Dual regulation of L-selectin (CD62L) by HIV-1: Enhanced expression by Vpr in contrast with cell-surface down-modulation by Nef and Vpu.

The HIV-1 accessory protein Vpr displays various activities that can favor viral replication such as G2 cell cycle arrest. Vpr also modulates host gene expression, although this property is poorly characterized. Here, we investigated the effect of Vpr on L-selectin (CD62L), which crucially controls leukocytes circulation and generation of immune responses against pathogens. We report that Vpr up-regulates CD62L mRNA level when individually expressed in Jurkat T cells as well as during HIV-1 infection of primary CD4+ T cells. Vpr mutant analysis and use of inhibitors suggest that the effect of Vpr on CD62L occurs independently of G2 arrest but requires activation of the ATR kinase. Yet, induction of CD62L expression by Vpr is contrasted by down-regulation of CD62L protein by Nef that, together with Vpu, induces a net reduction of cell-surface CD62L on HIV-1-infected cells, which may impact viral spread and evasion of immune responses.

NK cells of HIV-1-infected patients with poor CD4+ T-cell reconstitution despite suppressive HAART show reduced IFN-γ production and high frequency of autoreactive CD56bright cells.

HIV-1-infected patients failing to recover CD4+ T-cell count despite HAART (immunological non-responders, NRs), are at increased risk of disease progression and death. To better understand the NR status, we performed a comprehensive assessment of NK cells in NR patients as compared to immunologic responders. NRs exhibited an accumulation of CD56bright NK cells inversely correlated with CD4+ counts. Both CD56bright and CD56dim NK cells of NRs displayed unimpaired degranulation ability, but poorly responded to cytokine stimulation in terms of NKp44 up-regulation and IFN-γ production that may explain the susceptibility of NRs to infections and tumors. Notably, CD56bright NK cells from NRs showed higher cytotoxicity against autologous activated CD4+ T cells. Moreover, NRs had reduced Treg cell counts that showed an inverse correlation with autoreactive CD56bright cells. These data suggest that accumulation of CD56bright NK cells, possibly linked to decreased homeostatic control by Tregs, contributes to poor immune reconstitution in NRs.

Brief Report: L-Selectin (CD62L) Is Downregulated on CD4+ and CD8+ T Lymphocytes of HIV-1-Infected Individuals Naive for ART.

The expression of L-selectin (CD62L) in HIV-1 infection has not been extensively investigated. Here, we measured CD62L expression on T-cell subsets of HIV-1-infected individuals naive for antiretroviral therapy (ART-naive) or receiving therapy (ART), and seronegative control subjects (HIV-neg). We found reduced frequencies of CD62L(+) cells among CD4(+) and CD8(+) T cells from ART-naive as compared with ART and HIV-neg groups, particularly within naive and central memory subsets. CD62L expression on T cells inversely correlated with viral load and rapidly increased after ART initiation. Plasma sCD62L levels did not correlate with CD62L expression, being higher in all HIV-1-infected individuals as compared with HIV-neg subjects. Finally, CD62L downregulation was found associated with the expression of the CD38 activation marker in CD8(+) T cells, but not in CD4(+) T cells. We suggest that CD62L downregulation due to unconstrained HIV-1 replication may have important consequences for T-cell circulation and function and for disease progression.

Release of Soluble Ligands for the Activating NKG2D Receptor: One More Immune Evasion Strategy Evolved by HIV-1 ?

