RESUMO
In current sampling approaches, there exists a divergence between the surveillance of arthropod-borne and that of non-arthropod-borne viruses. It is commonly held that the collection of vector specimens applies only to arbovirus surveillance and that the surveillance of non-arboviruses must rely on traditional methods that involve the sampling of blood, faeces or saliva, or other examinations. The vector-based approach is a sampling method that has the ability to survey both arboviruses and non-arboviruses by distinguishing engorged vector specimens from entire vector samples. Accordingly, five arboviruses and three non-arboviruses were detected in a study using a vector-based approach conducted during 2012-2015. Hence, this report provides the first description of the Taiwanese vector species for the bovine arboviruses detected. The present investigations demonstrate that the vector-based approach applies not only to the surveillance of arboviruses, but also has potential as a possible tool for monitoring non-arboviruses on livestock farms in the future.
Assuntos
Infecções por Arbovirus/veterinária , Arbovírus/isolamento & purificação , Doenças dos Bovinos/virologia , Ceratopogonidae/virologia , Culicidae/virologia , Insetos Vetores/virologia , Animais , Infecções por Arbovirus/virologia , Bovinos , Mosquitos Vetores/virologia , TaiwanRESUMO
AIM: To test the measurement properties of the revised and updated Pediatric Quality of Life Inventory (PedsQL) 3.2 Diabetes Module originally developed in Type 1 diabetes in youth with Type 2 diabetes. METHODS: The PedsQL 3.2 Diabetes Module and PedsQL Generic Core Scales were administered in a field test study to 100 young people aged 9-25 years with Type 2 diabetes. Factor analysis was conducted to determine the factor structure of the items. RESULTS: The 15-item Diabetes Symptoms Summary Score and 12-item Type 2-specific Diabetes Management Summary Score were empirically derived through factor analysis. The Diabetes Symptoms and Type 2-specific Diabetes Management Summary Scores showed acceptable to excellent reliability across the age groups tested (α = 0.85-0.94). The Diabetes Symptoms and Type 2-specific Diabetes Management Summary Scores evidenced construct validity through large effect size correlations with the Generic Core Scales Total Scale Score (r = 0.67 and 0.57, respectively). HbA1c was correlated with the Diabetes Symptoms and Type 2-specific Diabetes Management Summary Scores (r = -0.13 and -0.22). Minimal clinically important difference (MCID) scores were 5.91 and 7.39 for the Diabetes Symptoms and Type 2-specific Diabetes Management Summary Scores. CONCLUSIONS: The PedsQL 3.2 Diabetes Module Diabetes Symptoms Summary Score and Type 2-specific Diabetes Management Summary Score exhibited satisfactory measurement properties for use as youth self-reported diabetes symptoms and diabetes management outcomes for clinical research and clinical practice for young people with Type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/epidemiologia , Nível de Saúde , Psicometria/métodos , Qualidade de Vida , Inquéritos e Questionários , Adolescente , Adulto , Fatores Etários , Idade de Início , Criança , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/psicologia , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/psicologia , Estudos de Viabilidade , Feminino , Humanos , Masculino , Medidas de Resultados Relatados pelo Paciente , Reprodutibilidade dos Testes , Inquéritos e Questionários/normas , Adulto JovemRESUMO
Thrips, the sole vector of plant Tospovirus, are major pests of many agricultural crops throughout the world. Molecular approaches have been applied in recent decades to identify these minute and morphologically difficult to distinguish insects. In this study, sequences of internal transcribed spacer 1 (ITS1) region of 15 agronomically important thrips, including several virus transmission species, have been analyzed in order to design species-specific primers for multiplex PCR and probes for microarray assay. That the ITS1 sequence distances within species were smaller than those among species suggests that the ITS1 fragment can be used for thrips species identification. The specificity and stability of these primers, combined with universal paired primers, were tested and verified in multiplex PCR. Using these specific primers as probes, microarray assay showed that PCR products of all thrips species hybridized consistently to their corresponding probes, though some signals were weak. We have demonstrated that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable, and cost-effective diagnostic tool for thrips species identification. Moreover, the DNA microarray assay is expected to extend into a reliable high-throughput screening tool for the vast numbers of thrips.
