Conjugation Mechanism for Pneumococcal Glycoconjugate Vaccines: Classic and Emerging Methods.

Licensed glycoconjugate vaccines are generally prepared using native or sized polysaccharides coupled to a carrier protein through random linkages along the polysaccharide chain. These polysaccharides must be chemically modified before covalent linking to a carrier protein in order to obtain a more defined polysaccharide structure that leads to a more rational design and safer vaccines. There are classic and new methods for site-selective glycopolysaccharide conjugation, either chemical or enzymatic modification of the polysaccharide length or of specific amino acid residues of the protein carrier. Here, we discuss the state of the art and the advancement of conjugation of S. pneumoniae glycoconjugate vaccines based on pneumococcal capsular polysaccharides to improve existing vaccines.

ISCOM-Matrices Nanoformulation Using the Raw Aqueous Extract of Quillaja lancifolia (Q. brasiliensis).

Quillaja saponins have an intrinsic capacity to interact with membrane lipids that self-assembles in nanoparticles (immunostimulating complexes or ISCOM-matrices) with outstanding immunoadjuvant activity and low toxicity profile. However, the expensive and laborious purification processes applied to purify Quillaja saponins used to assemble ISCOM-matrices show an important drawback in the large-scale use of this vaccine adjuvant. Thus, in this study, we describe a protocol to appropriately formulate ISCOM-matrices using the raw aqueous extract (AE) of Quillaja lancifolia leaves. In the presence of lipids, AE was able to self-assemble in nanostructures that resembles immunostimulating complexes (ISCOM). These negatively charged nanoparticles of approximately 40 nm were characterized by transmission electron microscopy and dynamic light scattering. In addition, well-known saponins with remarkable immunoadjuvant activity, as QS-21, were detected into nanoparticles. Thus, the easier, robust, cheaper, and environmentally friendly method developed here may be an alternative to the classical methods for ISCOM-matrices production that use high-purified saponins.

Polyvalent Bacterial Lysate Protects Against Pneumonia Independently of Neutrophils, IL-17A or Caspase-1 Activation.

Polyvalent bacterial lysates have been in use for decades for prevention and treatment of respiratory infections with reported clinical benefits. However, besides claims of broad immune activation, the mode of action is still a matter of debate. The lysates, formulated with the main bacterial species involved in respiratory infections, are commonly prepared by chemical or mechanical disruption of bacterial cells, what is believed influences the biological activity of the product. Here, we prepared two polyvalent lysates with the same composition but different method of bacterial cell disruption and evaluated their biological activity in a comparative fashion. We found that both bacterial lysates induce NF-kB activation in a MyD88 dependent manner, suggesting they work as TLR agonists. Further, we found that a single intranasal dose of any of the two lysates, is sufficient to protect against pneumococcal pneumonia, suggesting that they exert similar biological activity. We have previously shown that protection against pneumococcal pneumonia can also be induced by prior S. pneumoniae sub lethal infection or therapeutic treatment with a TLR5 agonist. Protection in those cases depends on neutrophil recruitment to the lungs, and can be associated with increased local expression of IL-17A. Here, we show that bacterial lysates exert protection against pneumococcal pneumonia independently of neutrophils, IL-17A or Caspase-1/11 activation, suggesting the existence of redundant mechanisms of protection. Trypsin-treated lysates afford protection to the same extent, suggesting that just small peptides suffice to exert the protective effect or that the molecules responsible for the protective effect are not proteins. Understanding the mechanism of action of bacterial lysates and deciphering the active components shall allow redesigning them with more precisely defined formulations and expanding their range of action.

Bacterial Lysates as Immunotherapies for Respiratory Infections: Methods of Preparation.

