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Biomedical subjects

P Verani

Publications and source records attributed to P Verani.

At least 37 records · Page 2Linked to original sources
PubMed1996

Sandfly fever viruses in Italy.

Two serologically distinct agents, the sandfly fever Sicilian and the sandfly fever Naples viruses, were isolated by Sabin from blood samples taken during an Italian epidemic of febrile illness. Since then, several different viruses have been isolated from sandflies and/or humans in both the Old and New World. Toscana virus, a new virus closely antigenically related to sandfly fever Naples virus, was isolated in 1971 from the sandfly Phlebotomus perniciosus in Italy. Extensive studies on the importance of Toscana virus as a human pathogen demonstrated its association with acute neurologic diseases. A serosurvey for the presence of antibodies to sandfly fever Sicilian, sandfly fever Naples and Toscana viruses indicated that, as in other Mediterranean areas, both sandfly fever Sicilian and sandfly fever Naples viral infections decreased or disappeared after the 1940s in countries performing insecticide-spraying malaria eradication campaigns. In contrast, clinical cases of aseptic meningitis or meningoencephalitis caused by Toscana virus are observed annually in Central Italy during the summer. Toscana virus may be present in other Mediterranean countries where sandflies of the genus Phlebotomus are present.

Animals↗
PubMed1996

The non-producer phenotype of the human immunodeficiency virus type 1 provirus F12/HIV-1 is the result of multiple genetic variations.

A cell clone (Hut-78/F12) chronically infected with a non-producer human immunodeficiency virus type 1 (HIV-1) variant showed an abnormal pattern of virus structural proteins and released no detectable virus particles. Exchanges of homologous parts of the F12/HIV provirus and a replication-competent HIV (strain NL4-3) were undertaken to define the genetic determinants of the F12/HIV phenotype. The non-infectious phenotype was reproduced by replacing an NL4-3 genomic fragment encoding the C terminus of gp 120 and the N terminus of gp41 with the corresponding parts of the F12/HIV provirus. Conversely, a much more extended genomic fragment (encompassing the vif, pol and env genes) was necessary to convert the F12/HIV phenotype. These results demonstrate that the F12/HIV non-producer phenotype is the result of mutations scattered along most of the genome, rendering the conversion to an infectious phenotype a very unlikely event. The F12/HIV genome is thus a reliable model for preclinical studies of anti-HIV gene therapy.

Defective Viruses↗
PubMed1996

Reversion of a human immunodeficiency virus type 1 integrase mutant at a second site restores enzyme function and virus infectivity.

The integration of a DNA copy of the retroviral RNA genome into the host cell genome is essential for viral replication. The virion-associated integrase protein, encoded by the 3' end of the viral pol gene, is required for integration. Stable virus-producing T-cell lines were established for replication-defective human immunodeficiency virus type 1 carrying single amino acid substitutions at conserved residues in the catalytic domain of integrase. Phenotypically reverted virus was detected 12 weeks after transfection with the integrase mutant carrying the P-109-->S mutation (P109S). Unlike the defective P109S virus, the revertant virus (designated P109SR) grew in CD4+ SupT1 cells. In addition to the Ser substitution at Pro-109, P109SR had a second substitution of Ala for Thr at position 125 in integrase. Site-directed mutagenesis was used to show that the P109S T125A genotype was responsible for the P109SR replication phenotype. The T125A substitution also rescued the in vitro enzyme activities of recombinant P109S integrase protein. P109S integrase did not display detectable 3' processing or DNA strand transfer activity, although 5 to 10% of wild-type disintegration activity was detected. P109S T125A integrase displayed nearly wild-type levels of 3' processing, DNA strand transfer, and disintegration activities, confirming that T125A is a second-site intragenic suppressor of P109S. P109S integrase ran as a large aggregate on a size exclusion column, whereas wild-type integrase ran as a monomer and P109S T125A integrase ran as a mixed population. Pro-109 and Thr-125 are not immediately adjacent in the crystal structure of the integrase catalytic domain. We suggest that the T125A substitution restores integrase function by stabilizing a structural alteration(s) induced by the P109S mutation.

Animals↗
PubMed1996

Anti-HIV viral interference induced by retroviral vectors expressing a nonproducer HIV-1 variant.

