Biomedical subjects
Publications and source records attributed to S Ghezzi.
An Italian multigenerational family with four members affected by an axonal Charcot-Marie-Tooth type 2D (CMT-2D) or distal spinal muscular atrophy (dSMA) phenotype with upper limb predominance, variable age at onset, degree of disability, and autosomal dominant inheritance is reported. A novel heterozygous missense GARS gene mutation (D500N) was identified.
Plasma total lipids, total cholesterol (cholesterol esters and free cholesterol) and oxysterol (mainly 7 beta-hydroxycholesterol (7 beta OH)) concentrations were significantly elevated in New Zealand rabbits fed a 2% cholesterol-containing diet with respect to controls fed the same diet without cholesterol. In addition, linoleic (18:2 n-6) and alpha-linolenic acid (18:3 n-3) plasma concentrations were significantly elevated in hypercholesterolemic rabbits, while concentrations of long-chain n-6 and n-3 derivatives were reduced. Studies in monocytic cell line THP-1 revealed that 7 beta OH markedly inhibited the conversion of 18:2 to 20:4 n-6 and of 18:3 to 22:6 n-3, indicating depression of the desaturation steps; in particular the inhibition was greater for the Delta 5 desaturation step. Furthermore, experiments of Real-Time PCR showed that 5-10 microM 7 beta OH decreased the Delta 5 gene expression. In conclusion, atherogenic oxysterols interfere with the production of long-chain polyunsaturated fatty acids from their precursors both in hypercholesterolemic rabbits and in cultured cells.
The ideal microbicide must fulfill a number of criteria including a broad and potent activity against transmission of HIV and other sexually transmitted agents in the absence of toxicity and inflammation. We have described that derivatives of K5 polysaccharide from Escherichia coli inhibit HIV entry in target cells. K5 derivatives have a structure that resembles that of heparin, but they are devoid of the anticoagulant activity typical of heparin. Moreover, in contrast to heparin, they inhibit a broad spectrum of HIV-1 laboratory-adapted and primary isolates that use either CCR5 or CXCR4 or both coreceptors in terms of their infection and replication in primary CD4+ lymphocytes and monocytes-derived macrophages (MDM). Therefore, these compounds could be developed as candidate microbicides for preventing sexual HIV transmission, a predominant modality of HIV spreading in both the developed and underdeveloped world.
In THP-1 cells, simvastatin decreases, in a concentration-dependent manner, cholesterol synthesis and increases linoleic acid (LA) conversion to its long-chain derivatives, in particular to arachidonic acid, activating delta6 and delta5 fatty acid (FA) desaturases. The intermediates in cholesterol synthesis, mevalonate and geranylgeraniol, partially reverse the effects of simvastatin on the LA conversion. The aims of this work were to evaluate: (i) the correlation between cholesterol synthesis and desaturase activity and (ii) the possible involvement of protein isoprenylation in desaturase activity, assessed through pharmacological treatments. THP-1 cells were incubated with [1-14C]LA or with [1-14C]di-homo-gamma-linolenic acid (DHGLA) and treated with simvastatin or with curcumin and nicardipine, inhibitors of desaturases. Curcumin was more active than nicardipine in inhibiting LA and DHGLA conversion: 20 microM curcumin, alone or with simvastatin, totally inhibited delta6 and delta5 desaturation steps; 10 microM nicardipine only partially inhibited the enzymes, being more active on delta5 desaturase. Simvastatin treatment decreased the incorporation of acetate in cholesterol (-93.8%) and cholesterol esters (-70.2%), as expected. Curcumin and nicardipine also decreased cholesterol synthesis and potentiated simvastatin. Finally, the isoprenylation inhibitors (perillic acid and GGTI-286) neither affected the conversion of LA nor inhibited the delta5 desaturase activity. In conclusion, our results indicate that there is no direct relationship between cholesterol synthesis and desaturase activity. In fact, simvastatin decreased cholesterol synthesis and enhanced LA conversion (mainly delta5 desaturation), whereas curcumin and nicardipin decreased delta5 desaturation, with a limited effect on cholesterol synthesis.
To evaluate the safety and efficacy of 3 regimens of intermittent subcutaneous (sc) interleukin (IL)--2 in a phase 2 study, 61 antiviral drug-experienced human immunodeficiency virus (HIV)--positive patients were randomly assigned to one of the following study arms: antiretroviral therapy (ART) plus IL-2 (12 million IU [MIU] by continuous intravenous infusion, followed by 7.5 MIU twice a day, sc, every 8 weeks); ART plus IL-2 (7.5 MIU twice a day, sc, every 8 weeks); ART plus IL-2 (3 MIU twice a day, sc, every 4 weeks); or ART alone. A significant increase of circulating CD4 cells was observed in IL-2--treated subjects, compared with those given ART alone. Low doses of IL-2 were better tolerated. Despite the incomplete suppression of viral replication, IL-2 with ART did not increase either plasma viremia or cell-associated HIV DNA levels. Low doses of intermittent sc IL-2 induced a stable increase of peripheral CD4 cells that was indistinguishable from those associated with higher, less well-tolerated doses of IL-2.