Bacterial lysates, prepared from the microorganisms most frequently involved in human Respiratory Tract Infections (RTIs) have been in the market for several decades, and at present, several different brands are available in many countries worldwide. They all claimed to exert local and systemic immunomodulatory effects but different clinical trials show disparate results between them. The lack of consistency of predicted therapeutic effects has undermined their clinical use and hampered licensing in several countries. One explanation for such lack of consistency in the results is that their methods of preparation are also very different. Here, we review the available literature describing methods of preparation of bacterial lysates, including patent disclosure documents. We found a great variety of methodologies of preparation and a lack of standardized procedures among them. The main conclusion of our study is that there is a clear need for standardized protocols of production to obtain comparable results in clinical trials worldwide.

Next-Generation Whole-Cell Pneumococcal Vaccine.

Streptococcus pneumoniae remains a major public health hazard. Although Pneumococcal Conjugate Vaccines (PCVs) are available and have significantly reduced the rate of invasive pneumococcal diseases, there is still a need for new vaccines with unlimited serotype coverage, long-lasting protection, and lower cost to be developed. One of the most promising candidates is the Whole-Cell Pneumococcal Vaccine (WCV). The new generation of whole-cell vaccines is based on an unencapsulated serotype that allows the expression of many bacterial antigens at a lower cost than a recombinant vaccine. These vaccines have been extensively studied, are currently in human trial phase 1/2, and seem to be the best treatment choice for pneumococcal diseases, especially for developing countries.

Optimal Conditions for Streptococcus pneumoniae Culture: In Solid and Liquid Media.

Control of Streptococcus pneumoniae is mainly achieved by the use of existing vaccines. Capsular polysaccharides are the major antigenic component and are also the main virulence factor.Capsular polysaccharides must fulfill requirements of purity, uniformity, and an accurate molecular weight to be used as vaccine antigens. Vaccine production largely relies on cultivation of the pathogen in appropriate conditions.Here we describe widely used techniques to culture S. pneumoniae based on solid or complex liquid media, which are successfully applied in the diagnosis of the pathogen and in development and production of S. pneumoniae vaccines. Furthermore, we present a new chemically defined medium that can be used at lab scale.

Concausas en lesionados con fracturas de miembros atendidos en el Departamento de Medicina Legal de Ciego de Ávila / Concauses in injured people with fractures of members treated at the Legal Medicine Department of Ciego de Ávila

Introducción: el delito de lesiones constituye una problemática en el Departamento de Medicina Legal de la provincia Ciego de Ávila dado que a un grupo de pacientes con fracturas de miembros se les prolonga el tiempo esperado de sanidad. Objetivo: caracterizar las concausas que demoraron la consolidación ósea en los lesionados con fracturas de miembros a los que se les atestó sanidad en el trienio 2014-2016. Método: se realizó una investigación descriptiva de tipo retrospectivo en el Departamento Provincial de Medicina Legal de Ciego de Ávila a una población de 82 lesionados de 16 años o más con fracturas de miembros cuya consolidación ósea demoró un tiempo mayor de 30 días. Los datos fueron extraídos de las historias clínicas individuales. Resultados: predominaron el grupo etario de 21 a 40 años (46,3 por ciento), el sexo masculino (75,61 por ciento) y el tipo de delito por riña (68,29 por ciento) sobre los accidentes de tránsito (31,71 por ciento). La localización predominante de las fracturas fue en huesos (57,32 por ciento) respecto a las articulaciones (42,68 por ciento), y dentro de las primeras la más frecuente fue en cúbito y radio. Predominaron las concausas preexistentes (62,20 por ciento) sobre las sobrevenidas (37,80 por ciento) y no se identificaron concomitantes. Dentro de las preexistentes, predominó la osteoporosis (29,27 por ciento) y entre las sobrevenidas, la infección (23,17 por ciento).Conclusiones: las concausas preexistentes predominaron sobre las sobrevenidas y no se registró concausa concomitante. Se halló una primacía de la osteoporosis como preexistente y de la infección como sobrevenida(AU)

