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Rapid advances in mesenchymal stem progenitor cells (MSPCs) have rendered impetus into the area of cell therapy and regenerative medicine. The main promise of future stem cell therapies is their reliance on autologous stem cells derived from adipose tissue, which also includes treatments of bone fractures and degeneration. The effectiveness of different electric devices utilized to reprogram MSPCs toward osteogenic differentiation has provided varying degrees of effectiveness for clinical use. Adipose tissue-derived MSPCs were flow-cytometrically characterized and further differentiated into osteoblasts by culturing either in growth medium with pro-osteogenic supplements or without supplements with alternating electromagnetic field (EMF) generated by IteraCoil. IteraCoil is a multi-solenoid coil with a specific complex geometry that creates a 3D-EMF with desired parameters without directly applying electrodes to the cells and tissues. The flow-cytometric analysis of highly enriched (≥95%) adipose-derived MSPCs (CD34

Background: Inhalable medication devices on the market deliver aerosolized drugs in a turbulent flow, which creates significant obstacles for reaching the lower lungs. The complexity of the interaction of external turbulent flow from an inhalation device with complex anatomy of the upper airways, including the oropharyngeal cavity, makes the fidelity of aerosolized medication delivery to the lungs extremely low. Unpredictable outcomes, waste, and side effects result from unintended upper airway drug deposition. Methods: Here we compared the efficiency of aerosolized medication (fluticasone) delivery via a novel Flow Modification Device (ModiFlow) to that of a Standard Spacer (SS) device. The ability of ModiFlow to minimize the turbulence in the flow was assessed preliminarily by measuring the length of the Laminar Outflow using video recording. Oral mucosal 3D tissue culture (SkinAxis) was used as a target for delivery, and was placed either “well within” the range of the length of the oro-pharyngeal cavity (5cm) or “well outside” of it (20cm) from exit points of each device. The efficiency of fluticasone delivery to the surface of tissue cultures was quantified by mass spectrometry. Results: The results of the study demonstrated a statistically significant advantage of ModiFlow over a Standard Spacer in delivering aerosolized fluticasone to target tissue at both distances. The difference in the efficiency of delivery between the two spacers was more pronounced at a longer distance.Conclusions: Lamination of the outflow using internal septi helps improve the delivery of aerosolized medication to target tissue despite an increased inner surface area of the spacer device, which also indicates lesser deposition of the medication on the walls of the test tube. This suggests that the use of ModiFlow will potentially result in the more efficient delivery of aerosolized medications to the lungs with lesser deposition in the oral cavity and fewer side effects.

End-stage liver disease is a common cause of non-AIDS-related mortality in HIV+ patients, despite effective anti-retroviral therapies (ARTs). HIV-1 infection causes gut CD4 depletion and is thought to contribute to increased gut permeability, bacterial translocation, and immune activation. Microbial products drain from the gut into the liver via the portal vein where Kupffer cells (KCs), the resident liver macrophage, clear translocated microbial products. As bacterial translocation is implicated in fibrogenesis in HIV patients through unclear mechanisms, we tested the hypothesis that HIV infection of KCs alters their response to LPS in a TLR4-dependent manner. We showed that HIV-1 productively infected KCs, enhanced cell-surface TLR4 and CD14 expression, and increased IL-6 and TNF-α expression, which was blocked by a small molecule TLR4 inhibitor. Our study demonstrated that HIV infection sensitizes KCs to the proinflammatory effects of LPS in a TLR4-dependent manner. These findings suggest that HIV-1-infected KCs and their dysregulated innate immune response to LPS may play a role in hepatic inflammation and fibrosis and represent a novel target for therapy.

