Effect of exercise duration on toluene-induced locomotor sensitization in mice: a focus on the Renin Angiotensin System.

RATIONALE: Exercise attenuates addictive behavior; however, little is known about the contribution of exercise duration to this positive effect. The Renin Angiotensin System (RAS) has been implicated both in addictive responses and in the beneficial effects of exercise; though, its role in the advantageous effects of exercise on toluene-induced addictive responses has not been explored. OBJECTIVES: To evaluate the impact of different exercise regimens in mitigating the expression of toluene-induced locomotor sensitization and to analyze changes in RAS elements' expression at the mesocorticolimbic system after repeated toluene exposure and following voluntary wheel running in toluene-sensitized animals. METHODS: Toluene-induced addictive-like response was evaluated with a locomotor sensitization model in mice. Toluene-sensitized animals had access to running wheels 1, 2, 4 or 24 h/day for 4 weeks; thereafter, locomotor sensitization expression was evaluated after a toluene challenge. RAS elements (ACE and ACE2 enzymes; AT1, AT2 and Mas receptors) expression was determined by Western blot in the VTA, NAc and PFCx of toluene-sensitized mice with and without exercise. RESULTS: Individual differences in toluene-induced locomotor sensitization development were observed. Access to wheel running 1 and 2 h/day reduced but 4 and 24 h/day completely blocked locomotor sensitization expression. Repeated toluene exposure changed RAS elements' expression in the VTA, NAc and PFCx, while exercise mainly modified ACE and AT1 in air-exposed and toluene-sensitized mice. CONCLUSIONS: Inhalant-exposed animals show different sensitization phenotypes. Exercise duration determined its efficacy to attenuate the addictive-like response. Toluene exposure and exercise each modified RAS, the latter also modifying toluene-induced changes.

New Formulation of a Subunit Vaccine Candidate against Lawsonia intracellularis Increases Humoral and Cellular Immune Responses.

Previously, we designed a subunit vaccine candidate based on three L. intracellularis antigens with promising results in pigs. In this study, antigens were produced individually to achieve an even antigen ratio in the formulation. The emulsion characterization included the drop size and the mechanical and thermal stability. Immune response was evaluated by indirect and sandwich ELISAs, qPCR, and flow cytometry. The vaccine candidate's safety was assessed by histopathology and monitoring the clinical behavior of animals. The average production yielded for the chimeric antigen as inclusion bodies was around 75 mg/L. The formulation showed mechanical and thermal stability, with a ratio Hu/Ho > 0.85 and a drop size under 0.15 nm. Antigens formulated at a ratio of 1:1:1 induced a significant immune response in inoculated pigs that persisted until the end of the experiment (week 14). The dose of 200 µg significantly activated cellular response measured by transcriptional and translational levels of cytokines. The cell proliferation assay revealed an increment of lymphocytes T CD4+ at the same dose. Animals gained weight constantly and showed proper clinical behavior during immunization assays. This research demonstrated the immunological robustness of the new subunit vaccine candidate against Porcine Proliferative Enteropathy evenly formulated with three chimeric antigens of L. intracellularis.

Captopril and losartan attenuate behavioural sensitization in mice chronically exposed to toluene.

Inhalants are consumed worldwide for recreational purposes. The main component found in many inhalants is toluene. One of the most deleterious behavioural effects caused by chronic exposure to inhalants is addiction. This response has been associated with activation of the mesolimbic dopaminergic pathway, and it is known that the renin angiotensin system plays a role in the modulation of this dopaminergic system. In the present work, we hypothesize that blockade of the RAS with angiotensin converting enzyme inhibitors or angiotensin II type 1 receptor blockers is able to attenuate the addictive response induced by toluene. We exposed mice to toluene for four weeks to induce locomotor sensitization. In the second phase of the work, captopril or losartan were administered for 20 days. Subsequently, the expression of behavioural sensitization was evaluated with a toluene challenge. To exclude false associations between the observed responses and treatments, motor coordination and blood pressure were analysed in animals treated with captopril or losartan. At the end of the behavioural studies, animal brains were harvested and Ang II/Ang-(1-7) and Ang-(1-7)/Ang II ratios were analysed in the nucleus accumbens (NAc) and prefrontal cortex (PFCx). The results showed that toluene induced behavioural sensitization, while captopril or losartan treatment attenuated the expression of this response. No significant differences were observed in motor coordination or blood pressure. Repeated toluene administration decreased Ang-(1-7)/Ang II ratio in the PFCx. On the other hand, treatment with captopril or losartan decreased the Ang II/Ang-(1-7) ratio and enhanced the Ang-(1-7)/Ang II ratio in the NAc. This work suggests that blockade of RAS attenuates the toluene-induced behavioural sensitization.

