RESUMEN
Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Leptospira interrogans/inmunología , Lipoproteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/inmunología , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Humanos , Leptospira interrogans/genética , Leptospirosis/inmunología , Leptospirosis/microbiología , Lipoproteínas/metabolismo , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TailandiaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) is separated into several subtypes and circulating recombinant forms (CRFs). Here, infections of 4 clinical isolates (0-47-1, CU98-26, CU98-28, and CU98-31) from Thailand were examined in human CD4(+) T-cell lines, MT-4 and MOLT-4. The CU98-26 isolates in both cells and 0-47-1 in MT-4 established chronic infections, as in control 2 subtype B isolates from Japan, while 0-47-1 in MOLT-4 caused a latent infection. In contrast, CU98-28 and CU98-31 established aberrant infections in both cells. Integrated provirus was detected in all the chronic infections, including 0-47-1 in both cells. In contrast, extrachromosomal circular forms of HIV-1 DNA were detected in CU98-28- and CU98-31-infected cells, whereas the amount of the integrated form was below the limit of detection. Interestingly, phylogenetic trees and sequencing revealed that all the Thai isolates, except 0-47-1, displayed CRF15_01B-like mosaic structures of CRF01_AE with subtype B-like sequences in several regions that were apparently different from those of the inocula in peripheral blood mononuclear cells. Thus, in the infections of most of the above Thai isolates it was suggested that a minor population with mosaic patterns having multiple breakpoints between CRF01_AE and subtype B in the inocula could be selected by the T-cell lines.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , Secuencia de Aminoácidos , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Genoma Viral , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Filogenia , TailandiaRESUMEN
Recombinant BCGs (rBCGs) containing extrachromosomal plasmids with different HIV-1 insert sequences: nef, env (V3J1 and E9Q), gag p17 or whole gag p55 were evaluated for their immunogenicity, safety and persistent infection in BALB/c mice. Animal injected with, rBCG-plJKV3J1, rBCG-pSO gag p17 or rBCG-pSO gag p55 could elicit lymphocyte proliferation as tested by specific HIV-1 peptides or protein antigen. Inoculation with various concentration of rBCG-pSO gag p55 generated satisfactory specific lymphocyte proliferation in dose escalation trials. The rBCG-pSO gag p55 recovered from spleen tissues at different time interval post-inoculation could express the HIV protein as determined by ELISA p24 antigen detection kit. This result indicated that the extrachromosomal plasmid was stable and capable to express Gag protein. It was also demonstrated that rBCGs did not cause serious pathological change in the inoculated animals. The present study suggested the role of BCG as a potential vehicle for using in HIV vaccine development.
Asunto(s)
Antígenos Virales/genética , Vacuna BCG , ADN Viral/genética , VIH-1/genética , Mycobacterium bovis/genética , Animales , Antígenos Virales/inmunología , Femenino , Recuento de Linfocitos , Ratones , Ratones Endogámicos BALB C , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Piel/patología , Bazo/inmunologíaRESUMEN
The human immunodeficiency virus Tat regulatory protein is essential for virus replication and for the efficient transcription of HIV-1 provirus, and in the pathogenesis of AIDS. The role of the tat gene was investigated in 300 samples. It was found that 71.7% were subtype CRF_01AE, 9.3% were subtype B, while 11.7 and 7.3% of them were cross-reactive and non-typeable, respectively. Moreover the results from peptide ELISA also showed that a low CD4 cell count was related to a low anti-Tat antibody (p < 0.05), which may be due to the progression of HIV-1, which can be found predominantly in AIDS patients. The results of nested PCR showed that the second Tat exon might also play a role in T-cell activation. Reverse transcription polymerase chain reaction (RT-PCR) was used to measure HIV-1 mRNA expression in PBMC. RT-PCR negative results were found mostly in the asymptomatic HIV-seropositive group (88%). HIV-1 mRNA expression was found to correlate with current immunologic status. The differences in Tat protein sequences from DNA sequencing between the patients who had anti-Tat antibody positive and anti-Tat antibody negative, were not significant (p > 0.05). These results suggested that the Tat amino acid sequences were conserved among each group of samples and did not change significantly compared with the consensus sequence in previous studies. Several factors make Tat an attractive target for vaccine design.