Assuntos
Controle de Insetos/métodos , Reação em Cadeia da Polimerase Multiplex , Análise de Sequência com Séries de Oligonucleotídeos , Tisanópteros/genética , Animais , Primers do DNA/genética , DNA Espaçador Ribossômico , Dados de Sequência Molecular , Análise de Sequência de DNA , Especificidade da Espécie , Taiwan , Tisanópteros/classificaçãoRESUMO
While morphological identification of thrips species has been difficult because of their minute size and a lack of easily recognizable characteristics, molecular identification based on the development of specific molecular markers can be easily and reliably carried out. Among the known molecular markers, the nuclear internal transcribed spacer (ITS) exhibits distinguishable variations among thrips species. In this study, sequences of ITS2 region of 10 agriculturally important thrips were established to design species-specific primers for polymerase chain reaction (PCR). ITS2 sequence variations within these species were far less than those among species, indicating the suitability of this marker for species-specific primers design. These primers, though with one or two sporadically variable positions, showed a good efficacy within species. The specificity of these primers, examined on thrips species belonging to 15 genera, proved satisfactory. Furthermore, a multiplex PCR was used successfully for identifying Frankliniella occidentalis (Pergande), an insect pest monitored for quarantine purpose, and three additional thrips also commonly found in imported agricultural products and field samples, i.e., Thrips tabaci Lindeman, Thrips hawaiiensis (Morgan), and Frankliniella intonsa (Trybom). This study has demonstrated that specific primers and multiplex PCR based on ITS2 are reliable, convenient, and diagnostic tool to discriminate thrips species of quarantine and agricultural importance.
Assuntos
DNA Intergênico/genética , Tisanópteros/classificação , Tisanópteros/genética , Animais , Primers do DNA/análise , Reação em Cadeia da Polimerase Multiplex , Filogenia , Quarentena , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Approximately one quarter of the identified human serpin genes are cancer-related and clustered mainly at two distinct loci: 6p25 and 18q21. We have studied a novel serpin gene cluster at 3q26 containing at least two recently identified members: the pancreas-specific protease inhibitor, pancpin (PI14), and the brain-associated protease inhibitor, neuroserpin (PI12). In this, unlike a previous study, both PI14 and PI12 at 3q26 were found to consist of 9 exons and 8 introns and to share a perfectly conserved gene organization whose pattern is very different from that of the ov-serpin family. This distinct pattern appears identical in the genomic structures of human plasminogen activator inhibitor-1 (PAI1) at 7q21 and protease nexin 1 (PI7) at 2q33-35, confirming that these four genes in three different chromosomes form a discrete subset within the serpin superfamily. As in the other three members whose gene expression is altered during tumorigenesis, PI12 expression was found to be down-regulated in tumor brain tissues and in two brain cancer cell lines: U-87 MG and H4. By screening genomic libraries, we isolated two overlapping clones showing that the marker SGC32223 (centromere) is located within intron F of PI12 and the marker WI-10077 (telomere) is located downstream of the 3'-flanking region of PI14. This finding indicates that the distance between human PI14 and PI12 is approximately 100 kb, and hence we speculate that other tissue-specific cancer-related serpin genes are likely to reside within this 3q26.1 cluster region.