IL-1β is an important mediator of innate inflammatory responses and has been shown to contribute to liver injury in a number of etiologies. HIV patients have increased necroinflammation and more rapid fibrosis progression in chronic liver injury compared to non-HIV-infected patients. As the resident liver macrophage is critical to the IL-1β response to microbial translocation in chronic liver disease, we aim to examine the impact of HIV-1 and LPS stimulation on the IL-1β response of the resident hepatic macrophages. We isolated primary human liver macrophages from liver resection specimens, treated them with HIV-1BaL and/or LPS ex vivo, examined the IL-1β response, and then studied underlying mechanisms. Furthermore, we examined IL-1β expression in liver tissues derived from HIV-1 patients compared to those with no underlying liver disease. HIV-1 up-regulated TLR4 and CD14 expression on isolated primary CD68+ human liver macrophages and contributed to the IL-1β response to LPS stimulation as evidenced by TLR4 blocking. Nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) was shown to be involved in the IL-1β response of liver macrophages to HIV-1 infection and NLRP3 blocking experiments in primary CD68+ liver macrophages confirmed the contribution of the NLRP3-caspase 1 inflammatory signaling pathway in the IL-1β response. High in situ IL-1β expression was found in CD68+ cells in human liver tissues from HIV-1-infected patients, suggesting a critical role of IL-1β responses in patients infected by HIV. HIV infection sensitizes the IL-1β response of liver macrophages to LPS through up-regulation of CD14 and TLR4 expression and downstream activation of the NLRP3-caspase 1 pathway. These findings have implications for enhanced immune activation in HIV+ patients and mechanisms for rapid fibrosis progression in patients with chronic liver injury. Resident liver macrophages infected by HIV-1 demonstrate an increased IL-1β response to LPS that is mediated by TLR4 and downstream activation of the caspase1-NLRP3 pathway.

During assembly and budding of retroviruses, host cell proteins are incorporated into viral particles. Identification of virion-associated proteins may help pinpoint key cellular components required for virus production and function. The cellular protein annexin 2 (Anx2) is incorporated into HIV-1 particles, and knockdown of Anx2 has been reported to cause defects in Gag processing and infectivity of HIV-1 particles in macrophages. Here, we tested whether Anx2 was required for HIV-1 production in other cell types capable of producing HIV-1 virions. Endogenous Anx2 levels were knocked down by ∼98% using lentivirus encoding short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting Anx2. Under these conditions, there was no reduction in HIV-1 virus-like particle (VLP) production in either COS-1, 293T, or Jurkat T cells or primary human monocyte-derived macrophages (MDMs). Murine embryonic fibroblasts derived from Anx2 −/− mice produced the same levels of VLPs as matched cells from wild-type mice. The calcium-mediated spike in VLP production still occurred in Anx2-depleted COS-1 cells, and there was no apparent alteration in the intracellular Gag localization. Overexpression of Anx2 in trans had no effect on Gag processing or VLP production. Neither Anx2 depletion nor Anx2 overexpression altered the infectivity of HIV-1 particles produced by COS-1 or 293T cells. However, supernatants containing virus from Anx2 siRNA-treated primary human MDMs exhibited decreased infectivity. These data indicate that Anx2 is not required for HIV-1 assembly or Gag processing but rather plays a cell type-dependent role in regulating production of infectious HIV-1 by macrophages.

Induction of type I interferons (IFN) is a central feature of innate immune responses to microbial pathogens and is mediated via Toll-like receptor (TLR)-dependent and -independent pathways. Prothymosin-α (ProTα), a small acidic protein produced and released by CD8 + T cells, inhibits HIV-1, although the mechanism for its antiviral activity was not known. We demonstrate that exogenous ProTα acts as a ligand for TLR4 and stimulates type I IFN production to potently suppress HIV-1 after entry into cells. These activities are induced by native and recombinant ProTα, retained by an acidic peptide derived from ProTα, and lost in the absence of TLR4. Furthermore, we demonstrate that ProTα accounts for some of the soluble postintegration HIV-1 inhibitory activity long ascribed to CD8 + cells. Thus, a protein produced by CD8 + T cells of the adaptive immune system can exert potent viral suppressive activity through an innate immune response. Understanding the mechanism of IFN induction by ProTα may provide therapeutic leads for IFN-sensitive viruses.