Asunto(s)
Genes tat/genética , Infecciones por VIH/genética , VIH-1/genética , VIH-1/patogenicidad , Replicación Viral/genética , Adulto , Anciano , Secuencia de Bases , Recuento de Linfocito CD4 , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Anticuerpos Anti-VIH/análisis , Humanos , Lactante , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero , Análisis de Secuencia de ADN , TailandiaRESUMEN
This preliminary study aimed to investigate sensitivity and specificity of a protein chip system for multi-tumor marker serodiagnosis of ten types of cancers, and to understand the possible clinical applications of this protein chip for the Thai population. The specific cancers diagnosed by this protein chip are lung, breast, liver, cervix, colo-rectal, stomach, ovary, esophagus, prostate and pancreas cancers. We analyzed 215 serum samples of which 165 were obtained from clinically confirmed cancer patients and 50 from healthy people with no evidence of cancer. The sensitivity and specificity of the protein chip were 82.4% and 94.0%, respectively. The success rate of the protein chip for detecting all 10 types of cancers varied from 57% to 100%. The value of the simultaneous measurement of multiple tumor markers using the protein chip for cancer screening lied in the higher sensitivity compared to using single tumor markers for each type of cancer. In short, protein chips may be useful in mass screening for cancer during health checkups as well as for metastasis follow-up of cancer patients.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias/sangre , Neoplasias/diagnóstico , Análisis por Matrices de Proteínas , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Carbohidratos Asociados a Tumores/sangre , Antígeno Carcinoembrionario/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/sangre , Pruebas Serológicas , Tailandia , alfa-Fetoproteínas/metabolismoRESUMEN
The third variable (V3) domain of the envelop (env) protein has been used for determining genetic subtype and phenotypic characteristics of human immunodeficiency virus type 1 (HIV-1) isolates. Based on the seroreactivity of the HIV-1 subtype by V3 peptide binding enzyme immunoassay (EIA) of 351 samples obtained in 1998 from HIV-1 infected individuals and AIDS patients, we found that 283 (80.6%) were subtype E, 20 (5.7%) were subtype B, 28 (8.0%) were cross-reactive between both types and 20 (5.7%) were non-typeable. The degree of seroreactivity of HIV-1 subtype E decreased significantly when the amino acid at the crown of the V3 loop was substituted from a GPGQ motif to GPGR motif. Interestingly, AIDS patients who had V3 sequences of subtype E as GPGR motif had a stronger immunoreactivity to GPGQ motif peptides than to GPGR motif peptides, in contradiction for their proviral sequences. The results suggested that mutations in the V3 loop may lead to a changed immunoreactivity that makes HIV-1 mutants unrecognizable or allow escape from the primary immune response by means of neutralizing sensitivity. In connection with vaccine development, it should be pointed out that the combination of V3 sequencing and peptide EIA could provide a novel approach to obtain a primarily infected virus sequence as a target for a preventive AIDS vaccine.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Seroprevalencia de VIH , VIH-1/inmunología , Síndrome de Inmunodeficiencia Adquirida/epidemiología , Síndrome de Inmunodeficiencia Adquirida/genética , Adulto , Secuencia de Aminoácidos , Reacciones Cruzadas/genética , ADN Viral/genética , Femenino , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Humanos , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fenotipo , TailandiaRESUMEN
Clinical isolates of human herpesvirus 7 (HHV-7) from the saliva of healthy individual were investigated for genetic variations in the regions of two immediate-early (IE) genes, the glycoprotein B (gB) and glycoprotein H (gH) genes, and in R2-repeat. The genomic DNA of 24 isolates from citizens of Thailand, Japan, and the United States was amplified to detect size variations in the IE-1 and IE-2 loci, but none was observed, suggesting that there was no deletion or insertion in these genes, in contrast with an IE gene of human herpesvirus 6 (HHV-6). The sequences of the gB gene from isolates acquired from 5 Japanese and 8 Thai subject were then compared with those of American strains JI and RK with respect to codons that are known to differentiate gB alleles. All the isolates were found to have gB allele C except for the JI strain, which has allele F. Variability was also observed in five specific gH codons, resulting in 6 different groups. The HHV-7 isolates might be classified into two major genetic variants by combining their gB and gH allelic groupings. In the present study, only JI belonged to variant 1, while the rest of the isolates appeared to belong to variant 2. In the R2-repeat region, size heterogeneities were observed among the 24 isolates, due to different repeat numbers (17, 15, 14, 13, or 12 repeats). Therefore, we used the R2-repeat to identify the origins of isolates in a study of HHV-7 transmission, and found HHV-7 to be transmitted within a family from both mothers and fathers to their children.