Introduction: the crime of injuries constitutes a problem at the Legal Medicine Department of Ciego de Ávila province, given that in a group of patients with fractures of the limbs, the expected time of healing is extended.Objective: to characterize the concauses that delayed bone consolidation in those injured with fractures of limbs who were tested for healing in the 2014-2016 triennium. Method: a retrospective descriptive investigation was carried out in the Provincial Department of Legal Medicine of Ciego de Ávila, to a population of 82 injured persons aged 16 years or older with fractures of limbs whose bone consolidation took more than 30 days. Data were extracted from individual medical records.Results: the age group of 21 to 40 years (46,3 percent), the male sex (75,61 percent) and the type of crime by quarrel (68,29 percent) predominated over traffic accidents (31,71 percent ). The predominant location of the fractures was in bones (57,32 percent) with respect to the joints (42,68 percent), and within the former the most frequent was in ulna and radius. Pre- existing concauses (62,20 percent) prevailed over those that occurred (37,80 percent) and no concomitants were identified. Among the preexisting ones, osteoporosis (29,27 percent) predominated and among those that occurred, infection (23,17 percent).Conclusions: pre-existing concauses predominated over those that occurred and there was no concomitant concause. A primacy of osteoporosis was found as preexisting and infection as an occurrence(AU)

Purification of Capsular Polysaccharides of Streptococcus pneumoniae: Traditional and New Methods.

Pneumonia caused by Streptococcus pneumoniae is a major bacterial disease responsible for many deaths worldwide each year and is particularly dangerous in children under 5 years old and adults over 50. The capsular polysaccharide (CPS) constitutes the outermost layer of the bacterial cell and is the main virulence factor. Regardless of whether pharmaceutical agents are composed of CPS alone or protein-conjugated CPS, CPS purification is essential for the development of vaccines against S. pneumoniae. These vaccines are effective and safe but remain quite expensive. This review describes the methods currently available for CPS purification. Advances in CPS purification methods are aimed at improvements in quality and yield and, above all, process simplification.

Treatment in a preventive cardiology clinic utilizing advanced practice providers effectively closes atherosclerotic cardiovascular disease risk-management gaps among a primary-prevention population compared with a propensity-matched primary-care cohort: A team-based care model and its impact on lipid and blood pressure management.

BACKGROUND: Advanced practice providers (APPs) can fill care gaps created by physician shortages and improve adherence/compliance with preventive ASCVD interventions. HYPOTHESIS: APPs utilizing guideline-based algorithms will more frequently escalate ASCVD risk factor therapies. METHODS: We retrospectively reviewed data on 595 patients enrolled in a preventive cardiology clinic (PCC) utilizing APPs compared with a propensity-matched cohort (PMC) of 595 patients enrolled in primary-care clinics alone. PCC patients were risk-stratified using Framingham Risk Score (FRS) and coronary artery calcium scoring (CACS). RESULTS: Baseline demographics were balanced between the groups. CACS was more commonly obtained in PCC patients (P < 0.001), resulting in reclassification of 30.6% patients to a higher risk category, including statin therapy in 26.6% of low-FRS PCC patients with CACS ≥75th MESA percentile. Aspirin initiation was higher for high and intermediate FRS patients in the PCC (P < 0.001). Post-intervention mean LDL-C, non-HDL-C, and triglycerides (all P < 0.05) were lower in the PCC group. Compliance with appropriate lipid treatment was higher in intermediate to high FRS patients (P = 0.004) in the PCC group. Aggressive LDL-C and non-HDL-C treatment goals (<70 mg/dL, P = 0.005 and < 130 mg/dL, P < 0.001, respectively), were more commonly achieved in high-FRS PCC patients. Median post-intervention SBP was lower among intermediate and low FRS patients (P = 0.001 and P < 0.001, respectively). Cumulatively, this resulted in a reduction in median post-intervention PCC FRS across all initial FRS risk categories (P < 0.001 for all). CONCLUSIONS: APPs within a PCC effectively risk-stratify and aggressively manage ASCVD risk factors, resulting in a reduction in post-intervention FRS.

Leaf saponins of Quillaja brasiliensis enhance long-term specific immune responses and promote dose-sparing effect in BVDV experimental vaccines.

