RESUMEN
AIMS/HYPOTHESIS: The effect of the benzopyran derivative T33, a novel non-thiazolidinedione agent, was studied on peroxisome proliferator-activated receptors (PPARs), insulin signalling and glucose uptake in adipocytes and skeletal muscle. We hypothesised that T33 could activate PPARgamma and exert a beneficial effect on insulin action on glucose uptake and lipid metabolism. MATERIALS AND METHODS: Using a cell-based reporter gene assay, T33 was identified as a PPARalpha/gamma dual agonist, which activated human PPARgamma and PPARalpha with EC50 values of 19 and 148 nmol/l, respectively. The effect of T33 on glucose metabolism was studied in cultured 3T3-L1 adipocytes and L6 myotubes. In vivo effects of T33 on skeletal muscle were determined in ob/ob mice treated with 8 mg/kg T33. The effect of T33 on metabolic abnormalities was observed in diet-induced obese mice. RESULTS: Exposure of 3T3-L1 adipocytes to T33 for 4 days increased basal and insulin-stimulated glucose uptake, with no effect noted in L6 myotubes. Treatment of ob/ob mice for 20 days with T33 normalised basal and insulin-stimulated glucose uptake and increased phosphorylation of Akt and p38 mitogen-activated protein kinase in skeletal muscle. In contrast, phosphorylation of AMP-activated protein kinase was unaltered. Moreover, T33 improved insulin sensitivity and lipid metabolism in diet-induced obese mice. CONCLUSIONS/INTERPRETATION: T33 is non-thiazolidinedione PPARalpha/gamma dual agonist which directly increases basal and insulin-stimulated glucose uptake in adipocytes and secondarily improves insulin action on insulin signalling and glucose metabolism in skeletal muscle from diabetic ob/ob mice.
Asunto(s)
Cumarinas/farmacología , Glucosa/metabolismo , Músculo Esquelético/metabolismo , Oxazoles/farmacología , PPAR alfa/agonistas , PPAR gamma/agonistas , Tiazolidinedionas/farmacología , Células 3T3 , Adenocarcinoma , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon , Genes Reporteros , Humanos , Ratones , Músculo Esquelético/efectos de los fármacos , RosiglitazonaRESUMEN
Viruses of the tick-borne encephalitis (TBE) antigenic complex, within the family Flaviviridae, cause a variety of diseases including uncomplicated febrile illness, encephalitis, meningo-encephalitis, hemorrhagic fever and chronic disease in humans, domesticated animals or wildlife species. TBE is a serious problem in Latvia with up to a 1,000 patients confirmed serologically annually 1994-1995. No previous data had been reported on the causative agent of TBE in Latvia. In the present study, a virus was isolated from serum of a patient with clinical symptoms of an acute TBE infection. Nucleotide sequence information obtained by direct reverse transcription-polymerase chain reaction (RT-PCR) and the serological characteristics of the isolated virus strain, designated TBE-Latvia-1-96, indicated a closer relationship to the Vasilchenko strain, isolated in Novosibirsk (Siberia, Russia), as compared to the western European or far eastern subtypes of TBE viruses. In a mouse neurovirulence assay, a significant difference in survival rates (days) was shown between Latvia-1-96 and the western European TBE virus subtype.
Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas/aislamiento & purificación , Encefalitis Transmitida por Garrapatas/virología , Proteínas del Envoltorio Viral/análisis , Animales , Anticuerpos Monoclonales , Arvicolinae , Encéfalo/virología , Chlorocebus aethiops , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/patogenicidad , Encefalitis Transmitida por Garrapatas/inmunología , Femenino , Humanos , Letonia , Ratones , Ratones Endogámicos , Filogenia , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Pruebas Serológicas , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , VirulenciaRESUMEN
A previously unknown picornavirus was isolated from bank voles (Clethrionomys glareolus). Electron microscopy images and sequence data of the prototype isolate, named Ljungan virus, showed that it is a picornavirus. The amino acid sequences of predicted Ljungan virus capsid proteins VP2 and VP3 were closely related to the human pathogen echovirus 22 (approximately 70% similarity). A partial 5' noncoding region sequence of Ljungan virus showed the highest degree of relatedness to cardioviruses. Two additional isolates were serologically and molecularly related to the prototype.