Over 40 million people are infected with human immunodeficiency virus (HIV) worldwide. Because of their shared routes of transmission, infection with hepatitis C virus (HCV) is common among patients with HIV infection, and approximately 30% of HIV-infected persons in the United States are also infected with HCV.1 The introduction of highly active antiretroviral therapy (HAART) in 1996 changed the natural history of HIV infection and resulted in a dramatic decline in most opportunistic infections. Given their increased survival, HCV-related liver disease has emerged as a major cause of morbidity and mortality among patients infected with HIV. Although the effects of HCV on HIV disease progression are less clear, several studies have demonstrated that HIV infection adversely impacts every stage in the natural history of HCV infection. Infection with HIV enhances HCV transmission, decreases the rates of spontaneous HCV clearance leading to higher rates of chronic hepatitis C infection,2 and is associated with higher HCV RNA loads.3 Once chronic infection is established, HIV/HCV-coinfected patients have higher necro-inflammatory activity on liver biopsies, faster rates of fibrosis progression, and earlier development of end-stage liver disease.4,5 Despite the significant adverse clinical consequences of HIV/HCV coinfection, the underlying molecular mechanisms by which HIV infection impacts HCV disease progression have not been clearly defined. While the host T cell responses appear to be crucial in promoting HCV clearance in acute infection and controlling viral replication and recurrence,6 ultimately these responses fail and the majority of patients become chronically infected. Epidemiological studies support a correlation between lower CD4 cell counts, HCV persistence, and progression of liver disease,4 suggesting that the immunosuppression associated with HIV infection partially contributes to the pathogenesis of chronic liver disease. Whereas higher HCV RNA loads in coinfected patients do not correlate with progression of liver disease, HIV RNA levels correlate with fibrosis progression rates in a dose-dependent fashion.7 Moreover, effective suppression of HIV replication by HAART has been associated with better outcomes.7 Taken together, these findings suggest a direct role for HIV on liver disease progression in coinfected patients. The hepatic stellate cell (HSC) is a central mediator in liver fibrosis. In its quiescent state, it is a vitamin A–rich cell that produces type IV collagen, the collagen characteristic of a normal basement membrane. With injury, such as in chronic hepatitis C, HSCs undergo a process of activation, rendering them susceptible to a variety of stimuli that yield a highly proliferative, contractile, and fibrogenic cell producing predominantly type I collagen, the collagen characteristic of the cirrhotic liver. Activated HSCs express both HIV chemokine coreceptors, chemokine (C-C motif) receptor 5 (CCR5)8 and cysteine-X-cysteine receptor 4 (CXCR4),9 and recent studies suggest effects of HIV envelope protein on HSC responses.10,11 To date there has been no evidence that activated HSCs are a cellular target for HIV infection to account for the accelerated fibrosis observed in coinfected patients. We report that activated HSCs are infectable by HIV, support viral gene expression, and are capable of transmitting infectious virus to susceptible lymphocytes through cell–cell contact. Furthermore, HIV infection of HSCs induces collagen I expression and secretion of the proinflammatory cytokine, monocyte chemoattractant protein 1 (MCP-1). These findings support direct profibrogenic and proinflammatory effects of HIV on stellate cells.

A persulfated molecular umbrella derived from one spermine, four lysine, and eight deoxycholic acid molecules was found to exhibit ionophoric activity, as shown by pH discharge and Na(+) and Cl(-) transport experiments. In sharp contrast, a moderately more hydrophilic analogue derived from cholic acid showed no such ionophoric activity. Both molecular umbrellas crossed liposomal membranes by passive transport with experimental rates that were similar. These findings show how the interactions between such amphomorphic molecules and phospholipid bilayers are a sensitive function of the umbrella's hydrophilic/lipophilic balance (HLB). They also raise the possibility of exploiting molecular umbrellas in fundamentally new ways.