Saponin-based adjuvants are promising adjuvants that enhance both humoral and T-cell-mediated immunity. One of the most used natural products as vaccine adjuvants are Quillaja saponaria bark saponins and its fraction named Quil A®. Despite that, its use has been restricted for human use due to safety issues. As an alternative, our group has been studying the congener species Quillaja brasiliensis saponins and its performance as vaccine adjuvants, which have shown to trigger humoral and cellular immune responses comparable to Quil A® but with milder side effects. Here, we studied a semi purified aqueous extract (AE) and a previously little characterized saponin-enriched fraction (QB-80) from Q. brasiliensis as vaccine adjuvants and an inactivated virus (bovine viral diarrhea virus, BVDV) antigen co-formulated in experimental vaccines in mice model. For the first time, we show the spectra pattern of the Q. brasiliensis saponins by MALDI-TOF, a novel and cost-effective method that could be used to characterize different batches during saponins production. Both AE and QB-80 exhibited noteworthy chemical similarities to Quil A®. In addition, the haemolytic activity and toxicity were assessed, showing that both AE and QB-80 were less toxic than Quil A®. When subcutaneously inoculated in mice, both fractions promoted long-term strong antibody responses encompassing specific IgG1 and IgG2a, enhanced the avidity of IgG antibodies, induced a robust DTH reaction and significantly increased IFN-É£ production in T CD4+ and T CD8+ cells. Furthermore, we have proven herein that AE has the potential to promote dose-sparing, substantially reducing the dose of antigen required for the BVDV vaccines and still eliciting a mixed Th1/Th2 strong immune response. Based on these results, and considering that AE is a raw extract, easier and cheaper to produce than commercially available saponins, this product can be considered as candidate to be escalated from experimental to industrial uses.

Intranasal administration of a polyvalent bacterial lysate induces self-restricted inflammation in the lungs and a Th1/Th17 memory signature.

Respiratory tract infections are among the most frequent infections in humans causing millions of deaths especially in children and the elderly. Antibiotics and vaccines are the main available tools of control, but resistant strains are continuously arising and available vaccines only account for few of many pathogens involved. Non-specific immunotherapies are an emerging alternative to induce protective immunity at the airways. Mucosally administered polyvalent bacterial lysates (PBLs) have been widely used for decades for prevention of respiratory diseases, but the bases of their proposed therapeutic effectiveness are still controversial. Here, we show that PBL engages a pro-inflammatory gene expression program in macrophages and epithelial cells, induces an acute lung inflammatory response and elicits full protection against pneumococcal pneumonia. Chronic lung inflammation may have pathological consequences, so the capacity to regain local homeostasis after treatment is central. We found that local inflammation is fully resolved and 30 days after treatment lungs become undistinguishable from naïve mice. Nevertheless, this process leaves an immunological imprinting with a Th1/Th17 memory phenotype that may be a marker of the protective immunity elicited. Increasing our understanding of the mechanism of action of PBLs may improve the efficiency of these immunotherapies and expand their range of action.

Entre la cocina y el laboratorio: la alimentación científica y las dietistas / Between the kitchen and the laboratory: the scientific diet and dietitians

Desde tiempos remotos la alimentación ha sido asociada con la salud. No obstante, la nutrición como ciencia moderna surgiría en Occidente a partir del desarrollo de las ciencias físicas y la medicalización de las sociedades ocurridas desde finales del siglo XVIII. El objetivo de este estudio fue analizar en este contexto los orígenes de la alimentación científica y su aplicación en los hospitales, en relación con la aparición de las dietistas en los ámbitos internacional y nacional. Se llevó a cabo una investigación histórico-documental a partir de fuentes cualitativas. Se encontró que la alimentación científica –planificada racionalmente según necesidades nutricionales, costo y palatabilidad– surgió a finales del siglo XIX ligada a la química y la fisiología. Su utilización terapéutica en los hospitales dio lugar a la profesión de dietista y a una nueva arquitectura hospitalaria. La llegada de estos avances a Argentina se vincula con la figura del médico porteño Pedro Escudero, quien creó el primer servicio de alimentación del país y el Instituto Municipal de Nutrición en 1928, y la primera Escuela de Dietistas de Latinoamérica en 1933. El establecimiento de la nutrición como ciencia modificó las representaciones de la alimentación que habían estado vigentes durante siglos; generó una nueva autoridad en la materia y originó la profesiónde dietista construida como auxiliar del médico con un carácter claramente feminizado. En Argentina, estos desarrollos se debieron a las relaciones políticas de Escudero y se produjeron con importantes diferencias entre la ciudad de Buenos Aires y el resto del país...

