RESUMEN
Simian immunodeficiency virus (SIV), which causes an acquired immunodeficiency syndrome in macaques, is a lentivirus that is morphologically, antigenically, genetically, and biologically similar to the human immunodeficiency virus (HIV). Because of these similarities, the SIV model represents a unique opportunity for in vitro and in vivo testing of antiretroviral agents. Since antiretroviral agents may exhibit different properties in different cells in vitro, more than one cell line may be necessary to evaluate the efficacy and modes of action of an antiretroviral agent. Initially we tested ten cell lines for their permissiveness to five SIV isolates. One B-cell line (AA-2) and one T-cell line (HuT 78) were selected to test antiretroviral agents since both were extremely permissive for SIVmac251, an isolate with a high rate of infectivity. Using this optimized in vitro testing protocol, we screened ten antiretroviral agents for their ability to inhibit SIV replication. Six of the compounds completely inhibited SIV viral antigen expression. Based on the selectivity index, 3'-azido-3'-dideoxythymidine, 3'-azido-2',3'-dideoxyuridine, and 3'-fluoro-3'-deoxythymidine appear to be the most efficacious antiretroviral agents against SIVmac251. Several different assays for determining viral antigen inhibition were conducted and the results of these assays were comparable. Our results demonstrate that the SIV in vitro model is a valuable screening tool for determining the efficacy and toxicity of new antiretroviral agents.
Asunto(s)
Antivirales/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Animales , Antígenos Virales/biosíntesis , Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Humanos , Macaca nemestrina , ADN Polimerasa Dirigida por ARN/metabolismo , Virus de la Inmunodeficiencia de los Simios/enzimología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Replicación Viral/efectos de los fármacosRESUMEN
A rapid, sensitive and specific antigen capture (AC) assay has been established for the detection of p27 core protein of SAIDS type-D retrovirus (SRV). SRV p27 antigen in test samples was identified on rabbit anti-p27 IgG-coated microtiter plates by the addition of biotinylated rabbit anti-p27 IgG. This assay was specific for the p27 core protein of SRV-1 and SRV-2 and provided semi-quantitative results in less than 7 hours. Results of the AC assay were highly correlated with those of reverse transcriptase (RT), immunofluorescence and immunoblotting assays. However, the AC assay was faster and more sensitive than the other three assays. The AC assay also provided a rapid diagnostic tool for the detection of SRV in plasma, serum and peripheral blood lymphocyte cocultures. In addition to mass screening of SRV infection in macaque colonies, the AC assay also will be valuable for monitoring the efficacy of antiretroviral agents against SRV in vitro and in vivo.
Asunto(s)
Antígenos Virales/análisis , Macaca nemestrina/microbiología , Macaca/microbiología , Enfermedades de los Monos/diagnóstico , Infecciones por Retroviridae/veterinaria , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Antígenos Virales/sangre , Línea Celular , Electroforesis en Gel de Poliacrilamida , Epítopos , Técnica del Anticuerpo Fluorescente , Immunoblotting , Macaca mulatta/microbiología , Infecciones por Retroviridae/diagnóstico , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificaciónRESUMEN
Four dosing regimens of 2',3'-dideoxycytidine (ddC) were administered intravenously for 10 to 28 days to 18 pigtailed macaques with simian acquired immunodeficiency syndrome. Ten macaques naturally infected with simian acquired immunodeficiency syndrome retrovirus serotype 2 (SRV-2), the etiologic agent of simian acquired immunodeficiency syndrome, received ddC by continuous intravenous infusion or by a daily bolus injection for 10 to 12 days. Another eight macaques that were negative for SRV-2 and antibody received ddC prophylaxis prior to challenge with virus and continued to receive ddC therapy for up to 28 days postchallenge. All monkeys treated with a continuous intravenous dose of ddC, which maintained plasma concentrations of ddC at levels known to inhibit SRV-2 in vitro, developed dose-related toxic effects, including leukopenia, anemia, lethargy, and decreased appetite. Monkeys treated with a daily bolus injection of ddC experienced more severe toxic effects than those on the continuous intravenous regimen, including exfoliative dermatitis and peripheral neuropathy. At the concentrations of ddC administered, no significant inhibition of SRV-2 replication was detected in naturally infected macaques. However, a prophylactic regimen of ddC did have an inhibitory effect on SRV-2. Our findings suggest that ddC may be valuable as a short-term prophylactic treatment rather than as a long-term therapy.
