Efecto del Bacilo Calmette-Guérin (BCG) sobre las Células Natural Killer y sus implicaciones en el tratamiento del cáncer de vejiga

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-06-2016Esta tesis tiene embargado el acceso al texto completo hasta el 24-03-2018La inmunoterapia con el bacilo Calmette-Guérin (BCG) se lleva utilizando con éxito para el tratamiento del cáncer de vejiga superficial desde hace 40 años, reduciendo la recurrencia en el 70% de los pacientes. A pesar de que todavía hay preguntas pendientes sobre su mecanismo de acción, está claro que tras la instilación de la micobacteria en la vejiga, se produce el reclutamiento de linfocitos y células mieloides, así como la liberación de citoquinas y quimioquinas. Se piensa que esta respuesta inmunitaria local es la que, en último término, será capaz de eliminar a las células tumorales. Las células Natural Killer (NK) son linfocitos citotóxicos que participan en la respuesta inmunitaria frente al cáncer y a infecciones producidas por distintos patógenos. Además, existen datos que demuestran que las células NK contribuyen al éxito de la inmunoterapia con BCG. En esta tesis, se han llevado, en paralelo, dos líneas de investigación para estudiar el papel de las células NK en la respuesta inmunitaria contra el cáncer de vejiga en el contexto del tratamiento con BCG: por un lado, se han realizado experimentos en un modelo in vitro y, por otro, se han analizado muestras de pacientes de cáncer de vejiga tratados con BCG. In vitro, se ha estudiado el reconocimiento de distintas líneas celulares tumorales uroteliales por las células NK aisladas de sangre periférica de donantes sanos, así como la manera en la que BCG afecta a ese reconocimiento. Si bien la micobacteria no produce cambios en las células tumorales uroteliales que modulen la respuesta de las células NK estimuladas con IL- 2, sí es capaz de producir la diferenciación de las células NK en presencia de otras células del sistema inmunitario. Así, la co-incubación de células de sangre periférica con BCG, genera una población de células NK CD56bright que, inesperadamente, expresa CD16, KIR y CD57 y que tiene una gran capacidad para responder frente a las células tumorales uroteliales. La expansión de esta nueva población de células NK, depende de la liberación de factores solubles producidos por otras células inmunitarias en respuesta a BCG. Por su parte, el estudio de las muestras de sangre y orina de pacientes reveló que, aunque a nivel periférico parece difícil poder detectar cambios debidos al tratamiento con BCG, a nivel local es posible detectar factores solubles que se liberan, al menos una semana después de la instilación, y que pueden estar implicados en la eficacia de la respuesta al tratamiento. La integración de ambos estudios ha permitido, a partir de los resultados obtenidos in vitro, proponer un modelo de la respuesta a la micobacteria, tanto a nivel celular como humoral, que puede verse reflejado en los factores solubles liberados en la orina de los pacientes. Se propone la realización de un estudio con un mayor número de pacientes que sería muy útil para, más adelante, poder determinar factores que permitan predecir la buena o mala respuesta de los pacientes en estadíos tempranos del tratamiento con BCG.Bacillus Calmette-Guérin (BCG) has been used to treat non-muscle invasive bladder cancer (NMIBC) since 1976, reducing recurrence in about 70% of patients. Although there are still open questions about its mechanism of action, the instillation of the mycobacteria into the bladder is known to result in the recruitment of lymphocytes and myeloid cells, as well as the release of cytokines and chemokines, and it is thought that this local immune response plays an important role in the elimination of the tumour. Natural Killer (NK) cells are cytotoxic lymphocytes important in immune responses against cancer and infections produced by different pathogens. Moreover, a variety of data from the literature strongly suggest that NK cells are important for the success of BCG immunotherapy. In this thesis, the role of NK cells in the immune response against bladder cancer triggered by BCG has been analysed by establishing an in vitro model system, and by the analysis ex vivo of peripheral blood and urine from bladder cancer patients undergoing BCG therapy. In vitro, the recognition of different bladder cancer cell lines by freshly isolated NK cells purified from healthy donors, as well as the effect of BCG on this recognition were studied. Although exposure to mycobacteria did not produce changes in the bladder tumour cells that modified their recognition by IL-2-activated NK cells, it was able to induce NK cell differentiation and activation in the presence of other immune cells. The co-incubation of peripheral blood mononuclear cells (PBMC) with BCG led to the expansion of a CD56bright NK cell population that, surprisingly, expressed CD16, KIR and CD57, and that could respond to the different bladder cancer cells. The expansion of these NK cells depended on the release of soluble factors by other immune cells responding to BCG. It proved difficult to detect significant changes in peripheral blood after intravesical instillation of BCG. However, the analysis, one week after BCG instillation, of the urine samples revealed the presence of a range of different soluble factors that could play a role in the efficacy of the response to the treatment. Finally, comparison of the data obtained in vitro and in vivo has allowed establishment of a model of the humoral and cellular response to the mycobacteria, as reflected in the soluble factors released in the urine of BCG-treated patients. It will be interesting to study in detail the release of these soluble factors in a larger cohort of patients to test whether they are useful biomarkers to predict the response of patients to treatment with BCG at early stages of the therapy

Pozzolanic materials to reduce CO2 emissions: local solutions for a global issue.

