having its junctional region close to the conserved cysteine in ECL2, functioned as coreceptor for both HIV-1BaL and HIV-1IIIB, but was not activated with RANTES or SDF-1beta. The results provide a basis for tailoring drugs that block viral entry through the two major coreceptors without interfering with their physiological function.

The C-C chemokines RANTES, MIP-1alpha, and MIP-1beta have been characterized as constituents of an HIV- and SIV-suppressive factor released by CD8+ cells. Furthermore, it has been demonstrated that chemokine receptors cooperate in HIV entry. However, these proteins are also known to have an effect on multiple intracellular signaling cascades that may affect the process of transcription. In the present study we demonstrate that treatment of CD4+ T cells with these chemokines or with cell supernatants from HTLV-I-immortalized CD8+ T cells results in significant reduction in the abundance of HIV-1-specific RNA as analyzed by Northern blot hybridization. To examine the possibility that such suppressive factors may inhibit HIV RNA transcription, we studied the effect of RANTES, the most effective HIV-suppressive chemokine, on basal and Tat-induced HIV-directed LTR expression of a reporter gene. Neither recombinant RANTES nor conditioned medium from CD8+ cells significantly altered HIV-1 LTR-directed chloramphenicol acetyltransferase expression in either transiently or stably transfected CD4+ T cell lines, either in the presence or in the absence of Tat. These results suggest that C-C chemokines do not inhibit viral RNA transcription.

Viral transcripts, particularly those of the regulatory genes (e.g., rev) in lymphocytic cells chronically infected with human immunodeficiency virus type 2, consist of two types, differing in the structure of the leader sequence derived from the 5' long terminal repeat (LTR). Some transcripts undergo a specific splicing event within the 5' LTR, removing an intron consisting of a part of the R region whereas others do not. Because this spliced-out R region is a part of the trans-activation response element (TAR), it could influence trans-activator (Tat)-mediated trans-activation of viral gene expression. Moreover, this part of the R region is predicted to contain a stable secondary structure that could affect the efficiency of translation of the transcripts without this splicing. Thus, the 5' LTR splicing could have important consequences for virus replication, latency, and pathogenicity.

Recently, we and others have characterized the HIV-suppressive activity of human β-defensins (hBD) 1–3. Our mechanistic studies indicate that hBD2 inhibits HIV through two distinct mechanisms. The first mechanism results in lower HIV infectivity. The second mechanism is intracellular, since it is observed even delaying treatment up to 2 hours after infection, which is not consistent with a mode of inhibition based on entry-fusion events. We hypothesized that this second antiviral mechanism is due to changes in the intracellular environment, possibly mediated by a cellular receptor. Treatment of cells in infectivity assays with an antibody specific for CCR6, a chemokine receptor that also functions as a receptor for hBD2, abrogated HIV inhibition by this defensin. Further, treatment of HIV-1 infectivity assays with MIP-3α, the cognate ligand for CCR6, resulted in inhibition of infection. Finally, single-cycle HIV infection is inhibited in cells that express CCR6 following transfection with an expression construct, but not in their CCR6low counterpart. Experiments performed using HIV pseudotyped with AMLV envelope showed that inhibition by hBD2 of HIV replication is sensitive to treatment with the Gi protein inhibitor pertussis toxin.CCR6 is an important receptor in the context of mucosal immunity, as it is expressed in CD4+CD45+CCR5+ lymphocytes, which are highly susceptible to HIV infection, and on dendritic cells, which are thought to play a crucial role in early events in HIV transmission. Therefore, an HIV suppressive mechanism targeting CCR6 is likely to play an important role in mucosal immunity to HIV.