Estrés oxidativo y expresión de proteínas relacionadas en enfermedades de alto riesgo cardiovascular: estudio especial de la hipertensión arterial

RESUMEN Entre las múltiples y variadas implicaciones fisiopatológicas del Estrés Oxidativo, los trastornos cardiovasculares son, sin duda, uno de los objetivos prioritarios y motivo de interés de un gran número de investigadores biomédicos. Uno de los principales factores de riesgo es el desarrollo de una serie de enfermedades frecuentes en nuestra población como son la hipertensión arterial y las dislipemias. Éstos trastornos, tienen una vinculación más o menos estrecha, directa o indirecta con el fenómeno que conocemos como estrés oxidativo y los 3 tipos de patologías poseen aumentados los niveles de EO de manera que se potencia el riesgo de aparición de eventos cardiovasculares. Objetivo de este estudio es comparar los niveles de EO en las diferentes patologías cardiovasculares seleccionadas (HTA con y sin Síndrome Metabólico asociado, HF y HFC) midiendo la respuesta de las células mononucleares frente al EO crónico a través de la medida de los niveles de ARNm de las actividades antioxidantes en cada una de ellas. Dicho estudio comparativo se va a realizar mediante el análisis de los siguientes parámetros oxidativos en orina, plasma y en células circulantes de los grupos seleccionados y el grupo control: 1.- Estudio de los productos de oxidación, marcadores de EO y metabolitos relacionados (GSH, GSSG, 8-oxo-dG nuclear, mitocondrial y urinaria, MDA, proteínas carboniladas y niveles de NO3/NO2 . 2.- Determinación de la actividad de los principales sistemas antioxidantes y prooxidante (SOD Cu-Zn y Mn, Cat, GPx, GSR y XO) . 3.- Cuantificación de los niveles de ARNm por RT-PCR cuantitativo a tiempo real, de los genes que codifican dichas actividades enzimáticas, además de OGG1 y G6PDH. CONCLUSIONES 1) En las tres patologías, HTA, HF y HFC, los resultados obtenidos sugieren que el EO representa un mecanismo común y subyacente en la patogenicidad de dichos procesos y posiblemente contribuya al desarrollo de complicaciones cardiovasculares, si bien, el mecanismo bioquímico-molecular responsable del proceso oxidativo presenta características diferenciales entre ellas, así como la intensidad de respuesta al EO. 2) Los niveles elevados de EO en estas patologías no dependen exclusivamente de un aumento en su producción ya que existe una menor actividad antioxidante. La disminución de los mecanismos antioxidantes se debe principalmente a: - Actividad reducida de los genes antioxidantes y del sistema GSH. - Menores niveles de ARNm, disminuyendo así su capacidad de producir enzimas y sistemas protectores frente a EO. 3) Los pacientes de HTA sin tratamiento se encuentran peor protegidos frente a EO que los controles, a pesar de la activación de algunos sistemas antioxidantes como el sistema Tiorredoxina y la enzima Manganeso Superóxido Dismutasa. 4) Los pacientes de HTA con y sin síndrome metabólico asociado no presentan diferencias significativas entre sí, lo que nos confirma que es la HTA el factor asociado al estrés oxidativo, siendo indiferentes las otras características que determinan el diagnóstico de síndrome metabólico. 6) La hipertensión arterial progresa con una marcada oxidación del material genético que se verifica por el aumento en la producción de la base modificada y mutagénica 8-oxo-dG como resultado del estrés oxidativo y de la disminución de enzima reparadora OGG1 en las células mononucleares de los pacientes hipertensos. 7) De los marcadores analizados, la liberación de 8-oxo-dG en orina es el parámetro más representativo del estrés oxidativo en la hipertensión arterial. 8) Los tres modelos de EO crónico analizados poseen un aumento en los niveles de EO pero distinta intensidad en su respuesta. Los pacientes se encuentran desprotegidos frente al EO. __________________________________________________________________________________________________The main aim of this study is to compare the EO levels in some different cardiovascular diseases, previously selected (control, HTA with and without an associate metabolic syndrome, HF and HFC), by measuring the response of mononuclear cells against the chronicle EO through the RNAm level in the antioxidant activity of each one. This comparative study is carried out in different groups by the analyse of different oxidative parameters in urine, plasma and circulated cells. 1.- Study of oxidation products , EO markers and related metabolites. 2.- Main antioxidant and prooxidant systems activity determination. 3.- RNAm level quantification by a real time quantitative RT-PCR. CONCLUSIONS: 1.- The results of the three pathologies (HTA, HF and HFC) propose that EO represents a common and underlying mechanism in their pathologies process and probably it contributes to develop cardiovascular complications, although the responsible biochemistry-molecular mechanism of the oxidative process shows many different features between them. 2.- The high levels of EO in these pathologies do not exclusively depends on an increase of its production because the oxidant activity is lower. 3.- HTA patients without a treatment are worse protected against EO that the controled ones, despite of the activation of some antioxidant systems like Tiorredoxine System and SOD Mn enzyme. 4.- HTA patients with and without the associated metabolic syndrome do not show great differences between them, so it confirms that HTA is the associated factor to the oxidative stress. 5.- The High blood pressure progresses with a high oxidation of the genetic material, that it is verified by the increase of the production of modify base and mutagenig 8- oxo-dG as a result of oxidative stress and the decrease of repairing enzyme OGG1 in the mononuclear cells. 6.- In analysed markers, the 8-oxo-dG liberation in urine is the most representative parameter of oxidative stress in high blood pressure. 7.- The three EO chronicle models analysed have an increase on EO level but different intensity in their response so patients are unprotected against EO