Asunto(s)
Arvicolinae/virología , Picornaviridae/clasificación , Secuencia de Aminoácidos , Animales , Antígenos Virales/inmunología , Secuencia de Bases , Cápside/genética , Proteínas de la Cápside , Cardiovirus/inmunología , Reacciones Cruzadas , Efecto Citopatogénico Viral , ADN Viral , Enterovirus Humano B/inmunología , Humanos , Datos de Secuencia Molecular , Picornaviridae/genética , Picornaviridae/aislamiento & purificación , Picornaviridae/ultraestructura , Homología de Secuencia de Aminoácido , Virión/ultraestructuraRESUMEN
Puumala (PUU) hantavirus is the aetiological agent of nephropathia epidemica (NE), a mild form of haemorrhagic fever with renal syndrome, which occurs in Fennoscandia, central Europe and Russia. In Norway, NE-like disease has been reported since 1946 and about 50 cases are diagnosed annually; however, the causative agent has not been characterized. In this study, a virus originating from bank voles (Clethrionomys glareolus) trapped near the town of Eidsvoll (Akershus county) was isolated and passaged in laboratory-bred bank voles. The bank vole strain was identified as a PUU virus by serological typing and by sequence analysis of the S and M gene segments. For comparison, complete or partial S sequences were determined for wild-type PUU strains from five locations in Sweden, two inhabited by the southern variant of bank vole present in Fennoscandia, and three by the northern variant. Phylogenetic analysis showed that Norwegian PUU strains are clustered together with Swedish strains from the first group forming a well-supported sublineage within the PUU genotype, distinct from other sublineages from northern Sweden, Finland, Russia and France. The results are consistent with the view of a complex evolutionary history of PUU strains in post-glacial Fennoscandia. Analyses of the current collection of nucleotide sequences suggest that PUU is the most variable genotype of the known hantaviruses.
Asunto(s)
Genoma Viral , Infecciones por Hantavirus/virología , Orthohantavirus/genética , Secuencia de Bases , Orthohantavirus/aislamiento & purificación , Infecciones por Hantavirus/epidemiología , Humanos , Datos de Secuencia Molecular , Noruega/epidemiología , Filogenia , Alineación de SecuenciaRESUMEN
Hantavirus infection was diagnosed serologically by mu-capture IgM and IgG ELISAs in hemorrhagic fever with renal syndrome (HFRS) patients admitted to Tuzla Hospital, Bosnia-Herzegovina. The results indicated that more than one hantavirus caused the outbreak. To address the question of which hantavirus serotypes were involved, sequentially drawn sera were analyzed by focus reduction neutralization test (FRNT) for antibodies against Puumala, Hantaan, Dobrava, and Seoul hantaviruses. The data revealed that acute- or early convalescent-phase sera, even when drawn as late as 3 weeks after the onset of disease, could not be used for typing of the causative hantavirus; a significant number of these samples showed similar reactivity of neutralizing antibodies to several different hantavirus serotypes. Moreover, although several acute-phase sera showed the highest FRNT titer to Hantaan virus, convalescent sera from these patients in all cases showed high specificity for Puumala or Dobrava viruses. This phenomenon, interpreted as a cross-neutralizing primary antibody response, makes several earlier reports concerning causative agents of HFRS questionable. Serological examination of small rodents trapped in the endemic area identified Puumala- and Dobrava-like virus infections. RT-PCR and sequencing of rodent lung samples identified Dobrava virus in one yellow-necked field mouse (Apodemus flavicollis). Cross-FRNT data, using polyclonal rabbit antibodies, clearly confirmed Dobrava virus as a unique hantavirus serotype. In conclusion, the results revealed that both Puumala- and Dobrava-like viruses caused HFRS in Bosnia-Herzegovina, whereas no signs of Hantaan or Seoul virus involvement were found.
Asunto(s)
Anticuerpos Antivirales/sangre , Fiebre Hemorrágica con Síndrome Renal/etiología , Fiebre Hemorrágica con Síndrome Renal/inmunología , Orthohantavirus/inmunología , Orthohantavirus/patogenicidad , Animales , Arvicolinae/virología , Bosnia y Herzegovina/epidemiología , Reacciones Cruzadas , Brotes de Enfermedades , Ensayo de Inmunoadsorción Enzimática , Orthohantavirus/genética , Fiebre Hemorrágica con Síndrome Renal/epidemiología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Datos de Secuencia Molecular , Muridae/virología , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , ARN Viral/genética , Conejos , Factores de TiempoRESUMEN
Reverse transcription-PCR was used to analyze specimens from 20 Finnish nephropathia epidemica (NE) patients hospitalized during the period from October 1994 to January 1995. Blood and/or urine sediment specimens from seven patients were found to be positive for the genome sequences of Puumala hantavirus (PUU). PCR positivity of the specimens from the patients correlated well with the HLA-DRB1*0301 and HLA B8 alleles, which previously were shown to associate with severe courses of NE. Genetic analysis of the partial M-and/or S-segment sequences obtained from three severely ill NE patients revealed three PUU strains related to but distinct from previously reported strains from Finland. The M-segment sequence of PUU from bank voles trapped near the probable site of infection for one of the patients showed 98.2% identity to that of the PUU strain obtained from the patient, suggesting a link between wild-type PUU from the natural focus and the NE case. The S-segment sequences from the patient and the bank voles, however, showed substantially lower identity (95.8%). As this difference in diversity for M and S genes (1.8 and 4.2%) is atypical for PUU genetic drift, one possibility is that the strain acquired at the putative place of infection is a reassortant one.