Human immunodeficiency virus (HIV) infection is associated with accelerated atherosclerosis and vasculopathy, although the mechanisms underlying these findings have not been determined. Hypotheses for these observations include: 1) an increase in the prevalence of established cardiac risk factors observed in HIV-infected individuals who are currently experiencing longer life expectancies; 2) the dyslipidemia reported with certain HIV anti-retroviral therapies; and/or 3) the proinflammatory effects of infiltrating HIV-infected monocytes/macrophages. An unexplored possibility is whether HIV itself can infect vascular smooth muscle cells (SMCs) and, by doing so, whether SMCs can accelerate vascular disease. Our studies demonstrate that human SMCs can be infected with HIV both in vivo and in vitro. The HIV protein p24 was detected by fluorescence confocal microscopy in SMCs from tissue sections of human atherosclerotic plaques obtained from HIV-infected individuals. Human SMCs could also be infected in vitro with HIV by a mechanism dependent on CD4, the chemokine receptors CXCR4 or CCR5, and endocytosis, resulting in a marked increase in SMC secretion of the chemokine CCL2/MCP-1, which has been previously shown to be a critical mediator of atherosclerosis. In addition, SMC proliferation appeared concentric to the vessel lumen, and minimal inflammation was detected, unlike typical atherosclerosis. Our data suggest that direct infection of human arterial SMCs by HIV represents a potential mechanism in a multifactorial paradigm to explain the exacerbated atherosclerosis and vasculopathy reported in individuals infected with HIV.

BACKGROUND Prothymosin α (ProTα) (isoform 2: iso2) is a widely distributed, small acidic protein with intracellular and extracellular-associated functions. Recently, we identified two new ProTα variants with potent anti-HIV activity from CD8+ T cells and cervicovaginal lavage. The first is a splice variant of the ProTα gene known as isoB and the second is the product of ProTα pseudogene 7 (p7). Similarly to iso2, the anti-HIV activity of both variants is mediated by type I IFN. Here we tested whether the immunomodulatory activity of isoB and p7 are also TLR4 dependent and determined their kinetic of release in response to HIV-1 infection. METHODS Type I, type III, TNF-α and IL-6 mRNA inducing activity was determined in macrophages from wild type and TLR4 knockout mice treated with recombinant ProTα variants. Supernatants from mock and HIV infected cells were analyzed by mass spectrometry in positive and negative modes for the presence of ProTα variants. In silico structural and functional analysis of ProTα variants were performed. RESULTS We show that both isoB and p7 upregulate IFN-β, IFN-λ1, IL-6, TNF-α and RANTES mRNAs in primary human macrophages. The potent stimulation of IFN-β by the recombinant ProTα variants in human macrophages is dependent on the TLR4 pathway, whereas the induction of TNF-α and IL-6 may also occur independently of TLR4, suggesting the interaction of ProTα variants with other signaling molecules/receptors. In silico analyses confirmed that the novel isoB and p7 variants are intrinsically disordered proteins, which lack the NLS and mass spectrometry showed release of ProTα variants within minutes post HIV-1 infection. These features are consistent with the function of ProTα variants as damage associate molecular patterns (DAMPs). CONCLUSIONS Our findings indicate that ProTα variants strongly inhibit viral replication mainly, but not exclusively, through TLR4 signaling and that they are released within minutes of viral infection suggesting that they may function as DAMPs.

The important role of CD8(+) T cells in controlling HIV-1 infection through the innate as well as the adaptive immune system is well established. In addition to the major histocompatibility complex (MHC)-dependent cytotoxic activity of CD8(+) T cells, they produce soluble factors that suppress HIV-1 replication in an MHC-independent manner. Several of those factors have been identified, including beta-chemokines, Rantes, MIP-1alpha, MIP-1beta, and MDC. We previously identified that prothymosin alpha (ProTalpha) in the conditioned medium of HVS transformed CD8(+) T cells was a potent inhibitor of HIV-1 replication following proviral integration. In this report we further characterize the anti-HIV-1 activity of ProTalpha by demonstrating its target-cell specificity, distinction from additional inhibitors of HIV-1 transcription in CD8(+) T cell supernatants, as well as the differential regulation of host cell antiviral genes that could impact HIV-1 replication. These genes include a number of transcription factors as well IFN-alpha-inducible genes including PKR, IRF1, and Rantes, in the absence of induction of IFN-alpha. These data suggest that the anti-HIV-1 activity of ProTalpha is mediated through the modulation of a number of genes that have been reported to suppress HIV-1 replication including the dysregulation of transcription factors and the induction of PKR and Rantes mRNA.

