ABSTRACT
In order to determine whether there is an association between the presence of Epstein-Barr virus (EBV) and mycosis fungoides (MF) disease progression, PCR was performed to detect the EBV status of 20 MF patients; six EBV-positive patients were found. EBV variants may differ in their biological properties, such as their ability to transform cells; therefore, the ability of these variants to immortalize B cells in vitro was analysed. Six continuously growing cell lines were obtained from prolonged cultures of unstimulated peripheral blood mononuclear cells that were taken from the six EBV-positive patients with MF. In order to characterize the EBV strains, EBNA-2 and LMP-1/LMP-2 gene polymorphisms in the six cell lines were also analysed. All patients were followed up for 10 years and it was noticed that EBV-positive patients had a poor prognosis with rapid disease progression and high mortality rates, compared to EBV-negative patients. EBV may therefore constitute a co-factor that accelerates the progression of disease.
Subject(s)
B-Lymphocytes/virology , Herpesvirus 4, Human/genetics , Mycosis Fungoides/virology , Polymorphism, Genetic , Aged , Aged, 80 and over , Amino Acid Sequence , Cell Line, Transformed , Cell Transformation, Viral , Cells, Cultured , Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Epstein-Barr Virus Nuclear Antigens/genetics , Epstein-Barr Virus Nuclear Antigens/metabolism , Female , Herpesvirus 4, Human/classification , Herpesvirus 4, Human/isolation & purification , Humans , Male , Middle Aged , Molecular Sequence Data , Mycosis Fungoides/physiopathology , Polymerase Chain Reaction , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Viral ProteinsSubject(s)
Chlamydia Infections/diagnosis , Chlamydia trachomatis/isolation & purification , Female Urogenital Diseases/microbiology , Immunoenzyme Techniques , Male Urogenital Diseases , Adolescent , Adult , Bacteriological Techniques , Female , Female Urogenital Diseases/diagnosis , Humans , Male , Middle Aged , Nucleic Acid Amplification Techniques , Predictive Value of Tests , Reagent Kits, Diagnostic , Sensitivity and SpecificityABSTRACT
We describe the specific interaction between high-purity recombinant human immunodeficiency virus (HIV) type 1 p17 and human gamma interferon (hIFN-gamma) proteins. This interaction was found to be dose dependent and to involve conformational epitopes on both sides. Specificity was confirmed by competition ELISA, using monoclonal antibodies (MAbs) to hIFN-gamma as specific reagents. By competition experiments we also identified the epitope(s) on the hIFN-gamma molecule involved in p17 binding, very close to the receptor binding site. The kinetic constants were determined by surface plasmon resonance (SPR) analysis. The affinity constant (KA) of the complex was 2.78 x 10(8) M-1, that is, the ratio between a low dissociation rate constant (Koff)(1 x 10(-5)sec-1) and a high association rate constant (Kon) (3 x 10(3) M-1sec-1). However, p17 did not displace the binding of hIFN-gamma to its cellular receptor, nor did it interfere with the capability of the lymphokine to induce de novo expression of HLA-DR antigens on human monocytic cells or to inhibit the proliferation of tumor cells.
Subject(s)
Gene Products, gag/metabolism , HIV Antigens/metabolism , Interferon-gamma/metabolism , Viral Proteins , Animals , Antibodies, Monoclonal/immunology , Binding, Competitive/immunology , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , HeLa Cells , Humans , Interferon-gamma/pharmacology , Kinetics , Rabbits , Recombinant Proteins , Sensitivity and Specificity , gag Gene Products, Human Immunodeficiency VirusABSTRACT
Cells infected with human immunodeficiency virus type 1 (HIV-1) develop viral antigens which can be detected by immunofluorescence. We developed a flow cytometric immunofluorescence assay (FIFA) which provides a quantitative analysis of HIV-1 p24 using a monoclonal antibody (mAb) as a specific reagent. The reduction of HIV p24 antigen expression in viral infected cells was then used to determine HIV neutralizing antibody titers in human sera. Results obtained by FIFA for detecting neutralizing antibodies against HIV-1, when compared with results obtained by indirect immunofluorescence assay (IFA), showed an overall index of agreement of 94.1%. The correlation between the neutralizing antibody titers obtained with each method was found to be highly significant (ro = 0.8; p < 0.01). The simple methodology and the adaptability of this FIFA to various assay conditions make it suitable for routine purposes and for assessing the efficacy of vaccination strategies.