Validation of Bact/Alert automathic system in the microbiological control of cell medicinal products of Advanced Therapies

Objetivo. El Control de calidad para demostrar que un producto está libre de agentes microbianos adventicios es un aspecto clave de control de procesos y evaluación de la calidad de todas las preparaciones medicinales celulares y en la ingeniería tisular. El objetivo de este estudio es validar el sistema de detección por hemocultivo BacT / ALERT para el control microbiológico de las células mesenquimales para terapia celular, según la Farmacopea Europea (EU.PH), 2.6.27. “Control microbiológico de productos celulares” (1). Método. Para el cálculo del límite de detección las botellas de hemocultivo fueron inoculadas e incubadas con 4 réplicas de 30 UFC, 5 réplicas de 15 UFC y 5 réplicas de 6 UFC de los microorganismos en ausencia de producto celular. Se llevaron a cabo también experimentos en presencia de producto con 400.000 células mesenquimales. Este método se ha comparado con el método de referencia de Esterilidad de la EU.PH (2). La especificidad se ensayó inoculando 5 réplicas con 400.000 células mesenquimales sin microorganismos. Resultados. Todas las botellas inoculadas con células mesenquimales sin microorganismos permanecieron negativas después de 7 días de incubación. Todas las botellas inoculadas con cepas bacterianas aerobias y anaerobias fueron detectadas como positivas por el sistema, en el caso del límite inferior (6 UFC) en menos de 36 horas. Se detectaron como positivas las botellas inoculadas con Candida albicans (6 UFC) en menos de 48 horas y con Aspergillus niger (6 UFC) en menos de 72 horas. No hubo diferencias notables en el tiempo de detección entre botellas inoculadas con y sin la presencia de células mesenquimales. Conclusión: El sistema de detección de hemocultivos Bact/Alert es un método fiable para la detección de la contaminación microbiana de medicamentos a base de células mesenquimales y cumple los requisitos de la UE PH, 2.6.27, para el control microbiológico de productos celulares.Objective. Quality control to demonstrate that a product is free from adventitious microbial agents is a key aspect of process control and quality evaluation of all cell medicinal preparations and in tisular engineering. Evaluate the validation of the BacT/ALERT Blood Culture System for the microbial control of mesenchymal cells for cell therapy according European Pharmacopoeia (EU.PH), 2.6.27. “Microbiological control of cellular products” (1). Method. Blood culture bottles were challenged with 4 replica of 30 cfu, 5 replica of 15 cfu and 5 replica of 6 cfu of the test microorganisms. Test were also carried out in the presence in each contaminated culture bottle of 400.000 mesenchymal cells. This method has been compared with the reference method for Sterility of the EU.PH (2). Specificity was tested inoculating 5 replicas of broth culture media with 400.000 cells without microorganisms. Results. All bottles challenged with mesenchymal cells without microorganisms remained negative after 7 days of incubation. All inoculated bottles with aerobic and anaerobic bacterial strains were flagged as positive for the system, in case of low inoculum (6 cfu) in less than 36 hours. Candida inoculated bottles (6 cfu) were detected in less than 48 hours and Aspergillus (6 cfu) in less than 72 hours. There were no significant differences in the detection time between bottles inoculated with and without the presence of mesenchymal cells. Conclusion: The BacT/ALERT blood culture detection system and is a reliable method for detection of microbial contamination of mesenchymal cells medicinal products that fulfils the requirements of the EU PH, 2.6.27, for the microbiological control of cellular products