Gonorrhea often occurs as a coinfection with human immunodeficiency virus (HIV). Lipooligosaccharide (LOS) is a component of the gonococcal outer membrane that induces innate immunity through engagement of Toll-like receptor 4 (TLR4). We investigated the effects that LOS from 5 different strains of Neisseria gonorrhoeae have on HIV infection and on HIV provirus in primary human macrophages. LOS-treated human primary macrophages developed resistance to new HIV infection as well as to HIV provirus. Gonococcal LOS from the 5 strains and lipopolysaccharide (LPS) from Escherichia coli showed no significant difference in their anti-HIV activities. Suppression of HIV provirus resulted from the induction of interferon (IFN)-beta and subsequent activation of signal transducer and activator of transcription 1. Neutralization of IFN-beta , but not IFN-alpha , via antibody significantly reduced the anti-HIV activity induced by LOS and LPS. We conclude that LOS expressed by various strains of N. gonorrhoeae induce specific innate immune responses through TLR4 signaling, resulting in anti-HIV activity in human primary macrophages in vitro.

Poly(styrene 4-sulfonate), cellulose sulfate, polymethylenehydroquinone, and PRO 2000 are sulfated or sulfonated polymers (SPs) under development as topical microbicides. They are presumed to work through similar mechanisms of action, although to date there has been no extensive comparison of their anti-human immunodeficiency virus activities. To determine whether any of these candidate microbicides offers a potential advantage, their in vitro activities, mechanisms of action, stabilities in biological secretions, and toxicities were compared. All four compounds were found to be active against X4, R5, and dualtropic primary isolates and against X4 and R5 laboratory-adapted strains in CD4 + T cells, macrophages, and single-coreceptor cell lines. Our single-cycle experiments using pseudotyped virus suggest that all four SPs function at the binding and entry stages of the viral life cycle but differ in degree of postentry effect. Surface plasmon resonance analyses demonstrate that SPs bind to X4 and R5 monomeric glycoprotein 120 with similar high binding affinities. When mixed with cervicovaginal lavage fluid, SPs maintain inhibitory activity at concentrations achievable in formulations.

Soluble factors from CD8(+) T cells and cervicovaginal mucosa of women are recognized as important in controlling human immunodeficiency virus type 1 (HIV-1) infection and transmission. Previously, we have shown the strong anti-HIV-1 activity of prothymosin α (ProTα) derived from CD8(+) T cells. ProTα is a small acidic protein with wide cell distribution, to which several functions have been ascribed, depending on its intracellular or extracellular localization. To date, activities of ProTα have been attributed to a single protein known as isoform 2. Here we report the isolation and identification of 2 new ProTα variants from CD8(+) T cells and cervicovaginal lavage with potent anti-HIV-1 activity. The first is a splice variant of the ProTα gene, known as isoform CRA_b, and the second is the product of a ProTα gene, thus far classified as a pseudogene 7. Native or recombinant ProTα variants potently restrict HIV-1 replication in macrophages through the induction of type I interferon. The baseline expression of interferon-responsive genes in primary human cervical tissues positively correlate with high levels of intracellular ProTα, and the knockdown of ProTα variants by small interfering RNA leads to downregulation of interferon target genes. Overall, these findings suggest that ProTα variants are innate immune mediators involved in immune surveillance.