A Hut-78 cell clone (F12) harboring a nonproducer human immunodeficiency virus (HIV-1) variant shows complete resistance to HIV-1 or HIV-2 superinfection. The F12-HIV provirus produces an altered HIV-1 protein pattern and cannot generate even immature viral particles. We demonstrated that HeLa CD4+ cells transfected with the F12-HIV genome resist HIV superinfection through a CD4-independent mechanism. As F12-HIV appears to be a useful system to induce anti-HIV intracellular immunization, we constructed various retroviral vectors containing the F12-HIV genome, modified by elimination of the F12 3'LTR and part of its nef gene, inserted 'antisense' with respect to the Moloney murine leukemia virus 5' LTR. Here we show that recombinant retroviral particles carrying the N2/F12-HIV nef- (as) construct can stably transduce both CEMss human cells and primary human peripheral blood lymphocytes, inducing the expression of the F12-HIV genome. These results could open the way to an anti-AIDS gene therapy strategy based on F12-HIV-induced intracellular immunization.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Hepatitis B and C viruses, human T-cell lymphotropic virus types I and II, and leukemias: a case-control study. The Italian Leukemia Study Group.

The relationship between acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myeloid leukemia (CML), and refractory anemia with excess of blasts (RAEB) and antibodies to human T-cell lymphotropic virus types I and II (HTLV-I and HTLV-II), and hepatitis B virus and hepatitis C virus (HCV) was investigated in a multicenter case-control study. There were 431 cases enrolled in the study at the time of diagnosis of hematological malignancies, and 862 controls ages 15 years or older were recruited in three hospitals. Antibodies to HTLV-I and HTLV-II, antibody to HCV, hepatitis B surface antigen, and antibody to hepatitis B core antigen were assayed. All cases and controls were negative for HTLV-1 antibodies; one case (1 of 431; 0.2%), and one control (1 of 862; 0.1%) were found positive for HTLV-II antibodies. A nonsignificant excess of risk for hepatitis B surface antigen was present among RAEB cases (odds ratio, 2.40; 95% confidence interval, 0.46--12) CML, (odds ratio, 2.70; 95% CI, 0.86--8.43), and between antibody of hepatitis B core antigen and AML (odds ratio, 1.40; 95% CI, 0.93-2.10). A weak, nonsignificant association was present between AML, acute lymphocytic leukemia, RAEB, and antibody to HCV. These preliminary results suggest a possible association (elevated odds ratios) between hepatitis B virus, AML, RAEB, and CML. However, because all confidence intervals overlapped the null value, these findings need to be confirmed in larger case-control studies.

Acute Disease↗
PubMed1995

Full expression of transfected nonproducer interfering HIV-1 proviral DNA abrogates susceptibility of human He-La CD4+ cells to HIV.

An Hut-78 cell clone (F12) harboring a nonproducer human immunodeficiency virus-1 (HIV) variant, and showing a complete resistance to HIV-1 or HIV-2 superinfection, was previously characterized. We demonstrated that the replication of the superinfecting HIVs is blocked at the retrotranscription step, despite the CD4 down-regulation, since HIVs are able to cross the Hut-78/F12 cell membrane. In order to establish if the expression of the HIV-1 variant (F12/HIV) could be per se sufficient to induce the homologous viral interference shown in the F12 cells, the whole F12/HIV provirus was cloned and transfected in He-La CD4+ cells. In F12/HIV expressing He-La CD4+ clones, both the viral proteins expressed and the HIV nonproducer phenotype remain unmodified compared to F12 cells. Furthermore, despite the full expression of CD4 HIV receptors, the life cycle of the superinfecting HIV could be either strongly inhibited or totally abolished, depending on the cell clone considered. The inhibition of the superinfecting HIV was also reproduced when an HIV infectious molecular clone was transfected in F12/HIV He-La CD4+ clones, thus indicating that a post-cDNA synthesis block may operate against the superinfecting HIV. These data demonstrate that HIV susceptibility could be abrogated in cells expressing the F12/HIV genome, even in absence of any CD4 down-regulation.

CD4 Antigens↗
PubMed1995

A nonproducer, interfering human immunodeficiency virus (HIV) type 1 provirus can be transduced through a murine leukemia virus-based retroviral vector: recovery of an anti-HIV mouse/human pseudotype retrovirus.

The expression of a human immunodeficiency virus (HIV) type 1 provirus (F12-HIV) cloned from a nonproducer, chronically infected CD4 down-regulated Hut-78 cell clone (F12) does not lead to the formation of viral particles and, upon transfection in HeLa CD4+ cells, confers resistance to HIV superinfection without affecting the CD4 receptor exposure. In an attempt to transfer the anti-HIV properties of F12-HIV into human primary cell, we constructed a Moloney murine leukemia virus-based retroviral vector containing an F12-HIV genome lacking the 3' long terminal repeat and part of the nef gene, which was expressed under the control of its 5' long terminal repeat. The F12-HIV genome was inserted in the orientation opposite to that of the murine leukemia virus transcriptional unit and was designated the N2/F12-HIV nef-antisense vector. Lymphoblastoid CEMss cells, as well as human peripheral blood lymphocytes, were successfully transduced by the recombinant retrovirus emerging from the producer PA317 clones. CEMss clones expressing the F12-HIV nef-antisense vector became resistant to HIV superinfection even at the highest utilized multiplicity of infection (10(5) 50% tissue culture infective doses per 10(6) cells). In transduced CEMss cells the viral interference induced by the F12-HIV expression is not due to CD4 HIV receptor down-regulation. Nonproducer, interfering HIV proviruses transduced into retroviral vectors may, therefore, provide an alternative strategy for the protection of CD4+ human primary cells from HIV infection, which strategy may be used in designating a safe and efficient gene therapy protocol for patients with AIDS.

