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Biomedical subjects

G B Rossi

Publications and source records attributed to G B Rossi.

At least 19 recordsLinked to original sources

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

Regulation and expression of a growth arrest-specific gene (gas5) during growth, differentiation, and development.

The growth arrest-specific gas5 gene was isolated from mouse genomic DNA and structurally characterized. The transcriptional unit is divided into 12 exons that span around 7 kb. An alternative splicing mechanism gives rise to two mature mRNAs which contain either 11 or 12 exons, and both are found in the cytoplasm of growth-arrested cells. In vivo, the gas5 gene is ubiquitously expressed in mouse tissues during development and adult life. In Friend leukemia and NIH 3T3 cells, the levels of gas5 gene mRNA were high in saturation density-arrested cells and almost undetectable in actively growing cells. Run-on experiments indicated that the gas5 gene is transcribed at the same level in both growing and arrested cells. On the other hand, in dimethyl sulfoxide-induced differentiating cells a sharp decrease in the rate of transcription was observed shortly before the cells reached the postmitotic stage. These results indicate that in density-arrested cells accumulation of gas5 mRNA is controlled at the posttranscriptional level while in differentiating cells expression is regulated transcriptionally.

3T3 Cells

Intracellular heme coordinately modulates globin chain synthesis, transferrin receptor number, and ferritin content in differentiating Friend erythroleukemia cells.

The effect of succinylacetone (SA), a highly specific inhibitor of ALA-dehydratase and heme synthesis, on hemoglobin (Hb) production, transferrin receptor (TfR), and ferritin expression was analyzed in differentiating Friend leukemia cells (FLC). This compound exerted a pronounced inhibitory effect not only on heme and Hb synthesis, but also on all the remaining above-mentioned parameters. In particular, SA induced: (1) a reduction of the level of alpha-globin mRNA; (2) a decreased number of exposed TfR molecules, without modification of their affinity for the ligand; (3) a reduced level of TfR RNA, without significant change of TfR gene transcription rate; and (4) a lower ferritin content. The addition of exogenous hemin to differentiating FLC exerted opposite effects, and particularly induced an increase of both the number of TfRs and ferritin content. These findings suggest that in erythroid cells optimal heme synthesis is required to coordinately sustain globin chains synthesis and TfR/ferritin production; thus, the intracellular heme level may represent a key regulatory factor in the Hb synthesis pathway.

Cell Differentiation

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

Human immunodeficiency virus type 1 gp120 mimics a hidden monomorphic epitope borne by class I major histocompatibility complex heavy chains.

Murine monoclonal antibodies (mAbs) M38 and L31 define two epitopes of a surface protein of activated lymphocytes and monocytes. It has been shown that M38 also defines a crossreactive epitope of human immunodeficiency virus type 1 (HIV-1) gp120 (Beretta et al., 1987. Eur. J. Immunol. 17: 1793). The mAb inhibits syncytia formation driven by HIV-1-infected cells. The surface protein was demonstrated to be a class I MHC alpha chain, by sequence analysis of the corresponding cDNA and by immunological means. The epitopes defined by mAbs M38 and L31 are monomorphic and hidden (i.e., inaccessible to antibodies) on native HLA molecules expressed by resting cells, but can be evidenced on denatured proteins by Western blot analysis. The two epitopes become accessible after activation processes have been implemented, likely reflecting a conformational alteration of alpha chains (such as that described by Schnabl et al. 1990. J. Exp. Med. 171:1431). Consistent with molecular data are the results of functional analysis, which indicate that the molecule recognized by M38 and L31 is a gate for pleiotropic negative signals, since the two mAbs were shown to inhibit monocyte antigen presentation and lymphocyte mitogenic proliferation, respectively.

Amino Acid Sequence

Posttranscriptional regulation of beta interferon expression in erythroid Friend cells treated with gamma interferon.

Treatment of Friend erythroleukemia cells (FLC) with gamma interferon (IFN-gamma) in the presence of anti-IFN-beta antibodies reduces the effectiveness of the antiviral state and the induction of 2'-5'-oligoadenylate synthetase activity, indicating that the antiviral activity of IFN-gamma in FLC is in part mediated by the production of IFN-beta. Accordingly, IFN-gamma induces a less pronounced antiviral state in FLC resistant to IFN-alpha/beta than in wild-type cells. Moreover, while results of run-on assays indicate that both IFN-alpha and -beta genes are constitutively transcribed in these cells, FLC treatment with IFN-gamma induces only IFN-beta mRNA accumulation. These results indicate that posttranscriptional mechanisms are involved in the regulation of IFN-beta and -alpha expression by IFN-gamma. The low amounts of the induced IFN-beta synergize with IFN-gamma in mounting the potent antiviral effect.

