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Biomedical subjects

G Palú

Publications and source records attributed to G Palú.

At least 19 recordsLinked to original sources
PubMed2000

Seroprevalence of herpes simplex virus type 2 infection among attendees of a sexually transmitted disease clinic in Italy. Italian Herpes Forum.

BACKGROUND AND OBJECTIVES: An increased prevalence of herpes simplex virus type 2 (HSV-2) infection has been recently observed in industrialized countries. GOAL: To determine HSV-2 seroprevalence in a high-risk population in Italy. STUDY DESIGN: A cross-sectional study was performed to ascertain the HSV-2 prevalence among 919 persons attending an STD clinic in northern Italy. A HSV-2-specific glycoprotein G-2-based immunoglobulin G enzyme-linked immunoabsorbent assay (Gull/Meridian ELISA; Meridian Diagnostics, Cincinnati, OH) was used and validated against Western blot analysis. RESULTS: A prevalence of 24.6% was found without differences between males and females. Seroprevalence increased with age and number of partners during the previous year. Compared with Western blot analysis, the Gull/Meridian ELISA showed a sensitivity of 91.9% and a specificity of 98%, and positive and negative predictive values of 93.9% and 97.4%, respectively. CONCLUSION: This is the first Italian survey of HSV-2 infection conducted with a properly validated, Food and Drug Administration-approved, type-specific serologic method in a high-risk population. It is likely that between one to three million adults are infected with HSV-2.

Adolescent↗
PubMed2000

Immunogenicity of the B monomer of Escherichia coli heat-labile toxin expressed on the surface of Streptococcus gordonii.

The B monomer of the Escherichia coli heat-labile toxin (LTB) was expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant bacteria expressing LTB were used to immunize BALB/c mice subcutaneously and intragastrically. The LTB monomer expressed on the streptococcal surface proved to be highly immunogenic, as LTB-specific immunoglobulin G (IgG) serum titers of 140,000 were induced after systemic immunization. Most significantly, these antibodies were capable of neutralizing the enterotoxin in a cell neutralization assay. Following mucosal delivery, antigen-specific IgA antibodies were found in feces and antigen-specific IgG antibodies were found in sera. Analysis of serum IgG subclasses showed a clear predominance of IgG1 when recombinant bacteria were inoculated subcutaneously, while a prevalence of IgG2a was observed upon intragastric delivery, suggesting, in this case, the recruitment of a Th1 type of immune response.

Animals↗
PubMed1999

Rational reconstitution of the immune repertoire in AIDS with autologous, antigen-specific, in vitro-expanded CD4 lymphocytes.

HIV infection leads to decrease of CD4 lymphocytes. In particular, loss of CD4 cells specific for opportunistic pathogens results in active opportunistic infections, that are the chief cause of morbidity and mortality in AIDS. Highly active anti-retroviral therapy (HAART) has been shown to have dramatic effects in a large fraction of treated individuals, such as decrease of viral load and increase of CD4 cells. It has not been clearly established, though, whether CD4 cells that appear during HAART represent a functional repertoire (i.e. a CD4 repertoire that encompasses all specificities, including T-cells responding to opportunistic pathogens, as in immunocompetent individuals) or an amplified mirror image of a defective repertoire. Therefore we propose that the immune repertoire can be reconstituted with autologous CD4 lymphocytes collected at early stages of infection, selected for specificity for opportunistic pathogens, expanded and stored for future use when laboratory or clinical signs indicate a depletion of antigen-specific CD4 cells. Since the reinfused CD4 cells are themselves susceptible to HIV infection-replication, we suggest that in vitro gene therapy of these cells may confer a genetic resistance that is permanently maintained in these cells.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Use of murine CXCR-4 as a second receptor by some T-cell-tropic human immunodeficiency viruses.

The human CXCR-4 molecule serves as a second receptor for primary, T-cell-tropic, and laboratory-adapted human immunodeficiency virus type 1 (HIV-1) isolates. Here we show that murine CXCR-4 can support the entry of some of these HIV-1 isolates. Differences between mouse and human CXCR-4 in the ability to function as an HIV-1 receptor are determined by sequences in the second extracellular loop of the CXCR-4 protein.

Animals↗
PubMed1996

Human immunodeficiency virus. Mutations in the viral protease that confer resistance to saquinavir increase the dissociation rate constant of the protease-saquinavir complex.