There have been increasing reports of acute coronary thrombotic events in patients with HIV. Although these clinical events have been attributed primarily to dyslipidemia associated with protease inhibitor therapy, autopsy studies in children with HIV suggest the presence of an underlying arteriopathy. This study demonstrates that the HIV envelope protein, gp120, activates human arterial smooth muscle cells to express tissue factor, the initiator of the coagulation cascade. The induction of tissue factor by gp120 is mediated by two biologically relevant coreceptors for HIV infection, CXCR4 and CCR5, and is also dependent on the presence of functional CD4. Induction of tissue factor by gp120 requires activation of mitogen-activating protein kinases, activation of protein kinase C, and generation of reactive oxygen species, signaling pathways that have protean effects on smooth muscle cell physiology. The activation of smooth muscle cells by gp120 may play an important role in the vascular, thrombotic, and inflammatory responses to HIV infection.

Background Breast cancer cell lines are widely used tools to investigate breast cancer biology and to develop new therapies. Breast cancer tissue contains molecularly heterogeneous cell populations. Thus, it is important to understand which cell lines best represent the primary tumor and have similarly diverse phenotype. Here, we describe the development of five breast cancer cell lines from a single patient’s breast cancer tissue. We characterize the molecular profiles, tumorigenicity and metastatic ability in vivo of all five cell lines and compare their responsiveness to 4-hydroxytamoxifen (4-OHT) treatment. Methods Five breast cancer cell lines were derived from a single patient’s primary breast cancer tissue. Expression of different antigens including HER2, estrogen receptor (ER), CK8/18, CD44 and CD24 was determined by flow cytometry, western blotting and immunohistochemistry (IHC). In addition, a Fuorescent In Situ Hybridization (FISH) assay for HER2 gene amplification and p53 genotyping was performed on all cell lines. A xenograft model in nude mice was utilized to assess the tumorigenic and metastatic abilities of the breast cancer cells. Results We have isolated, cloned and established five new breast cancer cell lines with different tumorigenicity and metastatic abilities from a single primary breast cancer. Although all the cell lines expressed low levels of ER, their growth was estrogen-independent and all had high-levels of expression of mutated non-functional p53. The HER2 gene was rearranged in all cell lines. Low doses of 4-OHT induced proliferation of these breast cancer cell lines. Conclusions All five breast cancer cell lines have different antigenic expression profiles, tumorigenicity and organ specific metastatic abilities although they derive from a single tumor. None of the studied markers correlated with tumorigenic potential. These new cell lines could serve as a model for detailed genomic and proteomic analyses to identify mechanisms of organ-specific metastasis of breast cancer.

Prothymosin α (ProTα) is a 12.5-kDa, highly acidic protein widely distributed in different cell types expressed intracellularly and extracellularly. ProTα does not contain a secretion-signal sequence and is released by a nonclassical secretory pathway with a cargo protein. New findings on the extracellular function of ProTα have yielded exciting insights into the cytokine-like functions of this host protein that stimulates type I interferon via Toll-like receptor 4. Here, we discuss the intracellular function of ProTα, how new findings of cytokine-like activities of ProTα aid our understanding of mechanisms that direct ProTα functions, and the potential application of these new insights to the development of immunotherapies.

In animal models of HIV-associated nephropathy, the expression of HIV regulatory genes in epithelial cells is sufficient to cause disease, but how the CD4-negative epithelial cells come to express HIV genes is unknown. Here, we co-cultured T cells infected with fluorescently tagged HIV with renal tubular epithelial cells and observed efficient virus transfer between these cells. The quantity of HIV transferred was much greater than that achieved by exposure to large amounts of cell-free virus and occurred without a requirement for CD4 or Env. The transfer required stable cell-cell adhesion, which could be blocked by sulfated polysaccharides or poly-anionic compounds. We found that the internalization of virus could lead to de novo synthesis of viral protein from incoming viral RNAs even in the presence of a reverse transcriptase inhibitor. These results illustrate an interaction between infected T cells and nonimmune cells, supporting the presence of virological synapses between HIV-harboring T cells and renal tubular epithelial cells, allowing viral uptake and gene expression in epithelial cells.