Asunto(s)
Infecciones por Retroviridae/tratamiento farmacológico , Retrovirus de los Simios/efectos de los fármacos , Zalcitabina/uso terapéutico , Animales , Antígenos Virales/análisis , Técnica del Anticuerpo Fluorescente , Macaca nemestrina , Infecciones por Retroviridae/patología , Retrovirus de los Simios/inmunología , Zalcitabina/toxicidadRESUMEN
A receptor binding the Fc region of equine immunoglobulin G (IgG) has been isolated from a heat-extracted preparation of a clinical isolate of Streptococcus zooepidemicus. This Fc receptor has a Mr of 45 x 10(3) and was occasionally seen as an apparent trimer of Mr 130 x 10(3). Antibodies prepared in horses against the receptor could be adsorbed to and eluted from whole live bacteria, confirming the surface location of this protein. Another 11 isolates of S. zooepidemicus from horses with pneumonia, abscesses or endometritis were tested for Fc-receptor activity. Although the Mr of the Fc receptors varied among isolates, their antigenicity was conserved. Thus, the Fc receptor is an attractive candidate for application in the diagnosis of, or protection against, infections with S. zooepidemicus.
Asunto(s)
Antígenos de Diferenciación/inmunología , Proteínas Bacterianas/inmunología , Inmunoglobulina G/inmunología , Receptores Fc/inmunología , Streptococcus/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos de Diferenciación/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Caballos/microbiología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/aislamiento & purificación , Receptores Fc/aislamiento & purificación , Receptores de IgG , Especificidad de la Especie , Streptococcus/análisisRESUMEN
Nonimmune binding of immunoglobulin to whole bacteria was quantitated for North American isolates of Haemophilus somnus recovered from cattle with pneumonia, reproductive failure (abortion), or thromboembolic meningoencephalitis or from the vagina or prepuce of carrier cattle. Quantitative binding activity covered a wide range, with most pathogenic and carrier isolates demonstrating significant immunoglobulin-Fc binding. Isolates for which Fc binding was not detectable were recovered only from the prepuces of asymptomatic bulls. Expression of Fc-binding activity correlated with the presence of the 41,000-molecular-weight protein (41K protein) and 270K protein. Isolates that lacked Fc-binding activity did not possess 41K or 270K protein. A 33K protein was detected in isolates that lacked Fc-binding activity but not in isolates that bound Fc.
Asunto(s)
Sitios de Unión de Anticuerpos , Portador Sano/veterinaria , Infecciones por Haemophilus/veterinaria , Haemophilus/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Aborto Veterinario/microbiología , Animales , Portador Sano/microbiología , Bovinos , Femenino , Haemophilus/aislamiento & purificación , Infecciones por Haemophilus/microbiología , Meningoencefalitis/microbiología , Meningoencefalitis/veterinaria , Neumonía/microbiología , Neumonía/veterinaria , Embarazo , Tromboembolia/microbiología , Tromboembolia/veterinariaRESUMEN
Monoclonal antibodies (MoAbs) to the major gag core protein p27 and a viral protein p44 of type D retrovirus (SRV-2) were produced and used in the detection of SRV-2 antigens in infected Raji cells and in tissues from macaques with simian acquired immunodeficiency syndrome (SAIDS) and retroperitoneal fibromatosis (RF). Anti-p44 MoAb showed inhibition of syncytium formation by both SRV-1- and SRV-2-infected Raji cells.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Antígenos Virales/análisis , Retrovirus de los Simios/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Western Blotting , Línea Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Productos del Gen gag , Hibridomas , Inmunohistoquímica , Pruebas de Neutralización , Proteínas de los Retroviridae/inmunologíaRESUMEN