Pilot study of cutaneous tolerability of fibrin-agarose substitutes in healthy volunteers

Objetivos: En el presente estudio se persigue comprobar posibles reacciones adversas, derivadas del uso tópico de láminas de fibrina-agarosa en el antebrazo de voluntarios sanos. Metodología: Se llevó a cabo un estudio experimental en siete voluntarios sanos, cinco varones y dos mujeres, que no presentaban ningún tipo de lesión cutánea visible. En el antebrazo de cada voluntario se colocaron dos láminas de fibrina-agarosa de 4 cm2 . Cada lámina se cubrió con un apósito impregnado y sobre una de las láminas se aplicó pomada antibiótica con mupirocina. Ambas láminas se cubrieron finalmente con un apósito protector y se mantuvieron en contacto directo sobre la piel durante 48 horas. Resultados: Los resultados determinaron que no se detectaron reacciones adversas después de 48 horas de evolución ni en los siguientes 7 días en ningún voluntario. Se observaron diferencias entre las dos láminas implantadas en cada voluntario, ya que al retirar el apósito cubierto con pomada antibiótica, la lámina presentaba un aspecto más hidratado que la que no llevaba pomada antibiótica. Conclusiones: El uso tópico de las láminas de fibrina-agarosa en voluntarios sanos no presenta reacciones adversas del tipo irritación o alergia al aplicarse directamente por vía tópica. Aunque el tamaño muestral del estudio es limitado, sugiere que la combinación de fibrina-agarosa se presenta como el biomaterial idóneo para el desarrollo de un modelo de piel artificial humana.Purpose: This study aims to analyse possible adverse reactions resulting from the topical use of fibrin-agarose substitutes in the forearm of healthy volunteers. Methods: An experimental study was carried out in seven healthy volunteers, five males and two females, who did not have any cutaneous lesion. Two fibrin-agarose substitutes of 4 cm2 were placed in the forearm of each volunteer. Each substitute was covered with an impregnated dressing and one of the substitutes was covered with antibiotic ointment (mupirocin). Both substitutes were finally covered with a protective dressing. The substitutes were maintained for 48 hours. Results: The results determined that no adverse reactions were detected in any volunteer after 48 hours and a week of evolution. Differences were observed between the two substitutes implanted in each volunteer, since when removing the covered dressing with antibiotic ointment, the substitute presented a more hydrated appearance than the one without antibiotic cream. Conclusions: The implant of fibrin-agarose substitutes in healthy volunteers does not present irritation or allergic type adverse reactions when they applied directly topically on the skin. Although the sample size is low, the fibrin-agarose combination is presented as the biomaterial suitable for the development of an artificial human skin model

Epithelial in vitro differentiation of human mesenchymal stem cells (hMSCs) from adipose tissue (AT) and bone marrow (BM): cellular characterization and study of HLA I and II expression