Caracterización de pacientes con intoxicación aguda por psicofármacos / Characterization of patients with acute poisoning due to psycho drugs

Se realizó un estudio descriptivo y transversal de 23 pacientes con intoxicación aguda por psicofármacos, atendidos en el Servicio de Cuidados Intensivos Emergentes del Hospital General Docente "Dr. Juan Bruno Zayas Alfonso" de Santiago de Cuba, desde octubre de 2010 hasta igual mes de 2011, con el objetivo de caracterizarles según algunas variables seleccionadas. La información fue procesada con el programa SPSS, versión 10.1. Se utilizó el porcentaje como medida de resumen y se aplicó la prueba de hipótesis de Ji al cuadrado para variables independientes. Predominaron los pacientes de 15 - 24 años (61,0 %), el sexo femenino y las intoxicaciones por psicofármacos combinados; también se obtuvo que 44,4 % de los afectados con intervalo asistencial de más de 6 horas presentaron complicaciones, mayoritariamente cardiovasculares.

A descriptive and cross-sectional study was carried out in 23 patients with acute poisoning due to psycho drugs, assisted in the Emergent Intensive Care Service from "Dr. Juan Bruno Zayas Alfonso" Teaching General Hospital in Santiago de Cuba, from October, 2010 to the same month, 2011, with the objective of characterizing them according to some selected variables. The information was processed with the program SPSS, version 10.1. The percentage was used as summary measure and the chi-square hypothesis test was applied for independent variables. Patients from 15-24 years (61.0 %), female sex and the poisonings due to combined psycho drugs prevailed; it was also obtained that 44.4 % of the affected patients with assistance interval of more than 6 hours presented with complications, most of them of cardiovascular type.

Caracterización de pacientes con intoxicación aguda por psicofármacos / Characterization of patients with acute poisoning due to psycho drugs

Se realizó un estudio descriptivo y transversal de 23 pacientes con intoxicación aguda por psicofármacos, atendidos en el Servicio de Cuidados Intensivos Emergentes del Hospital General Docente Dr Juan Bruno Zayas Alfonso de Santiago de Cuba, desde octubre de 2010 hasta igual mes de 2011, con el objetivo de caracterizarles según algunas variables seleccionadas. La información fue procesada con el programa SPSS, versión 10.1. Se utilizó el porcentaje como medida de resumen y se aplicó la prueba de hipótesis de Ji al cuadrado para variables independientes. Predominaron los pacientes de 15 - 24 años (61,0 por ciento), el sexo femenino y las intoxicaciones por psicofármacos combinados; también se obtuvo que 44,4 por ciento de los afectados con intervalo asistencial de más de 6 horas presentaron complicaciones, mayoritariamente cardiovasculares(AU)

A descriptive and cross-sectional study was carried out in 23 patients with acute poisoning due to psycho drugs, assisted in the Emergent Intensive Care Service from Dr Juan Bruno Zayas Alfonso Teaching General Hospital in Santiago de Cuba, from October, 2010 to the same month, 2011, with the objective of characterizing them according to some selected variables. The information was processed with the program SPSS, version 10.1. The percentage was used as summary measure and the chi-square hypothesis test was applied for independent variables. Patients from 15-24 years (61.0 percent), female sex and the poisonings due to combined psycho drugs prevailed; it was also obtained that 44.4 percent of the affected patients with assistance interval of more than 6 hours presented with complications, most of them of cardiovascular type(AU)

Features of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14.