To characterize the bovine immune response to an Haemophilus somnus antigen known to be recognized by convalescent-phase serum, we studied isotypic antibody titers to the 270-kilodalton protein, which we had previously shown to be an immunoglobulin Fc receptor. With a modified immunodot procedure, an immune response was detected after experimental H. somnus abortion, experimental H. somnus pneumonia, or vaccination with commercial H. somnus vaccine, with the greatest titer found within the immunoglobulin G2 isotype. With protein A peroxidase conjugate, which detects primarily bovine immunoglobulin G2, we showed that cattle with H. Somnus disease could be distinguished from clinically normal carriers, culture-negative cattle, or cattle with disease due to Pasteurella haemolytica or P. multocida. Little cross-reactivity between the 270-kilodalton Fc receptor antigen and antigens from other gram-negative bovine pathogens was seen. Thus, this antigen may be a useful diagnostic antigen.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Enfermedades de los Bovinos/inmunología , Infecciones por Haemophilus/veterinaria , Haemophilus/inmunología , Receptores Fc/inmunología , Aborto Veterinario/inmunología , Animales , Vacunas Bacterianas/inmunología , Portador Sano/inmunología , Portador Sano/veterinaria , Bovinos , Reacciones Cruzadas , Femenino , Infecciones por Haemophilus/inmunología , Immunoblotting , Técnicas para Inmunoenzimas , Inmunoglobulina G/biosíntesis , Masculino , Neumonía/inmunología , Neumonía/veterinaria , Embarazo , Vacunación/veterinariaRESUMEN
A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease.
Asunto(s)
Antígenos Bacterianos/genética , Genes Bacterianos , Haemophilus/genética , Antígenos de Superficie/inmunología , Membrana Celular/inmunología , Clonación Molecular , Enzimas de Restricción del ADN , ADN Bacteriano/genética , ADN Recombinante , Haemophilus/inmunología , Técnicas de Inmunoadsorción , Peso MolecularRESUMEN
In-vitro culture of Haemophilus somnus in liquid or solid media supplemented with bovine blood or serum resulted in non-immune binding of immunoglobulin (Ig) by the organism. This binding was independent of the antigen-combining site of the Ig molecule, since binding of an IgG preparation specific for the hapten dinitrophenol was unaffected by the presence of the homologous antigen. Quantitative comparison of the binding of Ig fragments Fab and Fc demonstrated that the non-immune binding occurred in the Fc region of bovine IgG. The isotypes of Ig that became bound to H. somnus included both bovine IgG subclasses (IgG1 and IgG2), which were bound equally, and bovine IgM.
Asunto(s)
Haemophilus/metabolismo , Inmunoglobulinas/metabolismo , Animales , Anticuerpos Antibacterianos/metabolismo , Especificidad de Anticuerpos , Bovinos , Dinitrofenoles/inmunología , Ensayo de Inmunoadsorción Enzimática , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Unión Proteica , Albúmina Sérica Bovina/inmunologíaRESUMEN
Receptors that bind the Fc region of bovine immunoglobulin (Ig) have been isolated from the culture supernatant of Haemophilus somnus by chromatography on a Sepharose 4B column. One receptor with a relative molecular weight of 41,000 weakly binds both bovine IgG subclasses, IgA and IgM, while three high molecular weight receptors (350,000, 270,000, and 120,000) strongly bind bovine IgG2, IgA, and IgM. All four Fc receptors are antigenically related and the 41,000 receptor appears to be a subunit of the high molecular weight receptors. In addition to bovine Ig, the purified 270,000 Fc receptor strongly binds horse IgG, rabbit IgG, pig IgG, cat IgG, dog IgG, and sheep IgG. The receptor also reacts weakly with mouse, rat, chicken, human, and guinea pig IgG and does not bind goat IgG. Fc receptors from 19 H. somnus isolates were compared. Variations in the molecular weight of the 41,000 protein were demonstrated among preputial isolates from asymptomatic carriers, but all other isolates appeared to have identically migrating proteins.
