Topical doxycycline monohydrate hydrogel 1% targeting proteases/PAR2 pathway is a novel therapeutic for atopic dermatitis.

Atopic Dermatitis (AD) is characterized by skin barrier disruption and an aberrant immune response. Doxycycline is tetracycline antibiotics broadly used systemically to treat inflammatory dermatologic conditions. Several studies have shown doxycycline has anti-inflammatory and pro-healing properties, mainly by blocking tissue proteolytic activity. It is our hypothesis that daily application of a novel doxycycline topical formulation in AD subjects will reduce severity of the disease, by blocking cutaneous proteases activity and restoring skin barrier function and inflammation. To test this hypothesis, we performed a proof of concept, open-label clinical study. Subjects enrolled in the study (n = 15) applied NanoDOX® Hydrogel 1% daily for 4 weeks on a chosen eczematous area. Investigational drug was well tolerated, and no local or systemic adverse events due to investigational drug were reported. Notably, a significant clinical improvement was observed based on a modified Eczema Area & Severity Index (EASI) score of the treated area from start of treatment to 14 and 28 days post-treatment (P < .001). A significant improvement of pruritus was also observed (P = .02). This proof of concept clinical trial is first to explore the impact of a non-systemic doxycycline treatment on AD patients. Our results provide evidence to investigate novel AD treatment strategies targeting cutaneous proteases activity.

HIV inhibits endothelial reverse cholesterol transport through impacting subcellular Caveolin-1 trafficking.

BACKGROUND: Human immunodeficiency virus (HIV) infection leads to decreased reverse cholesterol transport (RCT) in macrophages, and Nef mediated down-regulation and redistribution of ATP-binding cassette transporter A1 (ABCA1) are identified as key factors for this effect. This may partially explain the increased risk of atherosclerosis in HIV infected individuals. Since endothelial dysfunction is key in the initial stages of atherosclerosis, we sought to determine whether RCT was affected in human aortic endothelial cells (HAECs). RESULTS: We found that apoA-I does not significantly stimulate cholesterol efflux in HAECs while cholesterol efflux to high-density lipoprotein (HDL) was dramatically reduced in HAECs co-cultured with HIV infected cells. Studies with wild type and Nef defective HIV revealed no significant differences suggesting that multiple factors are working perhaps in concert with Nef to affect cholesterol efflux to HDL from HAECs. Interestingly, treating HAECs with recombinant Nef showed similar effect in HDL mediated cholesterol efflux as observed in HAECs co-cultured with HIV infected cells. Using a detergent-free based subcellular fractionation approach, we demonstrated that exposure of HAECs to HIV infected cells or Nef alone disrupts caveolin 1 (Cav-1) subcellular trafficking upon HDL stimulation. Moreover, Nef significantly enhanced tyrosine 14 phosphorylation of Cav-1 which may have an impact on recycling of Cav-1 and caveolae. CONCLUSION: These results suggest that HIV interferes with cholesterol efflux by HDL in HAECs through the disruption of Cav-1s' cellular distribution and that multiple factors are involved, possibly including Nef, for the inhibition of HDL mediated cholesterol efflux and alteration of cellular distribution of Cav-1.

Caveolin-1 reduces HIV-1 infectivity by restoration of HIV Nef mediated impairment of cholesterol efflux by apoA-I.

BACKGROUND: HIV infection results in inhibited cholesterol efflux by apolipoprotein A-I (apoA-I) in macrophages, and this impairment involves Nef mediated down-regulation and redistribution of ATP-binding cassette transporter A1 (ABCA-1). We investigated the effect of caveolin-1 (Cav-1) on the cholesterol efflux by apoA-I in HIV infected primary and THP-1 cell-differentiated macrophages as well as astrocyte derived glioblastoma U87 cells. RESULTS: Our results reveal that Cav-1 restores the Nef -mediated impairment of cholesterol efflux by apoA-I in both cell types. Co-immunoprecipitation studies indicate a physical association of Cav-1 and Nef. The level of ABCA-1 expression remains the same whether Cav-1 is over-expressed or not. In addition, we examined the cholesterol composition of HIV particles released from Cav-1 treated cells and identified that the cholesterol content is dramatically reduced. The infectivity level of these virus particles is also significantly decreased. CONCLUSIONS: These observations suggest that the interplay of Cav-1 with Nef and cholesterol subsequently counters Nef induced impairment of cholesterol efflux by apoA-l. The findings provide a cellular mechanism by which Cav-1 has an ability to restore HIV mediated impairment of cholesterol efflux in macrophages. This subsequently influences the cholesterol content incorporated into virus particles thereby inhibiting HIV infectivity and contributing to HIV's persistent infection of macrophages.

