RESUMEN
Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/β-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator β-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/β-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X β-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3β inhibitor (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80 percent of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38 percent of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64 percent, which are the dominant β-catenin signaling cells and decreased β-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/β-catenin signaling inhibitor NCTD.
Asunto(s)
Humanos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Indoles/antagonistas & inhibidores , Oximas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/antagonistas & inhibidores , beta Catenina/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Genes Reporteros/fisiología , Células Jurkat , Luciferasas/metabolismo , Plásmidos/efectos de los fármacos , Plásmidos/genética , Transfección/métodos , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Refractory and relapsed leukemia is a major problem during cancer therapy, which is due to the aberrant activation of Wnt/ß-catenin signaling pathway. Activation of this pathway is promoted by wingless (Wnt) proteins and induces co-activator ß-catenin binding to lymphoid enhancer factor (LEF)/T-cell factor protein (TCF). To provide a convenient system for the screening of anti-Wnt/ß-catenin agents, we designed a bi-functional pGL4-TOP reporter plasmid that contained 3X ß-catenin/LEF/TCF binding sites and a selectable marker. After transfection and hygromycin B selection, HEK 293-TOP and Jurkat-TOP stable clones were established. The luciferase activity in the stable clone was enhanced by the recombinant Wnt-3A (rWnt-3A; 100-400 ng/mL) and GSK3ß inhibitor (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO; 5 µM) but was inhibited by aspirin (5 mM). Using this reporter model, we found that norcantharidin (NCTD; 100 µM) reduced 80% of rWnt-3A-induced luciferase activity. Furthermore, 50 µM NCTD inhibited 38% of BIO-induced luciferase activity in Jurkat-TOP stable cells. Employing ³H-thymidine uptake assay and Western blot analysis, we confirmed that NCTD (50 µM) significantly inhibited proliferation of Jurkat cells by 64%, which are the dominant ß-catenin signaling cells and decreased ß-catenin protein in a concentration-dependent manner. Thus, we established a stable HEK 293-TOP clone and successfully used it to identify the Wnt/ß-catenin signaling inhibitor NCTD.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Indoles/antagonistas & inhibidores , Oximas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/antagonistas & inhibidores , beta Catenina/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Genes Reporteros/fisiología , Células HEK293 , Humanos , Células Jurkat , Luciferasas/metabolismo , Plásmidos/efectos de los fármacos , Plásmidos/genética , Transfección/métodos , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismoRESUMEN
To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F-->I, 21/21) and 212 (G-->S, 19/21) or (G-->N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G-->D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L-->F) and 129 (M-->I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W-->C), and 5/21 specimens showed another novel change at codon 115 (G-->A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.
Asunto(s)
Epítopos de Linfocito T/genética , Variación Genética , Antígeno HLA-A2/genética , Neoplasias Nasofaríngeas/virología , Proteínas de la Matriz Viral/genética , Adulto , Secuencia de Aminoácidos , Biopsia , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Epítopos de Linfocito T/análisis , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F arrow right I, 21/21) and 212 (G arrow right S, 19/21) or (G arrow right N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G arrow right D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L arrow right F) and 129 (M arrow right I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W arrow right C), and 5/21 specimens showed another novel change at codon 115 (G arrow right A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.
Asunto(s)
Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Epítopos de Linfocito T/genética , Variación Genética , /genética , Neoplasias Nasofaríngeas/virología , Proteínas de la Matriz Viral/genética , Secuencia de Aminoácidos , Biopsia , Epítopos de Linfocito T/análisis , Genotipo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
The seroprevalence of human herpesvirus 8 (HHV-8) was studied in 326 human immunodeficiency virus (HIV)-positive and -negative persons from Honduras; women constituted 77% (n = 251) of the subjects. Sera were tested for lytic HHV-8 antibodies by an IFA, and positive samples were confirmed by a radioimmunoprecipitation assay. Of the 326 persons tested, 58 (17.8%) had HHV-8 antibodies. Among the HIV-infected women, 22.7% were seropositive; 11.3% of the HIV-negative women were seropositive. HHV-8 seroprevalence was almost four times higher in HIV-positive female commercial sex workers (36%) than in HIV-negative female non-commercial sex workers (9.9%; odds ratio = 3.8, 95% confidence interval = 1.1-13; P = 0.01), suggesting that commercial sex work is a risk factor for HHV-8 infection. In the men studied, the overall HHV-8 seroprevalence was 22.6%, with a seropositivity rate of 28% for HIV-positive men compared with 12% for HIV-negative men.
Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/transmisión , Infecciones por Herpesviridae/transmisión , Herpesvirus Humano 8 , Enfermedades Virales de Transmisión Sexual/epidemiología , Infecciones Oportunistas Relacionadas con el SIDA/epidemiología , Infecciones Oportunistas Relacionadas con el SIDA/inmunología , Infecciones Oportunistas Relacionadas con el SIDA/virología , Anticuerpos Antivirales/sangre , Femenino , Infecciones por Herpesviridae/epidemiología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/inmunología , Honduras/epidemiología , Humanos , Masculino , Prevalencia , Factores de Riesgo , Enfermedades Virales de Transmisión Sexual/transmisiónRESUMEN
OBJECTIVE: To compare the efficacy of injections of 1.2 million units of benzathine penicillin G given every 3 weeks versus every 4 weeks for secondary prevention of rheumatic fever, based on the long-term outcome of patients receiving such prophylaxis. METHODS: A total of 249 consecutive patients with rheumatic fever, randomly assigned to either a 3-week or a 4-week regimen, were examined every 3 to 6 months, and followed for 794 and 775 patient-years, respectively. RESULTS: Compliance with each regimen was comparable: 83 (66.9%) of 124 patients in the 3-week group versus 92 (73.6%) of 125 patients in the 4-week group stayed in the program (p > 0.05). Streptococcal infections occurred less frequently in those receiving the 3-week regimen: 7.5 versus 12.6 per 100 patient-years (p < 0.01). Prophylaxis failed in 2 patients receiving the 3-week regimen and in 10 receiving the 4-week regimen (0.25 and 1.29 per 100 patient-years respectively; p = 0.015). Serum penicillin levels were adequate (> or = 0.02 micrograms/ml) in 100 (56%) of 179 samples obtained 21 days after penicillin injection in the 3-week regimen, and in 51 (33%) of 155 samples obtained 28 days after injection in the 4-week regimen (p < 0.01). Of 71 patients with mitral regurgitation in the 3-week regimen, 47 (66%) no longer had the murmur; of 87 patients in the 4-week regimen, 40 (46%) no longer had the murmur (p < 0.05). CONCLUSIONS: This 12-year controlled study indicates that the outcome of patients with rheumatic fever is better with a 3-week than with a 4-week penicillin prophylaxis regimen. Greater emphasis and more widespread use of the 3-week regimen should be recommended.
Asunto(s)
Penicilina G Benzatina/uso terapéutico , Fiebre Reumática/prevención & control , Cardiopatía Reumática/prevención & control , Adolescente , Adulto , Insuficiencia de la Válvula Aórtica/complicaciones , Insuficiencia de la Válvula Aórtica/epidemiología , Insuficiencia de la Válvula Aórtica/prevención & control , Niño , Preescolar , Esquema de Medicación , Femenino , Estudios de Seguimiento , Soplos Cardíacos/epidemiología , Soplos Cardíacos/etiología , Soplos Cardíacos/prevención & control , Humanos , Masculino , Insuficiencia de la Válvula Mitral/complicaciones , Insuficiencia de la Válvula Mitral/epidemiología , Insuficiencia de la Válvula Mitral/prevención & control , Cooperación del Paciente , Penicilina G Benzatina/sangre , Estudios Prospectivos , Recurrencia , Inducción de Remisión , Fiebre Reumática/complicaciones , Fiebre Reumática/epidemiología , Cardiopatía Reumática/complicaciones , Cardiopatía Reumática/epidemiología , Factores de Tiempo , Resultado del TratamientoRESUMEN
To compare the merits of 3-week versus 4-week injections of benzathine penicillin G in preventing recurrence of rheumatic fever, 179 patients aged 4 to 19 years were assigned to one of the two programs. Age, weight, cardiac status, and streptococcal infections among the patients and their family members studied in each program were comparable. Eight-two patients and their family members were monitored for streptococcal infections. Compliance in the two programs was comparable. Of the 63 patients who stayed in the 4-week program, RF recurred in six, as a result of prophylaxis failure in five and associated with partial compliance in one. Of the 90 patients in the 3-week program, RF recurred in one, associated with partial compliance; no failures occurred (P = 0.01). We recommended that for RF chemoprophylaxis in individuals at great risk, regardless of age, benzathine penicillin injections should be administered every 3 rather than every 4 weeks.