Asunto(s)
Genoma Viral , Antígenos HLA/genética , Infecciones por Hantavirus/virología , Orthohantavirus/genética , Adulto , Anciano , Animales , Femenino , Infecciones por Hantavirus/inmunología , Infecciones por Hantavirus/transmisión , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Roedores , Análisis de SecuenciaRESUMEN
Two monoclonal antibody escape virus mutants (MARs), rescued from a human MAb to glycoprotein 2 (G2) and a bank vole monoclonal antibody (MAb) directed to glycoprotein 1 (G1) of Puumala virus, strain Sotkamo, were produced by using a combination of neutralization tests and antigen detection. The MARs and the original virus were analyzed by nucleotide sequencing and the responsible mutations were defined and characterized. The G1 mutation was found to constitute an A to T nucleotide substitution, giving raise to an aspartic acid to valine mutation at residue 272, potentially increasing the hydrophobicity of this region. The G2 mutation was found to constitute a C to T substitution, altering the residue 944 from serine into the more hydrophobic phenylalanine and resulting in secondary structure alterations. The mutation was found to be in close vicinity to a glycosylation site. Synthetic peptides covering the regions of the native virus, defined by the MARs, were produced and evaluated for reactivity with the corresponding MAb. The peptides were not recognized by the MAbs, and did not inhibit the binding of the MAbs in competition assays. Sera from mice immunized with the peptides were not able to recognize the native protein. This indicates that the epitopes are non-linear and/or glycosylated in the native state, or alternatively, that the G1 and G2 MAbs binds to regions away from the mutations.
Asunto(s)
Antígenos Virales/inmunología , Epítopos/inmunología , Glicoproteínas/inmunología , Orthohantavirus/inmunología , Mutación Puntual , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Secuencia de Bases , Chlorocebus aethiops , ADN Viral , Procesamiento Automatizado de Datos , Epítopos/genética , Glicoproteínas/genética , Orthohantavirus/genética , Humanos , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Péptidos , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Células Vero , Proteínas del Envoltorio Viral/genéticaRESUMEN
Small mammals trapped in Sweden were analysed for specific antibody responses against three hantavirus serotypes and for the presence of viral antigen. To determine the genetic identity of viral RNA in lungs of seropositive bank voles (Clethrionomys glareolus), polymerase chain reactions and subsequent partial sequencing of both the M and S segments were employed. The sequences obtained were all identified as Puumala (PUU) virus, with a high degree of heterogeneity between the different geographical localities. Alignment of nucleotide and deduced amino acid sequences, together with phylogenetic analysis, showed that PUU viruses circulating in central Sweden were distinct from those in the northern region. The localization of the two distinct PUU virus genotypes was shown to correlate with the postglacial recolonization of Sweden by bank voles.
Asunto(s)
Variación Genética , Infecciones por Hantavirus/virología , Orthohantavirus/genética , ARN Viral/análisis , Animales , Anticuerpos Antivirales/sangre , Arvicolinae/virología , Chlorocebus aethiops , ADN Mitocondrial/análisis , Orthohantavirus/clasificación , Orthohantavirus/inmunología , Infecciones por Hantavirus/sangre , Infecciones por Hantavirus/inmunología , Infecciones por Hantavirus/patología , Filogenia , Suecia , Células VeroRESUMEN
Two hantavirus strains, MF43 and MF113, isolated from Microtus fortis trapped in the Khabarovsk region of far-eastern Russia, were analysed by direct nucleotide sequencing of PCR generated fragments of the M and S segments, by immunofluorescence and by focus reduction neutralization tests (FRNT). The nucleotide sequences revealed that the two isolates were closely related to each other but distinct from all other hantaviruses. Phylogenetic analysis of the M and S segments showed that the MF strains form a separate branch in the Hantavirus tree, positioned between the branches of Prospect Hill and Puumala viruses. The strains were shown to be serologically distinct from the other hantavirus serotypes by FRNT using immune rabbit sera. Puumala virus was the closest relative, both genetically and serologically. We propose that this new hantavirus serotype should be named Khabarovsk (KBR).
