RESUMEN
The main objectives of this study were to assess a dual molecular beacon approach for fast detection of Mycobacterium tuberculosis (MT). MT beacon (Tb-B) was designed to target the unique IS6110 (114 bp) and rpoB (215 bp) fragment of the MT (H37Ra) genome, and the two fragments were inserted into the PMD-19T vector after purification, by PCR and sequencing, to construct plasmids. Different dilutions of positive plasmid standards were used for dual molecular beacon RT-PCR of rpoB and IS6110, and standard curves were established.The results show that the dual molecular beacon of rpoB and IS6110 detecting MT was stable (CV is 1.91-2.68 %) with a high amplification efficiency (95.6 %). In addition, the strains of non MT did not generate fluorescence signals, while strains of MT did, indicating that the primers and molecular beacons were specific, and only MT complex was amplified. The linear range was wide (10(3)-10(11) copies/mL), and clinical specimens presenting different bacterial counts can be detected.
Asunto(s)
Sondas de ADN/genética , Mycobacterium tuberculosis/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Tuberculosis/diagnóstico , Proteínas Bacterianas/genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN , Genes Bacterianos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Datos de Secuencia Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Tuberculosis/microbiologíaRESUMEN
BACKGROUND: Asthma is a complex inflammatory disorder of the airways, and research on alternative therapeutic strategies has attracted attention. This study aimed at hypersusceptibility and toxicity of recombinant human phospholipase D2 (rhPLD2) in guinea pigs. We determined the behavioral responses in the model of immediate hypersensitivity animals and changes of eosinophil levels following use of the drugs. Special attention was given to the effects of rhPLD2 in vivo on the guinea pig with chronic persistent asthma and the mechanism involved. METHODS: To investigate the effect of rhPLD2 on the expression of protein kinase C (PKC), and to examine the activity of signal transducer and activator of transcription 1 and 5a in the lung of the guinea pig with chronic asthma. Guinea pigs with chronic asthma were divided into five groups: a saline group, a dexamethasone 5.0 mg group, and rhPLD2 (1.5, 2, or 3 mg) groups. Non-sensitized animals were as normal control group. PKC expression was measured by immunohistochemistry, alterations of STAT1 and STAT5a were detected by TransAM transcription factor assay kits. RESULTS: rhPLD2 (3.0 mg) decreased PKC expression to baseline and inhibited STAT1 activity compared with that of the saline group (p < 0.01). CONCLUSION: The rhPLD2 may suppress the chronic inflammatory reaction through down-regulating PKC expression and STAT1/STAT5a activity in the lung. The rhPLD2 may be a suitable therapeutic target for asthma.
Asunto(s)
Asma/tratamiento farmacológico , Fosfolipasa D/farmacología , Proteínas Recombinantes/farmacología , Animales , Asma/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Cobayas , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Proteína Quinasa C/biosíntesis , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT5/metabolismoRESUMEN
OBJECTIVE: To investigate the effect of low molecular weight potassium alginate (L-PA) on blood pressures in spontaneously hypertensive rats (SHRs) and its pharmacokinetics characteristics in mice. METHODS: The systolic blood pressure (SBP) was measured by tail-cuff method in conscious SHRs. Forty rats were randomly assigned to the following five groups: control, hydrochlorothiazide (HCT, 6.25 mg/kg), L-PA in low, middle or high dose groups (100, 250, 500 mg/kg). SHRs were intragastrically (i. g.) administrated once daily for 28 days. The SBP was measured once weekly during drug treatment, and 3 and 6 days after drug with drawal. KM mice were i. g. administered with 100 mg/kg (74 MBq/kg) of 3H-L-PA. Ten microl blood samples were obtained from the tail vein at 2, 5, 10, 20, 30 min and 1, 2, 4, 6, 12, 24, 48, 72, 96, 120 or 144 h after drug administration for measuring radioactivities. Pharmacokinetics parameters of the oral administration of L-PA were analysed with DAS 2.0 software. RESULTS: Twenty-one or 28 days after administration, the rats in the groups treated with HCT or L-PA at 100, 250 or 500 mg/kg had a significant decrease in SBP (P<0.01 vs control group). Three or 6 days after drug withdrawal, the antihypertensive effect of HCT disappeared (P>0.05), whereas the rats treated with 250 or 500 mg/kg L-PA still had lower SBP than the controls (P<0.01). The L-PA at a dose of 100 mg/kg also led to a significant decrease in SBP 3 days after drug withdrawal (P<0.05). The pharmacokinetics of L-PA (i. g.) was consistent with a two-compartment model, with 2.76 h of absorption half-life (t1/2, Ka), 42. 30 h of distributional half-life (t1/2alpha), 42. 31 h of elimination half-life (t1/2beta), and 36.28 h of terminal phase elimination half-life (t1/2z). CONCLUSION: Oral administration of L-PA has significant anti-hypertensive effect, which can be maintained to 6 days after drug withdrawal. The sustaining anti-hypertensive effect of L-PA is probably associated with its slow elimination in vivo.