The susceptibility of HIV-1 to chemokine-mediated inhibition may be lost as a consequence of the expanded usage of chemokine co-receptors frequently occurring in clade B isolates obtained from individuals with advanced disease. Since chemokine-based immune intervention is under intense investigation, it is crucial to determine its potential effect on primary dualtropic HIV isolates characterized by simultaneous utilization of CCR5 and CXCR4 chemokine co-receptors (R5X4 viruses). In the present study, the CCR5 binding chemokine regulated upon activation normal T cell expressed and secreted (RANTES) strongly inhibited the replication of two of eight primary R5X4 viruses in mitogen-activated primary peripheral blood mononuclear cells (PBMC). The CXCR4 antagonist AMD3100 efficiently suppressed the replication of other two HIV isolates, whereas the remaining four viruses were partially inhibited by treatment with either RANTES or AMD3100. The potency of chemokine-mediated inhibition was influenced by PBMC donor variability, but it was usually independent from the levels of expression of CCR5 or CXCR4. Dual co-receptor usage was maintained by the viruses after two serial passages on U87.CD4 astrocytic cell lines expressing exclusively either CCR5 or CXCR4. The gp120 env variable domains were sequenced before and after passages on U87.CD4 cells. Virus replication into U87.CD4-CXCR4 cells did not result in changes in the V3 region but perturbed the dominant env V4 sequence. Interestingly, double passage onto U87.CD4-CXCR4 cells determined the loss of susceptibility to RANTES inhibition. In conclusion, interference with CCR5 may efficiently inhibit the replication of at least some dualtropic HIV-1 strains, whereas forced CXCR4 usage may result in viral escape from CCR5-dependent inhibitory effects.
Intermittent administration of recombinant interleukin-2 (rIL-2) to individuals infected with human immunodeficiency virus (HIV) has been shown to raise and maintain the absolute number of circulating CD4(+) T cells to normal or near normal levels. One of the signaling pathways triggered by IL-2 is the Janus kinase-signal transducer and activator of transcription (JAK-STAT). In particular, IL-2 activates the tyrosine kinases JAK1 and JAK3 and the transcription factors STAT3 and STAT5. We have previously observed that most HIV(+) individuals, unlike healthy seronegative controls, show a constitutive activation of STAT1 and a C-terminal truncated isoform of STAT5 (STAT5 Delta). In the present study, we have analyzed the protein level and activation state of STAT5 isoforms expressed in peripheral blood mononuclear cells of two HIV-infected individuals who showed a good or a poor response to intermittent IL-2 administration, respectively, and of a single individual before and after initiation of Zidovudine monotherapy. We provide evidence that both therapeutic interventions enhanced the expression and activation of the C-terminal truncated isoform of STAT5 (STAT5 Delta) in vivo.
Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HIV) infected individuals characterized by stable or even increasing CD4+ T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8+ T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts. Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8+ population with memory CD45RO+ phenotype, with the former being CD28- and the latter CD28+. Longitudinal studies highlighted a relatively stable HIV-specific effector repertoire, viremia, and CD4+ T-cell counts, which were all correlated with maintenance of nonprogressor status. In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection.
The SDF-1 3'A allelic polymorphism has been reported to influence either positively or negatively the progression of human immunodeficiency virus type 1 (HIV-1) disease. Therefore, the SDF-1 genotype of 729 HIV-1-infected individuals pooled from 3 distinct cohorts was determined. A statistically nonsignificant association between the SDF1-3'A/3'A genotype and accelerated disease progression was evident among seroconverters (n=319), but a striking correlation of decreased survival after either diagnosis of AIDS according to the 1993 definition or loss of CD4(+) T cell counts <200 was observed. The relative hazards for SDF1-3'A/3'A homozygotes, compared with heterozygotes and wild-type homozygotes were 2.16 (P=.0047), for time from diagnosis according to the 1993 Centers for Disease Control and Prevention AIDS case definition (AIDS-'93) to death, and 3.43 (P=.0001), for time from CD4(+) T cells <200 to death. Because no difference in survival was observed after diagnosis according to AIDS-'87, the association of the SDF1-3'A/3'A genotype with the accelerated progression of late-stage HIV-1 disease appears to be explained for the most part by the loss of CD4(+) T lymphocytes.