Asunto(s)
Arvicolinae/virología , Orthohantavirus/clasificación , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Secuencia de Bases , Chlorocebus aethiops , ADN Viral , Orthohantavirus/genética , Orthohantavirus/inmunología , Orthohantavirus/aislamiento & purificación , Datos de Secuencia Molecular , Pruebas de Neutralización , Filogenia , Conejos , Federación de Rusia , Serotipificación , Células VeroRESUMEN
The Swedish Puumala (PUU) virus strain Vindeln 83-L20, isolated from a bank vole trapped in 1983 near Vindeln, Västerbotten county, Sweden, was characterized by nucleotide sequence analysis. The coding region of the M segment was determined by PCR followed by direct sequencing and the entire S segment was characterized by cloning and nucleotide sequence analysis. The genomic organization was found to be very similar to that of other PUU virus strains regarding open reading frames, polypeptide sizes and potential glycosylation sites. According to phylogenetic analysis 83-L20 was found to represent a new lineage within the Puumala virus serotype in the Hantavirus genus. The M segment sequence of 83-L20 was found to be more closely related to the Finnish PUU virus strains than to strains from Central Europe or from Russia. The evolutionary origin of the S segment was not as clearly resolved since the branching points of all PUU virus strains in the phylogenetic tree were nearly the same.
Asunto(s)
ADN Viral , Infecciones por Hantavirus/veterinaria , Orthohantavirus/genética , Secuencia de Aminoácidos , Animales , Arvicolinae , Secuencia de Bases , Chlorocebus aethiops , Orthohantavirus/química , Orthohantavirus/aislamiento & purificación , Infecciones por Hantavirus/virología , Datos de Secuencia Molecular , Filogenia , Suecia , Células VeroRESUMEN
Antigen capture enzyme-linked immunosorbent assays (ELISAs) based on detection of the viral nucleocapsid protein (N) were designed for rapid diagnosis of hantavirus infections. Several combinations of bank vole (Clethrionomys glareolus) monoclonal antibodies with various N-epitope specificities were used for the development of two double-antibody sandwich forms of ELISA; PUU virus AG-ELISA for an exclusive detection of Puumala-related viruses, and Hantavirus AG-ELISA for a more extensive detection of all serotypes of hemorrhagic fever with renal syndrome (HFRS) viruses. The biotin-streptavidin system, in combination with horseradish peroxidase, rendered the assays' sensitivities similar to or even higher than immunoblotting. Calculated detection limits for the PUU virus and the Hantavirus AG-ELISAs were 405 and 50 focus forming units or 80 and 10 infected Vero E6 cells, respectively. The assays were evaluated and found to be suitable for convenient and routine detection of hantaviruses in cell cultures and in infected animal tissue.
Asunto(s)
Anticuerpos Monoclonales , Arvicolinae/virología , Cápside/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Infecciones por Hantavirus/diagnóstico , Orthohantavirus/aislamiento & purificación , Proteínas del Núcleo Viral/análisis , Animales , Arvicolinae/inmunología , Línea Celular , Chlorocebus aethiops , Humanos , Immunoblotting , Pulmón/virología , Sensibilidad y Especificidad , Células VeroRESUMEN
A sensitive method based on PCR was developed for the detection of Puumala virus (PUU) in human samples. The assay was found to be specific for PUU-like strains and distinguished between these and hantaviruses of other serotypes. The detection limit was found to be 10(-5) focus-forming units. Clinical samples were collected from patients with nephropathia epidemica in Sweden and western Russia. Five whole blood samples collected from patients in Russia with the acute phase of disease were found to be positive by the PCR. All samples were negative for PUU antigen when examined by enzyme-linked immunosorbent assay. Virus isolation on Vero E6 cells from several of the acute-phase samples, including the 5 PCR-positive samples, was not successful. The amplified samples were subjected to direct nucleic acid sequencing for confirmation of identity. The sequences differed from each other and were closely related to the Russian bank vole isolate CG-1820, thereby indicating the origin of nephropathia epidemica. The PCR was used for amplification and subsequent nucleotide sequencing of eight PUU-like isolates with different geographic origins. The Swedish strains were more closely related to the Finnish PUU prototype strain, Sotkamo, than to the Russian isolates. Interestingly, a Belgian isolate, CG-13891, differed markedly from all other PUU strains.
