Development of a Serum-Free Medium To Aid Large-Scale Production of Mycoplasma-Based Therapies.

To assist in the advancement of the large-scale production of safe Mycoplasma vaccines and other Mycoplasma-based therapies, we developed a culture medium free of animal serum and other animal components for Mycoplasma pneumoniae growth. By establishing a workflow method to systematically test different compounds and concentrations, we provide optimized formulations capable of supporting serial passaging and robust growth reaching 60 to 70% of the biomass obtained in rich medium. Global transcriptomic and proteomic analysis showed minor physiological changes upon cell culture in the animal component-free medium, supporting its suitability for the production of M. pneumoniae-based therapies. The major contributors to growth performance were found to be glucose as a carbon source, glycerol, cholesterol, and phospholipids as a source of fatty acids. Bovine serum albumin or cyclodextrin (in the animal component-free medium) were required as lipid carriers to prevent lipid toxicity. Connaught Medical Research Laboratories medium (CMRL) used to simplify medium preparation as a source of amino acids, nucleotide precursors, vitamins, and other cofactors could be substituted by cysteine. In fact, the presence of protein hydrolysates such as yeastolate or peptones was found to be essential and preferred over free amino acids, except for the cysteine. Supplementation of nucleotide precursors and vitamins is not strictly necessary in the presence of yeastolate, suggesting that this animal origin-free hydrolysate serves as an efficient source for these compounds. Finally, we adapted the serum-free medium formulation to support growth of Mycoplasma hyopneumoniae, a swine pathogen for which inactivated whole-cell vaccines are available. IMPORTANCE Mycoplasma infections have a significant negative impact on both livestock production and human health. Vaccination is often the first option to control disease and alleviate the economic impact that some Mycoplasma infections cause on milk production, weight gain, and animal health. The fastidious nutrient requirements of these bacteria, however, challenges the industrial production of attenuated or inactivated whole-cell vaccines, which depends on the use of animal serum and other animal raw materials. Apart from their clinical relevance, some Mycoplasma species have become cellular models for systems and synthetic biology, owing to the small size of their genomes and the absence of a cell wall, which offers unique opportunities for the secretion and delivery of biotherapeutics. This study proposes medium formulations free of serum and animal components with the potential of supporting large-scale production upon industrial optimization, thus contributing to the development of safe vaccines and other Mycoplasma-based therapies.

Widespread ribosome stalling in a genome-reduced bacterium and the need for translational quality control.

Trans-translation is a ubiquitous bacterial mechanism of ribosome rescue mediated by a transfer-messenger RNA (tmRNA) that adds a degradation tag to the truncated nascent polypeptide. Here, we characterize this quality control system in a genome-reduced bacterium, Mycoplasma pneumoniae (MPN), and perform a comparative analysis of protein quality control components in slow and fast-growing prokaryotes. We show in vivo that in MPN the sole quality control cytoplasmic protease (Lon) degrades efficiently tmRNA-tagged proteins. Analysis of tmRNA-mutants encoding a tag resistant to proteolysis reveals extensive tagging activity under normal growth. Unlike knockout strains, these mutants are viable demonstrating the requirement of tmRNA-mediated ribosome recycling. Chaperone and Lon steady-state levels maintain proteostasis in these mutants suggesting a model in which co-evolution of Lon and their substrates offer simple mechanisms of regulation without specialized degradation machineries. Finally, comparative analysis shows relative increase in Lon/Chaperone levels in slow-growing bacteria suggesting physiological adaptation to growth demand.

Characteristics of patients with survival longer than 20 years following heart transplantation.