Assuntos
Cromossomos Humanos Par 3/genética , Família Multigênica/genética , Proteínas de Neoplasias , Inibidores de Serina Proteinase/genética , Sequência de Aminoácidos , Animais , Bacteriófago P1/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular , DNA Complementar/isolamento & purificação , Éxons , Humanos , Íntrons , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma , Estrutura Secundária de Proteína , Ratos , Inibidores de Serina Proteinase/metabolismo , Serpinas/genética , Serpinas/metabolismo , Células Tumorais CultivadasRESUMO
Because of an inadequate supply of potable water, villagers of Small Liu-Chiu Isle, Ping-Tung County, Taiwan, store water in containers supporting a large population of Aedes aegypti. In 1989-96, integrated control measures against Ae. aegypti were implemented on the basis of community participation. These measures included release of mosquito larvivorous fish in the drinking water storage facilities, application of larvicides to the water storage facilities in vegetable gardens, removal of discarded and unused containers and tires, improvement of household water storage facilities, and increase of potable water supply. Before implementation of the integrated control measures in 1988, 74% of the water-containing vessels were water storage facilities, and 24% of those were infested by Ae. aegypti. In 1989, the Breteau index for the entire island, indicating the average distribution density for larval Ae. aegypti, was 53.9, as compared to an index of 1.2 in 1996. In 4 villages located at the southwest and middle of the island, Ae. aegypti nearly became extinct because of the enthusiastic participation of the community. Before the implementation of integrated control, Ae. aegypti was the dominant species in containers both inside and outside the household, but after the integrated control, Aedes albopictus became predominant outside.
Assuntos
Aedes/virologia , Dengue/prevenção & controle , Insetos Vetores/virologia , Controle de Mosquitos , Animais , TaiwanRESUMO
A phase I/IIA clinical trial with the chimeric mouse-human monoclonal antibody CGP 47,439 to the principal neutralization determinant in the V3 region of human immunodeficiency virus type 1 (HIV-1) strain IIIB envelope protein gp120 is reported. The trial was an uncontrolled single-center, open-label, multidose tolerability, immunogenicity, and pharmacokinetic study in homosexual men with advanced HIV disease. Patient groups were formed on the basis of the reactivity of the antibody with the gp120 of their HIV-1 isolates. Intravenous infusions of 1, 10, and 25 mg of antibody were followed by seven escalated doses of 50, 100, and 200 mg, every 3 weeks. The antibody was well tolerated; no toxicity was observed. Some patients showed a transient but insignificant antibody response to the antibody with no apparent adverse reactions or accelerated elimination of it. Substantial serum levels of the antibody were maintained with a mean t1/2 beta of 8-16 days. A virus burden reduction was observed in some patients.
Assuntos
Síndrome da Imunodeficiência Adquirida/terapia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Anti-HIV/uso terapêutico , Proteína gp120 do Envelope de HIV/imunologia , HIV-1 , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/virologia , Adulto , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacocinética , Contagem de Linfócito CD4 , Estudos de Coortes , Anticorpos Anti-HIV/efeitos adversos , Proteína do Núcleo p24 do HIV/sangue , HIV-1/isolamento & purificação , HIV-1/fisiologia , Homossexualidade Masculina , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Testes de Neutralização , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
In a successful management program of dengue vectors, not only health education, source reduction or insecticide application should be conducted, but all basic information should also be manipulated properly and efficiently. This information includes the surveys of species, dispersal and dynamics of vectors, as well as the detection of breeding sources, and the records of dengue cases and epidemic periods. Most of the above information expressed as regionalized variables always varies spatially and/or temporally. However, due to the deficiency of topological information, the conventional database management system cannot efficiently analyze those dengue related data. Thus, we have applied the geographic information system (GIS) to the monitoring of dengue vectors. The purpose of this report is to introduce the basic concepts of GIS, to describe the framework of the prototype dengue vector monitoring system which was built using data collected from the Sanmin area, Kaoshiung city, Taiwan, and to indicate the possibility of using this system to manipulate spatially correlated data and support decision making in the control of dengue disease.