Asunto(s)
Infecciones por Hantavirus/virología , Orthohantavirus/genética , Orthohantavirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Animales , Antígenos Virales/aislamiento & purificación , Secuencia de Bases , Cartilla de ADN/genética , ADN Viral/genética , Europa (Continente) , Estudios de Evaluación como Asunto , Orthohantavirus/inmunología , Infecciones por Hantavirus/inmunología , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , ARN Viral/genética , ARN Viral/aislamiento & purificación , Federación de Rusia , Sensibilidad y Especificidad , Especificidad de la Especie , SueciaRESUMEN
A sensitive assay based on the polymerase chain reaction for the detection of Ockelbo virus RNA was developed. Two primer pairs from the gene coding for the E2 glycoprotein were chosen. By use of a nested strategy for the primers, as few as 1 to 10 PFU could be detected. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The primer pairs allowed amplification of several Ockelbo and Sindbis virus isolates but discriminated between these and other alphaviruses. Ockelbo virus RNA was detected in 4 of 10 skin biopsy specimens collected during the acute stage of the disease. The identities of the amplified products were confirmed by restriction endonuclease cleavage. Acute- and convalescent-phase sera as well as lymphocytes collected during the convalescent phase were negative by the polymerase chain reaction. No infectious virus could be recovered from any of the specimens.
Asunto(s)
Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Piel/microbiología , Infecciones por Togaviridae/microbiología , Togaviridae/aislamiento & purificación , Secuencia de Bases , Biopsia , Humanos , Datos de Secuencia Molecular , Togaviridae/genéticaRESUMEN
Human monoclonal antibodies (MAbs) against Puumala (PUU) virus were generated and characterized. Human spleen B lymphocytes were preselected for specific surface immunoglobulin (Ig) using magnetic beads coated with the viral glycoproteins, and subsequently immortalized by Epstein-Barr virus transformation. Four IgG-positive monoclonal lymphoblastoid cell lines (LCLs) were established and have remained stable MAb secretors for over 12 months. Analyses of the antigen and epitope specificities recognized by the MAbs showed overlapping binding patterns of four anti-glycoprotein 2-specific clones. Identical isotypes (IgGl lambda) and isoelectric points (9.2) of the four MAbs suggested that they were derived from the same original clone. The MAbs reacted with eight PUU virus-like strains, but were negative for Hantaan, Seoul, and Prospect Hill viruses in an immunofluorescence assay, indicating binding to a conserved epitope unique for strains associated with the European form of haemorrhagic fever with renal syndrome, nephropathia epidemica. The MAbs neutralized all investigated PUU virus-like strains in a focus reduction neutralization test. The MAb neutralizing activity was significantly enhanced in the presence of human or guinea-pig complement. To stabilize and increase antibody secretion and to reduce the demand for culture medium supplements (e.g. fetal calf serum), three of the monoclonal LCLs were fused with the non-secreting human x mouse partner SPAM-8. Several of the established human x (human x mouse) monoclonal triomas grew faster and produced larger amounts of MAbs when compared with the original LCLs.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Linfocitos B/inmunología , Fiebre Hemorrágica con Síndrome Renal/microbiología , Orthohantavirus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Linfocitos B/citología , Línea Celular Transformada , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Femenino , Herpesvirus Humano 4/genética , Humanos , Magnetismo , Persona de Mediana Edad , Muridae , Bazo/inmunologíaRESUMEN
Enzyme-linked immunosorbent assays for determination of antibodies directed to the nucleocapsid protein (N) or to either of the two envelope glycoproteins (G1 and G2) of Puumala virus were designed and evaluated. The assays were proven to be entirely restricted for each viral structural protein by biotin-labelled monoclonal antibodies. Sera from sequentially bled nephropathia epidemica patients (acute, convalescent, and 2-year sera) and sera from 10-20 year convalescents were examined for antibody specificity. All but one (n = 19) acute phase sera were shown to contain IgM antibodies directed to all three viral proteins. In the convalescent specimens the proportions of IgM to the different viral components were similar, but lower, when compared to the acute samples. Low levels of IgM against N and G2 were found in two out of ten 2-year sera. No virus-specific IgM were detected in sera drawn 10-20 years after infection. IgG antibodies to all three viral proteins were detected in all except one acute phase serum. The IgG response initially increased more rapidly to N, as compared to the anti-glycoprotein responses. The levels of glycoprotein-specific IgG were considerably increased 2 years after the disease, when compared to the levels detected in the convalescent specimens. The levels and specificities of IgG in very late convalescent sera (drawn 10-20 years after disease) resembled those detected 2 years after infection.