A trivalent TNF-R2 as a new tumor necrosis factor alpha-blocking molecule.

The neutralization of tumor necrosis factor alpha (TNFα) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNFα-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNFα and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNFα-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNFα was assessed. By microscale thermophoresis, the KD value for the interaction was 4.17 ± 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNFα-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNFα-antagonist agent.

Expression and characterization of a novel single-chain anti-vascular endothelial growth factor antibody in the goat milk.

Vascular endothelial growth factor (VEGF) has essential functions in angiogenesis, endothelial cell proliferation, migration, and tumor invasion. Different approaches have been developed to suppress tumor angiogenesis, which is considered a hallmark of cancer. Anti-VEGF monoclonal antibodies constitute an important strategy for cancer immunotherapy, which has been produced on several platforms. In this study, a novel single-chain anti-VEGF monoclonal antibody (scVEGFmAb) was produced in the goat mammary gland by adenoviral transduction. scVEGFmAb was purified by affinity chromatography. N-glycans were analyzed by exoglycosidase digestion and hydrophilic interaction ultra-performance liquid chromatography coupled to electrospray ionization mass spectrometry. The biological activity of scVEGFmAb was assessed by scratch and mouse aortic ring assays. scVEGFmAb was produced at 0.61 g/L in the goat milk, and its purification rendered 95 % purity. N-glycans attached to scVEGFmAb backbone were mainly neutral biantennary core fucosylated with Galß1,4GlcNAc motif, and charged structures were capped with Neu5Ac and Neu5Gc. The chimeric molecule significantly prevented cell migration and suppressed microvessel sprouting. These results demonstrated for the first time the feasibility of producing an anti-VEGF therapeutic antibody in the milk of non-transgenic goats with the potential to counteract tumor angiogenesis.

Design and characterization of a novel dimeric blood-brain barrier penetrating TNFα inhibitor.

Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.

In vivo modification of the goat mammary gland glycosylation pathway.

Complex recombinant glycoproteins produced as potential biopharmaceuticals in goat's milk have an aberrant pattern of N-glycosylation due to the lack of multi-antennary structures. Overexpression of glycosyltransferases may increase oligosaccharide branching of the desired glycoproteins. Here, human erythropoietin fused to human IgG Fc (EPO-Fc) was co-expressed with N-acetyl-glucosaminyltransferase-IVa (GnT-IVa) by adenoviral transduction in goat mammary gland to evaluate the in vivo modification of N-glycosylation pattern in this tissue. Adenoviral vectors, containing the EPO-Fc and GnT-IVa sequences were assembled for in vitro and in vivo expression in mammalian cell culture or in goat mammary gland. Protein detection was assessed by gel electrophoresis and western blot, and N-glycans were identified by HPLC and mass spectrometry. GnT-IVa overexpression and its colocalization with EPO-Fc in the Golgi apparatus of SiHa cells were demonstrated. N-glycan analysis of in vitro and in vivo expression of EPO-Fc modified by GnT-IVa (EPO-Fc/GnT-IVa) showed an increase in high molecular weight structures, which corresponded to tri- and tetra-antennary N-glycans in SiHa cells and mostly tri-antennary N-glycans in goat's milk from transformed mammary tissue. The results confirmed that successful modification of the goat mammary gland secretion pathway could be achieved by co-expressing glycoenzymes together with the glycoprotein of interest. This is the first report of modification of the N-glycosylation pattern in the goat mammary gland in vivo, and constitutes a step forward for improving the use of the mammary gland as a bioreactor for the production of complex recombinant proteins.

