RESUMEN
Recently, we reported that a DNA vaccine, composed of three copies of a self B cell epitope of amyloid-beta (Abeta(42)) and the foreign T-cell epitope, Pan DR epitope (PADRE), generated strong anti-Abeta immune responses in wild-type and amyloid precursor protein transgenic animals. Although DNA vaccines have several advantages over peptide-protein vaccines, they induce lower immune responses in large animals and humans compared with those in mice. The focus of this study was to further enhance anti-Abeta(11) immune responses by developing an improved DNA vaccination protocol of the prime-boost regimen, in which the priming step would use DNA and the boosting step would use recombinant protein. Accordingly, we generated DNA and recombinant protein-based epitope vaccines and showed that priming with DNA followed by boosting with a homologous recombinant protein vaccine significantly increases the anti-Abeta antibody responses and do not change the immunoglobulin G1 (IgG1) profile of humoral immune responses. Furthermore, the antibodies generated by this prime-boost regimen were long-lasting and possessed a higher avidity for binding with an Abeta(42) peptide. Thus, we showed that a heterologous prime-boost regimen could be an effective protocol for developing a potent Alzheimer's disease (AD) vaccine.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Inmunización Secundaria , Vacunas de ADN/inmunología , Vacunas de Subunidad/inmunología , Animales , Afinidad de Anticuerpos , Quimiocina CCL22/inmunología , Epítopos de Linfocito T/inmunología , Femenino , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/metabolismo , Vacunas contra la Malaria/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
The methanolic extract of roots of Cimicifuga racemosa and its methanolysis products have been analysed by GC-MS. 2-Hexylcyclopropaneoctanoic acid (9,10-methylenehexadecanoic acid) was found to be the marker most specific for the identification for this herb. For the phytochemical standardisation of Rhizoma Cimicifugae racemosae, validated methods for the quantitative analysis of formononetin (by TLC-fluorometry), of isoferulic acid (by GC-MS of the methyl ester), and of total triterpene glycosides (transformed to coloured complexes and measured photometrically) in roots of C. racemosa have been developed. The contents of formononetin, isoferulic acid and total triterpene glycosides (measured as actein) in the herb ranged from 0.0031 to 0.0035, from 1.22 to 1.35 and from 20.09 to 22.06 mg/g dry weight, respectively.
Asunto(s)
Cimicifuga/química , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Cinamatos/análisis , Cinamatos/química , Cromatografía de Gases y Espectrometría de Masas , Isoflavonas/análisis , Isoflavonas/química , Metanol , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Estándares de Referencia , Rizoma/química , Saponinas/análisis , Saponinas/química , Triterpenos/análisis , Triterpenos/químicaRESUMEN
Familial Mediterranean fever (FMF) is a recessively inherited inflammatory disorder, characterised by recurrent attacks of fever and serositis. Since nitric oxide (NO) is an important mediator of inflammation, the production of NO (assessed as the accumulation of nitrate and nitrite and measured by capillary electrophoresis) in blood plasma of FMF patients during acute attacks (active) and attack-free periods (inactive) of the disease has been determined and compared with NO levels found in healthy volunteers (control group C). Thirty-six FMF patients were involved in a placebo-controlled double-blind study (group A received the drug, group B the placebo) of the effects of Immuno-Guard, a novel herbal preparation which relieves the severity and longevity of FMF attacks on NO blood levels. Thirty-two FMF patients (group D) being permanently treated with colchicine were also examined with respect to their NO blood level. No significant differences were found between the NO levels in blood of inactive FMF patients and those of control group C, or between inactive colchicine-treated group D patients and inactive patients of groups A and B, a finding which is atypical for chronic inflammatory disorders. Significantly lower plasma NO levels were found in active FMF patients in groups A and B compared with inactive patients in those groups (p=0.031 and 0.036, respectively) and with patients of group D and the control group C (p=0.0235 and 0.0453, respectively). The decrease of NO in blood of FMF patients may trigger the generation of fever by initiating the production of pro-inflammatory IL-6. Plasma NO levels in inactive FMF patients were significantly increased during attack-free periods following treatment with Immuno-Guard. The preparation has a normalising effect both on NO and IL-6 blood levels in FMF patients during attacks, demonstrating a relationship between the beneficial effect of Immuno-Guard in reducing the severity of inflammatory attacks in FMF patients and the increase in NO blood levels.