Asunto(s)
Formación de Anticuerpos , Cápside/inmunología , Fiebre Hemorrágica con Síndrome Renal/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Orthohantavirus/inmunología , Insuficiencia Renal/inmunología , Proteínas del Núcleo Viral/inmunología , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Estudios de Seguimiento , Fiebre Hemorrágica con Síndrome Renal/sangre , Fiebre Hemorrágica con Síndrome Renal/fisiopatología , Humanos , Pruebas de Neutralización , Insuficiencia Renal/sangre , Insuficiencia Renal/etiología , Síndrome , Factores de Tiempo , Células VeroRESUMEN
The redox-active enzyme thioredoxin (Trx) is secreted by various virus-transformed cell lines of B- and T-cell origin and has been considered to play an autoregulatory role as a cofactor during cellular growth processes. We show in this paper that exposure of B lymphocytes from normal, healthy donors and B cells from B-type chronic lymphocytic leukemia (B-CLL) to Staphylococcus aureus Cowan I (SAC) induced expression of Trx mRNA. By combining SAC, or the phorbol ester TPA, with IL-2 and the conditioned medium of a T-cell hybridoma (BSF-MP6), we could strongly enhance the Trx expression. After [35S]methionine labeling of stimulated B-CLL cells in vitro, Trx was immunoprecipitated both from cell extracts and from the medium with antibodies against human placenta Trx. Secretion of newly synthesized Trx was also confirmed by a quantitative radioimmunoassay for human Trx. During 24 h cultivation experiments, treatment with SAC induced a 5-fold increase of the Trx content of normal B lymphocytes as well as in B-CLL cells. Approximately two-thirds of the total amount of the enzyme was released into the medium.
Asunto(s)
Linfocitos B/metabolismo , Activación de Linfocitos , Tiorredoxinas/metabolismo , Línea Celular , Células Cultivadas , Fase G1 , Humanos , Inmunoglobulinas/química , Tonsila Palatina , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/efectos de los fármacos , Staphylococcus aureusRESUMEN
An assay for detection of neutralizing antibodies to Puumala virus using 96-well microtiter plates (NT-ELISA) was developed and evaluated. The test proved to have similar sensitivity and specificity as an IgG ELISA and indirect immunofluorescence test, when screening 187 sera (with an antibody prevalence rate of 19%) from normal populations in an endemic area of Nephropathia epidemica (NE) in Sweden. NE-patients monitored for 2 years had neutralizing antibodies in early sera collected 1-4 days after the onset of disease with a continuous increase in neutralizing antibodies with time. Furthermore, high titers of neutralizing antibodies were detected 10-20 years post-infection. This neutralization assay was also evaluated as a screening method in the production of monoclonal antibodies. The format of the NT-ELISA makes it feasible to screen a large number of specimens with results similar to the standard plaque or focus-reduction neutralization tests.
Asunto(s)
Anticuerpos Antivirales/sangre , Pruebas de Neutralización/métodos , Orthohantavirus/inmunología , Virología/métodos , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/estadística & datos numéricos , Estudios de Evaluación como Asunto , Fiebre Hemorrágica con Síndrome Renal/inmunología , Humanos , Hibridomas/inmunología , Inmunoglobulina G/sangre , Pruebas de Neutralización/estadística & datos numéricos , Sensibilidad y Especificidad , Factores de Tiempo , Virología/estadística & datos numéricosRESUMEN
Different virus isolates causing hemorrhagic fever with renal syndrome (HFRS) were compared using a neutralization test. Patient convalescent sera and antisera prepared in rabbits were used to compare Puumala-related Hantavirus isolates from Finland, Sweden, Belgium, and the USSR. The majority of European isolates were indistinguishable from each other using both homologous rabbit antisera and patient convalescent sera. The European isolates of HFRS were also compared with prototype Hantaan (the etiologic agent of Korean hemorrhagic fever). The one-way cross-reaction between the Hantaan and Puumala viruses, previously described using human convalescent sera tested by indirect immunofluorescence and immunoprecipitation, was also seen by the neutralization test.