Abstract de una keynote invitada.In recent decades, the cement sector has been looking for solutions to reduce the carbon footprint, being one of the most promising strategies, the replacement of clinker with supplementary cementitious materials, SCMs. However, the main limitation of this approach is the availability of suitable SCMs. This work presents the study of three families of pozzolanic materials, Spanish calcined clays (CC), Natural Pozzolans (NP) and fly ashes (FA). The characterization of each family will be presented, with emphasis on the kaolinite content of the original clays and the amorphous contents of the natural pozzolans and fly ashes. The results of the pozzolanic prediction tests will be compared: strength activity index, SAI, and R3 test according to ASTM C1897-20. The SAI test has two important limitations: i) it gives false positives at 28 days, as does the addition of quartz (Qz) and ii) a minimum of 28 days is required to obtain the pozzolanic activity results. In addition, the R3 test has proved to be useful in ruling out inert additions, such as quartz. Moreover, it presents a very good correlation between the heat emitted and the combined water at 7 days and the amount of kaolinite in clays or amorphous in ashes. However, the absolute values of heat and combined water cannot be compared between different families. That is, in the calcined clay family, it can be inferred that if the kaolinite content is higher than 50 wt%, the heat released should be between 500-700 J/g, whereas a fly ash with an amorphous content of around 70 wt% will release between 200-250 J/g.PID2020-114650RB-I00 grant from Spanish government, Master Builders Solutions Deutschland GmbH (Germany) and Buzzi Unicem SpA (Italy) are thanked for the funding. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Multidisciplinary team of critically ill patient care: What is the contribution of the pharmacist?

Antecedentes y objetivo: La gravedad e inestabilidad de los pacientes, junto con el alto grado de complejidad de la medicación, hacen de las unidades de cuidados intensivos (UCI) un área crítica de problemas relacionados con la medicación. El objetivo de nuestro estudio fue analizar y evaluar la actividad clínica realizada por el farmacéutico clínico integrado en una UCI y conocer la opinión del personal. Material y método: Estudio descriptivo, prospectivo, de 42 meses de duración. El farmacéutico se integró en la actividad diaria del equipo multidisciplinar de una UCI de 12 camas perteneciente al Servicio de Anestesiología y Reanimación. Se registraron todas las intervenciones farmacoterapéuticas (IF) realizadas, el grado de aceptación, el método de comunicación y destinatario de la intervención, así como la evaluación clínica de las intervenciones aceptadas. Posteriormente, se realizó una encuesta al personal de la unidad sobre la seguridad del paciente y la influencia de la integración del farmacéutico en la unidad. Resultados: Se realizaron un total de 2399 IF con un 97,0% de aceptación. De estas, las mayoritarias fueron las relacionadas con la posología (37,8%) y las consultas al farmacéutico (25,7%). De las IF aceptadas, el 53,7% influyeron sobre la eficacia del tratamiento farmacológico y el 35,1% sobre la tolerancia. En la encuesta realizada al personal de la unidad para valorar la percepción de la integración del farmacéutico se obtuvo una valoración global de de 8,58 ± 1,40 sobre 10. Conclusiones: El farmacéutico hospitalario integrado en el equipo multidisciplinar de UCI puede aportar un valor añadido al proceso farmacoterapéutico del paciente crítico.Background and objective: The severity and instability of the patients, together with the high degree of complexity of the medication, make the intensive care units (ICU) a critical area of problems related to medication. The aim of our study was to analyze and assess the activity performed by the clinical pharmacist integrated in an ICU and to know the opinion of the staff about it. Material and method: A 42 month descriptive and prospective study was conducted. The pharmacist was integrated into the daily activity of the multidisciplinary team of a 12-bed ICU belonging to the Anaesthesiology and Resuscitation Department. Every pharmacotherapeutic intervention (PI) carried out, the degree of acceptance, the method of communication and the receiver of the intervention, as well as the clinical evaluation of the accepted interventions were recorded. Subsequently, a survey was carried out to the staff of the unit on the patient´s safety and the influence of the integration of the pharmacist in the unit. Results: A total of 2399 PIs were carried out with a 97.0% of acceptance. Of these, most were those related to posology (37.8%) and consultations with the pharmacist (25.7%). Among the accepted PIs, 53.7% had an influence on the efficacy of drug therapy, and 35.1% on treatment tolerance. In the survey to the unit´s staff in order to assess the perception of the pharmacist´s integration, an overall assessment of 8.58 ± 1.40 out of 10 was obtained. Conclusions: The hospital pharmacist integrated in the ICU multidisciplinary team can add value to the pharmacotherapeutic process of the critical patient

Equipo multidisciplinar de atención al paciente crítico: ¿qué aporta la integración del farmacéutico?