INTRODUCTION AND OBJECTIVES: The number of heart-transplant recipients exceeding 20 years of follow-up is steadily increasing. However, little is known about their functional status, comorbidities, and mortality. Identifying the predictors of prolonged survival could guide the selection of candidates for the low number of available donors. METHODS: Functional status, morbidities, and mortality of heart-transplant patients between 1984 and 1992 were analyzed. To identify predictors of 20-year survival, a logistic regression model was constructed using the covariates associated with survival in the univariate analysis. RESULTS: A total of 39 patients who survived 20 years (26% of patients transplanted before 1992) were compared to 90 recipients from the same period who died between 1 and 20 years post-transplantation. Major complications were hypertension, renal dysfunction, infections, and cancer. After a mean follow-up of 30 months, 6 survivors had died, yielding a mortality rate of 6% per year (vs 2.5%-3% in years 1-19). Causes of mortality were infection (50%), malignancy (33%), and allograft vasculopathy (17%). Long-term survivors were younger and leaner, and had nonischemic cardiomyopathy and lower ischemic time. Logistic regression identified recipient age <45 years (odds ratio=3.9; 95% confidence interval, 1.6-9.7; P=.002) and idiopathic cardiomyopathy (odds ratio=3; 95% confidence interval, 1.4-7.8; P=.012) as independent predictors for 20-year survival. CONCLUSIONS: One fourth of all heart-transplant patients in our series survived >20 years with the same graft, and most enjoy independent lives despite significant comorbidities. Recipient age <45 years and idiopathic cardiomyopathy were associated with survival beyond 2 decades. These data may help decide donor allocation.

Influence of hypothermia on right atrial cardiomyocyte apoptosis in patients undergoing aortic valve replacement.

BACKGROUND: There is increasing evidence that programmed cell death can be triggered during cardiopulmonary bypass (CPB) and may be involved in postoperative complications. The purpose of this study was to investigate whether apoptosis occurs during aortic valve surgery and whether modifying temperature during CPB has any influence on cardiomyocyte apoptotic death rate. METHODS: 20 patients undergoing elective aortic valve replacement for aortic stenosis were randomly assigned to either moderate hypothermic (ModHT group, n = 10, 28 degrees C) or mild hypothermic (MiHT group, n = 10, 34 degrees C) CPB. Myocardial samples were obtained from the right atrium before and after weaning from CPB. Specimens were examined for apoptosis by flow cytometry analysis of annexin V-propidium iodide (PI) and Fas death receptor staining. RESULTS: In the ModHT group, non apoptotic non necrotic cells (annexin negative, PI negative) decreased after CPB, while early apoptotic (annexin positive, PI negative) and late apoptotic or necrotic (PI positive) cells increased. In contrast, no change in the different cell populations was observed over time in the MiHT group. Fas expression rose after reperfusion in the ModHT group but not in MiHT patients, in which there was even a trend for a lower Fas staining after CPB (p = 0.08). In ModHT patients, a prolonged ischemic time tended to induce a higher increase of Fas (p = 0.061). CONCLUSION: Our data suggest that apoptosis signal cascade is activated at early stages during aortic valve replacement under ModHT CPB. This apoptosis induction can effectively be attenuated by a more normothermic procedure.

[Surgical treatment for hypertrophic obstructive cardiomyopathy]. / Tratamiento quirúrgico de la miocardiopatía hipertrófica obstructiva.

INTRODUCTION AND OBJECTIVES: Five percent of the patients with hypertrophic obstructive cardiomyopathy (HOCM) have symptoms unresponsive to medical treatment and are candidates for invasive therapy. The objective of this study was to analyze our results with surgical treatment of HOCM during the last 10 years. PATIENTS AND METHOD: Between July 1993 and January 2004 26 patients with HOCM refractory to drug therapy were operated on. An extended septal myectomy was performed, in combination with anterior mitral leaflet plication in 19 cases (73%) and with mitral valve replacement in 5 (19%). Evolution of the grade of dyspnea, left ventricle outflow tract gradient (LVOTG), mitral regurgitation, and systolic anterior motion after surgery was analyzed. RESULTS: Mean follow-up was 63 (37) months. After surgery, a significant reduction in LVOTG (from 96.5 to 19.5 mmHg; P<.001), grade of mitral regurgitation (from 2.54 to 0.69; P<.001) and systolic anterior motion (from 2.92 to 0.23; P<.001) was achieved, which led to improvement in functional class. Hospital mortality and need for pacemaker implantation due to complete heart block after surgery was 3.8% (n=1). There were no cases of iatrogenic ventricular septal defect or mitro-aortic valve injury. Actuarial survival at 5 years was 96% (4%). CONCLUSIONS: Surgery in patients with HOCM yields great clinical improvements with low morbidity and mortality. Simultaneous intervention for both myocardial and valvular components of the disease allows not only reduction in the LVOTG but also correction of mitral regurgitation and abolition of systolic anterior motion.