Assuntos
Dengue/prevenção & controle , Sistemas de Informação , Insetos Vetores , Controle de Mosquitos , Animais , Dengue/epidemiologia , Humanos , Taiwan/epidemiologiaRESUMO
Murine monoclonal antibody (MAb) G3-519 has been shown to recognize a conserved neutralizing epitope in the fourth constant (C4) region of the external glycoprotein gp120 of HIV-1. Inasmuch as this antibody effectively neutralized the infectivity of diverse HIV-1 isolates, it has been selected to be developed for passive immunization against HIV-1 infection in humans. In order to minimize the problem of immunogenicity of murine antibodies and to confer additional accessory immune functions, we have constructed mouse/human chimeric and humanized forms of the antibody. The chimeric antibody was constructed by cloning the murine variable regions and replacing the mouse constant regions with those from human Ig gamma 1,kappa. The humanized antibody was constructed using the human KAS variable region framework sequences as template. Engineering was guided by a three dimensional model of the murine variable region. The murine, chimeric and humanized forms of the antibody exhibited similar reactivity with the peptidic antigen in ELISA, and comparably neutralized the infectivity of HIV-1 in vitro. Taken together, our results show that the chimeric and humanized forms of G3-519 essentially retain the binding activity of the mouse parental antibody. Clinical development is planned to assess the prophylactic and therapeutic usefulness of these reshaped antibodies in humans.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Anti-HIV/biossíntese , HIV-1/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Clonagem Molecular , DNA/genética , Genes de Imunoglobulinas , Vetores Genéticos , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/terapia , Humanos , Hibridomas/imunologia , Imunização Passiva , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Vitamin K3 is known to inhibit the growth of various rodent and human tumor cells. However, the molecular mechanism of its action is still elusive. We have found that vitamin K3 induces cell cycle arrest and apoptotic cell death in nasopharyngeal carcinoma (NPC) cells, as evaluated by flow cytometry and DNA gel electrophoresis. Involvement of c-fos and c-myc proto-oncogenes and expression of their proto-oncoproteins in VK3-induced cell cycle arrest and apoptosis were also investigated. Northern blot analysis of NPC cells treated with 50 microM VK3 showed that c-fos was transiently induced for 60 min after treatment, while c-myc was persistently induced for 1-9 h after drug treatment. Western blot analysis also showed that c-Fos was induced at 4-6 h and at 1-4 h after treatment with 50 microM and 200 microM VK3 respectively, while c-Myc was induced at 1-6 h and at 4-6 h, respectively, after such treatments. These results suggest that the expression of c-fos and c-myc may play an important role in the signaling mechanism of VK3-induced growth arrest and apoptotic cell death.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma/genética , Genes fos , Genes myc , Neoplasias Nasofaríngeas/genética , Vitamina K/farmacologia , Actinas/genética , Carcinoma/patologia , Ciclo Celular/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Humanos , Neoplasias Nasofaríngeas/patologia , Células Tumorais CultivadasRESUMO
The effects of okadaic acid (OA), a potent and specific inhibitor of serine/threonine phosphatases 2A and 1, on the transient expression of a human hsp 70 promoter-linked chloramphenicol acetyltransferase gene transfected into N-18 mouse neuroblastoma cells were determined. Assays of reporter gene activity showed that nanomolar concentrations of OA markedly potentiated the heat-induced (but not the basal) expression of pHBCAT, a full-length hsp 70 promoter-driven chloramphenicol acetyltransferase gene construct. This effect of OA was dose-dependent and promoter-specific and appeared to be attributable to inhibition of protein phosphatase 2A as opposed to protein phosphatase 1. The ability of OA to potentiate the heat-induced expression of pHBCAT appeared to be a feature common to several different cell types examined. We propose that the heat-induced transcriptional activation of heat shock genes is associated with the phosphorylation of component(s) of the transcription complex and that OA enhances this phosphorylation, thereby potentiating the heat-induced hsp 70 promoter activity.
