Giuseppe Nunnari

IL-7 is a potent and proviral strain–specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART

The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.

Alpha interferon potently enhances the anti-human immunodeficiency virus type 1 activity of APOBEC3G in resting primary CD4 T cells

ABSTRACT The interferon (IFN) system, including various IFNs and IFN-inducible gene products, is well known for its potent innate immunity against wide-range viruses. Recently, a family of cytidine deaminases, functioning as another innate immunity against retroviral infection, has been identified. However, its regulation remains largely unknown. In this report, we demonstrate that through a regular IFN-α/β signal transduction pathway, IFN-α can significantly enhance the expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) in human primary resting but not activated CD4 T cells and the amounts of APOBEC3G associated with a low molecular mass. Interestingly, short-time treatments of newly infected resting CD4 T cells with IFN-α will significantly inactivate human immunodeficiency virus type 1 (HIV-1) at its early stage. This inhibition can be counteracted by APOBEC3G-specific short interfering RNA, indicating that IFN-α-induced APOBEC3G plays a key role in mediating this anti-HIV-1 process. Our data suggest that APOBEC3G is also a member of the IFN system, at least in resting CD4 T cells. Given that the IFN-α/APOBEC3G pathway has potent anti-HIV-1 capability in resting CD4 T cells, augmentation of this innate immunity barrier could prevent residual HIV-1 replication in its native reservoir in the post-highly active antiretroviral therapy era.

Hepatic echinococcosis: Clinical and therapeutic aspects

Echinococcosis or hydatid disease (HD) is a zoonosis caused by the larval stages of taeniid cestodes belonging to the genus Echinococcus. Hepatic echinococcosis is a life-threatening disease, mainly differentiated into alveolar and cystic forms, associated with Echinoccus multilocularis (E. multilocularis) and Echinococcus granulosus (E. granulosus) infection, respectively. Cystic echinococcosis (CE) has a worldwide distribution, while hepatic alveolar echinococcosis (AE) is endemic in the Northern hemisphere, including North America and several Asian and European countries, like France, Germany and Austria. E. granulosus young cysts are spherical, unilocular vesicles, consisting of an internal germinal layer and an outer acellular layer. Cyst expansion is associated with a host immune reaction and the subsequent development of a fibrous layer, called the pericyst; old cysts typically present internal septations and daughter cysts. E. multilocularis has a tumor-like, infiltrative behavior, which is responsible for tissue destruction and finally for liver failure. The liver is the main site of HD involvement, for both alveolar and cystic hydatidosis. HD is usually asymptomatic for a long period of time, because cyst growth is commonly slow; the most frequent symptoms are fatigue and abdominal pain. Patients may also present jaundice, hepatomegaly or anaphylaxis, due to cyst leakage or rupture. HD diagnosis is usually accomplished with the combined use of ultrasonography and immunodiagnosis; furthermore, the improvement of surgical techniques, the introduction of minimally invasive treatments [such as puncture, aspiration, injection, re-aspiration (PAIR)] and more effective drugs (such as benzoimidazoles) have deeply changed life expectancy and quality of life of patients with HD. The aim of this article is to provide an up-to-date review of biological, diagnostic, clinical and therapeutic aspects of hepatic echinococcosis.

Intensification and Stimulation Therapy for Human Immunodeficiency Virus Type 1 Reservoirs in Infected Persons Receiving Virally Suppressive Highly Active Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) has led to significant changes in mortality and morbidity in the human immunodeficiency virus type 1 (HIV-1) epidemic. Nevertheless, because of molecular mechanisms of viral persistence, HAART does not eradicate HIV-1. Didanosine and hydroxyurea were added to the antiretroviral regimens of 3 HIV-1-infected men who were receiving stable HAART and who had HIV-1 RNA levels <50 copies/mL at the initiation of the study protocol, as a novel intensification to attack cryptic viral replication; low-dose OKT3 was then administered, followed by a course of interleukin-2, to stimulate latent provirus. Replication-competent virus was undetectable after treatment, and plasma viral RNA was either undetectable or <5 copies/mL. In trial periods during which no antiretroviral therapy was administered, the patients developed plasma viral rebound. This translational approach combines novel intensification and stimulation therapy to deplete residual HIV-1 reservoirs. Additional experimental approaches must be developed if HIV-1 eradication is to become possible in patients receiving virally suppressive HAART.

