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Biomedical subjects

S Ugolini

Publications and source records attributed to S Ugolini.

15 recordsLinked to original sources
PubMed2001

Biology of T memory type 1 cells.

Engagement of inhibitory natural killer (NK) cell receptors for MHC class I molecules (NKR) can impair NK-cell activation programs. Inhibitory NKR thus confer to NK cells the capacity to discriminate between MHC class I+ and MHC class I- target cells, and are therefore involved in the control of NK-cell tolerance to self, as well as in the elimination of MHC class I- distressed cells by NK cells. In human and mouse, a subset of alphabeta T cells also express inhibitory NKR at their surface, but the biological function of inhibitory NKR on T cells remains to be precisely elucidated. We refer to these cells as T memory type 1 (Tm1) cells, and review here the phenotypic and functional features of this subset of memory-phenotype CD8+ alphabeta T cells. In vitro studies suggest that inhibitory NKR are involved in the peripheral control of T-cell self-tolerance. In vitro and in vivo analysis have revealed a novel biological function for inhibitory NKR when expressed on T cells. Indeed, engagement of inhibitory NKR on T cells provides them with survival signals against activation-induced cell death. Thus, sensing of self-MHC class I molecules by inhibitory NKR displayed on alphabeta T cells leads to the in vivo accumulation of Tm1 cells.

Animals↗
PubMed2001

Involvement of inhibitory NKRs in the survival of a subset of memory-phenotype CD8+ T cells.

Inhibitory natural killer receptors (NKRs) such as killer cell immunoglobulin-like receptors (KIRs) in humans and Ly49 molecules in mice are expressed on NK cells and recognize multiple major histocompatibility (MHC) class I proteins. In humans and mice, a subset of CD8+ T cells also expresses NKRs and harbors a memory phenotype. Using mice that are transgenic for KIR2DL3 and its cognate HLA-Cw3 ligand, we show that engagement of inhibitory NKRs selectively drives the in vivo accumulation of a subset of memory-phenotype CD8+ T cells that express the beta chain of the interleukin 2 receptor. In vitro, recognition of MHC class I molecules by inhibitory NKRs on T cells down-regulated activation-induced cell death. These results unveil an MHC class I-dependent pathway that promotes the survival of a subset of memory-phenotype CD8+ T cells and also reveal an unexpected biological function for inhibitory NKRs on T cells.

Animals↗
PubMed2000

Regulation of T cell function by NK cell receptors for classical MHC class I molecules.

Inhibitory receptors for MHC class I molecules were initially characterised on NK cells. Human and mouse NK cell receptors (NKRs) are also expressed on T cells, predominantly on a subset of memory-phenotype CD8(+) T cells. This review focuses on the precise determination of interactions between NKRs and MHC class I, as well as on the unexpected in vivo function of NKRs on T cells.

Animals↗
PubMed2000

Development, reliability and acceptability of a new version of the DSM-IV Social and Occupational Functioning Assessment Scale (SOFAS) to assess routine social functioning.

OBJECTIVE: Development of a scale to assess patients' social functioning, the Personal and Social Performance scale (PSP). METHOD: PSP has been developed through focus groups and reliability studies on the basis of the social functioning component of the DSM-IV Social and Occupational Functioning Assessment Scale (SOFAS). The last reliability study was carried out by 39 workers with different professional roles on a sample of 61 psychiatric patients admitted to the rehabilitation unit. Each patient was rated independently on the scale by the two workers who knew them best. RESULTS: The PSP is a 100-point single-item rating scale, subdivided into 10 equal intervals. The ratings are based mainly on the assessment of patient's functioning in four main areas: 1) socially useful activities; 2) personal and social relationships; 3) self-care; and 4) disturbing and aggressive behaviours. Operational criteria to rate the levels of disabilities have been defined for the above-mentioned areas. Excellent inter-rater reliability was also obtained in less educated workers. CONCLUSION: Compared to SOFAS, PSP has better face validity and psychometric properties. It was found to be an acceptable, quick and valid measure of patients' personal and social functioning.

Adult↗
PubMed1999

Constitutive cell surface association between CD4 and CCR5.

HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor. For most strains of HIV, this coreceptor is CCR5. Here, we provide evidence that CD4 is specifically associated with CCR5 in the absence of gp120 or any other receptor-specific ligand. The amount of CD4 coimmunoprecipitated with CCR5 was significantly higher than that with the other major HIV coreceptor, CXCR4, and in contrast to CXCR4 the CD4-CCR5 coimmunoprecipitation was not significantly increased by gp120. The CD4-CCR5 interaction probably takes place via the second extracellular loop of CCR5 and the first two domains of CD4. It can be inhibited by CCR5- and CD4-specific antibodies that interfere with HIV-1 infection, indicating a possible role in virus entry. These findings suggest a possible pathway of HIV-1 evolution and development of immunopathogenicity, a potential new target for antiretroviral drugs and a tool for development of vaccines based on Env-CD4-CCR5 complexes. The constitutive association of a seven-transmembrane-domain G protein-coupled receptor with another receptor also indicates new possibilities for cross-talk between cell surface receptors.

3T3 Cells↗
PubMed1999

Disruption and phenotypic analysis of six novel genes from chromosome IV of Saccharomyces cerevisiae reveal YDL060w as an essential gene for vegetative growth.

The disruption of six novel genes (YDL059c, YDL060w, YDL063c, YDL065c, YDL070w and YDL110c), localized on the left arm of chromosome IV in Saccharomyces cerevisiae, is reported. A PCR-based strategy was used to construct disruption cassettes in which the kanMX4 dominant marker was introduced between two long flanking homology regions, homologous to the promoter and terminator sequences of the target gene (Wach et al., 1994). The disruption cassettes were used to generate homologous recombinants in two diploid strains with different genetic backgrounds (FY1679 and CEN. PK2), selecting for geneticin (G418) resistance conferred by the presence of the dominant marker kanMX4. The correctness of the cassette integration was tested by PCR. After sporulation and tetrad analysis of the heterozygous deletant diploids, geneticin-resistant haploids carrying the disrupted allele were isolated. YDL060w was shown to be an essential gene for vegetative growth. A more detailed phenotypic analysis of the non-lethal haploid deletant strains was performed, looking at cell and colony morphology, growth capability on different media at different temperatures, and ability to conjugate. Homozygous deletant diploids were also constructed and tested for sporulation. Only minor differences between parental and mutant strains were found for some deletant haploids.

DNA Primers↗
PubMed1999

Antibody neutralization of HIV-1 and the potential for vaccine design.

Neutralisation by antibody is, for a number of viruses, an in vitro correlate for protection in vivo. For HIV-1 this is controversial. However, the induction of a potent anti-HIV neutralising antibody response remains one of the principal goals in vaccine development. A greater knowledge of the fundamental mechanisms underlying the neutralisation process would help direct research towards suitable vaccine immunogens. The primary determinant of HIV neutralisation appears to be antibody affinity for the trimeric envelope glycoprotein spike on the virion, suggesting that epitope-specific effects are secondary and implying a single, dominant mechanism of neutralisation. Antibody interference with virion attachment to the target cell appears to be a major mechanism of neutralisation by gp120-specific antibodies. This is probably achieved both by antibody-induced dissociation of gp120 from gp41 and by direct inhibition of virus binding to receptor-coreceptor complexes. A gp41-specific antibody neutralises by interfering with post-attachment steps leading to virus membrane fusion. Recent advances in structural analyses of the HIV envelope glycoproteins coupled with data obtained from antibody mapping and neutralisation studies allow a greater understanding of Env function and its inhibition. This in turn should lead to a more rational basis for vaccine design aimed at stimulating highly effective neutralising antibodies.

AIDS Vaccines↗
PubMed1999

HIV-1 attachment: another look.

HIV-1 attachment to host cells is generally considered to take place via high-affinity binding between CD4 and gp120. However, the binding of virion-associated gp120 to cellular CD4 is often weak, and most cell types that are permissive for HIV-1 infection express little CD4. Thus, other interactions between the virion and the cell surface could dominate the attachment process.

CD4 Antigens↗
PubMed1998

Interactions among HIV gp120, CD4, and CXCR4: dependence on CD4 expression level, gp120 viral origin, conservation of the gp120 COOH- and NH2-termini and V1/V2 and V3 loops, and sensitivity to neutralizing antibodies.

