Electromagnetic Fields Generated by the IteraCoil Device Differentiate Mesenchymal Stem Progenitor Cells Into the Osteogenic Lineage.

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- , CD73+ , CD90+ , and CD105+ ) was utilized for the study. Osteoblasts and chondrocyte differentiations were then assessed by specific staining and quantified using ImageJ (National Institutes of Health). The osteoblastic differentiation of MSPCs cultured in regular medium and exposed to EMF at 0.05 and 1 kHz frequencies was compared with MSPCs cultured in a pro-osteogenic supplemented medium. In this study, we demonstrated that EMF from IteraCoil might have affected the signaling pathways that induce the osteogenic differentiation of human adipose-derived MSPCs in the absence of exogenous osteogenic factors. Therefore, EMF-generated osteogenic differentiation of reprogrammed adipose-derived autologous MSPCs may treat the loss of osteoblasts and osteoporosis and open new avenues for the development of regenerative cellular therapy. © 2022 Bioelectromagnetics Society.

Prothymosin α variants isolated from CD8+ T cells and cervicovaginal fluid suppress HIV-1 replication through type I interferon induction.

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.

Prothymosin-alpha inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction.

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-alpha (ProTalpha), 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 ProTalpha 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 ProTalpha, retained by an acidic peptide derived from ProTalpha, and lost in the absence of TLR4. Furthermore, we demonstrate that ProTalpha 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 ProTalpha may provide therapeutic leads for IFN-sensitive viruses.

Virological synapses allow HIV-1 uptake and gene expression in renal tubular epithelial cells.

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.

Annexin 2 is not required for human immunodeficiency virus type 1 particle production but plays a cell type-dependent role in regulating infectivity.

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 approximately 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.

Human immunodeficiency virus (HIV)-1 infects human hepatic stellate cells and promotes collagen I and monocyte chemoattractant protein-1 expression: implications for the pathogenesis of HIV/hepatitis C virus-induced liver fibrosis.

UNLABELLED: Patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) develop more rapid fibrosis than those infected with HCV only. In HIV/HCV-coinfected patients, fibrosis progression correlates with HIV RNA levels, suggesting a direct role of HIV in liver fibrogenesis. Chemokine (C-C motif) receptor 5 (CCR5) and cysteine-X-cysteine receptor 4 (CXCR4), the two major coreceptors required for HIV entry into cells, are expressed on activated hepatic stellate cells (HSCs), the principle fibrogenic cell type in the liver. We therefore examined whether HIV can infect HSCs, explored the potential mechanisms of viral entry, and assessed the impact of infection as reflected by the ability of HSCs to transfer virus to T lymphocytes and elicit a proinflammatory and profibrogenic response. We report that the laboratory-adapted viruses HIV-IIIB (CXCR4-tropic or X4) and HIV-BaL (CCR5-tropic or R5) and primary HIV isolates can infect both a human stellate cell line, LX-2, and primary human HSCs. HIV entry and gene expression in HSCs was confirmed using HIV-green fluorescent protein (GFP) expression viral constructs in the presence or absence of the reverse-transcriptase inhibitor azidothymidine. CD4 expression on a subset of primary HSCs was demonstrated using fluorescence-activated cell sorting and immunofluorescence staining. Blocking experiments in the presence of anti-CD4, anti-CXCR4, and anti-CCR5 revealed that HIV entry into HSCs is predominantly CD4/chemokine coreceptor-independent. HIV infection promoted HSC collagen I expression and secretion of the proinflammatory cytokine monocyte chemoattractant protein-1. Furthermore, infected LX-2 cells were capable of transferring GFP-expressing virus to T lymphocytes in a coculture system. CONCLUSION: Taken together, our results suggest a potential role of HIV in liver fibrosis/inflammation mediated through effects on HSCs. The role of early highly active antiretroviral therapy initiation in patients with HIV/HCV coinfection warrants further investigation.

A fine line between molecular umbrella transport and ionophoric activity.

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.

HIV infection modulates IL-1ß response to LPS stimulation through a TLR4-NLRP3 pathway in human liver macrophages.

IL-1ß 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-1ß response to microbial translocation in chronic liver disease, we aim to examine the impact of HIV-1 and LPS stimulation on the IL-1ß 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-1ß response, and then studied underlying mechanisms. Furthermore, we examined IL-1ß 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-1ß 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-1ß 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-1ß response. High in situ IL-1ß expression was found in CD68+ cells in human liver tissues from HIV-1-infected patients, suggesting a critical role of IL-1ß responses in patients infected by HIV. HIV infection sensitizes the IL-1ß 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.