Background and objective: The severity and instability of the patients, together with the high degree of complexity of the medication, make the intensive care units (ICU) a critical area of ​​problems related to medication. The aim of our study was to analyze and assess the activity performed by the clinical pharmacist integrated in an ICU and to know the opinion of the staff about it. Material and method: A 42 month descriptive and prospective study was conducted. The pharmacist was integrated into the daily activity of the multidisciplinary team of a 12-bed ICU belonging to the Anaesthesiology and Resuscitation Department. Every pharmacotherapeutic intervention (PI) carried out, the degree of acceptance, the method of communication and the receiver of the intervention, as well as the clinical evaluation of the accepted interventions were recorded. Subsequently, a survey was carried out to the staff of the unit on the patient´s safety and the influence of the integration of the pharmacist in the unit. Results: A total of 2399 PIs were carried out with a 97.0% of acceptance. Of these, most were those related to posology (37.8%) and consultations with the pharmacist (25.7%). Among the accepted PIs, 53.7% had an influence on the efficacy of drug therapy, and 35.1% on treatment tolerance. In the survey to the unit´s staff in order to assess the perception of the pharmacist´s integration, an overall assessment of 8.58 ± 1.40 out of 10 was obtained. Conclusions: The hospital pharmacist integrated in the ICU multidisciplinary team can add value to the pharmacotherapeutic process of the critical patient.Antecedentes y objetivo: La gravedad e inestabilidad de los pacientes, junto con el alto grado de complejidad de la medicación, hacen de las unidades de cuidados intensivos (UCI) un área crítica de problemas relacionados con la medicación. El objetivo de nuestro estudio fue analizar y evaluar la actividad clínica realizada por el farmacéutico clínico integrado en una UCI y conocer la opinión del personal. Material y método: Estudio descriptivo, prospectivo, de 42 meses de duración. El farmacéutico se integró en la actividad diaria del equipo multidisciplinar de una UCI de 12 camas perteneciente al Servicio de Anestesiología y Reanimación. Se registraron todas las intervenciones farmacoterapéuticas (IF) realizadas, el grado de aceptación, el método de comunicación y destinatario de la intervención, así como la evaluación clínica de las intervenciones aceptadas. Posteriormente, se realizó una encuesta al personal de la unidad sobre la seguridad del paciente y la influencia de la integración del farmacéutico en la unidad. Resultados: Se realizaron un total de 2399 IF con un 97,0% de aceptación. De estas, las mayoritarias fueron las relacionadas con la posología (37,8%) y las consultas al farmacéutico (25,7%). De las IF aceptadas, el 53,7% influyeron sobre la eficacia del tratamiento farmacológico y el 35,1% sobre la tolerancia. En la encuesta realizada al personal de la unidad para valorar la percepción de la integración del farmacéutico se obtuvo una valoración global de de 8,58 ± 1,40 sobre 10. Conclusiones: El farmacéutico hospitalario integrado en el equipo multidisciplinar de UCI puede aportar un valor añadido al proceso farmacoterapéutico del paciente crítico

Guía del Trabajo Fin de Grado (TFG): Grado en Enfermería

La elaboración del TFG supone la aplicación de los conocimientos adquiridos durante los años académicos del grado cursado y, por tanto, debe reflejar la adquisición de competencias. El Grado de Enfermería oferta gran diversidad de temas para la elaboración del TFG, siendo el colectivo de profesorado implicado en su seguimiento y evaluación, numeroso y diverso. Con el objetivo de facilitar tanto su elaboración como su evaluación unificada a todos los estudiantes, se propone el diseño de un documento donde se recojan las diversas posibilidades y modelos de elaboración, así como la creación de un documento general de rúbrica donde se recojan todos los aspectos evaluables y su peso en la puntuación global. Con la finalidad de clarificar tanto los modelos de TFG como los criterios de evaluación, se ha elaborado un documento general de rúbrica donde se recogen los aspectos evaluables y su peso en la puntuación global. En su elaboración ha participado una red de profesorado y alumnado de la UA compuesto por ocho profesores, un representante del personal de administración y servicios y un estudiante del cuarto curso del grado en Enfermería. Está previsto que el documento elaborado sea único y válido tanto para el profesorado tutor como para el tribunal de evaluación. El documento provisional se divide en dos o tres bloques (para el tribunal o tutor, respectivamente) para conseguir un sistema de evaluación común y unificado para todos los departamentos participantes