Assuntos
Éteres Cíclicos/farmacologia , Proteínas de Choque Térmico/genética , Fosfoproteínas Fosfatases/antagonistas & inibidores , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Temperatura Alta , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Neuroblastoma , Ácido Okadáico , Fosforilação , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Proteínas Recombinantes/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
A summary of the properties of CGP 51901 is shown in Table 3. On the basis of its binding to IgE and IgE-secreting cells and its activity in vitro and in vivo, CGP 51901 is expected to be able to decrease serum IgE by direct clearance of IgE and by reduction of the numbers and productivity of IgE-secreting cells. The end result of reduction of IgE in the circulation and on mast cells is expected to be the attenuation of IgE-mediated reactions and the improvement in allergy symptoms. The effective serum concentration of CGP 51901 is expected to be in the range 1-10 micrograms/ml. Because CGP 51901 is an antibody specific for IgE, it is expected to be highly selective in its activity. Because IgE does not appear to be essential and because CGP 51901 has been rigorously tested to confirm its non-anaphylactic nature, this treatment is not expected to have any adverse effects. Therefore, CGP 51901 is expected to be safe and to have a good probability of being effective when it is tested in human clinical trials.
Assuntos
Anticorpos Anti-Idiotípicos/uso terapêutico , Hipersensibilidade Imediata/terapia , Imunoglobulina E/imunologia , Animais , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Humanos , Hipersensibilidade Imediata/imunologia , Camundongos , Camundongos Endogâmicos BALB CAssuntos
Infecções por HIV/prevenção & controle , HIV-1 , Imunização Passiva , Complexo Relacionado com a AIDS/complicações , Complexo Relacionado com a AIDS/terapia , Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/terapia , Animais , Modelos Animais de Doenças , Feminino , Infecções por HIV/complicações , Infecções por HIV/transmissão , Humanos , Recém-Nascido , Troca Materno-Fetal , Camundongos , Gravidez , Complicações Infecciosas na Gravidez/terapiaRESUMO
The pharmacokinetics of mouse V/human C (gamma 1, kappa) chimeric monoclonal antibody CGP 47 439 specific for the principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) was studied in patients with stage IV HIV-1 disease in an open-labeled phase I/IIA trial. Twelve male patients were enrolled and nine completed the study. Patients were divided into three groups according to the extent of CGP 47 439 to bind to gp120 from their viral isolates: undetectable for group 1, modestly reactive for group 2, and strongly reactive for group 3. A first dose of 1, 10, or 25 mg was administered by intravenous infusion to group 1, group 2 and group 3 patients, respectively. The patients then received seven doses of 50, 100, or 200 mg, respectively, every three weeks. CGP 47 439 serum concentrations were determined by an ELISA using monoclonal antibody AB19-4 specific for the idiotope of CGP 47 439. Half an hour after infusion only 25.5-36.1% of the administered antibody was found in the serum, reflecting its rapid distribution in the extravascular space and possibly binding to gp120 antigen in some of the patients. The terminal elimination half-life (T1/2) was 16.2 days in group 1 patients, 9.7 days in group 2 and in group 3 patients 7.5 days and 9.1 days. An antibody response to CGP 47 439 was not a factor in determining elimination rates, because only very low and transient responses were found in three patients. These results suggest that the reactivity of CGP 47 439 with HIV-1 gp120 contributed to its elimination in HIV-1 infected patients.