Edge-to-Edge tricuspid repair for redeveloped valve incompetence after DeVega's annuloplasty.

"Edge-to-edge" technique is a well-accepted procedure with excellent results for correction of mitral insufficiency. We describe a simple edge-to-edge combined with bicuspidalization repair method that was successfully applied in 2 patients for the treatment of redeveloped functional tricuspid regurgitation after previous annuloplasty. Significant improvement in symptoms and echocardiographic results were achieved.

Estudio randomizado doble ciego, comparando pirrolidona carboxilato de sodio con hidrocortisona al 1 por ciento, contra pirrolidona carboxilato de sodio / A randomized doble blind study comparing sodium salt of pyrrolidone carboxilic acid with hydrocortisone 1 per cent against sodium salt of pyrrolidone carboxylic acid

En un estudio efectuado en eccemas de diferente naturaleza, comparamos la eficacia de pirrolidona carbo xilato de sodio con hidrocortisona al 1 por ciento contra pirrolidona carboxilato de sodio simple en 100 pacientes observados a lo largo de dos semanas. Se evaluaron tanto la eficacia clínica, como la aceptación cosmética y los posibles efectos colaterales, al fin del tratamiento y una semana después. Con pirrolidona carboxilato de sodio con hidrocortisona al 1 por ciento los resultados se consideraron como muy buenos en 94 porciento, y moderados en 6 porciento. Con pirrolidona carboxilato de sodio los resultados fueron muy buenos en 40 porciento y moderados en 60 porciento. La aceptación cosmética fue excelente. No se encontraron efectos colaterales.

Recepción y atención masiva de bajas en un hospital / Reception and care of victims in an hospital

Se presenta la planificación de un Hospital Militar para enfrentar a una situación de catástrofes con atención de gran número de víctimas. Este Plan se divide en: 1) Comité de Planificación de Atención Hospitalaria, 2) Médico Jefe de Operaciones, 3) Política y Doctrinas, 4) Planificación de Atención Hospitalaria en Catástrofes. El Comité de Planificación es un asesor de las autoridades del Hospital con tareas específicas de organización, control y evaluación. El Médico Jefe de Operaciones debe cumplir varios requisitos y sus funciones más importantes las constituyen la decisión de poner en marcha la fase operativa, el ejercicio del mando del personal médico y paramédico y, el disponer los cambios de la organización del Plan según situaciones. El Plan de Atención hospitalaria en catástrofes debe estar escrito en forma clara y precisa y se puede subdividir para su comprensión en varios capítulos

Descripción de un método anatómo clínico para alivio del dolor en el síndrome de ATM / Description of an anatomoclinical method for alleviation of ATM syndrome pain

Se tratan 20 pacientes con Síndrome de ATM, que presentan dolor al inicio del tratamiento, con estimulación eléctrica transcutánea. Se usa un criterio anatómico, basado en un estudio previo en cadáveres, para la ubicación de los electrodos sobre ramas cutáneas del Trigémino y plexo cervical. Se obtiene un resultado de un 70 por ciento de alivio total después de 3 sesiones consecutivas de una hora cada una, en días diferentes. El 30 por ciento de los pacientes restantes también presentó analgesia, pero en un grado menor, sin efectos colaterales negativos