Development and characterization of murine monoclonal antibody specific for the P1,4 Por A proteins from strain B: 4: P1,(7b),4, of Neisseria meningitidis / s. t

Neisseria meningitidis isolates are conventionally classified by serosubtyping that characterizes the reactivities of the PorA outer membrane protein variable-region epitopes with monoclonal antibodies. Porins are outer membrane proteins (OMPs) of N. meningitidis serogroup B and have attracted study principally for two reasons: their use in the classification of meningococcal isolates into serotype and subtype and as potential components of vaccines against this important pathogen. New murine hybridomas, secreting specific monoclonal antibodies against PorA serotype P1.4 of N. meningitidis serogroup B, were generated using conventional hybridoma procedures. The monoclonal antibodies obtained were characterized by Western blot and whole cell ELISA, using reference strains from different N. meningitidis serotypes and subtypes. All monoclonal antibodies belong to isotype IgG1. Others hybridomas producing MAbs against PorB and FrpB were also obtained(AU)

Los aislamientos de Neisseria meningitidis se clasifican convencionalmente por serosubtipos. Su reactividad se realiza entre el epítope de la región variable de la proteína de membrana externa PorA con anticuerpos monoclonales. Las porinas, proteínas de membrana externa de N. meningitidis del serogrupo B, son atractivas para su estudio principalmente por la clasificación en serotipo y subtipo de los aislamientos del meningococo y como posibles componentes de vacunas contra este importante agente patógeno. Se generaron nuevos hibridomas murinos secretores de anticuerpos monoclonales específicos contra la proteína PorA subtipo P1.4 de N. meningitidis del serogrupo B, mediante los procedimientos convencionales de hibridomas. Los anticuerpos monoclonales, pertenecientes al isotipo IgG1, fueron caracterizados mediante Western blot y ELISA de células enteras. Se utilizaron cepas de referencia de diferentes serotipos y subtipos de N. meningitidis y se obtuvieron hibridomas productores de anticuerpos monoclonales contra otras proteínas como PorB y FrpB(AU)

Randomized, double-blind, placebo-controlled trial to evaluate the safety and immunogenicity of live oral cholera vaccine 638 in Cuban adults.

A randomized, double-blind, placebo-controlled clinical trial was conducted to evaluate the safety, reactogenicity and the immunogenicity of a 2 x 10(9)CFU dose of the 638 lyophilized live attenuated cholera vaccine for oral administration, formulated and produced at Finlay Institute, City of Havana, Cuba. Thirty-six healthy female and male adult volunteers from 18 to 40 years old were involved, clinically examined and laboratory tested after the informed consent signature. Adverse events were monitored and seroconversion rates and geometrical mean titer (GMT) of vibriocidal antibodies were tested in volunteer's sera samples. Neither serious adverse events nor other damages to the volunteers due to vaccine or placebo feeding were reported during the clinical follow-up period of this study; none of the adverse events registered within the first 72 h after inoculation were life-threatening for volunteers. Neither severe nor moderate adverse events were reported. Sixty-one percent of subjects showed mild expected adverse events in an interval lower than 24h up to the first 72 h, 75% of these in the vaccinated group and 18% in the placebo group. Fourteen days after inoculation the GMT of vibriocidal antibodies in the vaccine group significantly increased in comparison to the placebo group. All subjects in the vaccine group (24) seroconverted (100%). Results show that this vaccine is safe, well tolerated and immunogenic in healthy female and male volunteers.

[Erectile dysfunction as a marker of silent cardiovascular disease in type-2 diabetic patients in Spain. The DIVA (DIabetes and VAscular disease) study]. / Disfunción eréctil como marcador de vasculopatía en la diabetes mellitus tipo 2 en España. Estudio DIVA.