Mutations in the human immunodeficiency virus (HIV) protease (L90M, G48V, and L90M/G48V) arise when HIV is passaged in the presence of the HIV protease inhibitor saquinavir. These mutations yield a virus with less sensitivity to the drug (L90M > G48V >> L90M/G48V). L90M, G48V, and L90M/G48V proteases have 1/20, 1/160, and 1/1000 the affinity for saquinavir compared to WT protease, respectively. Therefore, the affinity of mutant protease for saquinavir decreased as the sensitivity of the virus to saquinavir decreased. Association rate constants for WT and mutant proteases with saquinavir were similar, ranging from 2 to 4 x 10(7) M-1 s-1. In contrast, the dissociation rate constants for WT, L90M, G48V, and L90M/G48V proteases complexed with saquinavir were 0.0014, 0.019, 0.128, and 0. 54 s-1, respectively. This indicated that the reduced affinity for mutant proteases and saquinavir is primarily the result of larger dissociation rate constants. The increased dissociation rate constants may be the result of a decrease in the internal equilibrium between the bound inhibitor with the protease flaps up and the bound inhibitor with the flaps down. Interestingly, the affinity of these mutant proteases for VX-478, ABT-538, AG-1343, or L-735,524 was not reduced as much as that for saquinavir. Finally, the catalytic constants of WT and mutant proteases were determined for eight small peptide substrates that mimic the viral cleavage sites in vivo. WT and L90M proteases had similar catalytic constants for these substrates. In contrast, G48V and L90M/G48V proteases had catalytic efficiency (kcat/Km) values with TLNF-PISP, RKIL-FLDG, and AETF-YVDG that were 1/10 to 1/20 the value of WT protease. The decreased catalytic efficiencies were primarily the result of increased Km values. Thus, mutations in the protease decrease the affinity of the enzyme for saquinavir and the catalytic efficiency with peptide substrates.

Antiviral Agents↗
PubMed1996

Use of cis- and trans-acting viral regulatory sequences to improve expression of human immunodeficiency virus vectors in human lymphocytes.

We compared the efficiency of human immunodeficiency virus (HIV-1) vectors that express a marker gene (chloramphenicol acetyltransferase, CAT) using different promoter elements. In one vector, CAT was expressed under the control of an internal murine leukemia virus (MuLV) long terminal repeat (LTR). In other vectors, CAT production was regulated by the HIV-1 LTR; these vectors also contained the HIV-1 tat gene and pol sequences reported to exert cis-acting positive effects on reverse transcription or gene expression. Vectors employing the Tat-driven HIV-1 LTR exhibited up to 500-fold greater CAT expression in Jurkat lymphocytes or human peripheral blood mononuclear cells compared with vectors using the internal MuLV LTR element as a promoter. This difference was not due to improved packaging of the vector RNA into virions, but to an improved level of gene expression in the target cells. Target cell CAT expression was two- to threefold higher for the vector containing the pol sequences and was only slightly less than that seen for a trans-complemented envdeleted provirus. These results indicate that defective HIV-1 vectors with efficiencies of gene transfer and expression comparable with that of HIV-1 itself are feasible.

Cells, Cultured↗
PubMed1994

A bioactive fullerene peptide.

The highly hydrophobic C60 (buckminsterfullerene) was water solubilized by covalently linking the synthon 1,2-dihydro-1,2-methanofullerene [60]-61-carboxylic acid to the alpha-amino group of the hydrophilic 4-8 sequence of peptide T, known to display potent human monocyte chemotaxis. The resulting compound, characterized by a variety of analytical techniques, including a UV spectrum in aqueous solution, exhibits remarkable chemotactic potency, comparable to that of the parent pentapeptide. Furthermore, this fullerene-peptide conjugate inhibits, albeit weakly, HIV-1 protease.

Amino Acid Sequence↗
PubMed1994

Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) can be used to generate recombinant viral vectors for delivery of heterologous genes to human CD4-positive lymphocytes. To define the cis-acting sequences required for efficient gene transfer, a number of HIV-1 vectors containing a previously identified packaging signal, long terminal repeats, and additional gag, pol, and env viral sequences were designed. By providing the viral proteins in trans, recombinant viruses were generated and analyzed for their abilities to transfer genes into human T lymphocytes. Inclusion of up to 653 nucleotides derived from the 5' end of the gag gene in the vector improved the efficiency of gene transfer, but inclusion of additional gag or pol sequences did not further improve this efficiency. The increased efficiency of gene transfer associated with the inclusion of 5' gag sequences in the vector arose, at least in part, from an increase in the packaging of vector RNA. The presence of the Rev-responsive element (RRE) increased the efficiency of transfer of vectors containing significant lengths of gag sequence, as expected from the Rev requirement for nucleus-to-cytoplasm transport of unspliced vector RNA containing intact packaging signals. However, the presence of a RRE did not affect the transfer efficiency of smaller vectors lacking significant lengths of gag sequences, arguing against a specific role for the RRE in packaging or vector transfer. These results contribute to an understanding of the minimal cis-acting sequences that operate in the context of HIV-1 vectors for delivering genes into human lymphocytes.

