Anna Miserocchi

HIV-1 Tat protein enhances RANKL/M-CSF-mediated osteoclast differentiation.

Impaired osteoblast/osteoclast cross-talk and bone structure homeostasis resulting in osteopenia/osteoporosis are often observed in HIV seropositive patients but the causal mechanisms remain unsettled. This study analyzed the biological effects of Tat on peripheral blood monocyte-derived osteoclast differentiation. Tat enhances osteoclast differentiation and activity induced by RANKL plus M-CSF treatment increasing both the mRNA expression of specific osteoclast differentiation markers, such as cathepsin K and calcitonin receptor, and TRAP expression and activity. These Tat-related biological effects may be related, at least in part, to the induction of c-fos expression and AP-1 activity. c-fos up-regulation was triggered by Tat when cell cultures were co-treated with RANKL/M-CSF and an analysis of c-fos promoter with c-fos deletion mutant constructs disclosed specific c-fos promoter domains targeted by Tat. Together, these results show that Tat may be considered a viral factor positively modulating the osteoclastogenesis and then bone resorption activity suggesting a pathogenetic role of this viral protein in the HIV-related osteopenia/osteoporosis.

Peptide-derivatized SB105-A10 dendrimer inhibits the infectivity of R5 and X4 HIV-1 strains in primary PBMCs and cervicovaginal histocultures.

Peptide dendrimers are a class of molecules that exhibit a large array of biological effects including antiviral activity. In this report, we analyzed the antiviral activity of the peptide-derivatized SB105-A10 dendrimer, which is a tetra-branched dendrimer synthetized on a lysine core, in activated peripheral blood mononuclear cells (PBMCs) that were challenged with reference and wild-type human immunodeficiency virus type 1 (HIV-1) strains. SB105-A10 inhibited infections by HIV-1 X4 and R5 strains, interfering with the early phases of the viral replication cycle. SB105-A10 targets heparan sulfate proteoglycans (HSPGs) and, importantly, the surface plasmon resonance (SPR) assay revealed that SB105-A10 strongly binds gp41 and gp120, most likely preventing HIV-1 attachment/entry through multiple mechanisms. Interestingly, the antiviral activity of SB105-A10 was also detectable in an organ-like structure of human cervicovaginal tissue, in which SB105-A10 inhibited the HIV-1ada R5 strain infection without altering the tissue viability. These results demonstrated the strong antiviral activity of SB105-A10 and suggest a potential microbicide use of this dendrimer to prevent the heterosexual transmission of HIV-1.

Effects of human immunodeficiency virus on the erythrocyte and megakaryocyte lineages

Anaemia and thrombocytopenia are haematological disorders that can be detected in many human immunodeficiency virus (HIV)-positive patients during the development of HIV infection. The progressive decline of erythrocytes and platelets plays an important role both in HIV disease progression and in the clinical and therapeutic management of HIV-positive patients. HIV-dependent impairment of the megakaryocyte and erythrocyte lineages is multifactorial and particularly affects survival, proliferation and differentiation of bone marrow (BM) CD34+ haematopoietic progenitor cells, the activity of BM stromal cells and the regulation of cytokine networks. In this review, we analyse the major HIV-related mechanisms that are involved in the genesis and development of the anaemia and thrombocytopenia observed in HIV positive patients.

HIV-1 and recombinant gp120 affect the survival and differentiation of human vessel wall-derived mesenchymal stem cells

BackgroundHIV infection elicits the onset of a progressive immunodeficiency and also damages several other organs and tissues such as the CNS, kidney, heart, blood vessels, adipose tissue and bone. In particular, HIV infection has been related to an increased incidence of cardiovascular diseases and derangement in the structure of blood vessels in the absence of classical risk factors. The recent characterization of multipotent mesenchymal cells in the vascular wall, involved in regulating cellular homeostasis, suggests that these cells may be considered a target of HIV pathogenesis. This paper investigated the interaction between HIV-1 and vascular wall resident human mesenchymal stem cells (MSCs).ResultsMSCs were challenged with classical R5 and X4 HIV-1 laboratory strains demonstrating that these strains are able to enter and integrate their retro-transcribed proviral DNA in the host cell genome. Subsequent experiments indicated that HIV-1 strains and recombinant gp120 elicited a reliable increase in apoptosis in sub-confluent MSCs. Since vascular wall MSCs are multipotent cells that may be differentiated towards several cell lineages, we challenged HIV-1 strains and gp120 on MSCs differentiated to adipogenesis and endotheliogenesis. Our experiments showed that the adipogenesis is increased especially by upregulated PPARγ activity whereas the endothelial differentiation induced by VEGF treatment was impaired with a downregulation of endothelial markers such as vWF, Flt-1 and KDR expression. These viral effects in MSC survival and adipogenic or endothelial differentiation were tackled by CD4 blockade suggesting an important role of CD4/gp120 interaction in this context.ConclusionsThe HIV-related derangement of MSC survival and differentiation may suggest a direct role of HIV infection and gp120 in impaired vessel homeostasis and in genesis of vessel damage observed in HIV-infected patients.