Human immunodeficiency virus (HIV) infection is associated with accelerated atherosclerosis and vasculopathy, although the mechanisms underlying these findings have not been determined. Hypotheses for these observations include: 1) an increase in the prevalence of established cardiac risk factors observed in HIV-infected individuals who are currently experiencing longer life expectancies; 2) the dyslipidemia reported with certain HIV anti-retroviral therapies; and/or 3) the proinflammatory effects of infiltrating HIV-infected monocytes/macrophages. An unexplored possibility is whether HIV itself can infect vascular smooth muscle cells (SMCs) and, by doing so, whether SMCs can accelerate vascular disease. Our studies demonstrate that human SMCs can be infected with HIV both in vivo and in vitro. The HIV protein p24 was detected by fluorescence confocal microscopy in SMCs from tissue sections of human atherosclerotic plaques obtained from HIV-infected individuals. Human SMCs could also be infected in vitro with HIV by a mechanism dependent on CD4, the chemokine receptors CXCR4 or CCR5, and endocytosis, resulting in a marked increase in SMC secretion of the chemokine CCL2/MCP-1, which has been previously shown to be a critical mediator of atherosclerosis. In addition, SMC proliferation appeared concentric to the vessel lumen, and minimal inflammation was detected, unlike typical atherosclerosis. Our data suggest that direct infection of human arterial SMCs by HIV represents a potential mechanism in a multifactorial paradigm to explain the exacerbated atherosclerosis and vasculopathy reported in individuals infected with HIV.

CD8 + T lymphocytes control human immunodeficiency virus type 1 (HIV-1) infection by a cytotoxic major histocompatibility complex-restricted pathway as well as by secretion of noncytotoxic soluble inhibitory factors. Several components of CD8 + cell supernatants have been identified that contribute to the latter activity. In this study we report that prothymosin alpha (ProTα), a protein found in the cell culture medium of the herpesvirus saimiri-transformed CD8 + T-cell line, K#1 50K, has potent HIV-1-inhibitory activity. Depletion of native ProTα from an HIV-1-inhibitory fraction of CD8 + cell supernatants removes the inhibitory activity, supporting its role in inhibition via soluble mediators. ProTα is an abundant, acidic peptide that has been reported to be localized in the nucleus and associated with cell proliferation and activation of transcription. In this report we demonstrate that ProTα suppresses HIV-1 replication, its activity is target cell specific, and inhibition occurs following viral integration. Native and recombinant ProTα protein potently inhibit HIV-1 long terminal repeat (LTR)-driven gene expression in macrophages. Furthermore studies using different promoters in lentiviral vectors (cytomegalovirus and phosphoglycerate kinase) revealed that suppression of viral replication by ProTα is not HIV LTR specific.

CD8+T lymphocytes can inhibit human immunodeficiency virus type 1 (HIV-1) replication by secreting a soluble factor(s) known as CD8+T-lymphocyte antiviral factor (CAF). One site of CAF action is inhibition of HIV-1 RNA transcription, particularly at the step of long terminal repeat (LTR)-driven gene expression. The inhibitory effect of CAF on HIV-1 LTR activation is mediated through STAT1 activation. A recent study reports that α-defensins 1 to 3 account for CAF activity against HIV-1. Here, we address whether α-defensins, particularly α-defensin-1, contribute to CAF-mediated inhibition of HIV-1 transcription. Both recombinant α-defensin-1 and CAF derived from herpesvirus saimiri (HVS)-transformed CD8+cells inhibited HIV-1 infection and gene expression. For both factors, the inhibition of HIV-1 infection did not occur at the level of viral entry. Pretreatment of cells with α-defensin-1 followed by a washing out prior to infection blocked infection by HIV-1, indicating that direct inactivation of virions was not required for its inhibitory effect. In contrast to CAF, α-defensin-1 did not inhibit phorbol myristate acetate- or Tat-mediated HIV-1 LTR activation in a transient transfection system, nor did it activate STAT1 tyrosine phosphorylation. Furthermore, α-defensins 1 to 3 were below the level of detection in a panel of HVS-transformed CD8+cells with potent HIV-1 inhibitory activity and a neutralizing antibody against α-defensins 1 to 3 did not reverse the inhibitory effect of CAF on HIV-1 gene expression in infected cells and on HIV-1 LTR activation in transfected cells. Taken together, our results suggest that α-defensin-1 inhibits HIV-1 infection following viral entry but that α-defensins 1 to 3 are not responsible for the HIV-1 transcriptional inhibition by CAF.