Asunto(s)
Alginatos/farmacología , Alginatos/farmacocinética , Antihipertensivos/farmacología , Antihipertensivos/farmacocinética , Hipertensión/tratamiento farmacológico , Alginatos/uso terapéutico , Animales , Antihipertensivos/uso terapéutico , Ácido Glucurónico/farmacocinética , Ácido Glucurónico/farmacología , Ácido Glucurónico/uso terapéutico , Ácidos Hexurónicos/farmacocinética , Ácidos Hexurónicos/farmacología , Ácidos Hexurónicos/uso terapéutico , Hipertensión/metabolismo , Masculino , Ratones , Distribución Aleatoria , Ratas , Ratas Endogámicas SHRRESUMEN
Our previous studies have shown that recombinant human phospholipase D2 (rhPLD2) plays a modulator role on NF-κB and PKC signaling pathways. It also inhibits IL-5-induced inflammatory response in chronic asthmatic guinea pigs. Additionally, increasing evidence also has revealed that the adoptive transfer of induced regulatory T cells (Tregs) may be a therapeutic solution to airway allergic diseases. To investigate the epigenetic, transcriptomic and phenotypic variability of Treg population in an ovalbumin (OVA)-induced airway inflammation model derived from the induction of rhPLD2, OVA-induced asthmatic murine model is used in this study. The lung inflammation, eosinophil infiltration, the differentiation and proliferation of T helper cells and the amplification of Tregs were examined in this mouse model with and without rhPLD2 induction. Our data showed that rhPLD2 administration in asthmatic mice significantly increases CD4+CD25+ Foxp3+ Treg cell numbers and alleviates lung inflammation. The addition of rhPLD2 in vitro enhanced the demethylation of Treg-specificdemethylated region (TSDR) in iTregs, suggesting that rhPLD2 protein may be involved in improving the quality and quantity of Treg cells that eventually significantly reduces lung inflammation in asthmatic murine model. These results suggest that rhPLD2 could have a clinical impact treating patients with allergic airway inflammation via promoting and stabilizing iTreg differentiation and function.
Asunto(s)
Asma/tratamiento farmacológico , Asma/inmunología , Factores de Transcripción Forkhead/metabolismo , Inflamación/tratamiento farmacológico , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Pulmón/patología , Fosfolipasa D/uso terapéutico , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Asma/patología , Islas de CpG/genética , Metilación de ADN/genética , Modelos Animales de Enfermedad , Eosinófilos/patología , Humanos , Ratones Endogámicos BALB C , Modelos Biológicos , Fosfolipasa D/farmacología , Estabilidad Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
This article is to investigate the effect of human recombinant phospholipase D2 (rhPLD2) in vivo on the expression of nuclear transcription factor p65 in chronic asthma of guinea pigs. After treating the guinea pigs with chronic asthma by rhPLD2, the crude nuclear extraction was assayed with TransAM Transcription Factor Assay Kit for the activity of pulmo tissue nuclear transcription factor p65. Compared with the healthy guinea pigs, the activity of nuclear transcription factor p65 in guinea pigs of chronic asthma is much higher than that of control groups. Our results showed that rhPLD2 markedly depressed the activity of p65 when the guinea pigs were attacked by chronic asthma.