BACKGROUND AND OBJECTIVES: Erectile dysfunction (ED) is a sign of vascular disease in type 2 diabetic patients. The present subanalysis of the DIVA Registry, whose main objective was to estimate the prevalence of clinical vascular disorder and silent vascular disorder, as well as risk factors in type 2 diabetic patients treated in Spain, aims to analyze the relationship between those data and the prevalence of ED in these patients. PATIENTS AND METHODS: A total of 2444 type 2 diabetic patients (56% male; mean age 65.2 years) attended by 387 cardiologists and endocrinologists at ambulatory care were included. RESULTS: Coronary heart disease was present in 37% of the patients, cerebrovascular disease in 12%, and peripheral arterial disease in 13%. Forty percent of male patients had ED (according to the IIEF criteria), although in this group, as compared to those patients without ED, the prevalence of cardiovascular disease and signs of subclinical vascular disorder (microalbuminuria and abnormal ankle/brachial index (ABI)) was higher. The only independent predictor of ED was left ventricular hypertrophy (OR 5.2; 95% CI: 1.1-24.1; P=.03), with the ABI <0,9 being of borderline significance (OR 5.9; 95% CI: 0.9-39.9; P=.06). Poor glycemic and lipemic control (P<.05 in both cases) as well as cerebrovascular and peripheral arterial disease (P<.01 in both cases) and renal dysfunction (P<.001) were all more frequent among patients with severe ED. CONCLUSIONS: Forty percent of diabetic patients suffer from ED. The results of this study suggest that ED may be considered as an atherosclerosis marker and could be included in algorithms for risk stratification and subclinical vascular disorder detection.

Disfunción eréctil como marcador de vasculopatía en la diabetes mellitus tipo 2 en España. Estudio DIVA / Erectile dysfunction as a marker of silent cardiovascular disease in type-2 diabetic patients in Spain. The DIVA (Diabetes and Vascular disease) study

Fundamento y objetivo: la disfunción eréctil (DE) es un signo de enfermedad vascular en los sujetos con diabetes mellitus tipo 2 (DM2). El objetivo principal del estudio DIVA (por las letras iniciales de diabetes y vasculopatía) fue estimar la prevalencia de vasculopatía clínica y asintomática, así como la prevalencia de factores de riesgo en los sujetos diabéticos atendidos por especialistas en España; en él se analizó la relación de los datos citados con la prevalencia de DE. Pacientes y método: registro transversal compuesto por 2.444 sujetos consecutivos (56% varones con una edad media de 65,2 años) diagnosticados de DM2, atendidos en consulta por 387 cardiólogos y endocrinólogos. Resultados: el 37% de los sujetos presentaba cardiopatía isquémica, el 12% presentaba enfermedad cerebrovascular (ECRV) y el 13% presentaba arteriopatía periférica. El 40% de los varones tenía DE (según criterios del Índice internacional de la función eréctil), aunque en comparación con los grupos que no presentaban DE, en este grupo la enfermedad cardiovascular era significativamente más prevalente, así como los signos de vasculopatía subclínica (albuminuria e índice tobillo-brazo [ITB] anormal). El único factor predictor de DE independiente de otras variables de confusión fue la hipertrofia ventricular izquierda (riesgo relativo [RR] de 5,2; intervalo de confianza [IC] del 95%: 1,1¿24,1; p=0,03); el ITB fue menor que 0,9 de significación limítrofe (RR de 5,9; IC de 95%: 0,9¿39,9; p=0,06). El mal control glucémico y lipídico (p<0,05 en ambos casos) así como la presencia de ECRV, de enfermedad arterial periférica (p<0,01 en ambos casos) y de disfunción renal (p<0,001) eran más prevalentes en sujetos con DE grave. Conclusiones: el 40% de los varones diabéticos presenta DE. Los resultados de este estudio demuestran que la DE puede considerarse como marcador de aterosclerosis e incluirse en los algoritmos de estratificación de riesgo y detección de vasculopatía asintomática (AU)