Increasing lines of evidence indicate that NKG2D, an activating receptor of natural killer (NK) and CD8(+) T cells, plays an important role in immune responses against HIV-1. Through its ability to recognize a diverse array of ligands (NKG2DLs) induced by cell 'stress' such as viral infection, NKG2D delivers activating and co-stimulatory signals resulting in cytotoxicity and release of cytokines. Therefore, HIV-1 and other viruses have evolved clever mechanisms to counteract NKG2D-dependent immune responses. While, on one hand, the HIV-1 Vpr protein up-regulates NKG2DLs expression by activating the DNA damage response (DDR) pathway, other viral proteins (Nef and Vif) have developed the capacity to reduce NKG2DLs expression levels. In addition, recent evidences suggest that HIV-1-infected CD4(+) T cells may release NKG2DLs, particularly MICA, in soluble form, a phenomenon that has the potential to down-modulate NKG2D on circulating lymphocytes and allow evasion of NKG2D-mediated immune responses. Indeed, despite controversial, lower NKG2D expression was found on both NK and CD8(+) T cells in HIV-1-infected patients. This review discusses recent advances in the understanding of how HIV-1 affects the NKG2D/NKG2DLs system, with a special focus on virus-induced release of soluble NKG2DLs and its functional implications for the immune surveillance of the infected host.

Expression and Function of NKG2D Is Impaired in CD8+ T Cells of Chronically HIV-1-Infected Patients Without ART.

OBJECTIVES: Increasing line of evidence indicates that the NKG2D-activating receptor plays a relevant role in the effector functions of cytotoxic lymphocytes. In this study, we investigated the expression and function of NKG2D in CD8⁺ T cells from chronically HIV-1-infected patients with or without antiretroviral therapy (ART). METHODS: We measured by flow cytometry the expression of NKG2D on CD8⁺ T-cell subsets of ART-naive and ART patients as well as seronegative healthy subjects (HIV-neg). An intrapatient analysis before and after ART initiation was also performed. Results were correlated with viral load, CD4⁺ T-cell counts, markers of immune activation (CD38, sCD14), and soluble NKG2D ligands (sMICA and sULBP2). The function of NKG2D on CD8⁺ T cell cytotoxicity was tested by ex vivo degranulation assays. RESULTS: We showed that NKG2D was downregulated on all CD8⁺ T-cell subsets of ART-naive patients. The expression of NKG2D on CD8⁺ T cells inversely correlated with viral load and CD38 expression but not with plasma levels of sMICA and sULBP2. Importantly, we found that NKG2D-mediated costimulation of CD8⁺ T-cell lytic activity was strongly reduced in ART-naive patients if compared with HIV-neg and ART subjects. Finally, intrapatient analysis demonstrated that effective anti-HIV-1 therapy restores NKG2D expression and NKG2D-induced cytotoxicity by CD8⁺ T cells. CONCLUSIONS: These data underscore that NKG2D downregulation contributes to impaired CD8⁺ T-cell responses in untreated HIV-1 infection and have implications for monitoring immune functions and response to treatments, and for the development of novel anti-HIV-1 strategies combining ART with drugs that stimulate NKG2D expression and function.

Behavioral and emotional profile and parental stress in preschool children with autism spectrum disorder.

Parents of children with autism spectrum disorder (ASD) were shown to experience more stress than parents of typically developing peers, although little is known about risk factors predicting stress in this population. The aim of this study was to evaluate parental stress levels and behavioral and emotional problems in a sample of preschool children with ASD as compared to typically developing (TD) peers and to investigate the role of several factors, including the severity of autistic symptoms, adaptive skills, cognitive abilities and behavioral and emotional problems, on parental stress. Results confirmed that parents of children with ASD experience higher stress levels than parents of TD and that children with ASD show more behavioral and emotional problems than controls. Moreover, our results showed that behavioral and emotional problems are strong predictors of parental stress, while stress related to a parent-child dysfunctional relationship was associated with daily living and communication skills as well as cognitive abilities. Findings revealed different behavioral and emotional problems affecting parental stress in ASD and TD samples. No association between the severity of autism symptoms and parental stress was detected. These results suggest that dysfunctional behaviors in preschool children with ASD have a strong impact on parental stress, profoundly affecting the well-being of the entire family. Therefore, strategies aimed at the early detection and management of these behavioral and emotional problems are crucial in order to prevent parental stress and to develop the most appropriate treatment interventions.

HIV-1 Nef and Vpu Interfere with L-Selectin (CD62L) Cell Surface Expression To Inhibit Adhesion and Signaling in Infected CD4+ T Lymphocytes.