A Kaposi's sarcoma-associated herpesvirus-encoded ortholog of microRNA miR-155 induces human splenic B-cell expansion in NOD/LtSz-scid IL2Rγnull mice.

MicroRNAs (miRNAs) are small noncoding RNA molecules that function as posttranscriptional regulators of gene expression. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), a B-cell-tropic virus associated with KS and B-cell lymphomas, encodes 12 miRNA genes that are highly expressed in these tumor cells. One viral miRNA, miR-K12-11, shares 100% seed sequence homology with hsa-miR-155, an oncogenic human miRNA that functions as a key regulator of hematopoiesis and B-cell differentiation. So far, in vitro studies have shown that both miRNAs can regulate a common set of cellular target genes, suggesting that miR-K12-11 may mimic miR-155 function. To comparatively study miR-K12-11 and miR-155 function in vivo, we used a foamy virus vector to express the miRNAs in human hematopoietic progenitors and performed immune reconstitutions in NOD/LtSz-scid IL2Rγ(null) mice. We found that ectopic expression of miR-K12-11 or miR-155 leads to a significant expansion of the CD19(+) B-cell population in the spleen. Subsequent quantitative PCR analyses of these splenic B cells revealed that C/EBPß, a transcriptional regulator of interleukin-6 that is linked to B-cell lymphoproliferative disorders, is downregulated when either miR-K12-11 or miR-155 is ectopically expressed. In addition, inhibition of miR-K12-11 function using antagomirs in KSHV-infected human primary effusion lymphoma B cells resulted in derepression of C/EBPß transcript levels. This in vivo study validates miR-K12-11 as a functional ortholog of miR-155 in the context of hematopoiesis and suggests a novel mechanism by which KSHV miR-K12-11 induces splenic B-cell expansion and potentially KSHV-associated lymphomagenesis by targeting C/EBPß.

Caveolin 1 inhibits HIV replication by transcriptional repression mediated through NF-κB.

Caveolin 1 (Cav-1), the scaffold protein of a specific membrane lipid raft called caveolae, has been reported to suppress HIV-1 replication. However, the mechanism by which Cav-1 inhibits HIV replication remains unclear. In this study, we investigated the mechanism by which Cav-1 inhibits HIV replication at the level of gene expression. Our results show that Cav-1 represses viral gene expression and that this suppression involves the NF-κB pathway. We used several approaches in different cell types, including primary CD4(+) T cells and macrophages, to demonstrate the role of nuclear factor κB (NF-κB) in Cav-1-mediated inhibition of viral expression. A mutational analysis of the cis-acting element shows that the two NF-κB sites in the U3 region of the long terminal repeat (LTR) are critical for Cav-1-mediated inhibition of viral expression. In the presence of Cav-1, phosphorylation of IKKß, IKKα, IκBα, and NF-κB p65 is dramatically reduced, while viral gene expression is suppressed. In addition, translocation of NF-κB p65 to the nucleus decreases substantially in the presence of Cav-1. Furthermore, significant inhibition of NF-κB activation and binding to target DNA are evident in the presence of Cav-1. These results establish evidence that Cav-1 inhibits HIV replication by transcriptional repression of viral gene expression and contributes to HIV's persistent infection of macrophages.

HIV infection upregulates caveolin 1 expression to restrict virus production.