Background and objectives: Erectile dysfunction(ED) is a sign of vascular disease in type 2 diabetic patients. The presents ubanalys is of the DIVA Registry, whos emain objective wast o estimate the prevalence of clinical vascular disorder and silent vascular disorder, as well as risk factors in type2diabetic patients treated in Spain, aims to analyze the relationship between those data and the prevalence of ED in these patients. Patients and Methods: A total of 2444 type 2 diabetic patients (56%male; meanage 65.2 years) attended by 387 cardiologists and endocrinologists at ambulatory care were included. Results: Coronary heart disease was present in 37%of the patients, cerebrovascular disease in12%,and peripheral arterial disease in 13%. Forty percent of male patients had ED (according to the IIEF criteria),although in this group, as compared to those patients without ED, the prevalence of cardiovascular disease and signs of subclinical vascular disorder (microalbuminuria and abnormalankle/brachialindex (ABI)) was higher. The only independent predictor of ED was left ventricular hypertrophy (OR5.2;95%CI: 1.1–24.1; P ¼ .03), with the ABI o0,9 being of border line significance (OR5.9;95%CI:0.9–39.9;P ¼ .06).Poor glycemic and lipemic control (Po.05 in both cases)as well as cerebrovascular and peripheral arterial disease (Po.01inbothcases) and renaldys function (Po.001)were all more frequent among patients with severe ED. Conclusions: Forty percent of diabetic patients suffer from ED. The results of this study suggest that EDmay be considered as an at hero sclerosis marker and could be included in algorithms for risk stratification and subclinical vascular disorder detection (AU)

Purificación de lipopolisacárido de Neisseria meningitidis a partir de unafracción colateral del proceso de producción de VA-MENGOC-BC® / Purification of Neisseria meningitidis lipopolysaccharide starting from a collateral fraction of the process of the antimeningococcal vaccine, VA-MENGOC-BC production process

El trabajo tuvo como objetivo purificar lipopolisacáridos (LPS) de Neisseria meningitidis a partir de una fraccióncolateral del proceso de producción de la vacuna antimeningocócica VA-MENGOC-BC®, el sobrenadante que seobtiene del paso de ultracentrifugación durante el proceso de extracción de las proteínas de la membrana externa delmeningococo. La purificación se realizó mediante precipitación con etanol al 80 por ciento, extracción de las proteínas con fenol al 90 porciento entre 65-70 ºC y ultracentrifugación fraccionada a 105,000 g. Se obtuvieron tres lotes de LPS, en total 1,069 g, con un contenido de proteínas, ácidos nucleicos y ácido sálico respecto al LPS de 0,5 por ciento, 0,3 por ciento y 2,2 por ciento (m/m),respectivamente. La evaluación por cromatografía mostró una alta integridad molecular, con valores de constante de distribución reproducibles (0,36-0,38) y una posible asociación del ácido siálico al LPS. Se apreció homogeneidad en el perfil electroforético de los tres lotes y alta actividad endotóxica. El LPS purificado fue identificado fundamentalmente como del inmunotipo L3,7,9. El procedimiento de purificación empleado permite aprovechar una fracción colateral del proceso de producción de la vacuna, es escalable, no incluye métodos cromatográficos, y posibilita la obtención de gran cantidad de LPS de Neisseria meningitidis, no disponible en el mercado, con elevada pureza y alta actividad endotóxica(AU)

The work aimed at purifying lipopolysaccharides (LPS) of Neisseria meningitidis from a collateral fraction of the antimeningococcal BC vaccine, VAMENGOC-BC®. production process, the supernatant obtained from the ultra centrifugation stage during the proteins extraction process of the meningococcus outer membrane. The purification was carried out by precipitation 80 percent ethanol, protein extraction with 90 percent phenol from 65-70 ºC and fractional ultra centrifugation at 105.000 g. Three lots of LPS were obtained, in total 1.069 g, with a content of proteins, nucleic acids and sialic acid in respect to the LPS of 0.5 percent, 0.3 percent and 2.2percent (m/m) respectively. The assessment by chromatography showed a high molecular integrity. with constant valves of reproducible distribution (Kd 0.36 0.38) and a possible sialic acid association to the LPS. Homogeneitywas observed in the electrophoretic profile of the three lots and a high endotoxic activity. The purified LPS was mainly identified as the inmunotype L3,7,9. The purification procedure used allows making use of a collateral fraction of the vaccine production process, it is scalable, it does not include chromatographic methods and makes easy the obtainment of large quantityof LPS of Neisseria meningitidis, wihich it is non available on the market, with high purity and high endotoxic activity(AU)

Immunogenicity and safety of three doses of a bivalent (B:4:p1.19,15 and B:4:p1.7-2,4) meningococcal outer membrane vesicle vaccine in healthy adolescents.