Certain chemokines inhibit HIV replication through binding to cell surface receptors which also act as viral coreceptors. Based on our previous observations that HIV-1 Tat can interact with alpha- and beta-chemokine receptors, we investigated the potential effect of extracellular Tat (ecTat) on infection and replication of CCR5-dependent (R5) and CXCR4-using (X4) HIV-1 strains in primary activated peripheral blood mononuclear cells (PBMC) of uninfected donors. Receptor desensitization and binding competition studies were used to determine chemokine receptor binding by ecTat. Standard HIV replication assays based on reverse transcriptase (RT) activity determination in culture supernatants of PBMC and real time PCR for HIV-1 gag DNA were used to determine potential effects on early (entry or RT) steps of infection. ecTat bound to CXCR4 expressing monocytes and mitogen-activated PBMC, and competed with the natural ligand of CXCR4, SDF-1alpha (stromal cell-derived factor-1alpha) in calcium mobilization assays. EcTat inhibited replication of the X4 HIV-1 (LAI/IIIB strain) in activated PBMC at concentrations close to those of SDF-1alpha, whereas it only modestly interfered with R5 HIV-1 (BaL) replication in PBMC. Both SDF-1alpha and ecTat inhibited accumulation of X4 HIV-1 gag DNA, indicating interference with viral entry and/or RT. Our data show the surprising and counter-intuitive observation that ecTat selectively represses X4 HIV replication. This could favour spreading of R5 viruses, a condition observed in vivo immediately after transmission and in the early asymptomatic phase of infection.
Explore the source record for details and available documents.
We investigated the role of different CC chemokines, including regulated upon activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-lalpha (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1), and MCP-3 on virus replication in cultures established from CD8+ T cell-depleted peripheral blood mononuclear cells (PBMC) of HIV-infected individuals that were either cocultivated with allogeneic T cell blasts (ATCB) of uninfected individuals or directly stimulated by mitogen plus interleukin-2. RANTES was the only chemokine that showed a clear-cut suppressive effect on HIV replication in both culture systems, although inhibitory effects were frequently also observed with MIP-1alpha, MCP-3, and, occasionally, with MCP-1. In contrast, MCP-1 frequently enhanced HIV production in most patients' cultures or cocultures that were characterized by secreting relatively low levels (<20 ng/mL) of MCP-1. When CD8-depleted PBMC of HIV+ individuals were cocultivated with ATCB of uninfected healthy donors, a positive correlation was observed between MCP-1 concentrations and the enhancement of HIV-1 replication occurring after depletion of CD8+ cells from donors' cells. Depletion of CD14+ cells (monocytes) from ATCB resulted in the down-regulation of virus replication during co-cultivation with CD8-depleted PBMC of infected individuals. Of interest, MCP-1 up-regulated HIV production in these CD14-depleted ATCB cocultures. Altogether these observations suggest that MCP-1 may represent an important factor enhancing HIV spreading, particularly in anatomical sites, such as the brain, where infection of macrophages and microglial cells plays a dominant role.
Leader binding protein-1 (LBP-1)/late SV40 factor (LSF) and ying yang-1 (YY1) transcription factors are involved in the regulation of HIV expression. In particular, YY1 and LBP-1 have been shown to cooperate in repressing HIV-1-long terminal repeat reporter gene expression by in vitro cotransfection experiments. However, no information is available on the levels of expression and activation of these transcription factors in PBMC of HIV-infected individuals. Therefore, we have evaluated the expression and DNA binding activity of YY1 and LBP-1 (LSF) in PBMC of HIV-infected individuals before, during, and after administration of IL-2 in association with antiretroviral therapy (ART), a regimen under consideration for broad clinical use in this disease based on its ability to stably raise the absolute number of circulating CD4+ T lymphocytes. Both YY1- and LBP-1 (LSF)-DNA binding were profoundly down-modulated during administration of IL-2/ART, and a proteolytic activity probably responsible for the reduced expression of the two cellular transcription factors was found activated in PBMC of individuals receiving the immunotherapeutic regimen. This study is the first evidence of modulation of cellular transcription factors following IL-2/ART administration and provides a potential correlate of the transient raises in plasma viremia early reported in patients receiving IL-2 in the absence of ART, thus underscoring the importance of always administering this cytokine to HIV-infected individuals together with potent antiretrovirals.