Influence of prothymosin-alpha on HIV-1 target cells.

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.

Frontline Science: HIV infection of Kupffer cells results in an amplified proinflammatory response to LPS.

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.

Prothymosin-α Variants Elicit Anti-HIV-1 Response via TLR4 Dependent and Independent Pathways.

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-ß, IFN-λ1, IL-6, TNF-α and RANTES mRNAs in primary human macrophages. The potent stimulation of IFN-ß 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.

Multiple breast cancer cell-lines derived from a single tumor differ in their molecular characteristics and tumorigenic potential.

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 Fluorescent 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.

X4 Human immunodeficiency virus type 1 gp120 promotes human hepatic stellate cell activation and collagen I expression through interactions with CXCR4.

BACKGROUND & AIMS: Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the in vitro impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the in vivo expression of gp120 in HIV/HCV coinfected livers. METHODS: Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/- either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies. RESULTS: X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers. CONCLUSIONS: X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.

Intracellular and extracellular cytokine-like functions of prothymosin α: implications for the development of immunotherapies.

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.

Human peritoneal macrophages from ascitic fluid can be infected by a broad range of HIV-1 isolates.

Macrophages are major HIV target cells. They support both productive and latent HIV-1 infection. Susceptibility of primary macrophages to HIV depends on the anatomical location and activation state of the cells. We demonstrate that peritoneal macrophages (PMs) are abundant in ascitic fluid of patients with liver cirrhosis and are susceptible to HIV-1 infection. PMs expressed CD68, a differentiation marker, exhibited phagocytic activity, and survived in culture for 2 months without additional growth factors. Freshly isolated PMs were susceptible to HIV-1 R5 strains but not to X4-T-cell line-adapted strains. Interestingly, after 7 days in culture, PMs acquired susceptibility to X4-T-cell line-adapted strains. HIV entry inhibitors, TAK779 and AMD3100, blocked HIV infection of PMs, indicating that infection by R5 and X4 strains was mediated by CCR5 and CXCR4, respectively. Although PMs did not express detectable cell surface levels of CXCR4 and CCR5, they did express mRNAs of these HIV coreceptors and responded to stimulation by their natural ligands, SDF-1alpha and RANTES. PMs were susceptible to HIV-1 X4, R5, and X4R5 primary isolates. PMs after 7 days in culture produced greater amounts of X4 and X4R5 HIV than freshly isolated PMs. The day-7 PMs were more susceptible to R5 infection in a single-cycle infection assay, but there was no increase in viral production in a multiple-round infection assay. The level of CXCR4 mRNA and production of CC-chemokines (MIP-1alpha, MIP-1beta, and RANTES) increased significantly during 7 days in culture. Our results indicate that PMs are susceptible to receptor-mediated infection by a broad range of HIV strains. These primary macrophages could provide a valuable system for investigating the role of primary macrophages in HIV pathogenesis.

Human immunodeficiency virus (HIV) infects human arterial smooth muscle cells in vivo and in vitro: implications for the pathogenesis of HIV-mediated vascular disease.

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.

Gonococcal lipooligosaccharide suppresses HIV infection in human primary macrophages through induction of innate immunity.

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.

Novel function of prothymosin alpha as a potent inhibitor of human immunodeficiency virus type 1 gene expression in primary macrophages.

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 (ProTalpha), 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 ProTalpha from an HIV-1-inhibitory fraction of CD8(+) cell supernatants removes the inhibitory activity, supporting its role in inhibition via soluble mediators. ProTalpha 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 ProTalpha suppresses HIV-1 replication, its activity is target cell specific, and inhibition occurs following viral integration. Native and recombinant ProTalpha 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 ProTalpha is not HIV LTR specific.

Candidate sulfonated and sulfated topical microbicides: comparison of anti-human immunodeficiency virus activities and mechanisms of action.

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.

CAF-mediated human immunodeficiency virus (HIV) type 1 transcriptional inhibition is distinct from alpha-defensin-1 HIV inhibition.

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 alpha-defensins 1 to 3 account for CAF activity against HIV-1. Here, we address whether alpha-defensins, particularly alpha-defensin-1, contribute to CAF-mediated inhibition of HIV-1 transcription. Both recombinant alpha-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 alpha-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, alpha-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, alpha-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 alpha-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 alpha-defensin-1 inhibits HIV-1 infection following viral entry but that alpha-defensins 1 to 3 are not responsible for the HIV-1 transcriptional inhibition by CAF.