Asunto(s)
Fiebre Mediterránea Familiar/sangre , Fiebre Mediterránea Familiar/tratamiento farmacológico , Óxido Nítrico/sangre , Extractos Vegetales/uso terapéutico , Enfermedad Aguda , Adolescente , Niño , Preescolar , Colchicina/uso terapéutico , Femenino , Humanos , Interleucina-6/sangre , Masculino , Nitratos/sangre , Nitritos/sangreRESUMEN
The immunomodulatory properties of a diterpene lactone andrographolide and Kan Jang--a standardized fixed combination of Andrographis paniculata extract SHA-10 and Eleutherococcus senticosus extract SHE-3 were investigated. Their role on spontaneous and phytohemagglutinin (PHA)-induced proliferation of human peripheral blood lymphocytes (PBL) and on production of interferon-gamma (INF-gamma) and tumor necrosis factor-alpha (TNF-alpha) were determined in vitro. Proliferation of PBL induced by PHA was enhanced by co stimulation with andrographolide and Kan Jang. At the same time andrographolide and Kan Jang inhibit spontaneous proliferation of PBL in vitro. These preparations also have effect on the formation of INF-gamma, TNF-alpha and some immune activation markers such as neopterin (Neo), beta-2-microglobulin (beta2MG), and soluble receptor for interleukin-2 (sIL-2R or sCD25) in blood cells culture. Andrographolide and Kan Jang stimulate the INF-gamma, Neopterin and beta2MG formation, but do not have any significant effect on the production of INF-gamma and Neopterin in PHA stimulated blood cells. An opposite effect on these immune makers was observed in the PHA-stimulated blood cells: both andrographolide and Kan Jang increase the formation of TNF-alpha and beta2MG in cultivated whole blood cells. Thus, andrographolide and Kan Jang can have an in vitro effect on the activation and proliferation of immunocompetent cells as well on the production of key cytokines and immune activation markers. The results show an overall higher effect of the fixed combination as compared with the equivalent amount of the pure substance andrographolide. The data are consistent with results from clinical studies of Kan Jang and contributed to a better understanding of these results.
Asunto(s)
Andrographis , Diterpenos/farmacología , Eleutherococcus , Linfocitos/efectos de los fármacos , Extractos Vegetales/farmacología , Biomarcadores , Células Sanguíneas/citología , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Células Cultivadas , Citocinas/biosíntesis , Glucósidos/farmacología , Humanos , Interferón gamma/biosíntesis , Interferón gamma/efectos de los fármacos , Lignanos , Linfocitos/citología , Linfocitos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
A new method of analysis of vanillin, syringaldehyde, coniferaldehyde, and sinapaldehyde in brandy and wine by high-performance capillary electrophoresis is described. Electrophoretic mobility of these compounds is achieved by a borate buffer at pH 9.3. At this pH, the sensitivity of UV detection of these phenolic aldehydes also increases. UV absorptions at 348, 362, 404, and 422 nm were selected for monitoring vanillin, syringaldehyde, coniferaldehyde, and sinapaldehyde, respectively. This procedure was performed simultaneously during one run using a diode array detector. Samples of brandy or wine were analyzed directly without concentration, extraction, or any other preliminary treatment of the test sample. The limits of detection were found to be 0.275, 0.1425, 0.1475, and 0.1975 ppm for syringaldehyde, coniferaldehyde, sinapaldehyde, and vanillin, respectively, which is acceptable for analysis of both brandy and wine aged in oak barrels. The method has been shown to be linear in a range from 0.3 to 57 mg/L. Recoveries ranged between 99.9% and 107.7% for all of the compounds tested. Repeatability and reproducibility of the method were high. The relative standard deviation was consequently approximately 3% and also between 4.47% and 6.89% for all tested compounds. The method is useful for the identification of counterfeit brandy, which is easy to recognize by the absence of sinapaldehyde, syringaldehyde, and coniferaldehyde, which are not detectable in false brandy.
Asunto(s)
Bebidas Alcohólicas/análisis , Aldehídos/análisis , Vino/análisis , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Indicadores y Reactivos , Espectrofotometría UltravioletaRESUMEN
Validated analytical methods (HPLC, CE and GC-MS) for determining the amount of andrographolide (AND) in the blood plasma of rats and human volunteers following the oral administration of Andrographis paniculata extract (APE) and Andrographis paniculata fixed combination Kan Jang tablets were developed and used for the pharmacokinetic study. Andrographolide was quickly and almost completely absorbed into the blood following the oral administration of APE at a dose of 20 mg/kg body wt. in rats. Its bio-availability, however, decreased four-fold when a 10-times-higher dose was used. Since a large part (55 %) of AND is bound to plasma proteins and only a limited amount can enter the cells, the pharmacokinetics of AND are described well by a one-compartment model. Renal excretion is not the main route for eliminating AND. It is most likely intensely and dose dependently metabolized. Following the oral administration of four Kan Jang tablets (a single therapeutic dose, equal to 20 mg of AND) to humans, maximum plasma levels of approximately 393 ng/ml (approx. 1.12 microM) were reached after 1.5-2 hours, as quantified using a UV diode-array detection method. Half-life and mean residence times were 6.6 and 10.0 hours, respectively. AND pharmacokinetics in humans are explained well by an open two-compartment model. The calculated steady state plasma concentration of AND for multiple doses of Kan Jang (after the normal therapeutic dose regimen, 3 x 4 tablets/day, about 1 mg AND/kg body wt./day) was approximately 660 ng/ml (approx. 1.9 microM), enough to reveal any anti-PAF effect, particularly after drug uptake when the concentration of AND in blood is about 1342 ng/ml (approx. 3.8 microM, while for anti-PAF effect EC50 - 5 microM).