Infection by the human immunodeficiency virus (HIV) either upregulates or downregulates the expression of several cytokines and interferons (IFNs) that use the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway for signal transduction. However, very little is known on the state of activation of the JAK/STAT pathway after HIV infection either in vivo or in vitro. In this regard, we report here that a constitutive activation of a C-terminal truncated STAT5 (STAT5triangle up) and of STAT1alpha occurs in the majority ( approximately 75%) of individuals with progressive HIV disease. We have further demonstrated that, among peripheral blood mononuclear cells (PBMCs), STAT5triangle up is activated preferentially in CD4(+) T cells. In contrast to a published report, expression of STATs from PBMCs of infected individuals was comparable with that of seronegative donors. In addition, in vitro infection of mitogen-activated PBMCs with a panel of laboratory-adapted and primary HIV strains characterized by differential usage of chemokine coreceptors did not affect STAT protein levels. However, enhanced activation of STAT was observed after in vitro infection of resting PBMCs and nonadherent PBMCs by different viral strains. Thus, constitutive STAT activation in CD4(+) T lymphocytes represents a novel finding of interest also as a potential new marker of immunological reconstitution of HIV-infected individuals.
We have recently described a significant correlation between human immunodeficiency virus-1 (HIV-1) RNA replication and monocyte chemotactic protein-1 (MCP-1) levels in the cerebrospinal fluid (CSF) of individuals with the acquired immunodeficiency syndrome (AIDS) with HIV encephalitis (E). Because local macrophages (microglia) are the cells predominantly infected in the brain, we investigated whether in vitro HIV infection affects MCP-1 production in mononuclear phagocytes (MP). MCP-1 secretion and expression were consinstently upregulated over constitutive levels in human monocyte-derived macrophages (MDM) infected with the M-tropic R5 BaL strain of HIV-1. HIV replication was required for this effect, as demonstrated by the absence of chemokine upregulation after infection in the presence of 3'-azido-3'-deoxythimidine (AZT) or cell-exposure to heat-inactivated (triangle up degrees ) virus. MCP-1 induction was not restricted to HIV-1 BaL, but was also observed during productive infection of MDM with two primary isolates differing for entry coreceptor usage and of U937 cells with the X4 HIV-1 MN strain. Based on the observation that exogenous HIV-1 Tat induced MCP-1 expression in astrocytes, we also investigated its role in MDM and U937 cells. Exogenous Tat induced MCP-1 production from MDM in a concentration-dependent manner, however, it was not effective on uninfected U937 cells or on the chronically infected U937-derived cell line U1. Transfection of Tat-expressing plasmids moderately activated HIV expression in U1 cells, but failed to induce MCP-1 expression in this cell line or in uninfected U937 cells. HIV replication-dependent expression of MCP-1 in MP may be of particular relevance for the pathogenesis of HIV infection in nonlymphoid organs such as the brain.
The immunosuppressive and antiinflammatory cytokine interleukin (IL) 10 selectively upregulates the expression of the CC chemokine receptors CCR5, 2, and 1 in human monocytes by prolonging their mRNA half-life. IL-10-stimulated monocytes display an increased number of cell surface receptors for, and better chemotactic responsiveness to, relevant agonists than do control cells. In addition, IL-10-stimulated monocytes are more efficiently infected by HIV BaL. This effect was associated to the enhancement of viral entry through CCR5. These data add support to an emerging paradigm in which pro- and antiinflammatory molecules exert reciprocal and opposing influence on chemokine agonist production and receptor expression.
Explore the source record for details and available documents.
Seven of 112 hemophiliacs infected with human immunodeficiency virus type-1 (HIV-1) before 1986 through contaminated plasma products are currently healthy, with CD4 T-cell counts above 500 cells/microL, and have never received antiretroviral therapy (long-term nonprogressors [LTNPs]). Seven age and sex-matched hemophiliacs infected in the same period but who have progressive HIV disease (progressors) and one additional slow-progressing individual were also studied. One hundred-fold, 20-fold, and 10-fold lower levels of full-length HIV RNA in plasma, peripheral blood mononuclear cells (PBMCs), and proviral DNA in PBMCs, respectively, were found in LTNPs compared with progressors. Plasma and cell-associated HIV RNA and proviral DNA were lower in LTNPs who tested negative for viral isolation from PBMCs or who were positive only after removal of CD8+ cells. No substantial differences were observed in the in vitro production of chemokines including RANTES, MIP-1 alpha, MIP-1 beta, MCP-1, and interleukin-8 (IL-8) in supernatants of activated PBMCs or CD8-depleted PBMCs of LTNPs, even when HIV isolation was simultaneously accomplished exclusively after removal of CD8+ cells. Low levels of HIV load and replication in peripheral blood are the strongest correlates of nonprogression in this small number of infected hemophiliacs.