Diseño y validación de la escala pronóstica cubana PREDICMED para estratificar el riesgo de mediastinitis postoperatoria / Design and validation of the Cuban prognostic scale PREDICMED to stratify the risk of postoperative mediastinitis

RESUMEN Introducción: La predicción de fenómenos en las ciencias médicas mediante escalas pronósticas constituye una herramienta valiosa en la actualidad y deben incluirse en el proceso de toma de decisiones. Pronosticar la mediastinitis postoperatoria permite disponer de recursos para su prevención. Objetivo: Construir una escala pronóstica para estratificar el riesgo de padecer mediastinitis postoperatoria. Método: Se realizó un estudio de casos y controles para los factores de riesgo de mediastinitis postoperatoria en el Cardiocentro Ernesto Guevara de Santa Clara, Cuba. Luego de la regresión logística se obtuvo el modelo y, a partir de este, se incluyeron y ponderaron los predictores para obtener la escala cubana pronóstica de mediastinitis postoperatoria: PREDICMED, que se validó por diversos métodos. Resultados: Esta escala se obtuvo con seis predictores y dos estratos de riesgo. Se analizó su rendimiento mediante ajuste, calibración y determinación de su poder discriminante, con buenos resultados. Se realizó validación interna por el método de división de datos y se comparó su capacidad en ambos subconjuntos (desarrollo y validación) sin diferencias. Se probó su buena validez de constructo, al no existir diferencias entre las probabilidades predichas y las observadas. También se analizó su validez de contenido mediante expertos. Por último, se determinó su validez de criterio al comparar con otra escala similar (MEDSCORE). PREDICMED presentó muy buena capacidad discriminatoria (área bajo la curva 0,962) y elevados valores de sensibilidad (84,62%) y especificidad (92,31%). Conclusiones: La escala pronóstica cubana PREDICMED, para estratificar el riesgo de mediastinitis postoperatoria, mostró buenos parámetros de validación y logró estratificar el riesgo en no alto y alto.

ABSTRACT Introduction: Phenomena prediction through prognostic scales is a valuable tool in medical sciences nowadays and it should be included in the decision-making process. Predicting postoperative mediastinitis allows to count on resources for its prevention. Objective: To build a prognostic scale to stratify the risk of suffering from postoperative mediastinitis. Method: A case-control study for the risk factors of postoperative mediastinitis was carried out at the Cardiocentro Ernesto Guevara from Santa Clara, Cuba. After the logistic regression, the model was obtained and from it, the predictors to obtain the Cuban prognostic scale of postoperative mediastinitis PREDICMED were included and weighted, which was validated through several methods. Results: This scale was obtained, counting on six predictors and two risk strata. Its performance was analyzed through adjustment, calibration and determination of its discriminating capacity, showing good results. Internal validation was carried out through the data division method and its capacity was compared in both subsets (development and validation) showing no differences. Its good construct validity was demonstrated, since there were no differences between the predicted and the observed probabilities. Its contents validity was also analyzed by experts. Finally, its criteria validity was determined when compared with another similar scale (Medscore). PREDICMED showed a very good discriminatory capacity (area under the curve 0.962) as well as high values of sensitivity (84.62%) and specificity (92.31%). Conclusions: The Cuban prognostic scale PREDICMED, to stratify the risk of postoperative mediastinitis showed good validation parameters and it was able to stratify the risk in not high and high.

New insights on the interaction mechanism of rhTNFα with its antagonists Adalimumab and Etanercept.

TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS-PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10-9 M and >10-8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.

Fluorescence-assisted sequential insertion of transgenes (FASIT): an approach for increasing specific productivity in mammalian cells.

Currently, the generation of cell lines for the production of recombinant proteins has the limitation of unstable gene expression due to the repeat-induced gene silencing or the loss of transgene copies resulting from recombination events. In this work, we developed a new strategy based on the sequential insertion of transgenes for generating stable clones producing high levels of a chimeric human follicle-stimulating hormone (hscFSH). Gene insertion was done by transducing HEK-293 cells with a lentiviral vector containing a bicistronic transcriptional unit for expressing hscFSH and GFP genes. Clone selection was performed by flow cytometry coupled to cell sorting, and the GFP gene was further removed by CRE-mediated site-specific recombination. High-producing clones of hscFSH were obtained after three rounds of lentiviral transduction. Expression levels increased in a step-wise manner from 7 to 23 pg/cell/day, with a relatively constant rate of 7 pg/cell/day in each round of transduction. The GFP gene was successfully removed from the cell genome without disturbing the hscFSH gene expression. Clones generated using this approach showed stable expression levels for more than two years. This is the first report describing the sequential insertion of transgenes as an alternative for increasing the expression levels of transformed cell lines. The methodology described here could notably impact on biotechnological industry by improving the capacity of mammalian cells to produce biopharmaceuticals.