UNLABELLED: Leukocyte recirculation between blood and lymphoid tissues is required for the generation and maintenance of immune responses against pathogens and is crucially controlled by the L-selectin (CD62L) leukocyte homing receptor. CD62L has adhesion and signaling functions and initiates the capture and rolling on the vascular endothelium of cells entering peripheral lymph nodes. This study reveals that CD62L is strongly downregulated on primary CD4(+) T lymphocytes upon infection with human immunodeficiency virus type 1 (HIV-1). Reduced cell surface CD62L expression was attributable to the Nef and Vpu viral proteins and not due to increased shedding via matrix metalloproteases. Both Nef and Vpu associated with and sequestered CD62L in perinuclear compartments, thereby impeding CD62L transport to the plasma membrane. In addition, Nef decreased total CD62L protein levels. Importantly, infection with wild-type, but not Nef- and Vpu-deficient, HIV-1 inhibited the capacity of primary CD4(+) T lymphocytes to adhere to immobilized fibronectin in response to CD62L ligation. Moreover, HIV-1 infection impaired the signaling pathways and costimulatory signals triggered in primary CD4(+) T cells by CD62L ligation. We propose that HIV-1 dysregulates CD62L expression to interfere with the trafficking and activation of infected T cells. Altogether, this novel HIV-1 function could contribute to virus dissemination and evasion of host immune responses. IMPORTANCE: L-selectin (CD62L) is an adhesion molecule that mediates the first steps of leukocyte homing to peripheral lymph nodes, thus crucially controlling the initiation and maintenance of immune responses to pathogens. Here, we report that CD62L is downmodulated on the surfaces of HIV-1-infected T cells through the activities of two viral proteins, Nef and Vpu, that prevent newly synthesized CD62L molecules from reaching the plasma membrane. We provide evidence that CD62L downregulation on HIV-1-infected primary T cells results in impaired adhesion and signaling functions upon CD62L triggering. Removal of cell surface CD62L may predictably keep HIV-1-infected cells away from lymph nodes, the privileged sites of both viral replication and immune response activation, with important consequences, such as systemic viral spread and evasion of host immune surveillance. Altogether, we propose that Nef- and Vpu-mediated subversion of CD62L function could represent a novel determinant of HIV-1 pathogenesis.

Combination of the CCL5-derived peptide R4.0 with different HIV-1 blockers reveals wide target compatibility and synergic cobinding to CCR5.

R4.0, a synthetic CCL5/RANTES-derived peptide, exerts potent anti-HIV-1 activity via its nonactivating interaction with CCR5, the major HIV-1 coreceptor. CCR5 chronic activation may promote undesirable inflammatory effects and enhance viral infection; thus, receptor antagonism is a necessary requisite. HIV-1 gp120, CCL5, and maraviroc dock on CCR5 by sharing two receptor sites: the N terminus and the second extracellular loop. In combination studies, R4.0, CCL5, and maraviroc exhibited concomitant interactions with CCR5 and promoted synergic inhibition of HIV-1 in acute-infection assays. Furthermore, various degrees of additive/synergic HIV-1 inhibition were observed when R4.0 was tested in combination with drugs and lead compounds directed toward different viral targets (gp120, gp41, reverse transcriptase, and protease). In combination with tenofovir, R4.0 provides cross-clade synergic inhibition of primary HIV-1 isolates. Remarkably, an in vitro-generated maraviroc-resistant R5 HIV-1 strain was inhibited by R4.0 comparably to the wild-type strain, suggesting the presence of viral resistance barriers similar to those reported for CCL5. Overall, R4.0 appears to be a promising lead peptide with potential for combination in anti-HIV-1 therapy and in microbicide development to prevent sexual HIV-1 transmission.

The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222.

Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.

The human immunodeficiency virus type 1 Vpr protein upregulates PVR via activation of the ATR-mediated DNA damage response pathway.

Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses. Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr protein in Jurkat T cells increases cell-surface and total PVR levels. Analysis of mutated Vpr variants indicated that Vpr uses the same protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase. Moreover, we found that PVR upregulation by Vpr relied on the ability of the protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest. Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4(+) T lymphocytes and inhibits the PVR downregulating activity of the viral Nef protein.

Paediatric non-alcoholic Fatty liver disease: impact on patients and mothers' quality of life.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is one of the causes of fatty liver in adults and is currently the primary form of chronic liver disease in children and adolescents. However, the psychological outcome (i.e. the behavioural problems that can in turn be related to psychiatric conditions, like anxiety and mood disorders, or lower quality of life) in children and adolescents suffering of NAFLD has not been extensively explored in the literature. OBJECTIVES: The present study aims at evaluating the emotional and behavioural profile in children suffering from NAFLD and the quality of life in their mothers. PATIENTS AND METHODS: A total of 57 children (18 females/39 males) with NAFLD were compared to 39 age-matched control children (25 females/14 males). All participants were submitted to the following psychological tools to assess behavior, mood, and anxiety: the Multidimensional Anxiety Scale for Children (MASC), the Child Behavior Checklist (CBCL), and the Children's Depression Inventory (CDI). Moreover, the mothers of 40 NAFLD and 39 control children completed the World Health Organization Quality of Life-BREF (WHOQOL-BREF) questionnaire. RESULTS: NAFLD children scored significantly higher as compared to control children in MASC (P = 0.001) and CDI total (P < 0.001) scales. The CBCL also revealed significantly higher scores for NAFLD children in total problems (P = 0.046), internalizing symptoms (P = 0.000) and somatic complaints (P < 0.001). The WHOQOL-BREF revealed significantly lower scores for the mothers of NAFLD children in the overall perception of the quality of life (P < 0.001), and in the "relationships" domain (P = 0.023). CONCLUSIONS: Increased emotional and behavioural problems were detected in children with NAFLD as compared to healthy control children, together with an overall decrease in their mothers' quality of life. These results support the idea that these patients may benefit from a psychological intervention, ideally involving both children and parents, whose quality of life is likely negatively affected by this disease.

Longitudinal neuropsychological profile in a patient with triple a syndrome.

Triple A syndrome is an autosomal recessive disorder characterized by the triad of adrenocorticotropic hormone resistant adrenal insufficiency, achalasia, and alacrima. Our aim was to describe the neuropsychological characteristics and the cooccurring psychopathological and neurological disorders in an Italian male child suffering from Triple A syndrome at the time of admission (T0) and after one year of follow-up (T1). Many difficulties were observed in the motor domain, as well as in manual dexterity and static/dynamic balance domains of the motor task over time. In sharp contrast with previous literature reports on frequent mild cognitive dysfunction in patients with Triple A syndrome, our child did not show any mental retardation. By contrast, he showed an average IQ at T0 with a slight improvement at T1. To our knowledge, this report is the first describing neuropsychological profile and co-occurring psychopathological problems in a child with Triple A syndrome. Considering that the Triple A syndrome is a progressive disorder which can take years to develop the full-blown clinical picture, these patients require periodical medical controls. Moreover, assessment of neuropsychological and psychopathological features should be performed in patients with this disease, in order to underline the variability of this syndrome.

Enhancement of anti-HIV-1 activity by hot spot evolution of RANTES-derived peptides.

CCR5, the major HIV-1 coreceptor, is a primary target for HIV-1 entry inhibition strategies. CCL5/RANTES, a natural CCR5 ligand, is one of the most potent HIV-1 entry inhibitors and, therefore, an ideal candidate to derive HIV-1 blockers. Peptides spanning the RANTES N-loop/ß1-strand region act as specific CCR5 antagonists, with their hydrophobic N- and C termini playing a crucial role in virus blockade. Here, hydrophobic surfaces were enhanced by tryptophan substitution of aromatic residues, highlighting position 27 as a critical hot spot for HIV-1 blockade. In a further molecular evolution step, C-terminal engraftment of RANTES 40' loop produced a peptide with the highest solubility and anti-HIV-1 activity. These modified peptides represent leads for the development of effective HIV-1 inhibitors and microbicides.