Unraveling the effect of silent, intronic and missense mutations on VWF splicing : contribution of next generation sequencing in the study of mRNA

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. identifier:02869074

Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.This work was supported by: Spanish National Research Council (CSIC-202020E079, CSIC-COVID19-028); Madrid Regional Government “IMMUNOTHERCAN” [S2017/BMD-3733-2 (MVG)]; Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU, RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (EMGC); PI19/00549 (AA)]; “La Caixa Bank Foundation” (HR17-00016), Fondo Supera COVID (CRUE-Banco de Santander), both to FSM.Peer reviewe

Bead-assisted SARS-CoV-2 multi-antigen serological test allows effective identification of patients

Many new aspects of COVID-19 disease, including different clinical manifestations, have been identified during the pandemic. The wide array of symptoms and variation in disease severity after SARS-CoV-2 infection might be related to heterogeneity in the immune responses of different patients. Here we describe a new method for a simple multi-antigen serological test that generates a full picture of seroconversion in a single reaction. The assay is based on the detection by flow cytometry of multiple immunoglobulin classes (isotypes) specific for four SARS-CoV-2 antigens: the Spike glycoprotein (one of the highly immunogenic proteins), its RBD fragment (the major target for neutralising antibodies), the nucleocapsid protein and the main cysteine-like protease. Until now, most diagnostic serological tests measured antibodies to only one antigen and some patients seemed to not make any antibody response. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. Combining all the four antigens and using machine learning techniques, it was possible to clearly discriminate between patients and healthy controls with 100% confidence. Further, combination of antigens and different immunoglobulin isotypes in this multi-antigen assay improved the classification of patients with mild and severe disease. Introduction of this method will facilitate massive screenings of patients to evaluate their immune response. It could also support vaccination campaigns both to select non-immune individuals and to distinguish infected patients from vaccine responders.This work was supported by the Spanish National Research Council (CSIC, project numbers 202020E079 and CSIC-COVID19-028) and grants from Madrid Regional Government “IMMUNOTHERCAN” [S2017/BMD-3733-2 (MVG)]; the Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU): RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (JMRF); PI19/00549 (AA)]. The study was also funded by “La Caixa Banking Foundation” (HR17-00016 to FSM) and Fondo Supera COVID (CRUE-Banco de Santander) to FSM.N

COVID-19 outbreaks in a transmission control scenario: challenges posed by social and leisure activities, and for workers in vulnerable conditions, Spain, early summer 2020

Severe acute respiratory syndrome coronavirus 2 community-wide transmission declined in Spain by early May 2020, being replaced by outbreaks and sporadic cases. From mid-June to 2 August, excluding single household outbreaks, 673 outbreaks were notified nationally, 551 active (>6,200 cases) at the time. More than half of these outbreaks and cases coincided with: (i) social (family/friends’ gatherings or leisure venues) and (ii) occupational (mainly involving workers in vulnerable conditions) settings. Control measures were accordingly applied

Prodromal symptoms and the duration of untreated psychosis in first episode of psychosis patients: what differences are there between early vs. adult onset and between schizophrenia vs. bipolar disorder?

To assess the role of age (early onset psychosis-EOP < 18 years vs. adult onset psychosis-AOP) and diagnosis (schizophrenia spectrum disorders-SSD vs. bipolar disorders-BD) on the duration of untreated psychosis (DUP) and prodromal symptoms in a sample of patients with a first episode of psychosis. 331 patients with a first episode of psychosis (7–35 years old) were recruited and 174 (52.6%) diagnosed with SSD or BD at one-year follow-up through a multicenter longitudinal study. The Symptom Onset in Schizophrenia (SOS) inventory, the Positive and Negative Syndrome Scale and the structured clinical interviews for DSM-IV diagnoses were administered. Generalized linear models compared the main effects and group interaction. 273 AOP (25.2 ± 5.1 years; 66.5% male) and 58 EOP patients (15.5 ± 1.8 years; 70.7% male) were included. EOP patients had significantly more prodromal symptoms with a higher frequency of trouble with thinking, avolition and hallucinations than AOP patients, and significantly different median DUP (91 [33–177] vs. 58 [21–140] days; Z = − 2.006, p = 0.045). This was also significantly longer in SSD vs. BD patients (90 [31–155] vs. 30 [7–66] days; Z = − 2.916, p = 0.004) who, moreover had different profiles of prodromal symptoms. When assessing the interaction between age at onset (EOP/AOP) and type of diagnosis (SSD/BD), avolition was significantly higher (Wald statistic = 3.945; p = 0.047), in AOP patients with SSD compared to AOP BD patients (p = 0.004). Awareness of differences in length of DUP and prodromal symptoms in EOP vs. AOP and SSD vs. BD patients could help improve the early detection of psychosis among minors