Caveolin 1 (Cav-1) is a major protein of a specific membrane lipid raft known as caveolae. Cav-1 interacts with the gp41 of the human immunodeficiency virus (HIV) envelope, but the role of Cav-1 in HIV replication and pathogenesis is not known. In this report, we demonstrate that HIV infection in primary human monocyte-derived macrophages (MDMs), THP-1 macrophages, and U87-CD4 cells results in a dramatic upregulation of Cav-1 expression mediated by HIV Tat. The activity of p53 is essential for Tat-induced Cav-1 expression, as our findings show enhanced phosphorylation of serine residues at amino acid positions 15 and 46 in the presence of Tat with a resulting Cav-1 upregulation. Furthermore, inhibition of p38 mitogen-activated protein kinase (MAPK) blocked phosphorylation of p53 in the presence of Tat. Infection studies of Cav-1-overexpressing cells reveal a significant reduction of HIV production. Taken together, these results suggest that HIV infection enhances the expression of Cav-1, which subsequently causes virus reduction, suggesting that Cav-1 may contribute to persistent infection in macrophages.

Caveolin-1 modulates HIV-1 envelope-induced bystander apoptosis through gp41.

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4(+) T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4(+) T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.

The foamy virus genome remains unintegrated in the nuclei of G1/S phase-arrested cells, and integrase is critical for preintegration complex transport into the nucleus.

Foamy viruses are a member of the spumavirus subfamily of retroviruses with unique mechanisms of virus replication. Foamy virus replication is cell cycle dependent; however, the genome is found in the nuclei of cells arrested in the G(1)/S phase. Despite the presence of genome in the nuclei of growth-arrested cells, there is no viral gene expression, thus explaining its dependency on cell cycle. This report shows that the foamy virus genome remains unintegrated in G(1)/S phase-arrested cells. The foamy virus genome is detected by confocal microscopy in the nuclei of both dividing and growth-arrested cells. Alu PCR revealed foamy virus-specific DNA amplification from genomic DNA isolated in cycling cells at 24 h postinfection. In arrested cells no foamy virus DNA band was detected in cells harvested at 1 or 7 days after infection, and a very faint band that is significantly less than DNA amplified from cycling cells was observed at day 15. After these cells were arrested at the G(1)/S phase for 1, 7, or 15 days they were allowed to cycle, at which time foamy virus-specific DNA amplification was readily observed. Taken together, these results suggest that the foamy virus genome persists in nondividing cells without integrating. We have also established evidence for the first time that the foamy virus genome and Gag translocation into the nucleus are dependent on integrase in cycling cells, implicating the role of integrase in transport of the preintegration complex into the nucleus. Furthermore, despite the presence of a nuclear localization signal sequence in Gag, we observed no foamy virus Gag importation into the nucleus in the absence of integrase.

Activity of TAR in inducible inhibition of HIV replication by foamy virus vector expressing siRNAs under the control of HIV LTR.

In this report we describe foamy virus vectors with conditional expression of short interfering RNAs (siRNAs) in HIV infected cells. Short hairpin RNAs (shRNAs) based on two targets in the 5' end of the untranslated region and one in the rev gene flanked with 5' and 3' microRNA 30 (miR30) sequences were synthesized and placed under the control of an HIV promoter for Tat-mediated expression. HIV permissive cells were transduced with foamy virus vectors containing each hybrid shRNA expression cassette and tested for their efficacy on the inhibition of HIV replication. Effective Tat dependent expression of the shRNAs, as well as GFP placed downstream each shRNA was evident. In addition the results show inhibition of HIV replication by greater than 98%. Interestingly, transduction of cells with a vector lacking an shRNA also revealed GFP expression in the presence of Tat with similar levels of inhibition of virus replication. When the TAR region was removed from this vector there was neither reduction in virus replication nor Tat-induced GFP expression. These results suggest that TAR in the vector, which Tat interacts to promote expression of the shRNA, is a potent inhibitor of virus replication. Previous studies with TAR regulated expression of antiviral genes ignore the contribution of TAR in the repression of virus replication. Interpretation of effective inhibition of HIV replication by antiviral genes located downstream of TAR while neglecting the efficacy of a potent repression by TAR is misleading.

Establishing Restricted Expression of Caveolin-1 in HIV Infected Cells and Inhibition of Virus Replication.