The effect of several cultivation conditions on the kinetics of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14 was studied. The presence in the supernatant of serotype-specific CPS (capsular polysaccharide) during growth was followed by size-exclusion HPLC and, in parallel, confirmed by using a specific latex reagent. The agitation level did not affect the production behaviour, whereas pH maintenance above 6 strongly enhanced both growth and CPS production throughout the cultivation period in flasks. Production of high-molecular-mass polysaccharide was found to be maximal between 5 and 6 h of cultivation, at the end of the exponential phase. By laser light scattering, 90% of this purified CPS product showed a M(w) (molecular mass) range from 350 to 1500 kDa, with an average M(w) of 921 kDa. Extending the culture to 24 h gave rise to a clear shift of the M(w) distribution of the polysaccharide to values lower than 100 kDa. These findings may have strong implications for the large-scale manufacture of the polysaccharide and the associated conjugate vaccine.

Preparative purification of soybean agglutinin by affinity chromatography and its immobilization for polysaccharide isolation.

Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5+/-3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity.

Quantitative determination of pneumococcal capsular polysaccharide serotype 14 using a modification of phenol-sulfuric acid method.

The capsular polysaccharide of Streptococcus pneumoniae, serotype 14, is part of every pneumococcal vaccine presently in the market or under development. A strategy for the quantitative determination of this polysaccharide by the phenol-sulfuric acid method is described. The modality of acid addition is shown to be the critical step for obtaining reproducible test results between different technicians. Raising the incubation temperature above 80 degrees C increased the consistency of the method by more than 60% regardless of the acid addition modality, but at the expense of some loss of sensitivity. Incubation at 110 degrees C was found necessary to obtain reproducible results within 3% for this technique, which was used to follow the enrichment of the polysaccharide during the last steps of purification. A model mixture of the component polysaccharide sugars provided an adequate and economic standard to construct the calibration curve for this assay, with absorbance reading either in the reaction tubes or in a microplate. A similar procedure may be applied to the determination of other bacterial polysaccharides as well.

Melanoma extracutáneo de cabeza y cuello. Estudio clínico patológico de 19 casos / Head and neck extracutaneous melanoma. Clinical pathological study of 19 cases

El presente es un estudio, descriptivo y retrospectivo en el que se analizan las características clínicas y patológicas de 19 melanomas extracutáneos localizados en la región de la cabeza y el cuello. El 63 por ciento se presentó en varones y 37 por ciento en mujeres. El promedio de edad fue de 55 años, con límites entre 18 y 84 años. Las principales manifestaciones clínicas fueron aumento de volumen local (73 por ciento) e hiperpigmentación (16 por ciento). La localización más frecuente fue la cavidad oral (37 por ciento), fosas nasales (26 por ciento) y órbita (21 por ciento). En el 21 por ciento se efectuó el diagnóstico clínico correcto. El tratamiento más frecuente fue el quirúrgico (32 por ciento). El 37 por ciento de los casos presentaron metástasis. Con respecto al análisis microscópico de los casos, los datos más relevantes fueron que el 32 por ciento presentaban actividad de unión, el 37 por ciento presentaron patrón de crecimiento difuso, el 26 por ciento están formados por células epitelioides y el 89 por ciento de los casos presentaron pigmento melánico.

Rol del enfermero especialista en salud mental en los programas de prevencion del maltrato fisico y psicologico al menor por parte de padres y adultos / Rol of nurse specialist in mental health in programs of prevention of child physical and psychological abuse by parents and adults

Con el proposito de establecer las funciones del enfermero especialista en salud mental en la prevencion del maltrato al menor se establecio un flujograma de actividades para el profesional de enfermeria en educacion, asistencia administracion e investigacion tanto a nivel institucional como de la comunidad. Se recomienda implementar este perfil ocupacional para verificar su viabilidad en la practica..