AGRADECIMIENTOS Laboratorio de Citogenética del servicio de Análisis Clínicos del Hospital Universitario Virgen de las Nieves. Servicio de Análisis Clínicos (Sección de Citometría/Biopatología tumoral) del Hos- pital Universitario Virgen de las Nieves.Introducción: Las células troncales mesenquimales derivadas de tejido adiposo o médula ósea constituyen uno de los tratamientos de terapia celular más utilizados en los ensayos clínicos actuales por su capacidad inmunomoduladora. Además, por su potencial de diferenciación a células epiteliales pueden ser utilizadas en ingeniería tisular incorporadas a tejidos artificiales como la piel o córnea, sustituyendo a las células epiteliales autólogas de estos tejidos. Es necesario realizar una correcta caracterización de estas células diferenciadas y estudiar el efecto de la diferenciación en la expresión del HLA de clase I y II. Objetivos: Caracterizar y realizar los controles de calidad GMP en dos líneas de células mesenquimales troncales humanas de distintos orígenes (tejido adiposo y médula ósea) tras diferenciarlas a células epiteliales in vitro, y analizar si se modifica la expresión de los marcadores HLA I y II antes y después del proceso diferenciador. Metodología: Se ha realizado el aislamiento y expansión de las dos líneas celulares de células mesenquimales troncales a partir del tejido fuente y se ha procedido a su diferenciación in vitro a células epiteliales mediante medios de cultivos suplementados con factores de crecimiento específico. Se han realizado controles de calidad siguiendo los requerimientos de las normas de correcta fabricación y se ha estudiado por citometría de flujo la expresión de HLA tipo I y II antes y después del proceso diferenciador. Finalmente se ha comprobado mediante estudio histológico e inmunohistoquímico las características de las células diferenciadas. Resultados: Se han aislado dos líneas de células mesenquimales troncales de tejido adiposo y médula ósea que cumplen los controles de calidad propuestos. Tras el proceso diferenciador in vitro, las células mesenquimales troncales humanas no expresan marcadores HLA (I y II) importantes en la respuesta inmune, pero sí expresan débilmente proteínas relacionadas con los principales estratos epiteliales (CK5, CK6 y CK14). Conclusión: La ausencia de expresión de marcadores de HLA I y II por citometría de flujo en las células diferenciadas favorecería su uso con carácter alogénico en la construcción de piel y córneas humanas por ingeniería de tejidos, sin embargo, son necesarios más estudios que confirmen estos resultados preliminares y protocolos que optimicen el proceso diferenciador in vitro de las células mesenquimales troncales.Background: Human mesenchymal stem cells derived from adipose tissue and bone marrow are one of the most common cell therapy procedures used in recent clinical trials due to their immunomodulation capacity. Furthermore, for their epithelial differentiation potential can be used in tissue engineering, incorporated in artificial tissues such as skin and cornea, replacing autologous epithelial cells. It is necessary to make a correct cellular characterization of differentiated cells and to study the effect in HLA I and II expression. Objetives: Characterization and quality controls under GMP conditions of in vitro differentiated human mesenchymal stem cells from different sources (adipose tissue and bone marrow) to epithelial lineage, and study of HLA I and II expression before and after differentiation. Methods: Isolation and expansion of two human mesenchymal stem cells lines from their tissues of origin and in vitro differentiation to epithelial cells using culture mediums supplemented with specific growth factors. Quality controls according Good Manufacturing Practices have been made and HLA I and II expression before and after differentiation have been studied. Finally, characteristics of differentiated cells have been demonstrated by histological and immunohistochemical analysis. Results: Two human mesenchymal stem cells lines from adipose tissue and bone marrow have been isolated complying with the proposed quality controls. After in vitro differentiation, human mesenchymal stem cells do not express HLA (I and II) markers, which are important in immune response, but weakly express proteins related to main epithelial layers of human skin (CK5, CK6 and CK14). Conclusion: The absence of expression of HLA I and II by flow cytometry in differentiated cells would promote the use of them with allogenic character to construct human skin and cornea by tissue engineering, however, more studies and protocols are required to confirm these preliminary results and to optimize in vitro differentiation of human mesenchymal stem cells.FIS ISC-III and FEDER PI13/0257

Optimization of human keratinocyte culture to develop an artificial human skin model: cell alternatives as feeder layer of Advanced Therapies