Assuntos
Anticorpos Monoclonais/sangue , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1 , Proteínas Recombinantes de Fusão/farmacocinética , Adulto , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/sangue , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/imunologiaRESUMO
We examined the effects of okadaic acid (OA), a potent and specific inhibitor of serine phosphatases 2A and 1, on the transient expression of an hsp 70 promoter-reporter gene construct in IMR-90 human diploid lung fibroblasts. We showed that OA markedly potentiated the heat-induced but not the basal expression of pHBCAT, a full-length human hsp-70-promoter-driven CAT gene construct. This effect of OA was dose and time dependent and promoter specific. Importantly, the potentiating effects of OA appeared to be independent of the binding of the activated heat shock transcription factor (HSTF) to its consensus DNA sequence, the heat shock element (HSE). Thus, OA had no effect on the HSTF DNA-binding activity as measured by mobility shift assay, and mutation of the HSE sequence did not obliterate the stimulatory effects of OA on reporter gene expression under a heat shock condition, although heat shock by itself was without effect. Analysis of the status of phosphorylation of the largest subunit of RNA polymerase II provided evidence that this effect of OA is attributable, at least in part, to the increased phosphorylation of RNA polymerase II. These results provided evidence that the heat-induced hsp 70 promoter activity is negatively regulated by serine phosphatases. We propose that the heat-induced transcriptional activation of hsps is associated with phosphorylation of component(s) of the transcription complex; one of the likely candidates being the transcriptionally engaged RNA polymerase II. OA, by inhibiting phosphatase 2A and 1 activity, enhanced this phosphorylation and potentiated the transcriptional activation of hsps.
Assuntos
Éteres Cíclicos/farmacologia , Proteínas de Choque Térmico/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Temperatura Alta , Humanos , Dados de Sequência Molecular , Ácido Okadáico , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Proteína Fosfatase 2 , TransfecçãoRESUMO
Three human immunodeficiency virus type 1 (HIV-1) mutants were constructed with mutations in their protease genes: AH2-pSVL, with an in-phase deletion; BH27-pSVL, with an out-of-phase deletion creating a stop codon immediately after the deletion site; and CA-pSVL, with a point mutation creating an Asp-to-Ala substitution at the putative protease active site. The wild-type, HXB2-pSVL, and the mutated viral genomes were used to transfect COS-M6 cells and to produce virions. Immunoblotting assays with a monoclonal antibody (MAb) specific for p24 showed that all three mutant contained a gag precursor, Pr56gag, with AH2 and CA expressing an extra band of about 160 kDa. Similar assays with a MAb specific for HIV-1 reverse transcriptase (RT) also revealed a 160-kDa protein from AH2 and CA virions and two mature p66 and p51 RT subunits from HXB2 virions. In addition, HXB2, AH2, and CA but not BH27 virions exhibited RT activity. The same protein in the 160-kDa band seemed to possess both p24 and RT components, since the MAb against p24 was able to immunoadsorb RT antigen and enzymatic activity. These results indicate that the HIV-1 gag-pol fusion protein produced in mammalian cells expressed significant RT activity.
Assuntos
Proteínas de Fusão gag-pol/metabolismo , HIV-1/genética , Transfecção , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Proteínas de Fusão gag-pol/genética , Produtos do Gene gag/imunologia , Antígenos HIV/imunologia , Proteína do Núcleo p24 do HIV , HIV-1/enzimologia , HIV-1/imunologia , Humanos , Immunoblotting , Dados de Sequência Molecular , Mutação , Oligonucleotídeos , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas do Core Viral/imunologiaRESUMO