3T3 Cells↗
PubMed1995

Arbovirus surveillance in Italy.

A high number of different arboviruses have been demonstrated to be present in Italy, due to the coexistence of climates that are characteristic of continental areas in the North of the country and of subtropical areas in the South. Viruses circulating in Central Europe, such as the Tick-Borne Encephalitis (TBE) virus, are present in Central and Northern Regions, whereas viruses circulating in Mediterranean areas, such as the Sandfly fever viruses are present in Central and Southern Italy. Viruses have been isolated from mosquitoes, ticks, sandflies and vertebrates (birds, rodents, humans). For some of them an association with human disease has been demonstrated.

Animal Population Groups↗
PubMed1993

Homologous superinfection of both producer and nonproducer HIV-infected cells is blocked at a late retrotranscription step.

An HUT-78 cell clone (F12) chronically infected by a nonproducer HIV-1 variant (Federico et al., (1989) AIDS Res. Hum. Retroviruses 5, 385-396) is fully resistant to superinfection with HIV-1 or HIV-2. We demonstrate that, in spite of the down-regulation of CD4 receptors, superinfecting-HIV-1 and -HIV-2 cross the F12 plasma membrane (even in the presence of OKT4A monoclonal antibodies) but fail to complete retrotranscription. We utilized a series of polymerase chain reaction primers designed to detect certain steps in the reverse transcription process. Superinfecting-HIV-1 (an African strain) and -HIV-2 are detectable using primers specific for env (for HIV-1), 5' LTR (the R and U5 regions), and vpx (for HIV-2). No amplification is visible when primers amplifying either HIV-2 gag or the "primer binding site" region of 5' LTR of HIV-2 are used. DNA-PCR performed on DNAse-pretreated HIV-1 and HIV-2 stocks failed to show any amplification. This rules out that any extra- or intravirion viral DNA contamination may have interfered with our results. In addition, no DNA amplification was observed in F12 and HUT-78 cells exposed to heat-inactivated HIV-2. Finally, when the nonproducer F12 cells as well as control CEMss cells are transfected with the HIV-1 infectious molecular clone pNL4-3, progeny infectious virus is obtained. These findings indicate that reverse transcription of HIVs superinfecting F12 cells is prevented from completing viral DNA synthesis. A similar block occurs in HIV-1-infected producer cells. When integration of the HIV genome into the F12 genome is achieved via transfection of a molecular clone, the virus life cycle can proceed as in control CEMss cells.

Base Sequence↗
PubMed1993

HIV variability and perspectives for a vaccine.

An overview of efforts to induce neutralizing antibodies in order to develop an effective vaccine against AIDS is presented. The principal Neutralizing Determinant (PND) on the HIV-1 envelope is described. PND variability and the induction of neutralizing antibodies by synthetic peptides representing PND are discussed. The use of a cocktail of different peptides representing the PND sequences of the majority of HIV-1 isolates, as well as the construction of hybrid immunogens containing PND of several viral isolates, could overcome the problems related to PND variability. A different approach based on the possibility of inducing a type of intracellular immunity is also discussed: a cellular clone (F12) obtained in our laboratory from Hut-78 cells infected with supernatant of cultured lymphocytes from an HIV-infected patient, does not release viral particles despite the presence of a full-length HIV-1 provirus. Moreover, F12 cells are fully resistant against superinfection with any HIV-1 or HIV-2 isolates. We are now attempting to reproduce the homologous viral interference by transferring the F12/HIV genome of the clone into HIV-susceptible cells in order to render these cells resistant to HIV infection.

AIDS Vaccines↗
PubMed1993

In vitro spontaneous production of anti-SIV antibodies is a reliable tool in the follow-up of protection of SIV-vaccinated monkeys.