Agradecimientos: Servicio de Medicina Nuclear del Complejo Hospitalario Universitario de GranadaObjetivos: En el presente estudio se persigue optimizar el cultivo de queratinocitos para desarrollar un modelo de piel artificial humana. Para ello, se utilizan como capa alimentadora células de origen humano: fibroblastos dérmicos humanos y células mesenquimales troncales derivadas de tejido adiposo. Los resultados obtenidos se comparan con los fibroblastos 3T3, capa alimentadora de origen murino utilizada desde hace décadas. Metodología: Se llevó a cabo un estudio experimental, utilizando células de origen humano y células de origen murino subletalmente irradiadas, como capa alimentadora para el establecimiento del cultivo de queratinocitos. Se evaluó la tasa de expansión celular y la tasa de duplicación en el pase celular de queratinocitos y en la recuperación celular final que se llevó a cabo a las 3 semanas de cultivo; así como el rendimiento celular y la viabilidad celular, que también se evaluaron en el procesamiento inicial. Resultados: Los resultados determinan que los fibroblastos dérmicos humanos irradiados y las células mesenquimales troncales derivadas de tejido adiposo pueden actuar como capa alimentadora promoviendo la adhesión y la expansión celular de los queratinocitos. Los fibroblastos dérmicos humanos proporcionan resultados equiparables a los obtenidos con los fibroblastos 3T3 murinos. Conclusiones: Los fibroblastos dérmicos humanos irradiados proporcionan una capa alimentadora funcional que permite la expansión in vitro de manera eficaz de los queratinocitos que se van a utilizar con fines clínicos para el desarrollo de un modelo de piel artificial humana.Purpose: This study aims to optimize keratinocyte culture to develop an artificial human skin model. For this purpose, human cells are used as feeder layer: human dermal fibroblasts and adipose derived mesenchymal stem cells. The results obtained are compared with 3T3 fibroblasts, murine feeder layer used for decades. Methods: We conducted an experimental study using human and murine sub-lethally irradiated cells as feeder layer for the establishment of keratinocyte culture. Cell expansion rate and doubling rate were evaluated in the keratinocyte cell passage and in the final cell recovery (was carried out at 3 weeks). The yield and viability of keratinocytes were also evaluated in the initial processing. Results: The results determine that irradiated human dermal fibroblasts and irradiated adipose derived mesenchymal stem cells can act as feeder layer promoting adhesion and expansion of keratinocytes. Human dermal fibroblasts provide comparable results to those obtained with murine 3T3 fibroblasts. Conclusions: Irradiated human dermal fibroblasts provide a functional feeder layer which allows effectively in vitro expansion of keratinocytes to be used for clinical purposes for the development of an artificial human skin model

Barriers to health care services for migrants living with HIV in Spain

BACKGROUND: In Spain, migrants are disproportionately affected by HIV and experience high rates of late diagnosis. We investigated barriers to health care access among migrants living with HIV (MLWH) in Spain. METHODS: Cross sectional electronic survey of 765 adult HIV-positive migrants recruited within 18 health care settings between July 2013 and July 2015. We collected epidemiological, demographic, behavioral and clinical data. We estimated the prevalence and risk factors of self-reported barriers to health care using multivariable logistic regression. RESULTS: Of those surveyed, 672 (88%) had information on health care access barriers: 23% were women, 63% from Latin America and Caribbean, 14% from Sub-Saharan Africa and 15% had an irregular immigration status. Men were more likely to report barriers than women (24% vs. 14%, P = 0.009). The main barriers were: lengthy waiting times for an appointment (9%) or in the clinic (7%) and lack of a health card (7%). Having an irregular immigration status was a risk factor for experiencing barriers for both men (OR: (4.0 [95%CI: 2.2–7.2]) and women (OR: 10.5 [95%CI: 3.1–34.8]). Men who experienced racial stigma (OR: 3.1 [95%CI: 1.9–5.1]) or food insecurity (OR: 2.1 [95%CI: 1.2–3.4]) were more likely to report barriers. Women who delayed treatment due to medication costs (6.3 [95%CI: 1.3–30.8]) or had a university degree (OR: 5.8 [95%CI: 1.3–25.1]) were more likely to report barriers. CONCLUSION: Health care barriers were present in one in five5 MLWH, were more common in men and were associated to legal entitlement to access care, perceived stigma and financial constraints

Mesenchymal stromal cells in human immunodeficiency virus infected patients with discordant immune response: Early results of a phase I/II clinical trial