Inhibition of HIV-1 infection by human α-defensin-5, a natural antimicrobial peptide expressed in the genital and intestinal mucosae.

BACKGROUND: α-defensin-5 (HD5) is a key effector of the innate immune system with broad anti-bacterial and anti-viral activities. Specialized epithelial cells secrete HD5 in the genital and gastrointestinal mucosae, two anatomical sites that are critically involved in HIV-1 transmission and pathogenesis. We previously found that human neutrophil defensins (HNP)-1 and -2 inhibit HIV-1 entry by specific bilateral interaction both with the viral envelope and with its primary cellular receptor, CD4. Despite low amino acid identity, human defensin-5 (HD5) shares with HNPs a high degree of structural homology. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that HD5 inhibits HIV-1 infection of primary CD4(+) T lymphocytes at low micromolar concentration under serum-free and low-ionic-strength conditions similar to those occurring in mucosal fluids. Blockade of HIV-1 infection was observed with both primary and laboratory-adapted strains and was independent of the viral coreceptor-usage phenotype. Similar to HNPs, HD5 inhibits HIV-1 entry into the target cell by interfering with the reciprocal interaction between the external envelope glycoprotein, gp120, and CD4. At high concentrations, HD5 was also found to downmodulate expression of the CXCR4 coreceptor, but not of CCR5. Consistent with its broad spectrum of activity, antibody competition studies showed that HD5 binds to a region overlapping with the CD4- and coreceptor-binding sites of gp120, but not to the V3 loop region, which contains the major determinants of coreceptor-usage specificity. CONCLUSION/SIGNIFICANCE: These findings provide new insights into the first line of immune defense against HIV-1 at the mucosal level and open new perspectives for the development of preventive and therapeutic strategies.

IL-7 induces expression and activation of integrin α4ß7 promoting naive T-cell homing to the intestinal mucosa.

Interleukin-7 (IL-7) is a nonredundant cytokine that plays a critical role in T-cell homeostasis and promotes immunologic reconstitution in lymphopenic hosts. Here, we show that IL-7, at doses that reflect suprahomeostatic concentrations achieved in lymphopenic hosts, is a potent and selective inducer of the gut-homing integrin α4ß7 in human T cells, as documented both ex vivo and in vivo in patients enrolled in a clinical trial of IL-7 treatment. Induction of α4ß7 by IL-7 occurs primarily in naive T cells and is associated with functional activation of the integrin, as indicated by increased binding activity for the specific α4ß7 ligand, MAdCAM-1. The physiologic relevance of these findings was validated by the preferential homing of IL-7-treated naive human T cells to the intestinal compartment in humanized NOD/SCID/IL-2 receptor-γ(null) (NSG) mice. We also show that IL-7 triggers a peculiar activation program in naive T cells, characterized by the acquisition of memory-like phenotypic features and proliferation uncoupled from expression of classic T-cell activation markers. These findings provide a mechanism for the transient in vivo depletion of circulating T cells after IL-7 administration and suggest that intestinal homing and memory-like conversion of naive T cells are critical steps in the IL-7-driven immunologic reconstitution of lymphopenic hosts.

Treatment with IL-7 prevents the decline of circulating CD4+ T cells during the acute phase of SIV infection in rhesus macaques.