2',5'-Oligoadenylate Synthetase

Metabolic and structural effects of HIV infection in human peripheral blood mononuclear cells can be monitored with 1H NMR spectroscopy.

Infection of human peripheral blood lymphocytes by human immunodeficiency virus type 1 (HIV-1) was investigated by means of 1H nuclear magnetic resonance spectroscopy, taking advantage of the presence of signals from fluid lipid domains in the membrane of stimulated lymphocytes. A transient decrease of the lipid methylene signal intensity was observed at the time of HIV internalization, monitoring a general rearrangement of membrane structure associated with virus entry. A similar effect was also observed a few days after infection, when HIV particles are released by infected cells as demonstrated by high reverse transcriptase activity in cell supernatant. Signals arising from choline-based metabolites were also affected by HIV infection, indicating a possible slowing down of phospholipid synthesis.

Cell-Free System

A full-length murine 2-5A synthetase cDNA transfected in NIH-3T3 cells impairs EMCV but not VSV replication.

Treatment of cells with interferons (IFNs) induces resistance to virus infection. The 2'-5'oligo A (2-5A) synthetase/RNase L is one of the pathways leading to translation inhibition induced by IFN treatment. A murine cDNA encoding the 43-kDa 2-5A synthetase was cloned and sequenced. NIH-3T3 cell clones transfected with this cDNA expressed the enzymatic activity to various extents and exhibited resistance to encephalomyocarditis virus (EMCV) but not to vesicular stomatitis virus replication. The specific resistance to EMCV can be attributed to 2-5A synthetase.

2',5'-Oligoadenylate Synthetase

Spectrum of biological activity of interferons.

The interferons comprise a group of proteins first identified by their ability to protect cells against virus infections but also capable of influencing cellular physiology. They are synthesized and secreted by a variety of cell types in response to various inducers. Their effects include antiviral action, inhibition of cell proliferation, modulation of cell differentiation and activation of various cell types in immune system. This review aims to summarize the current state of biology of interferon action with special emphasis on those aspects related to the use of these molecules in antitumoral therapy. The antitumor effects of IFNs results from pleiotropic IFN activity exerted either directly on tumor cells (i.e. antiproliferative effects, effects on oncogene expression, on cell differentiation and enhanced expression of cell surface antigens), or via indirect effects (i.e. activation of effector mechanisms of the host as modulation of the expression of the major histocompatibility antigens, effects on macrophages, NK, T and B cells).

Animals

[The AIDS project].

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Acquired Immunodeficiency Syndrome

Serological survey of human immunodeficiency virus (HIV) in Ethiopia.

The presence of anti-human immunodeficiency virus 1 antibodies was tested in 5,565 serum samples from Ethiopia of which 5,265 were collected from military recruits in the framework of a hepatitis B (HBV) seroepidemiological study performed on a national scale in 1985-1986; the remaining were 300 sera from a population of outpatients belonging to the Arsi region. Of the 5,565 sera, 121 (2.1%) were found to be repeatedly reactive by enzyme-linked immunosorbent assay (ELISA) test for HIV-1 antibodies, but these reactivities were confirmed by Western Blot (WB) assay in only four cases (0.07%) and by ENVACOR (confirmatory competitive ELISA) in three samples. Twenty-three sera were positive by WB to one or two bands related to core proteins but were all negative by ENVACOR. However, according to accepted criteria for positivity, these sera must be regarded as indeterminant reactors. A sample of 409 sera, both reactive and nonreactive by HIV-1 ELISA, were further tested for antibodies to HIV-2 by ELISA. Reactive sera were analysed by WB and by radioimmunoprecipitation assay (RIPA) using 35S-cysteine metabolically labelled SIVmac (HTLV-IV) infected cell lysates. Only 11 sera were found to be slightly reactive in ELISA, but this was not confirmed by WB or RIPA. Data indicate that HIV infection was not widespread in the general population of Ethiopia up to 1986.

AIDS Serodiagnosis

Prevalence of HBeAg, anti-HBe serological markers and HBV-DNA in asymptomatic carriers in Ethiopia.