To assess the reliability of the spontaneous in vitro synthesis of simian immunodeficiency virus (SIV)-specific antibodies as a marker in the monitoring of protection in SIV-vaccinated animals, Macaca fascicularis monkeys were immunized with formalin-inactivated SIVmac251 or SIVmac251/32H, and challenged with human-derived (SIVmac251/32H) or monkey-derived live SIV. As judged by virus isolation and polymerase chain reaction (PCR) techniques, immunized animals were protected against human-derived SIV challenge, and no spontaneous in vitro synthesis of anti-SIV antibody was observed in nonstimulated peripheral blood mononuclear cell cultures over a 4-month follow-up. On the contrary, human cell-grown SIVmac251 immunization did not afford protection against monkey-derived SIV, and all the animals became infected and showed spontaneous in vitro synthesis of anti-SIV antibodies. These data demonstrate that lack of protection in SIV-vaccinated monkeys is strictly associated with PBMC ability of spontaneously produce anti-SIV antibodies in vitro following challenge, and suggest that this parameter might also constitute a reliable marker for monitoring protection in large-scale HIV vaccination and immunotherapy programs.

Animals↗
PubMed1993

A recombinant retrovirus carrying a non-producer human immunodeficiency virus (HIV) type 1 variant induces resistance to superinfecting HIV.

A human immunodeficiency virus (HIV) type 1-infected Hut-78 cell clone (F12) shows a peculiar phenotype: it exhibits an altered viral protein pattern, is a nonproducer and is resistant to homologous superinfection. To determine whether this phenotype is dependent upon the expression of the HIV-1 genome integrated therein, the SstI/SstI F12 provirus [deprived of HIV long terminal repeats (LTRs)] was cloned and inserted in the pLj retroviral vector bearing the neomycin (neo) and Geneticin resistance gene. CD4+ HIV-susceptible CEMss cells (a CEM clone able to form large syncytia 2 to 3 days post-HIV infection) were infected with the recombinant retroviruses rescued from the F12/HIV-pLj-transfected (in either sense or antisense orientation) amphotropic packaging cells PA 317. Neo sense resistant gene clones showed approximately 10 copies of viral DNA/cell (without detectable major deletions) only in episomal form, low viral RNA expression and a viral protein pattern characterized by an uncleaved gp160, no gp41 and little, if any, p55 gag precursor (as in F12 cells). Superinfection of these F12/HIV DNA-engineered clones with HIV-1 resulted in a significant reduction in the yield of superinfecting HIV. This effect (more pronounced when the clones were maintained under neo selective pressure) was observed in all five retrovirus-infected clones exhibiting the presence and expression of sense episomal F12/HIV DNA but not in two clones bearing an antisense F12/HIV DNA or in one clone bearing only the pLj vector. These results indicate that bio-engineered human CD4+ cells expressing the F12/HIV genome exhibit a significant resistance to HIV superinfection.

Antigens, Viral↗
PubMed1992

Extrachromosomal human immunodeficiency virus type 1 DNA forms in fresh peripheral blood lymphocytes and in two interleukin-2-independent T cell lines derived from peripheral blood lymphocytes of an asymptomatic seropositive subject.

Two immature T cell lines (FT1 and FT4) were established after in vitro cloning of peripheral blood lymphocytes (PBLs) from an asymptomatic human immunodeficiency virus type 1 (HIV-1) seropositive, human T cell-lymphotropic virus type 1 seronegative homosexual subject. Although derived from a limiting dilution cell cloning assay, these cell lines were not recloned for this study. Their growth was independent of exogenous interleukin-2. Both cell lines were able to form colonies when cloned in agar, but failed to form solid tumours when injected into nude mice. FT lines belong to the very immature T cell lineage as they exhibit rearranged TCR genes but no expression of T cell membrane antigens, including CD2, CD3, CD4, CD6, CD7 and CD8. They also contain an HIV-1 genome that was detected only in an extra-chromosomal DNA form, even after several passages in vitro. The presence of unintegrated viral DNA was also detected by polymerase chain reaction analysis in the same sample of fresh uncultured PBLs. Furthermore, despite the absence of CD4 expression, both T cell lines were susceptible to CD4-independent HIV-1 superinfection (lack of superinfection inhibition in the presence of OKT4A monoclonal antibodies).

Adult↗
PubMed1991

Interaction of HIV-1 with susceptible lymphoblastoid cells. 1H NMR studies.

Different strains of HIV susceptible lymphoblastoid cells have been infected by HIV-1 and examined by means of 1H NMR spectroscopy at different times after infection, taking advantage of the presence of high resolution lipid signals from the plasma membrane of tumor cells. A transient decrease in intensity of fatty acid signals, originated by changes in membrane structure, has been observed early after viral infection. Marked alterations in membrane-dependent steps of phospholipid synthesis can also be inferred by the observed transient depression in peaks from choline-based metabolites. Spectral modifications deriving from changes in lipid metabolism are also produced both in infected cells a few days after infection and in permanently infected cells. 1H NMR can, therefore, monitor structural and metabolic effects induced by HIV infection.

Cell Membrane↗