Caracterización clínico-epidemiológica con enfoque quirúrgico de la endocarditis infecciosa en la región central de Cuba / Clinico-epidemiological characterization with a surgical approach of infective endocarditis in the central region of Cuba

RESUMEN Introducción: La endocarditis infecciosa no ha disminuido su incidencia y mortalidad en los últimos 30 años. Objetivo: Caracterizar los pacientes operados por diagnóstico de endocarditis infecciosa activa. Método: Se realizó un estudio descriptivo retrospectivo que incluyó a 109 pacientes con diagnóstico de esta enfermedad y fueron intervenidos quirúrgicamente en el Cardiocentro Ernesto Che Guevara (Cuba) desde julio de 2010 hasta junio de 2018. La información se obtuvo de las historias clínicas y el informe operatorio. Resultados: La endocarditis infecciosa sobre válvula protésica fue diagnosticada en 15 pacientes (13,8%), pero la más frecuente fue la relacionada con la colonización de dispositivos intracardíacos (51,4%), razón por la que el procedimiento quirúrgico más utilizado fue el cambio de sistema de estimulación. La principal indicación quirúrgica fue la infección incontrolada en 63 casos (57,8%), seguida por la insuficiencia cardíaca (38 pacientes; 34,9%) y en 67 casos (61,5%) los hemocultivos resultaron negativos. La sustitución de la válvula nativa por una prótesis mecánica se realizó en 28 pacientes (25,7%), de los cuáles 15 (13,8%) fueron en posición mitral. El recambio de una prótesis mecánica por otra de igual características se llevó a cabo en 14 (12,8%) casos, 8 (7,3%) de ellos mitrales. La mortalidad global fue de 17,4%, más evidente en aquellos con endocarditis sobre válvula protésica izquierda (7/15). Conclusiones: Predominaron el sexo masculino y la edad mayor de 60 años, así como la presencia de vegetaciones sobre dispositivos intracardíacos y los hemocultivos negativos. La endocarditis sobre válvula protésica se asoció significativamente con la mortalidad.

ABSTRACT Introduction: Infective endocarditis has not decreased its incidence and mortality in the last 30 years. Objective: To characterize patients operated on due to diagnosis of active infective endocarditis. Method: A retrospective, descriptive study was carried out, which included 109 patients with a diagnosis of this disease, who underwent surgery at Cardiocentro Ernesto Che Guevara (Cuba), from July 2010 to June 2018. The information was obtained from the medical records and the surgical report. Results: Prosthetic valve infective endocarditis was diagnosed in 15 patients (13.8%), but the most frequent was the one related to intracardiac device colonization (51.4%), reason why the most used surgical procedure was the change of the electrical stimulation system. The main surgical indication was the uncontrolled infection in 63 cases (57.8%), followed by heart failure (38 patients; 34.9%) and in 67 cases (61.5%), the blood cultures were negative. The native valve replacement for a mechanical prosthetic valve was performed in 28 patients (25.7%), of which 15 (13.8%) were in mitral position. The replacement of a mechanical prosthetic valve for another with the same characteristics was carried out in 14 (12.8%) cases, 8 (7.3%) of them were mitral. Overall mortality was of 17.4%, more evident in those with left-side prosthetic valve endocarditis (7/15). Conclusions: Male gender and age over 60 years old predominated, as well as the presence of vegetations on intracardiac devices, and negative blood cultures. The prosthetic valve endocarditis was significantly associated with mortality.