Data on the distribution of HBV-DNA and other serological markers of hepatitis B virus infection in a population of asymptomatic carriers in Ethiopia are reported. As compared to data from other countries of similar or lower levels of endemicity, it has been found that HBV-DNA prevalence and its correlation with HBeAg/anti-HBe status is similar to that of northern Europe. HBV-DNA is present in 84% of HBe Ag-positive sera but in only 4% of anti-HBe-positive sera, where the lowest concentration of DNA (less than 5 pgr/20 microliters) was found. The trend of increasing prevalence of serological markers with age seems to indicate a considerable horizontal transmission still present in this age range (18-30). In keeping with data of other authors a 3% of HBV-DNA positivity in anti HBc only positive sera was found. No regional or ethnical differences of HBV-DNA positive sera across the country were observed. There is also no evidence of any correlation between HBV-DNA prevalence and HBsAg subtypes ad and ay.

Adolescent

Natural resistance in mice against Friend leukemia cells. II. Studies with in vivo passaged interferon-sensitive and interferon-resistant cell clones.

Experiments were designed to test the presence of antitumor natural resistance (NR) in DBA/2 mice against highly oncogenic in vivo passaged histocompatible Friend leukemia cells (FLC-V). NR was measured in vivo as rapid clearance of radiolabeled cells from different organs or as growth inhibition in lethally irradiated mice. Interferon-sensitive (745) or interferon-resistant (3C18) lines were used. Organ clearance studies showed that young recipients eliminate cells more rapidly than old mice. Moreover, depressive (e.g., cyclophosphamide or carrageenen) or enhancing (e.g., poly (I:C) or Friend leukemia virus infection) agents of NR function modulate accordingly leukemia cell clearance. Similar results were obtained testing tumor growth in lethally irradiated hosts, although modulating agents were substantially less effective in this system. Both FLC-745-V and FLC-3C18-V lines were equally susceptible to NR. Therefore, these data provide further support to the hypothesis that exogenous IFN capable of suppressing the growth of both lines could act via enhancement of the NR function.

Animals

Interferons in cell growth and development.

Interferons (IFNs), besides inducing an antiviral state in uninfected cells, are also natural regulatory molecules. They play a key role in the regulation both of cell growth and differentiation, and of development. Up- or down-regulation of oncogenes by IFNs may be one of the mechanisms by which these molecules affect cell physiology. The list of IFN-inducible proteins continues to grow rapidly and future research should identify among these the mediators of the biological effects of IFNs.

Animals

Natural resistance in mice against Friend cells injected intravenously. III. Comparison between in vivo and in vitro passaged interferon-sensitive (745) and interferon-resistant (3Cl8) cell clones.

In vitro (FLC-Vt) or in vivo (FLC-V) passaged Friend erythroleukaemia cells of DBA/2 origin were tested for susceptibility to natural resistance (NR) in vivo or to NK cell activity in vitro. Scarcely oncogenic FLC-Vt cells were highly susceptible to in vivo NR (measured as rapid organ clearance or growth inhibition in lethally irradiated mice) or to in vitro NK attack. Conversely, highly oncogenic FLC-V cells were weakly susceptible to NR and to NK as well. These data seem to point out that natural immunity, which is up-regulated by endogenous or exogenous interferons, can play a significant role in surveillance against mouse leukaemic cells of retrovirus origin.

Animals

Biologic and molecular characterization of producer and nonproducer clones from HUT-78 cells infected with a patient HIV isolate.

HUT-78 cells were infected with a reverse transcriptase (RT)-positive supernatant of a culture of peripheral blood lymphocytes (PBL) from an AIDS patient and then cloned. Of these clones, two have been isolated and characterized. Clone D10 is persistently and productively infected with an HIV variant. The clone F12, in spite of the presence of an integrated full-length HIV provirus, does not release virus particles in the medium. D10 and F12 clones substantially differ in terms of protein pattern; that is, D10 is super-imposable to infected HUT-78 cells, whereas F12 exhibits a decreased uncleaved p55 gag precursor and the presence of uncleaved gp160 and of a unique p19, although they do not show qualitative or quantitative differences in viral RNA synthesis. Restriction patterns of F12 proviral DNA do not show major genomic deletions. These results indicate that F12 clone cells carry an HIV genome with minor mutations that probably affect the correct production of viral proteins at a posttranscriptional level. In addition, the F12 clone is resistant to high-multiplicity superinfection with HIV-1 or HIV-2.

Cells, Cultured