Closed circular (cc) forms of extrachromosomal HIV DNA are detected in patients with high viral loads; however, it is unclear whether these forms remain if virus replication is suppressed to undetectable levels by combination antiretroviral therapy. A nested primer polymerase chain reaction amplification assay was used to detect the presence of ccDNA containing two long terminal repeat sequences (2-LTR) in PBMC of patients with low or undetectable plasma HIV RNA. Fifty percent of patients with plasma RNA levels

CD8+T lymphocytes can suppress human immunodeficiency virus type 1 (HIV-1) replication by secreting a soluble factor(s) known as CD8+T-lymphocyte antiviral factor (CAF). One site of CAF action is inhibition of HIV-1 RNA transcription, particularly at the step of long terminal repeat (LTR)-driven gene expression. However, the mechanism by which CAF inhibits LTR activation is not understood. Here, we show that conditioned media from several herpesvirus saimari-transformed CD8+T lymphocytes inhibit, in a time- and dose-dependent manner, the replication of HIV-1 pseudotype viruses that express the envelope glycoproteins of vesicular stomatitis virus (HIV-1VSV). The same conditioned media also inhibit phorbol myristate acetate-induced activation of the HIV-1 LTR and activate the signal transducer and activator of transcription 1 (STAT1) protein. We have obtained direct evidence that STAT1 is necessary for CAF-mediated inhibition of LTR activation and HIV-1 replication. Thus, the inhibitory effect of CAF on HIV-1VSVreplication was abolished in STAT1-deficient cells. Moreover, CAF inhibition of LTR activation was diminished both in STAT1-deficient cells and in cells expressing a STAT1 dominant negative mutant but was restored when STAT1 was reintroduced into the STAT1-deficient cells. We also observed that CAF induced the expression of interferon regulatory factor 1 (IRF-1), and that IRF-1 gene induction was STAT-1 dependent. Taken together, our results suggest that CAF activates STAT1, leading to IRF-1 induction and inhibition of gene expression regulated by the HIV-1 LTR. This study therefore helps clarify one molecular mechanism of host defense against HIV-1.

The ability of viruses and bacteria to interact with the extracellular matrix plays an important role in their infectivity and pathogenicity. Fibronectin is a major component of the extracellular matrix in lymph node tissue, the main site of HIV deposition and replication during the chronic phase of infection. Therefore, we asked whether matrix fibronectin (FN) could affect the ability of HIV to infect lymphocytes. To study the role of matrix FN on HIV infection, we used superfibronectin (sFN), a multimeric form of FN that closely resembles in vivo matrix FN. In this study we show that HIV-1IIIB efficiently binds to multimeric fibronectin (sFN) and that HIV infection of primary CD4+ lymphocytes is enhanced by >1 order of magnitude in the presence of sFN. This increase appears to be due to increased adhesion of viral particles to the cell surface in the presence of sFN, followed by internalization of virus. Enzymatic removal of cell surface proteoglycans inhibited the adhesion of HIV-1IIIB/sFN complexes to lymphocytes. In contrast, Abs to integrins had no effect on binding of HIV-1IIIB/sFN complexes to lymphocytes. The III1-C peptide alone also bound HIV-1IIIB efficiently and enhanced HIV infection, although not as effectively as sFN. HIV-1IIIB gp120 envelope protein binds to the III1-C region of sFN and may be important in the interaction of virus with matrix FN. We conclude that HIV-1IIIB specifically interacts with the III1-C region within matrix FN, and that this interaction may play a role in facilitating HIV infection in vivo, particularly in lymph node tissue.