BACKGROUND: Caveolin-1 (Cav-1) is the major protein of the caveolae and plays a role in multiple cellular functions and implicated to have anti-HIV activity. Regulated expression of Cav-1 is important for safe and effective use in order to exploit Cav-1 for HIV therapeutic applications. METHODS: A series of Cav-1 and GFP expression vectors were constructed under the control of the HIV LTR for conditional expression or CMV promoter and the expression of Cav-1 was monitored in the presence or absence of Tat or HIV infection in order to establish the restricted expression of Cav-1 to HIV infected cells. RESULTS: Cav-1 expression was evident under the control of the HIV LTR in the absence of Tat or HIV infection as demonstrated by immunoblot. Placing two internal ribosomal entry sequences (IRES) and a Rev response element, RRE (5'~ LTR-IRES-GFP-RRE-IRES-Cav-1~3') resulted in no expression of Cav-1 in the absence of Tat with effective expression in the presence of Tat. Transduction of HIV permissive cells with this construct using a foamy virus vector show that Cav-1 was able to inhibit HIV replication by 82%. Cells that received LTR-IRES-GFP-RRE-IRES-Cav-1 remain healthy in the absence of Tat or HIV infection. CONCLUSION: These results taken together reveal the inclusion of two IRES establishes a significant reduction of leak through expression of Cav-1 in the absence of Tat or HIV infection. Such regulated expression will have therapeutic application of Cav-1 for HIV infection as well as broad applications which can be beneficial for other host-targeted interventions as therapeutics.

Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs.

Viral vectors available for gene therapy are either inefficient or suffer from safety concerns for human applications. Foamy viruses are non-pathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. In this report, we describe the use of simian foamy virus type 1 (SFV-1) vector to examine the efficacy of therapeutic genes. Hairpin short-interfering RNA (siRNA) that targets the simian immunodeficiency virus (SIV) rev/env was placed under the control of the PolIII U6 snRNA promoter for expression and screened for silencing target genes using cognate target-reporter fusions. We have identified an effective siRNA (designated R2) which reduces the rev and env gene expression by 89% and 95%, respectively. Using the simian foamy virus type 1 (SFV-1) based vector, we delivered the PolIII expressed R2 siRNA into cultured cells and challenged with SIV. The results show that the R2 siRNA is a potent inhibitor of SIV replication as determined by p27 expression and reverse transcriptase assays. Vectors based on a non-pathogenic SFV-1 vector may provide a safe and efficient alternative to currently available vectors, and the SIV model will help devise protocols for effective anti-HIV gene therapy.

A minimal genome simian foamy virus type 1 vector system with efficient gene transfer.

Foamy viruses have several inherent features for the opportunity to develop efficient and versatile vectors for gene therapy. We have constructed a series of vectors and helper plasmids based on simian foamy virus type 1 (SFV-1) to establish the minimum vector genome required for efficient gene transduction. To characterize the efficiency of gene transduction by these vectors, the green fluorescent protein (GFP) coding sequence is linked to the human cytomegalovirus immediate gene promoter. Several deletion analyses of SFV-1 vectors revealed that the minimum genome with efficient GFP transduction contained the 5' untranslated region extending to the first 637 nucleotides of the gag gene, a 596 nucleotides of pol sequence from position 3137-3733, the 3' pol region at position 5200-5693, the 3' end polypurine tract, and the 3' LTR. An additional 1131 nucleotides can be removed from the 3' end LTR without affecting the efficiency of vector transduction. SFV-1 vector can therefore accommodate a minimum 8930 base-size heterologous DNA fragment. Furthermore, the efficiency of SFV-1 vector transduction was analyzed using different packaging plasmids. GFP transduction with packaging plasmid that contained the 5' R-U5 region of the LTR was compared with helper plasmids that had deletions in this region except for 22 nucleotides (positions 21-41), the first 61, 77, or 140 nucleotides of the R of the LTR. Transduction efficiencies were significantly reduced with the deletion mutations implicating that for optimum SFV-1 vector productions a packaging construct that includes the 5' R-U5 is required.