Although treatment with interleukin-7 (IL-7) was shown to transiently expand the naïve and memory T-cell pools in patients with chronic HIV-1 infection receiving antiretroviral therapy (ART), it is uncertain whether a full immunologic reconstitution can be achieved. Moreover, the effects of IL-7 have never been evaluated during acute HIV-1 (or SIV) infection, a critical phase of the disease in which the most dramatic depletion of CD4(+) T cells is believed to occur. In the present study, recombinant, fully glycosylated simian IL-7 (50 µg/kg, s.c., once weekly for 7 weeks) was administered to 6 rhesus macaques throughout the acute phase of infection with a pathogenic SIV strain (mac251); 6 animals were infected at the same time and served as untreated controls. Treatment with IL-7 did not cause clinically detectable side effects and, despite the absence of concomitant ART, did not induce significant increases in the levels of SIV replication except at the earliest time point tested (day 4 post-infection). Strikingly, animals treated with IL-7 were protected from the dramatic decline of circulating naïve and memory CD4(+) T cells that occurred in untreated animals. Treatment with IL-7 induced only transient T-cell proliferation, but it was associated with sustained increase in the expression of the anti-apoptotic protein Bcl-2 on both CD4(+) and CD8(+) T cells, persistent expansion of all circulating CD8(+) T-cell subsets, and development of earlier and stronger SIV Tat-specific T-cell responses. However, the beneficial effects of IL-7 were not sustained after treatment interruption. These data demonstrate that IL-7 administration is effective in protecting the CD4(+) T-cell pool during the acute phase of SIV infection in macaques, providing a rationale for the clinical evaluation of this cytokine in patients with acute HIV-1 infection.

Brief report: peculiar evolution of autistic behaviors in two unrelated children with brachidactyly-mental retardation syndrome.

Brachidactyly-Mental Retardation (BDMR) Syndrome (MIM 600430) is associated with terminal deletions at chromosome 2q37 and a limited number of studies also reported an association between 2q37 â†’ qter deletion and autism. Herein we describe two cases of autism in unrelated children with BDMR Syndrome, showing physical, cognitive, behavioral, and disease natural history homologies, with a very prominent social impairment in the first 4 years of life. At follow-up evaluations, spanning a 5-years period, both children experienced a progressive reduction of the autistic symptoms, besides retaining compromised cognitive ability. This report supports the hypothesis that genes in the 2q37 region may contribute to the etiology of autism, leading, however, to a peculiar evolution of the disease, with symptoms severity decreasing over time.

Interleukin 7 reduces the levels of spontaneous apoptosis in CD4+ and CD8+ T cells from HIV-1-infected individuals.

Apoptosis has been suggested as one of the major mechanisms of CD4+ T cell depletion during the course of HIV type 1 (HIV-1) infection. Here, we show that interleukin 7 (IL-7), a nonredundant cytokine that plays essential roles in the generation and homeostasis of the T cell compartment of the immune system, exerts strong antiapoptotic effects ex vivo on both CD4+ and CD8+ T cells derived from HIV-1-infected subjects. The level of IL-7-mediated reduction of apoptosis was inversely correlated with the number of circulating CD4+ T cells, indicating a higher sensitivity to IL-7 effects in patients with more advanced disease. The antiapoptotic effect of IL-7 was uncoupled from the induction of cellular proliferation or endogenous HIV-1 replication. These results provide a further rationale for consideration of IL-7 as an agent of immune reconstitution in HIV-1 infection.

Alpha-defensins block the early steps of HIV-1 infection: interference with the binding of gp120 to CD4.

Alpha-defensins are antibiotic peptides that act as natural inhibitors of HIV-1 infection. However, the mechanisms of such inhibition are still unclear. Here we demonstrate that alpha-defensins block the earliest steps in the viral infectious cycle, as documented using an HIV-1 envelope-mediated cell-fusion assay. A broad-spectrum inhibitory activity was observed on primary and laboratory-adapted HIV-1 isolates irrespective of their coreceptor specificity and genetic subtype. A primary mechanism of such inhibition was identified as the ability of alpha-defensins to bind specifically both to the primary HIV-1 cellular receptor, CD4, and to the viral envelope glycoprotein, gp120. Moreover, treatment of CD4+ T cells with alpha-defensins caused a dramatic downmodulation of CD4 expression. By monoclonal antibody competition, the regions of interaction with alpha-defensins were mapped to the D1 domain of CD4 and to a surface contiguous to the CD4- and coreceptor-binding sites of gp120. Consistent with these findings, alpha-defensins inhibited the binding of gp120 to CD4. These data demonstrate that alpha-defensins specifically block the initial phase of the HIV infectious cycle and modulate the expression of CD4, a critical receptor in the physiology of T-cell activation.