Between 15% and 30% of HIV-infected subjects fail to increase their CD4+ T-cell counts despite continuous viral suppression (immunological nonresponders [INRs]). These subjects have a higher morbidity and mortality rate, but there are no effective treatments to reverse this situation so far. This study used data from an interrupted phase I/II clinical trial to evalu ate safety and immune recovery after INRs were given four infusions, at baseline and at weeks 4, 8, and 20, with human allogeneic mesenchymal stromal cells from adipose tissue (Ad-MSCs). Based on the study design, the first 5 out of 15 INRs recruited received unblinded Ad-MSC infusions. They had a median CD4+ nadir count of 16/μL (range, 2-180) and CD4+ count of 253 cells per microliter (171-412) at baseline after 109 (54-237) months on antiretroviral treatment and 69 (52-91) months of continuous undetectable plasma HIV RNA. After a year of follow-up, an independent committee recommended the suspension of the study because no increase of CD4+ T-cell counts or CD4+ /CD8+ ratios was observed. There were also no significant changes in the phenotype of different immunological lympho cyte subsets, percentages of natural killer cells, regulatory T cells, and dendritic cells, the inflammatory parameters analyzed, and cellular associated HIV-DNA in peripheral blood mononuclear cells. Furthermore, three subjects suffered venous thrombosis events directly related to the Ad-MSC infusions in the arms where the infusions were performed. Although the current study is based on a small sample of participants, the findings suggest that alloge neic Ad-MSC infusions are not effective to improve immune recovery in INR patients or to reduce immune activation or inflammation. ClinicalTrials.gov identifier: NCT0229004. EudraCT number: 2014-000307-26.Instituto de Salud Carlos IIIRed de Investigación en SIDAAndalusian Regional Ministry of Health and Familie

Mesenchymal stromal cells in human immunodeficiency virus‐infected patients with discordant immune response: Early results of a phase I/II clinical trial

Between 15% and 30% of HIV‐infected subjects fail to increase their CD4+ T‐cell counts despite continuous viral suppression (immunological nonresponders [INRs]). These subjects have a higher morbidity and mortality rate, but there are no effective treatments to reverse this situation so far. This study used data from an interrupted phase I/II clinical trial to evaluate safety and immune recovery after INRs were given four infusions, at baseline and at weeks 4, 8, and 20, with human allogeneic mesenchymal stromal cells from adipose tissue (Ad‐MSCs). Based on the study design, the first 5 out of 15 INRs recruited received unblinded Ad‐MSC infusions. They had a median CD4+ nadir count of 16/μL (range, 2‐180) and CD4+ count of 253 cells per microliter (171‐412) at baseline after 109 (54‐237) months on antiretroviral treatment and 69 (52‐91) months of continuous undetectable plasma HIV‐RNA. After a year of follow‐up, an independent committee recommended the suspension of the study because no increase of CD4+ T‐cell counts or CD4+/CD8+ ratios was observed. There were also no significant changes in the phenotype of different immunological lymphocyte subsets, percentages of natural killer cells, regulatory T cells, and dendritic cells, the inflammatory parameters analyzed, and cellular associated HIV‐DNA in peripheral blood mononuclear cells. Furthermore, three subjects suffered venous thrombosis events directly related to the Ad‐MSC infusions in the arms where the infusions were performed. Although the current study is based on a small sample of participants, the findings suggest that allogeneic Ad‐MSC infusions are not effective to improve immune recovery in INR patients or to reduce immune activation or inflammation. ClinicalTrials.gov identifier: NCT0229004. EudraCT number: 2014‐000307‐26.Andalusian Network for the Design and Translation of Advanced Therapies; Andalusian Regional Ministry of Health and Families, Grant/Award Number: 201600073585‐tra; Red de Investigación en SIDA, Grant/Award Number: RD16/0025/0020‐ISCIII‐FEDER; Instituto de Salud Carlos III, Grant/Award Number: PI15/01041

Impact of late presentation of HIV infection on short-, mid- and long-term mortality and causes of death in a multicenter national cohort : 2004-2013

To analyze the impact of late presentation (LP) on overall mortality and causes of death and describe LP trends and risk factors (2004-2013). Cox models and logistic regression were used to analyze data from a nation-wide cohort in Spain. LP is defined as being diagnosed when CD4 < 350 cells/ml or AIDS. Of 7165 new HIV diagnoses, 46.9% (CI:45.7-48.0) were LP, 240 patients died.First-year mortality was the highest (aHR = 10.3[CI:5.5-19.3]); between 1 and 4 years post-diagnosis, aHR = 1.9(1.2-3.0); an

Revista Temas Agrarios Volumen 26; Suplemento 1 de 2021

1st International and 2nd National Symposium of Agronomic Sciences: The rebirth of the scientific discussion space for the Colombian Agro.1 Simposio Intenacional y 2 Nacional de Ciencias Agronómicas: El renacer del espacio de discusión científica para el Agro colombiano