The binding of HIV-derived recombinant soluble (s)gp120 to the CD4(+)/CXCR4(+) A3.01 T cell line inhibits the binding of the CXCR4-specific monoclonal antibodies 12G5, which interacts with the second extracellular loop, and 6H8, which binds the NH2 terminus. We have used this as an assay to analyse the interaction of recombinant sgp120 from diverse viral origins with CXCR4. The strength of the interaction between sgp120 and CXCR4 correlated with sgp120 affinity for the CD4-CXCR4 complex, and the interaction of sgp120MN and sgp120IIIB with CXCR4 was highly dependent on the level of CD4 expressed on a variety of different T cell lines. sgp120 from X4, R5X4, and R5 viruses interacted with CXCR4, although the R5 sgp120-CXCR4 interactions were weaker than those of the other gp120s. The interaction of sgp120IIIB or sgp120MN with CXCR4 was inhibited by neutralizing monoclonal antibodies that prevent the sgp120-CD4 interaction but also by antibodies specific for the gp120 V2 and V3 loops, the CD4-induced epitope and the 2G12 epitope, which interfere weakly or not at all with CD4-sgp120 binding. The binding to A3.01 cells of wild-type sgp120HxB2, but not of sgp120 deleted in the COOH and NH2 termini, interfered with 12G5 binding in a dose-dependent manner. Further deletion of the V1 and V2 loops restored CXCR4 binding activity, but additional removal of the V3 loop eliminated the gp120-CXCR4 interaction, without decreasing the affinity between mutated sgp120 and CD4. Taken together, these results demonstrate that the interactions between sgp120 and CXCR4 are globally similar to those previously observed between sgp120 and CCR5, with some apparent differences in the strength of the sgp120-CXCR4 interactions and their dependence on CD4.

Antibodies, Monoclonal↗
PubMed1998

Human immunodeficiency virus type 1 attachment to HeLa CD4 cells is CD4 independent and gp120 dependent and requires cell surface heparans.

The binding of human immunodeficiency virus type 1 (HIV-1) (Hx10) virions to two different cell lines was analyzed by using a novel assay based on the detection, by anti-HLA-DR-specific antibodies, of HLA-DR+ virus binding to HLA-DR- cells. Virion attachment to the CD4+-T-cell line A3.01 was highly CD4 dependent in that it was potently inhibited by CD4 monoclonal antibodies (MAbs), and little virus binding to the CD4- sister A2.01 line was observed. By contrast, virion binding to HeLa cells expressing moderate or high levels of CD4 was equivalent to, or lower than, binding to wild-type CD4- HeLa cells. Moreover, several CD4 MAbs did not reduce, but enhanced, HIV-1 attachment to HeLa-CD4 cells. CD4 was required for infection of HeLa cells, however, demonstrating a postattachment role for this receptor. MAbs specific for the V2 and V3 loops and the CD4i epitope of gp120 strongly inhibited virion binding to HeLa-CD4 cells, whereas MAbs specific for the CD4bs and the 2G12 epitopes enhanced attachment. Despite this, all gp120- and gp41-specific MAbs tested neutralized infectivity on HeLa-CD4 cells. HIV-1 attachment to HeLa cells was only partially inhibited by MAbs specific for adhesion molecules present on the virus or target cells but was completely blocked by polyanions such as heparin, dextran sulfate, and pentosan sulfate. Treatment of HeLa-CD4 cells with heparinases completely eliminated HIV attachment and infection, strongly implicating cell surface heparans in the attachment process. CD4 dependence for HIV-1 attachment to target cells is thus highly cell line specific and may be replaced by other ligand-receptor interactions.

Antibodies, Monoclonal↗
PubMed1997

Inhibition of virus attachment to CD4+ target cells is a major mechanism of T cell line-adapted HIV-1 neutralization.

Antibody-mediated neutralization of human immunodeficiency virus type-1 (HIV-1) is thought to function by at least two distinct mechanisms: inhibition of virus-receptor binding, and interference with events after binding, such as virus-cell membrane fusion. Here we show, by the use of a novel virus-cell binding assay, that soluble CD4 and monoclonal antibodies to all confirmed glycoprotein (gp)120 neutralizing epitopes, including the CD4 binding site and the V2 and V3 loops, inhibit the adsorption of two T cell line-adapted HIV-1 viruses to CD4+ cells. A correlation between the inhibition of virus binding and virus neutralization was observed for soluble CD4 and all anti-gp120 antibodies, indicating that this is a major mechanism of HIV neutralization. By contrast, antibodies specific for regions of gp120 other than the CD4 binding site showed little or no inhibition of either soluble gp120 binding to CD4+ cells or soluble CD4 binding to HIV-infected cells, implying that this effect is specific to the virion-cell interaction. However, inhibition of HIV-1 attachment to cells is not a universal mechanism of neutralization, since an anti-gp41 antibody did not inhibit virus-cell binding at neutralizing concentrations, implying activity after virus-cell binding.