Subject(s)
Flow Cytometry/methods , Fluorescent Antibody Technique , HIV Antibodies/blood , HIV Core Protein p24/analysis , HIV Infections/immunology , HIV-1/immunology , Antibodies, Monoclonal , Cell Line , Cell Separation , Humans , Neutralization Tests , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
We describe a 65-year-old woman born and living in Northern Italy affected by non tropical spastic tetraparesis and her asymptomatic husband presenting HTLV-1 sequences in their lymphocytes detected by polymerase chain reaction (PCR). We discuss the significance of the case and the mechanism involved in HTLV-1 infection and the relationship with neurological disorders, stressing that this case is the first reported in Italy.
Subject(s)
HTLV-I Infections/microbiology , Human T-lymphotropic virus 1/isolation & purification , Lymphocytes/microbiology , Paralysis/microbiology , Blotting, Southern , Blotting, Western , Cells, Cultured , DNA Probes , DNA, Viral/blood , DNA, Viral/isolation & purification , Enzyme-Linked Immunosorbent Assay , Female , Genes, env , Genes, pX , Genes, pol , HTLV-I Antibodies/blood , HTLV-I Antigens/blood , HTLV-I Infections/diagnosis , Human T-lymphotropic virus 1/genetics , Human T-lymphotropic virus 1/immunology , Humans , Male , Middle Aged , Paralysis/diagnosis , Polymerase Chain ReactionABSTRACT
A stable hybridoma cell line secreting specific antibodies against human interferon-gamma (IFN-gamma) and designated IGMB-14 has been established. It belongs to the IgG1, kappa subclass and it reacts in Western blot with the 28 kDa and 56 kDa polypeptides present in two different affinity purified IFN-gamma preparations. Peripheral blood mononuclear cells (PBMC) from a healthy individual, stimulated in vitro by PHA, were analysed for IFN-gamma production both when viable and following fixation. The presence of cytoplasmic or surface IFN-gamma was visualized by an indirect immunofluorescence assay using monoclonal antibody (MAb) IGMB-14 and a single laser FACS-III fluorescence-activated cell sorter. The staining permitted the detection of newly synthesized cytoplasmic IFN-gamma molecules in lymphocytes at day 1 after PHA stimulation and surface IFN-gamma at day 2. IFN-gamma was expressed on almost all the CD4+ lymphocytes as shown by a double staining technique. The specificity of the reaction was confirmed by Western blots and abolishing IFN-gamma staining by pretreatment of MAb IGMB-14 with IFN-gamma. The presence of surface IFN-gamma was also visualized on freshly isolated PBMC from two patients suffering from measles and AIDS but not on PBMC from a healthy individual. The experiments showed that this immunofluorescent method is useful for the detection, enumeration, and phenotypic characterization of IFN-gamma-producing cells in vitro and, in addition, for evaluating the presence of PBMC expressing IFN-gamma on their surface during a viral disease.
Subject(s)
Antibodies, Monoclonal , Flow Cytometry , Interferon-gamma/isolation & purification , Lymphocytes/analysis , Animals , Antigen-Antibody Reactions , Blotting, Western , Fluorescent Antibody Technique , Humans , Hybridomas/analysis , Interferon-gamma/immunology , Lymphocyte Activation , Lymphocytes/immunology , Mice , Mice, Inbred BALB CABSTRACT
Pseudomonas aeruginosa strains resistant to ciprofloxacin were obtained from parental strains by serial transfer through subinhibitory concentrations of the drug. They showed reduced virulence for mice, and also increased sensitivity to aminoglycosides.
Subject(s)
Ciprofloxacin/pharmacology , Pseudomonas aeruginosa/pathogenicity , Aminoglycosides , Animals , Anti-Bacterial Agents/pharmacology , Drug Resistance, Microbial , Female , Lethal Dose 50 , Male , Mice , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Species Specificity , VirulenceABSTRACT
Productive infection of permissive cell cultures by HIV has been detected by different assays of which the measurement of reverse transcriptase (RT) activity has been considered highly specific and sensitive. Here we describe the production and characterization of a mouse hybridoma cell line, MB12, secreting monoclonal antibodies to HIV p24, the major core protein, and the use of this monoclonal antibody to develop a type specific indirect liquid competitive radioimmunoassay (RIA) capable of providing earlier detection of the replicating virus than the RT assay. This assay also provides a quantitative analysis of HIV p24, which can be used to study the viral replication in permissive cell cultures. The ease of methodology and the adaptability of the competitive RIA to various assay conditions make this immunoassay suitable for the study of HIV expression in infected cell cultures.