HIV-related mechanisms in atherosclerosis and cardiovascular diseases.

HIV-infected patients have a significantly higher risk of developing cardiovascular events during the progression of HIV disease. Atherosclerosis, myocardial infarction, cerebrovascular injury, pulmonary hypertension and thrombosis are consistently described in both combined antiretroviral therapy (cART)-treated and naive HIV-positive patients as major clinical complications. Recent studies indicate that the pathogenesis of cardiovascular lesions in HIV-positive patients is related to direct and indirect effects of HIV infection on vessel structures, independently of traditional risk factors. HIV infection strongly interferes with the biology of several cellular targets such as macrophage and endothelial cells. Moreover, HIV induces a profound derangement of lipid metabolism and inflammatory cytokine networks that are directly involved in atherogenesis and progressive impairment of the cardiovascular system. In this review, we discuss these major HIV-related mechanisms able to promote atherosclerosis and cardiovascular diseases in HIV-positive patients.

Analysis of the effects of HIV‐1 Tat on the survival and differentiation of vessel wall‐derived mesenchymal stem cells

HIV infection is an independent risk factor for atherosclerosis development and cardiovascular damage. As vessel wall mesenchymal stem cells (MSCs) are involved in the regulation of vessel structure homeostasis, we investigated the role of Tat, a key factor in HIV replication and pathogenesis, in MSC survival and differentiation. The survival of subconfluent MSCs was impaired when Tat was added at high concentrations (200–1,000 ng/ml), whereas lower Tat concentrations (1–100 ng/ml) did not promote apoptosis. Tat enhanced the differentiation of MSC toward adipogenesis by the transcription and activity upregulation of PPARγ. This Tat‐related modulation of adipogenesis was tackled by treatment with antagonists of Tat‐specific receptors such as SU5416 and RGD Fc. In contrast, Tat inhibited the differentiation of MSCs to endothelial cells by downregulating the expression of VEGF‐induced endothelial markers such as Flt‐1, KDR, and vWF. The treatment of MSCs with Tat‐derived peptides corresponding to the cysteine‐rich, basic, and RGD domains indicated that these Tat regions are involved in the inhibition of endothelial marker expression. The Tat‐related impairment of MSC survival and differentiation might play an important role in vessel damage and formation of the atherosclerotic lesions observed in HIV‐infected patients. J. Cell. Biochem. 113: 1132–1141, 2012.

Prevalence of R5 strains in multi-treated HIV subjects and impact of new regimens including maraviroc in a selected group of patients with CCR5-tropic HIV-1 infection

OBJECTIVES Maraviroc currently represents an important antiretroviral drug for multi-experienced and viremic HIV patients. This study focused on two main points: (1) determining the prevalence of R5 and X4 HIV strains in antiretroviral-experienced patients using two main tests currently in use to determine viral tropism, and (2) the follow-up to 3 years of a limited number of patients who started a new antiretroviral protocol including maraviroc. METHODS A group of 56 HIV patients, previously multi-treated, were first analyzed by genotyping assay and Trofile™ to establish their eligibility for maraviroc treatment. In addition, 25 subjects selected to follow a new therapeutic protocol including a CCR5 antagonist were monitored by HIV RNA viral load and CD4+ cell count. RESULTS The determination of viral tropism showed a large percentage of patients with an R5 profile (72% by genotyping assay and 74% by Trofile). The follow-up of most (21 out 25) patients who started the new antiretroviral protocol showed an undetectable viral load throughout the observation period, accompanied by a major improvement in CD4 cell count (cells/mm(3)) (baseline: median CD4 cell count 365, interquartile range (IQR) 204-511; 12 months: median value 501, IQR 349-677, p=0.042; 24 months: median value 503, IQR 386-678, p=0.026; 36 months: median value 601, IQR 517-717, p=0.001). Among the four non-responder subjects, two showed a lack of drug compliance and two switched from R5 to X4. CONCLUSION Although our patient cohort was small, the results showed a high prevalence of R5 viral strains in multi-experienced patients. As well as showing the advantages of genotyping, which can be performed in plasma samples with low viral load replication, the follow-up of HIV patients selected for an alternative drug protocol, including a CCR5 antagonist, showed a persistent undetectable viral replication and a good recovery of CD4 cell count in most treated HIV patients.