CD4-Positive T-Lymphocytes↗
PubMed1993

Polymerase chain reaction amplification and restriction enzyme typing as an accurate and simple way to detect and identify human papillomaviruses.

A simple and economic method for the detection and identification of human papillomaviruses (HPV) is described. The method has been developed with cloned HPV DNA and DNA from clinical samples. Genomic fragments were obtained from several different HPV types, including the ones most frequently encountered in the genital tract by polymerase chain reaction (PCR) amplification directed by degenerate general primers. The amplification fragments were identified by a form of miniature fingerprinting, with a set of restriction enzymes that gave a unique digestion pattern for each HPV type. Different strategies are proposed, based on PCR and restriction analysis, and this approach to identification was compared with more classic methods such as Southern hybridisation.

Condylomata Acuminata↗
PubMed1992

Inhibition of replication of HIV-1 by retroviral vectors expressing tat-antisense and anti-tat ribozyme RNA.

A ribozyme was constructed that specifically cleaves RNA that contains the first coding exon of the tat gene of HIV-1. This anti-tat ribozyme was incorporated into a Moloney murine leukemia virus vector. A sequence containing only the 48-nucleotide antisense region of the ribozyme was also inserted into the retroviral vector. Human T-cell lines constitutively producing the tat-antisense and the anti-tat ribozyme RNA were created by transduction into Jurkat cells. When challenged with HIV-1, both the tat-antisense and anti-tat ribozyme-producing cells inhibited the replication of HIV-1. The antisense vector conferred a greater resistance to HIV-1 replication than did the anti-tat ribozyme vector.

Base Sequence↗
PubMed1991

Cellular uptake of phosphonylmethoxyalkylpurine derivatives.

The cellular uptake of the phosphonylmethoxyalkylpurine derivatives HPMPA and PMEA has been studied in H9 cells. The two compounds exhibited an identical pattern of permeation in this cell line. Uptake did not occur via the nucleoside transport system, but through a different mechanism which, for its slow kinetics and temperature-dependence, is compatible with an endocytosis-like process. The amount of cell-associated drug increased up to one hour post-incubation.

Adenine↗
PubMed1990

Relevance of ionic effects on norfloxacin uptake by Escherichia coli.

The uptake of the quinolone drug norfloxacin by Escherichia coli was investigated at initial rate kinetics at different pH and monovalent/divalent metal ion concentration. The results support a simple diffusion mechanism for quinolone incorporation into cells. The uptake process decreases under acidic conditions. The presence of Na+ or K+ ions does not affect the results to an appreciable extent, whereas divalent ions cause a dramatic decrease in drug incorporation. The antibacterial activity, evaluated under identical experimental conditions, shows a direct relationship with the uptake data. As a general explanation for the above results it is suggested that the ability of the drug to penetrate into cells is a function of its net charge. The molecule in the zwitterionic form exhibits maximum permeation properties, whereas the uptake is remarkably reduced when the drug bears a net charge as a result of ionization or complex formation with bivalent ions. These results allow further insight into the mechanism of quinolone access to the intracellular compartment.

Biological Transport↗
PubMed1990

Effect of herpes simplex virus type 1 infection on nucleoside transport in HeLa S3 cells.

The initial velocity of thymidine uptake was measured in HeLa S3 cells infected with herpes simplex virus type 1 (HSV-1). The rate of nucleoside influx into the cells was shown to increase from as early as 1 h post-infection (p.i.) up to 8 h p.i. This increased uptake was shown to be attributable to a progressively increasing contribution from passive diffusion superimposed upon normal transport. Thus, the specific nucleoside transport system was still operating with unaltered kinetic parameters 8 h after infection. Despite the inhibition of host cell protein synthesis and its replacement by the synthesis of virus-specified proteins, the numbers and affinity of the nucleoside transporters in cells 8 h after infection were virtually unchanged. The increased transport of thymidine in infected cultures was resistant to the nucleoside transport inhibitor dipyridamole, and was correlated with entry of a normally impermeant solute (sucrose) into infected cells. These data suggest that the system for the carrier-mediated facilitated diffusion of nucleosides remains intact in HSV-infected cells, but that progressively increasing passive diffusion takes place. Passive diffusion is the major process operating late after virus infection.