An experimental bivalent meningococcal outer membrane vesicle (OMV) vaccine (B:4:P1.19,15 and B:4:P1.7-2,4) has been developed to provide wide vaccine coverage particularly of the circulating strains in Europe. A randomized, controlled phase II study (study identification number, 710158/002; ClinicalTrials.gov identifier number, NCT00137917) to evaluate the immunogenicity and safety of three doses of the OMV vaccine when given to healthy 12- to 18-year-olds on a 0-2-4 month (n = 162) or 0-1-6 month schedule (n = 159). A control group received two doses of hepatitis A and one of conjugated meningococcal serogroup C vaccine on a 0-1-6 month schedule (n = 157). Immune response, defined as a fourfold increase in serum bactericidal titer using a range of vaccine-homologous or PorA-related and heterologous strains, was determined for samples taken before and 1 month after vaccination; assays were performed at two laboratories. As measured at the GlaxoSmithKline (GSK) laboratory, the OMV vaccine induced an immune response against homologous or PorA-related strains (in at least 51% of subjects against strains of serosubtype P1.19,15 and at least 66% against strains of serosubtype P1.7-2,4) and against a set of three heterologous strains (in 28% to 46% of subjects). Both laboratories showed consistent results for immune response rates. The OMV vaccine had a similar reactogenicity profile for each schedule. Pain preventing normal activities occurred in approximately one-fifth of the subjects; this was significantly higher than in the control group. The immune responses induced by the bivalent OMV vaccine demonstrated the induction of bactericidal antibodies against the vaccine-homologous/PorA-related strains but also against heterologous strains, indicating the presence of protective antigens in OMVs and confirming the potential of clinical cross-protection.

Proteliposome-derived Cochleate as an immunomodulator for nasal vaccine.

Proteoliposome (PL) has been recently used as a protective intramuscular (i.m.) anti-meningococcal BC vaccine. It induces a preferential Th 1 type of immune response. Nevertheless, mucosal protection is mainly mediated by IgA antibody response, which is not usually induced by i.m. vaccination route. IgA antibody production needs the stimulation of Th3 subpopulation, which is also related to the induction of small dose tolerance. We hypothesized that PL-derived Cochleate can induce a specific mucosal IgA and systemic IgG antibody responses. We could show that mice immunized with two or three intranasal doses of PL-derived Cochleate developed significantly increased levels of local anti PL IgA and systemic IgG antibody responses. Thus, our results suggest that PL-derived Cochleate can be used as a promising immunomodulator and delivery system for the development of mucosal, particularly nasal vaccines.

Scale up of proteoliposome derived Cochleate production.

Cochleate are highly stable structures with promising immunological features. Cochleate structures are usually obtaining from commercial lipids. Proteoliposome derived Cochleate are derived from an outer membrane vesicles of Neisseria meningitidis B. Previously, we obtained Cochleates using dialysis procedures. In order to increase the production process, we used a crossflow system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The raw material and solutions used in the production process are already approved for human application. This work demonstrates that CFS is very efficient process to obtain Cochleate structures with a yield of more than 80% and the immunogenicity comparable to that obtained by dialysis membrane.

Outer membrane vesicles of the VA-MENGOC-BC vaccine against serogroup B of Neisseria meningitidis: Analysis of protein components by two-dimensional gel electrophoresis and mass spectrometry.

Neisseria meningitidis is a Gram-negative bacterium responsible for significant mortality worldwide. While effective polysaccharides-based vaccines exist against serogroups A, C, W135, and Y, no similar vaccine is suitable for children under 4 years against disease caused by serogroup B strains. Therefore, major vaccine efforts against this serogroup are based on outer membrane vesicles (OMVs), containing major outer membrane proteins. The OMV-based vaccine produced by the Finlay Institute in Cuba (VA-MENGOC-BC) contributed to the rapid decline of the epidemic in this Caribbean island. While the content of major proteins in this vaccine has been discussed, no detailed work of an outer membrane proteomic map of this, or any other, commercially available OMV-derived product has been published so far. Since OMVs exhibit a large bias toward a few major proteins and usually contain a high content of lipids, establishing the adequate conditions for high resolution, 2-DE of this kind of preparation was definitely a technical challenge. In this work, 2-DE and MS have been used to generate a proteomic map of this product, detailing the presence of 31 different proteins, and it allows the identification of new putative protective protein components it contains.

Tiazolidindionas: fármacos para la prevención cardiovascular / Thiazolidindiones: drugs for cardiovascular prevention

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