IFI16 Expression Is Related to Selected Transcription Factors during B-Cell Differentiation

The interferon-inducible DNA sensor IFI16 is involved in the modulation of cellular survival, proliferation, and differentiation. In the hematopoietic system, IFI16 is consistently expressed in the CD34+ stem cells and in peripheral blood lymphocytes; however, little is known regarding its regulation during maturation of B- and T-cells. We explored the role of IFI16 in normal B-cell subsets by analysing its expression and relationship with the major transcription factors involved in germinal center (GC) development and plasma-cell (PC) maturation. IFI16 mRNA was differentially expressed in B-cell subsets with significant decrease in IFI16 mRNA in GC and PCs with respect to naïve and memory subsets. IFI16 mRNA expression is inversely correlated with a few master regulators of B-cell differentiation such as BCL6, XBP1, POU2AF1, and BLIMP1. In contrast, IFI16 expression positively correlated with STAT3, REL, SPIB, RELA, RELB, IRF4, STAT5B, and STAT5A. ARACNE algorithm indicated a direct regulation of IFI16 by BCL6, STAT5B, and RELB, whereas the relationship between IFI16 and the other factors is modulated by intermediate factors. In addition, analysis of the CD40 signaling pathway showed that IFI16 gene expression directly correlated with NF-κB activation, indicating that IFI16 could be considered an upstream modulator of NF-κB in human B-cells.

Cold Atmospheric Plasma Treatment of Infected Skin Tissue: Evaluation of Sterility, Viability, and Integrity

Sterilization of equipment and tissues is a common clinical practice: there are different chemical, mechanical, and electromagnetic aseptic techniques for inactivating microorganisms. In particular, skin tissue banks are investigating new methods to efficiently decolonize skin tissues, while preserving their structural features. In recent years, cold atmospheric plasma (CAP) has demonstrated bactericidal, virucidal, and fungicidal properties, due to the generation of reactive species and charged particles. For this reason, the aim of this paper is to demonstrate that the implementation of a dielectric barrier discharge (DBD) treatment in air can effectively decontaminate skin tissue from Staphylococcus aureus, retaining cell viability and skin integrity. Fresh skin samples, taken from multitissue donors, were contaminated with Staphylococcus aureus and treated with a DBD source, to verify the level of bacterial decontamination induced by plasma. Cell viability and structural properties of skin tissue were investigated using MTT assay and hematoxylin-eosin staining, respectively. Our results show that CAP can sterilize skin tissue with a bacterial load up to 103 CFU/cm2; moreover, it does not affect cell viability, and no loss of skin structural properties was observed. Thus, CAP treatment could be considered an innovative method for decolonization of human skin, without inducing any microscopic tissue damage, while keeping good cell viability.

Non-equilibrium atmospheric pressure plasma as innovative method to crosslink and enhance mucoadhesion of econazole-loaded gelatin films for buccal drug delivery

In this paper we developed an innovative, effective and rapid one-step approach to crosslink mucoadhesive gelatin films for buccal drug delivery. The method, which involves the application of non-equilibrium pressure plasma for 3 or 5 minutes/side, was compared with a classical approach based on the use of a chemical crosslinking agent, namely genipin. Econazole nitrate (ECN), an imidazole antifungal agent used for the treatment of skin infections and mucosal candidiasis, was selected as model drug. X-Ray Diffraction characterization performed on the drug-containing gelatin films revealed that ECN undergoes to a topotactic transformation into Econazole (EC) immediately after mixing with gelatin suggesting the occurrence of an acid-base reaction between drug and gelatin during film processing. Plasma treatment, as well as genipin crosslinking, did not provoke any further variation of EC structure. However, plasma exposure significantly improved films adhesiveness and allowed to reach mucoadhesive strength values more than double with respect to those obtained with genipin, ascribable to the presence of polar and hydrophilic groups on the plasma treated films surface. A residence time of at least 48 h was obtained by properly selecting the plasma exposure times. These results, together with the in-vitro data showing retention of antifungal efficacy against a strain of Candida albicans, demonstrated that plasma treatment was a valid and rapid alternative, easy to scale-up, to chemical crosslinking methods for the production of highly mucoadhesive gelatin-based films.