Asunto(s)
Asma/tratamiento farmacológico , Asma/fisiopatología , Pulmón/metabolismo , Fosfolipasa D/farmacología , Proteínas Recombinantes/farmacología , Factor de Transcripción ReIA/metabolismo , Animales , Antiasmáticos/farmacología , Enfermedad Crónica , Modelos Animales de Enfermedad , Cobayas , Humanos , Pulmón/efectos de los fármacos , Masculino , Transducción de Señal/efectos de los fármacosRESUMEN
Available evidence indicates that brown algae may be beneficial for the treatment of high blood pressure. Our recent study demonstrated that low molecular mass potassium alginate (L-PA), one of the major polysaccharides extracted from brown algae, decreased systolic blood pressure (SBP) in spontaneous hypertensive rats. The present study investigated the effects of L-PA on deoxycorticosterone acetate (DOCA) salt-induced hypertension in rats. Hypertension was induced by biweekly subcutaneous injections of 50mg/kg DOCA plus 1% NaCl in drinking water. The control group received saline injections. L-PA (250 or 500 mg/kg), KCl (239 mg/kg), or volume-matched solvent was administered orally once daily for 30 days. DOCA salt administration significantly increased SBP, sodium excretion, serum sodium content, circulating plasma volume (CPV), plasma atrial natriuretic peptide (ANP) content, heart and renal weight indices, and mortality and decreased plasma aldosterone (ALD) and serum potassium levels in the vehicle-treated DOCA salt group compared with the control group. However, L-PA dose-dependently normalized the above changes induced by DOCA salt, with the exception of further increasing sodium excretion, while KCl did not affect the changes caused by DOCA salt, with the exception of slightly ameliorating hypokalemia and mortality. These findings suggest that L-PA may offer a novel form of potassium supplementation with greater antihypertensive and sodium excretion actions than KCl and may likely be beneficial for the primary prevention and treatment of hypertension and its cardiovascular sequelae.
Asunto(s)
Alginatos/farmacología , Antihipertensivos/farmacología , Hipertensión/tratamiento farmacológico , Phaeophyceae/química , Alginatos/administración & dosificación , Alginatos/aislamiento & purificación , Animales , Antihipertensivos/administración & dosificación , Antihipertensivos/aislamiento & purificación , Presión Sanguínea/efectos de los fármacos , Desoxicorticosterona , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ácido Glucurónico/administración & dosificación , Ácido Glucurónico/aislamiento & purificación , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/administración & dosificación , Ácidos Hexurónicos/aislamiento & purificación , Ácidos Hexurónicos/farmacología , Hipertensión/fisiopatología , Masculino , Potasio/metabolismo , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Sodio/metabolismoRESUMEN
In the present study, we investigated the role of recombinant human phospholipase D2 (rhPLD2) on proliferation and apoptosis in human leukemia HL-60 cells which induced by camptothecin. Our research demonstrated that various concentrations of rhPLD2 inhibit the growth of HL-60 cells in a dose-dependent manner, and rhPLD2 plus camptothecin can produce a synergistic effect on growth inhibition of HL-60 cells in vitro. So, we conclude that rhPLD2 alone cannot induce apoptosis in HL-60 cells, but it can potentiate the apoptosis of HL-60 cells induced by camptothecin. Similarly, we show that both rhPLD2 and standard PLD were able to enhance camptothecin-induced apoptosis of HL-60 cells.