Primer separador retráctil de pulmón para facilitar la disección extrapleural de la arteria torácica interna / First retractable lung spreader to facilitate extrapleural dissection of the internal thoracic artery

RESUMEN La disección de la arteria torácica interna es uno de los procedimientos más definitorios de la cirugía de revascularización miocárdica, pues se ha demostrado que la calidad de vida y la evolución a corto y largo plazo de los pacientes dependerán de las características morfológicas del injerto. Hace más de 25 años se propuso la utilización de un separador pulmonar maleable para facilitar la disección de la arteria torácica interna, especialmente su segmento proximal, pero nunca se ha fabricado ese dispositivo. Se presenta un separador pulmonar manufacturado a partir de dispositivos en desuso, empleados en cirugía coronaria. Se comentan y discuten las principales ventajas demostradas durante más de tres años en cerca de un centenar de pacientes.

ABSTRACT Internal thoracic artery dissection is one of the most defining procedures of coronary artery bypass grafting, as it has been demonstrated that the quality of life and short-long term outcome of patients will depend on the morphological characteristics of the graft. More than 25 years ago, the use of a malleable lung spreader was proposed to facilitate the internal thoracic artery dissection especially its proximal segment, but that device has never been manufactured. A lung spreader, assembled from disused devices, used in coronary artery revascularization is presented. The main advantages confirmed over more than three years in nearly a hundred patients are discussed and commented upon.

Rara coincidencia de dos tumores: mixoma cardíaco e hipernefroma. Presentación de un paciente / Rare coincidence of two tumors: cardiac myxoma and hypernephroma. A case report

RESUMEN La asociación de dos tumores de diferente origen en un mismo paciente es cada vez más frecuente en la práctica clínica actual. Se presenta un caso con una rara asociación de mixoma cardíaco e hipernefroma, previamente tratado. Ante la presencia de manifestaciones neurológicas, inicialmente se planteó el diagnóstico de metástasis cerebral del tumor renal; luego, con el hallazgo ecocardiográfico de una masa intracardíaca, se pensó en la posibilidad de trombo, por lo que se decidió practicar cirugía cardíaca con carácter urgente para resecarla. El estudio anatomopatológico confirmó la existencia de un mixoma cardíaco, por lo que se trata de un enfermo con dos enfermedades tumorales cuya coincidencia ha sido pocas veces descrita.

ABSTRACT The association of two tumors of different origin in the same patient is becoming more frequent in the current clinical practice. Here is presented a case with a rare association of cardiac myxoma and hypernephroma, previously treated. Due to the presence of neurological symptoms, there was initially set a diagnosis of brain metastasis from the renal tumor; then, with the echocardiographic finding of an intracardiac mass, the possibility of thrombus was considered, therefore a cardiac surgery was decided to be performed urgently in order to resect it. The pathologic examination confirmed the existence of a cardiac myxoma, then, we have a patient with two tumor diseases whose coincidence has been rarely described.

Automatized colon polyp segmentation via contour region analysis.

The increasing use of colorectal cancer screening programs has contributed to the growing number of colonoscopies performed by health centers. Hence, in recent years there has been a tendency to develop medical diagnosis support tools in order to assist specialists. This research has designed an automatized polyp detection system that allows a reduction in the rate of missed polyps that can lead to interval cancer; one of the main risks existing in colonoscopy. A characterization has therefore been made of the shape, color and curvature of edges and their regions, enabling the segmentation of polyps present in colonoscopy images. A 90.53% polyp detection rate has been achieved using the designed system, and 76.29% and 71.57% segmentation quality for the Annotated Area Covered and Dice Coefficient indicators respectively. This system aims to offer assistance with medical diagnosis that has a positive impact on patient health.

Patient prognosis based on feature extraction, selection and classification of EEG periodic activity.

Periodic activity in electroencephalography (PA-EEG) is shown as comprising a series of repetitive wave patterns that may appear in different cerebral regions and are due to many different pathologies. The diagnosis based on PA-EEG is an arduous task for experts in Clinical Neurophysiology, being mainly based on other clinical features of patients. Considering this difficulty in the diagnosis it is also very complicated to establish the prognosis of patients who present PA-EEG. The goal of this paper is to propose a method capable of determining patient prognosis based on characteristics of the PA-EEG activity. The approach, based on a parallel classification architecture and a majority vote system has proven successful by obtaining a success rate of 81.94% in the classification of patient prognosis of our database.