Asunto(s)
Haemophilus/ultraestructura , Receptores Fc/aislamiento & purificación , Epítopos , Inmunoglobulina G/clasificación , Especificidad de la EspecieRESUMEN
Haemophilus somnus expresses two types of receptors that bind to the Fc region of bovine IgG, IgA and IgM. In this study, the relationship between these two types of Fc receptors is characterized. The high molecular mass receptors (350, 270 and 120 kDa) were secreted into the culture medium and were also in the insoluble protein fraction of the culture medium. The 41 kDa Fc receptor, which is a major outer-membrane protein, was only present in the insoluble protein fraction. Peptide mapping of the two types of Fc receptors suggests that the 41 kDa receptor is related to the high molecular mass receptor complex. Disulphide linkage is unlikely to be the mechanism of association of the 41 kDa receptor with the high molecular mass receptors since reducing agents had no effect on separating the individual receptors. Although the 41 kDa receptor is a major protein in the outer membrane of H. somnus, it does not react with convalescent bovine sera in Western blots. In contrast, convalescent bovine sera reacts intensely with the high molecular mass receptors in Western blots.
Asunto(s)
Proteínas Bacterianas/análisis , Haemophilus/análisis , Receptores Fc/análisis , Haemophilus/inmunología , Mapeo PeptídicoRESUMEN
Certain group A streptococcal strains have been reported to express two distinct type II receptors that bind to the Fc region of human IgG. In this study, we have isolated and characterized these two type II Fc receptors and characterized their reactivity with differing species of IgG. The type IIa receptor was found to be a 56,000 molecular weight protein which binds human IgG1, IgG2 and IgG4, in addition to pig and rabbit IgG. The type IIb receptor was found to be a 38,000 molecular weight protein that bound exclusively to human IgG3. Neither the type IIa nor the type IIb receptor bound to goat, cow, dog, rat, or sheep IgG. Monospecific polyclonal antibodies were prepared against both the type IIa and type IIb Fc receptors. These antibodies demonstrated that the type IIa and type IIb were antigenically closely related and could not be distinguished from each other on the basis of their reactivity with either antibody. The distribution of type IIa and type IIb Fc receptors on a variety of different nephritogenic and non-nephritogenic group A streptococcal strains was documented.
Asunto(s)
Receptores Fc/aislamiento & purificación , Streptococcus pyogenes/inmunología , Cromatografía de Afinidad , Inmunoglobulina G/clasificación , Inmunoglobulina G/metabolismo , Peso Molecular , Nefritis/microbiología , Receptores Fc/análisis , Receptores Fc/inmunología , Receptores de IgG , Especificidad de la EspecieRESUMEN
Fifteen bacterial isolates (12 streptococcal and 3 staphylococcal strains) from patients with bacterial endocarditis were screened for a variety of surface receptors, in an attempt to identify a common feature that might contribute to their ability to attach to and colonize damaged heart tissue. The bacterial receptors screened for, using a dot-blot autoradiographic procedure, included those for the Fc region of human IgG, fibrinogen, fibronectin and human C1q. Bacteria with receptors for each of the probes used could be identified, but no common receptor was present on all endocarditis-causing strains.
Asunto(s)
Sitios de Unión , Endocarditis Bacteriana/inmunología , Receptores de Hialuranos , Glicoproteínas de Membrana , Infecciones Estafilocócicas/inmunología , Infecciones Estreptocócicas/inmunología , Proteínas Portadoras , Humanos , Proteínas Mitocondriales , Glicoproteínas de Membrana Plaquetaria/análisis , Receptores de Complemento/análisis , Receptores Fc/análisis , Receptores de Fibronectina , Receptores de IgG , Receptores Inmunológicos/análisisRESUMEN
A group A streptococcal strain rich in Fc receptors was selected by an immunoblotting technique and used as the source for isolation of a functionally active Fc receptor. A variety of extraction techniques were compared including (1) heat extraction at neutral, acid or alkaline pH, (2) treatment with the enzymes mutanolysin, hyaluronidase, trypsin, papain or phage lysin, or (3) autoclaving or heating in the presence of sodium dodecyl sulfate. The most homogeneous receptor was recovered following heat extraction and contained two molecular weight forms. The major form had a molecular weight of 56 000 daltons and the minor form had a molecular weight of 38 000 daltons. These two proteins could be isolated without loss of activity by binding to and elution from a column of immobilized human IgG. An antibody prepared against a single form of the affinity purified receptor demonstrated reactivity with both molecular weight forms of the heat extracted receptor. The group A receptor was found to be both antigenically and physicochemically distinct from either the type I receptor found on the majority of Staphylococcus aureus strains or the type III Fc receptors found on the majority of group C streptococcal strains.