Antibodies, Blocking↗
PubMed1997

HIV-1 gp120 induces an association between CD4 and the chemokine receptor CXCR4.

For efficient entry into target cells, certain T cell-tropic HIV-1 isolates require both CD4 and the coreceptor CXCR4. However, the molecular interactions among CD4, CXCR4, and the HIV-1 envelope glycoproteins are only now being elucidated. Here we show that the binding of soluble gp120 from one macrophage-tropic and four T cell-tropic viruses to a CD4+, but not to a CD4-, T cell line, decreased the binding of an mAb specific for CXCR4 to its epitope, implying an interaction among gp120, CD4, and CXCR4. To confirm such an interaction, we conducted double- and triple-color confocal laser scanning microscopy on CD4+/CXCR4+ cells and determined the extent of CD4 and CXCR4 colocalization by a semiquantitative analysis. In the absence of gp120, a low level of constitutive colocalization between CD4 and CXCR4 was observed. Treatment with T cell-tropic-derived gp120 and, to a lesser extent, macrophage-tropic-derived gp120, increased the colocalization of CD4 with CXCR4, and triple staining indicated that gp120 was associated with the CD4-CXCR4 complexes. Cocapping of the gp120-CD4-CXCR4 complexes at 37 degrees C resulted in the cointernalization of a proportion of the gp120-CXCR4 complexes into intracellular vesicles. These data demonstrate that the binding of gp120 to CD4+ T cells induces the formation of a trimolecular complex consisting of gp120, CD4, and the HIV-1 coreceptor molecule CXCR4.

Animals↗
PubMed1996

The red/white colony color assay in the yeast Saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells.

In the yeast Saccharomyces cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment, while epistatic mutations in the same pathway, i.e. ade8, preclude this phenomenon, resulting in normal white colonies. The shift in color from red to white (or vice versa) with a combination of appropriate wild-type and mutant alleles of the adenine-pathway genes has been widely utilized as a non-selective phenotype to visualise and quantify the occurrence of various genetic events such as recombination, conversion and aneuploidy. It has provided an invaluable tool for the study of gene dosage and plasmid stability. In competition experiments between disrupted ade2, ade8-18 transformants carrying either a functional or non-functional episomal ADE8 gene, we verified that white ade8 ade2 cells show a remarkable selective advantage over red ade2 cells, with important implications on the use of this assay for the monitoring of genetic events. The accumulation of the red pigment in ade2 cells is likely to be the cause for impaired growth in these cells.

Adenine↗
PubMed1993

High-level heterologous gene expression in Saccharomyces cerevisiae from a stable 2 microns plasmid system.

The best candidate for a high-copy-number and mitotic stability expression system in yeast is the endogenous 2 microns plasmid. Nevertheless, derivatives of the 2 microns plasmid typically exhibit lower copy numbers and require selection for adequate maintenance within cells. We report the construction and utilization of an efficient heterologous gene expression system containing a 4.5-kb inducible expression cassette inserted into the 2 microns plasmid and selected in cells utilizing a carrier plasmid which is subsequently lost via FRT/Flp recombination. The non-selectable 2 micron plasmid, containing the cassette, was found to be stably maintained in cells, without selection, at high copy number. The dynamics of resolution and partitioning of this plasmid were analyzed during the course of 50 generations of growth under non-selective conditions. The heterologous lacZ reporter gene coding for beta-galactosidase (beta Gal) is driven by the hybrid, galactose-inducible promoter GAL10::pMF alpha 1. Upon induction, beta Gal was secreted into the periplasm and culture supernatant at levels which could be detected directly from Coomassie blue-stained SDS-PAGE. Furthermore, plasmid-containing cells could be maintained directly on rich YPD medium and identified either by utilizing XGal or by observing inhibition of colony growth on YPGal solid medium. The cassette was designed for direct, high-level, inducible expression of cloned genes downstream from the MF alpha 1 signal sequence, with or without a C-terminal lacZ fusion. This vector represents the first demonstration of a non-selectable, mitotically stable, episomal plasmid system capable of expressing recombinant proteins at high levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