Affinity Labels↗
PubMed1989

Acyclovir resistance in herpes simplex virus type 1: biochemical and functional studies on the thymidine kinase of the highly resistant R100 strain.

The biochemical and functional properties of the thymidine kinase (TK) of the herpes simplex virus type 1 mutant R100, that is highly resistant to 9-(2-hydroxyethoxymethyl)guanine (acyclovir), are reported in comparison with the properties of its parental strain, wt. The mutant induced the production of a TK activity that accounted for only 10% of the wt one. This feature was not apparently related to a defective expression of the TK gene but it was rather connected to some functional characteristics of R100 enzyme. Although affinities of this enzyme for ATP and thymidine were unchanged, apparent Vmax values for thymidine were much reduced. In addition, affinities for antiviral analogues acyclovir, 9-(1,3-dihydroxymethyl)guanine (DHPG), 5-(2-bromovinyl)2'-deoxyuridine (BVdU), and 5-iodo-2'deoxycytidine (IdCyd) were drastically diminished (between 50-fold and more than 100-fold). This mutation therefore seems to affect the active site of the enzyme which is involved in the catalytic conversion of thymidine and in the binding of the analogues. The above features of HSV-1 R100 seem quite distinct from those of previously described HSV-1 resistant mutants.

Acyclovir↗
PubMed1988

Preliminary characterization of a mutant of herpes simplex virus type 1 selected for acycloguanosine resistance in vitro.

In this paper we report on the preliminary characterization of a mutant of herpes simplex virus type 1 (HSV-1) selected for acycloguanosine (acyclovir, ACV) resistance in vitro. The ACVr virus was examined for a series of parameters that include chemosensitivity assay, thymidine kinase (TK) activity, in vitro and in vivo growth, and mutation mapping. The data obtained indicate that a mutated TK gene is responsible for the ACVr phenotype. A distinctive feature of this mutant is the high level of resistance exhibited to ACV (100 microM) and the concomitant presence of a functional TK activity. Such a property makes this virus useful as a model for the study of viral resistance to nucleoside-type analogues in HSV.

Acyclovir↗
PubMed1980

Thymidine phosphorylase: a possible marker of cell maturation in acute myeloid leukaemia.

Thymidine Phosphorylase activity has been measured in two populations of human Acute Myeloid Leukaemia cells (AML) and in normal human macrophages. During short-term culture in vitro of AML cells the activity of this enzyme increases progressively and approaches that of normal non-dividing macrophages at a time when the leukaemia cells in culture show evidence of maturation as assayed by functional, enzymatic and morphological criteria. It is suggested that the level of Thymidine Phosphorylase activity may be a marker of maturation in AML.

Cells, Cultured↗
PubMed1979

Patterns of maturation in short-term culture of human acute myeloid leukaemic cells.

Leukaemic cells taken from the blood of patients with acute myelogenous leukaemia (AML) frequently proliferate in suspension culture without the addition of growth factors for a limited period only. After a 6--10-fold increase in total cells, cell numbers remain constant for a time and finally decline. The main cause for this limited growth in vitro is not, initially at least, cell death leading to a steady state, but maturation associated in its final stages with cessation of DNA synthesis. Two populations of AML cells from Patients St and Wi respectively were studied, and progressive maturation towards mature leucocytes was demonstrated by the gradual acquisition in culture by the growing blast cells of intracellular enzymes (lysozyme, arginase, acid phosphatase and esterase being measured), surface markers (Fc and C3 receptors), of lactoferrin by Wi cells and of colony-stimulating activity by St cells, as well as changes in Ia antigens, phagocytic properties, morphology and adhesiveness to plastic. With St cells, which carried a characteristic chromosome marker, maturation terminated in cells with the characteristic properties of macrophages. At an intermediate stage, non-adherent and still-dividing St cells acquired Fc and C3 receptors and enzymes characteristic of monocytes. Wi cells progressively became neutrophil-like, and again there was an intermediate population of dividing cells which had Fc and C3 receptors and proteins such as lactoferrin and esterases. characteristic of neutrophils.

Cell Adhesion↗