Asunto(s)
Receptores Fc/aislamiento & purificación , Streptococcus pyogenes/análisis , Unión Competitiva , Fraccionamiento Químico , Electroforesis en Gel de Poliacrilamida , Calor , Inmunoelectroforesis , Inmunoglobulina G/metabolismo , Peso Molecular , Receptores Fc/metabolismo , SolubilidadRESUMEN
Receptors that bind to the Fc region of all four human IgG subclasses have been described on a number of strains of group A streptococci. In this study, we have demonstrated that these immunoglobulin binding properties are mediated by two distinct Fc receptors. The first receptor, with a Mr of approximately 56,000, binds to human IgG1, IgG2, and IgG4, but not to IgG3. A second receptor, with a Mr of approximately 38,000, binds exclusively to human immunoglobulins of the IgG3 subclass.
Asunto(s)
Inmunoglobulina G/metabolismo , Receptores Fc/análisis , Streptococcus pyogenes/metabolismo , Animales , Reacciones Antígeno-Anticuerpo , Pollos , Colodión , Electroforesis en Gel de Poliacrilamida , Humanos , Peso Molecular , Proteínas de Mieloma/metabolismo , Papel , Receptores Fc/clasificación , Receptores Fc/inmunología , Receptores de IgG , Proteína Estafilocócica A/inmunología , Proteína Estafilocócica A/metabolismoRESUMEN
Binding of radiolabeled human IgG to bacteria expressing type I, type II, or type III Fc receptors in the presence of glycyl-glycine, glycyl-tyrosine, glycyl-histidine, glycyl-leucine, or glycyl-phenylalanine was studied. No inhibition of labeled human IgG binding to type I or type III Fc receptor positive bacteria was observed by any of the dipeptides. Inhibition of binding of labeled human IgG1, IgG2, and IgG4, but not IgG3, indicated the presence of two distinct Fc receptors associated with the type II Fc receptor-positive group A streptococcal strain.
Asunto(s)
Dipéptidos/metabolismo , Inmunoglobulina G/metabolismo , Receptores Fc/metabolismo , Staphylococcus aureus/inmunología , Streptococcus pyogenes/inmunología , Streptococcus/inmunología , Receptores de IgGRESUMEN
The expression and stability of receptors for the Fc region of human IgG on the surface of group A streptococci was studied. Two strains were sequentially passed in mice 22 times. The Fc-receptor expression on one group A strain, 529, was unaltered while the expression of Fc receptor on a second, 64, was enhanced and approached the level of Fc-receptor expression of the protein A-rich Staphylococcus aureus Cowan strain. The level of Fc-receptor expression on this organism remained stable for over 18 months of laboratory subculture. Mouse passage did not result in the production of a soluble Fc receptor from either of the streptococcal strains. Heat extraction of the Fc-receptor-positive group A strain resulted in solubilization of an Fc-receptor activity which was functionally distinct from either staphylococcal protein A or the Fc receptor isolated from a group C streptococcus.
Asunto(s)
Receptores Fc/inmunología , Infecciones Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C/microbiología , Receptores Fc/aislamiento & purificaciónRESUMEN
We have purified an acid-soluble DNA endonuclease, termed nuclease gamma, from Ustilago cell extracts. The enzyme is nearly homogeneous, purified 1700-fold. The protein appears to be globular with a molecular weight in the range 17,000 to 21,000. It requires a divalent cation and is optimally active at slightly alkaline pH. The enzyme prefers duplex DNA as substrate but will slowly cleave single-stranded DNA. Cleavage of covalently closed duplex DNA is unaltered by changes in superhelix density. Divalent cations direct the mode by which the enzyme cleaves duplex DNA. When Mg2+ or Ca2+ is added, the enzyme nicks one strand of the duplex. When Mn2+, Co2+, or Zn2+ is added, the enzyme can introduce double strand breaks. Oligonucleotides terminated with 5'-phosphoryl and 3'-hydroxyl groups are the products of hydrolysis. DNA fragments generated can be religated to linear pBR322 DNA with completely base-paired ends.