Human Immunodeficiency Virus Type 1 Enters Primary Human Brain Microvascular Endothelial Cells by a Mechanism Involving Cell Surface Proteoglycans Independent of Lipid Rafts

ABSTRACT Several studies have reported a crucial role for cholesterol-enriched membrane lipid rafts and cell-associated heparan sulfate proteoglycans (HSPGs), a class of molecules that can localize in lipid rafts, in the entry of human immunodeficiency virus type 1 (HIV-1) into permissive cells. For the present study, we examined the role of these cell surface moieties in HIV-1 entry into primary human brain microvascular endothelial cells (BMVECs), which represent an important HIV-1 central nervous system-based cell reservoir and a portal for neuroinvasion. Cellular cholesterol was depleted by exposure to β-cyclodextrins and 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitors (statins), the loss of cholesterol was quantitated, and disruption of membrane rafts was verified by immunofluorescence. Nevertheless, these treatments did not affect binding of several strains of HIV-1 virions to BMVECs at 4°C or their infectivities at 37°C. In contrast, we confirmed that cholesterol depletion and raft disruption strongly inhibited HIV-1 binding and infection of Jurkat T cells. Enzymatic digestion of cell-associated HSPGs on human BMVECs dramatically inhibited HIV-1 infection, and our data from quantitative HIV-1 DNA PCR analysis strongly suggest that cell-associated chondroitin sulfate proteoglycans greatly facilitate infective entry of HIV-1 into human BMVECs. These findings, in combination with our earlier work showing that human BMVECs lack CD4, indicate that the molecular mechanisms for HIV-1 entry into BMVECs are fundamentally different from that of viral entry into T cells, in which lipid rafts, CD4, and probably HSPGs play important roles.

Residual HIV-1 disease in seminal cells of HIV-1-infected men on suppressive HAART: latency without on-going cellular infections.

Background HIV-1-infected men on suppressive highly active antiretroviral therapy (HAART) have a reduction of viral replication in vivo, but HIV-1 RNA is still detectable by certain ultrasensitive reverse transcriptase–PCR assays in blood plasma. Replication-competent virus can also be isolated from both peripheral blood mononuclear cells (PBMC) and seminal cells of these patients. Despite HAART, on-going in vivo infection of HIV-1-seropositive patients’ PBMC was demonstrated by the detection of episomal HIV-1 moieties, known as HIV-1 two-long terminal repeat (2-LTR) DNA circles. Methods The present study analyzes whether new cellular infections occur in vivo in seminal cells of HIV-1-infected men on suppressive HAART. PBMC and seminal cells were isolated from a cohort of HIV-1-seropositive men taking suppressive HAART (< 50 copies HIV RNA/ml blood plasma). Viral growth assays were performed in vitro, as well as semi-quantitative PCR to detect HIV-1 2-LTR circular DNA in PBMC and seminal mononuclear cells. Results Viral growth in vitro was demonstrated in 16 out of 28 (57%) patients’ PBMC, and in five patients’ seminal cells (18%). Although 18 patients’ PBMC were positive for HIV-1 2-LTR DNA circles, importantly, 2-LTR circular DNA was not detected in any semen sample, even when replication-competent HIV-1 virus had been recovered from a patients seminal cells by viral co-culture assays. Conclusions The current study suggests that in HIV-1-infected men treated with suppressive HAART, new cellular infections occur in PBMC, but that new infections do not take place in seminal cells in vivo. Thus, these findings suggest that mainly latent HIV-1 occurs in seminal cells of men on suppressive HAART, which may be a compartment-specific mechanism of residual HIV-1 disease.

IL-7 as a potential therapy for HIV-1-infected individuals.

Highly active antiretroviral therapy (HAART), although effective in ameliorating the quality of life of HIV-1-infected individuals and their survival, has not been able to eradicate HIV-1. In fact, when HAART is interrupted, HIV-1 plasma viral load rebounds from viral reservoirs such as resting CD4+ T lymphocytes, monocytes and macrophages, remaining a major obstacle in attempting HIV eradication. Different therapeutic strategies have been attempted, such as structured treatment interruption (STI), immunotherapy (interleukin [IL]-2 and anti-CD3 antibodies [e.g., OKT3]), to try to stimulate HIV-1 out of latency along with antiretroviral intensification therapy. IL-7, a pleiotropic cytokine, bears diverse immune properties and plays a major role in T cell homeostasis. Moreover, IL-7 has recently been investigated as a possible immune adjuvant as well as a viral strain-specific inducer of HIV-1 replication. In fact, IL-7 was shown not only to be more effective than IL-2 in stimulating HIV-1 replication from resting CD4+ T lymphocytes ex vivo, but also to selectively induce a specific HIV-1 viral strain as compared with IL-2, suggesting the potential need for different viral inducers if complete eradication is to be achieved. In this present review, different immunological and virological properties of IL-7 are discussed, along with the possibility of its use as part of a combined antiretroviral-immune rationally based HIV-1 eradication approach.