A murine mAb BAT123 (Ab1) directing to the principal neutralization site of human T cell leukemia virus (HTLV)-IIIB gp120 (amino acid residue 308-322) was used to generate syngeneic anti-Id mAb (Ab2). Among the Ab2, a mAb AB19-4 was characterized by both serologic and biologic methods to be paratope-specific (Ab2 beta), bearing the internal image of the neutralization site. AB19-4 was found to bind specifically to BAT123 and also to its mouse-human chimeric form in ELISA. The binding of AB19-4 to BAT123 was specifically inhibited by HTLV-IIIB gp120 and the synthetic epitope peptides of HTLV-IIIB and HTLV-IIIMN defined by BAT123. AB19-4 also inhibited the binding of BAT123 to HTLV-IIIB-infected H9 cells in flow cytometric studies. Polyclonal goat and sheep antisera against HTLV-IIIB gp120 reacted specifically with AB19-4, suggesting that AB19-4 may recognize cross-species idiotopes. Rabbits immunized with purified AB19-4 generated anti-anti-Id antibodies (Ab3) that reacted specifically with HTLV-IIIB gp120 and the BAT123-binding epitope peptides of HTLV-IIIB and HTLV-IIIMN. The Ab3 bound to H9 cells infected by HTLV-IIIB or HTLV-IIIMN and inhibited the infection of CEM cells by HTLV-IIIB or HTLV-IIIMN, whereas BAT123 also bound H9 cells infected by HTLV-IIIB or HTLV-IIIMN but neutralized only HTLV-IIIB. Our data suggest that AB19-4 mimics the neutralization site on HIV-1 gp120 defined by BAT123. The induction of immunity to HIV using internal-image Ab2 to HIV-neutralizing antibodies may provide a viable approach for developing effective vaccines for AIDS.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Anti-HIV/biossíntese , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas Sintéticas/imunologia , Vacinas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Hibridomas , Testes de Neutralização , CoelhosRESUMO
Two murine monoclonal antibodies, G3.519, recognizing the CD4-binding region, and BAT123, a variable region of gp120 of human immunodeficiency virus, were chemically coupled to pokeweed antiviral protein isolated from seeds (PAP-S). The immunoconjugates were purified by Sephacryl S-200 gel filtration and Mono S ion exchange chromatography. Immunoconjugate G3.519-PAP-S specifically killed human T cells, H9, infected with three diverse HIV-1 strains, HTLV-IIIB, -IIIMN, and -IIIRF. Inhibition of thymidine incorporation by the immunoconjugate was concentration-dependent, with the ID50 ranging from 1.4 x 10(-10) M to 1.7 x 10(-9) M. Immunoconjugate BAT123-PAP-S was effective in killing H9 cells infected with HTLV-IIIB (ID50 = 4.3 x 10(-11) M) and -IIIMN (ID50 = 4.7 x 10(-10) M), but not -IIIRF. Both immunoconjugates did not inhibit thymidine incorporation in uninfected H9 cells up to a concentration of 5.3 x 10(-8) M, and their cytotoxic activities could be competitively blocked by the respective unconjugated antibodies. The immunoconjugates retained the ability to neutralize HIV virions to infect T cells and to prevent the syncytium formation. These in vitro studies suggest that the use of immunoconjugates capable of killing HIV-infected T cells and neutralizing virus may provide an alternative treatment for HIV-infected persons.
Assuntos
Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Imunotoxinas/imunologia , N-Glicosil Hidrolases , Linfócitos T/microbiologia , Anticorpos Monoclonais/uso terapêutico , Sobrevivência Celular , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Imunotoxinas/isolamento & purificação , Técnicas In Vitro , Testes de Neutralização , Proteínas de Plantas/administração & dosagem , Proteínas Inativadoras de Ribossomos Tipo 1 , Especificidade da Espécie , Linfócitos T/citologiaRESUMO
Murine mAb BAT123, which was made against the envelope glycoprotein gp120 of HTLV-IIIB strain of HIV type 1 (HIV-1), is capable of neutralizing HTLV-IIIB in vitro. It also inhibits the fusion between uninfected CD4+ cells and HIV-1-infected cells to form syncytia. As a step to explore the potential utility of the anti-HIV antibody in vivo, we have constructed a mouse-human chimeric antibody by rDNA techniques. The chimeric antibody, which bears the variable domains of mouse antibody BAT123 and constant domains Cr1 and C kappa of human Ig retains the Ag specificity of BAT123 as determined by its reactivity with HIV-1-infected H9 cells, gp120 in Western blot analysis, and the oligopeptide recognized by BAT123. The antiviral activities of the chimeric antibody in neutralizing HIV-1 infection as well as inhibiting the syncytia formation are also found identical to those of the parent murine antibody. Moreover, in the presence of human blood mononuclear cells, the chimeric antibody but not BAT123 (mouse IgG1) induces antibody-dependent cellular cytotoxicity. The findings point to the potential usefulness of the chimeric antibody in treating patients infected with HIV-1.