Pathogenesis of HIV-1 Infection Within Bone Marrow Cells

Mononuclear phagocytic cells and CD4+ T lymphocytes represent the major targets for infection by HIV-1 in vivo. The most severe pathogenic features associated with HIV-1 infection can be attributed to malfunction or premature death of these cells that are of hematopoietic origin. Patients with acquired immunodeficiency syndrome (AIDS), suffer from many hematologic disorders, particularly those persons with long-term infection of HIV-1. These disorders include anemia, lymphocytopenia, thrombocytopenia and neutropenia. The mechanisms that lead to the induction of these disorders are multi-factorial. However, sufficient evidence has accumulated which suggests that HIV-1 infection of cells within the microenvironment of the bone marrow can lead to the induction of hematopoietic deficits. Most studies indicate that marrow-derived hematopoeitic stem cells cannot be infected by HIV-1 until they undergo modest differentiation in order to express the appropriate receptors to enable virus entry and subsequent replication. Some cells within the mixed environment of the marrow stroma appear to support HIV-1 replication however. These cells include marrow microvascular endothelial cells, sometimes referred to as blanket cells, stromal fibroblasts, as well as mononuclear phagocytes. Our recent experiments suggest that the HIV-1 accessory protein, Vpr, plays some role in the activation of marrow-derived mononuclear phagocytes which appears to result in premature phagocytosis of non-adherent marrow cells present in the in vitro cultures. This phenomenon could account, in part, for the induction of cytopenias that are typical of individuals infected by HIV-1.

Exogenous IL-7 induces Fas-mediated human neuronal apoptosis: potential effects during human immunodeficiency virus type 1 infection

The use of exogenous cytokines is part of translational immune-antiretroviral approaches to induce immune reconstitution and possibly eliminate the persistence of human immunodeficiency virus type 1 (HIV-1) in virally suppressed infected individuals on highly active antiretroviral therapy (HAART). Recently, our laboratories demonstrated that interleukin-7 (IL-7) has significant efficiency in stimulating HIV-1 replication from proviral latency in CD4+ T lymphocytes of infected patients. The authors now investigated the possible role of IL-7 in HIV-1-associated dementia (HAD). The authors demonstrated that the IL-7 receptor is expressed on both human neurons (i.e., differentiated NT2 cells) and human astrocytes, with relatively higher mRNA levels in neurons. The translational protein levels of IL-7 receptor α were not proportional to those of the mRNA levels in these central nervous system (CNS)-based cell types. Exogenous IL-7 was observed to only slightly down-regulate IL-7 receptor α expression on both neurons and astrocytes, as assayed by Western blotting. Instead of promoting survival, surprisingly, exogenous IL-7 induced neuronal apoptosis, as detected by TUNEL assays. Furthermore, IL-7 augmented neuronal apoptosis induced by HIV-1 gp120. Human apoptosis genomic microarray analyses of IL-7-treated human neurons showed up-regulated expression of proapoptotic genes: protein kinases, caspase-10, FAST kinase, tumor necrosis factor (TNF) receptor, and BCL2-antagonist of cell death. These data suggest that IL-7 leads to neuronal apoptosis by a molecular mechanism(s) that occurs via Fas-mediated activation-induced cell death. These studies may therefore not only be key in evaluating the potential use of IL-7 in vivo as a therapeutic modality, but also suggest that IL-7, which is increased endogenously in HIV-1-infected individuals late in disease, may be involved in the neuronal apoptosis demonstrated during HAD.

Pentoxifylline suppresses transduction by HIV-1-based vectors.

Pentoxifylline, a caffeine-related compound, was shown to suppress human immunodeficiency virus type 1 (HIV-1) replication. This effect is thought to be mediated by inhibition of tumor necrosis factor-alpha (TNFα)-mediated long-terminal repeat (LTR)-driven expression. We now demonstrate that pentoxifylline efficiently inhibits transduction by HIV-1-based vectors. This latter effect is independent of LTR-driven expression, and correlates with a reduced efficiency of the completion of the integration process in infected cells. Finally, the effect of pentoxifylline is dramatically reduced in cells expressing a dominant negative ATR protein, and in primary human cells that exhibit low level of ATR activity, suggesting that the effect of pentoxifylline on HIV-1 transduction and replication is at least partly mediated by suppression of the ATR kinase.