HSF1: Primary Factor in Molecular Chaperone Expression and a Major Contributor to Cancer Morbidity

Heat shock factor 1 (HSF1) is the primary component for initiation of the powerful heat shock response (HSR) in eukaryotes. The HSR is an evolutionarily conserved mechanism for responding to proteotoxic stress and involves the rapid expression of heat shock protein (HSP) molecular chaperones that promote cell viability by facilitating proteostasis. HSF1 activity is amplified in many tumor contexts in a manner that resembles a chronic state of stress, characterized by high levels of HSP gene expression as well as HSF1-mediated non-HSP gene regulation. HSF1 and its gene targets are essential for tumorigenesis across several experimental tumor models, and facilitate metastatic and resistant properties within cancer cells. Recent studies have suggested the significant potential of HSF1 as a therapeutic target and have motivated research efforts to understand the mechanisms of HSF1 regulation and develop methods for pharmacological intervention. We review what is currently known regarding the contribution of HSF1 activity to cancer pathology, its regulation and expression across human cancers, and strategies to target HSF1 for cancer therapy.Fil: Prince, Thomas L.. Geisinger Clinic. Department of Molecular Functional Genomics; Estados UnidosFil: Lang, Benjamin J.. Harvard Medical School; Estados UnidosFil: Guerrero Gimenez, Martin Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Fernandez Muñoz, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ackerman, Andrew. Geisinger Clinic. Department of Molecular Functional Genomics; Estados UnidosFil: Calderwood, Stuart K.. Harvard Medical School; Estados Unido

Changes in prolactin receptor location in prostate tumors

Introduction: Prolactin (PRL) binds its receptor (PRLR) and stimulates cell proliferation, differentiation and survival in prostate cancer (PCa) cell lines via STAT5a, MAPK and AKT. Objective: To evaluate the expression of PRL and PRLR in normal and tumor prostate tissues with different Gleason patterns. Methods: Samples of normal, benign prostatic hyperplasia and PCa with different Gleason patterns were selected from radical prostatectomy. The intensity, location and percentage of stained cells for PRL and PRLR were evaluated by Immunohistochemistry. Co-localization was observed by confocal microscopy. Results: PRL was expressed diffusely and with a mild intensity in the cytoplasm of normal and tumor prostate luminal cells. Its expression only augmented in the Gleason 3 pattern (p 0.0001). The immunostaining intensity and the percentage of positive cells for PRLR did not vary between normal and tumor tissues. However, the location of the PRLR was modified by the tumorigenic process. In non-tumor tissues, PRLR expression was mostly in plasma membrane in the apical zone of epithelial cells. In tumor tissues, it was expressed in intracellular vesicles. The co-localization of PRL and PRLR was demonstrated in normal and tumor tissues suggesting that PRL could be acting in an autocrine and paracrine manner. Conclusion: PRL and its receptor were present in the cytoplasm of the epithelial cells of the normal and tumor prostate gland. In tumor tissues, the change in the location and appearance of cryptic PRLRs that store PRL may keep active the different signaling pathways related to cell proliferation and survival.Fil: López Fontana, Gastón. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentina. Clinica Andina de Urología; ArgentinaFil: Rey Echalecu, Lara Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Santiano, Flavia Eliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: López Fontana, José Rodrigo. Clinica Andina de Urología; ArgentinaFil: López Laur, José Daniel. Clinica Andina de Urología; ArgentinaFil: Zyla, Leila Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Valdemoros, Paula. Laboratorio de Anatomía Patológica Vega Valdemoros; ArgentinaFil: Guerrero Gimenez, Martin Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Fernandez Muñoz, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Gomez, Silvina Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Bruna, Flavia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Guglielmi, Juan Manuel. Clinica Andina de Urología; ArgentinaFil: Caron, Ruben Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: López Fontana, Constanza Matilde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentin

CD32 Expression is not Associated to HIV-DNA content in CD4 cell subsets of individuals with Different Levels of HIV Control

A recent study has pointed out to CD32a as a potential biomarker of HIV-persistent CD4 cells. We have characterized the level and phenotype of CD32+ cells contained in different subsets of CD4 T-cells and its potential correlation with level of total HIV-DNA in thirty HIV patients (10 typical progressors naive for cART, 10 cART-suppressed patients, and 10 elite controllers). Total HIV-DNA was quantified in different subsets of CD4 T-cells: Trm and pTfh cells. Level and immunephenotype of CD32+ cells were analyzed in these same subsets by flow cytometry. CD32 expression in Trm and pTfh subsets was similar in the different groups, and there was no significant correlation between the level of total HIV-DNA and the level of CD32 expression in these subsets. However, total HIV-DNA level was correlated with expression of CD127 (rho = -0.46, p = 0.043) and of CCR6 (rho = -0.418, p = 0.027) on CD32+ cells. Our results do not support CD32 as a biomarker of total HIV-DNA content. However, analyzing the expression of certain markers by CD32+ cells could improve the utility of this marker in the clinical setting, prompting the necessity of further studies to both validate our results and to explore the potential utility of certain markers expressed by CD32+ cells.We would like to thank all patients and healthy donors who participated in the study. This study has been funded by projects CP14/00198, PI16/01769, and RD16/0025/0013 integrated in the State Plan for Scientific and Technical Research and Innovation and co-funded by ISCIII-Sub-Directorate General for Research Assessment and Promotion and European Regional Development Fund (ERDF). N Rallon is a Miguel Servet investigator from the Spanish Carlos III Institute of Health (ISCIII), grant CP14/00198, Madrid, Spain. Maria Angeles Navarrete-Munoz was funded by RD16/0025/0013 and the Intramural Research Scholarship from IIS-FJD. Clara Restrepo was funded by project RD16/0025/0013. M Garcia is a predoctoral student co-funded by CP14/00198 project and the Intramural Research Scholarship from IIS-FJD.S

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

SPINK7 expression changes accompanied by HER2, P53 and RB1 can be relevant in predicting oral squamous cell carcinoma at a molecular level

The oral squamous cell carcinoma (OSCC), which has a high morbidity rate, affects patients worldwide. Changes in SPINK7 in precancerous lesions could promote oncogenesis. Our aim was to evaluate SPINK7 as a potential molecular biomarker which predicts OSCC stages, compared to: HER2, TP53, RB1, NFKB and CYP4B1. This study used oral biopsies from three patient groups: dysplasia (n = 33), less invasive (n = 28) and highly invasive OSCC (n = 18). The control group consisted of clinically suspicious cases later to be confirmed as normal mucosa (n = 20). Gene levels of SPINK7, P53, RB, NFKB and CYP4B1 were quantified by qPCR. SPINK7 levels were correlated with a cohort of 330 patients from the TCGA. Also, SPINK7, HER2, TP53, and RB1, were evaluated by immunohistofluorescence. One-way Kruskal–Wallis test and Dunn's post-hoc with a p < 0.05 significance was used to analyze data. In OSCC, the SPINK7 expression had down regulated while P53, RB, NFKB and CYP4B1 had up regulated (p < 0.001). SPINK7 had also diminished in TCGA patients (p = 2.10e-6). In less invasive OSCC, SPINK7 and HER2 proteins had decreased while TP53 and RB1 had increased with respect to the other groups (p < 0.05). The changes of SPINK7 accompanied by HER2, P53 and RB1 can be used to classify the molecular stage of OSCC lesions allowing a diagnosis at molecular and histopathological levels.Fil: Pennacchiotti, Graciela Laura. Universidad de Chile; ChileFil: Valdes Garrido, Fabio. Instituto Nacional del Cáncer; ChileFil: González Arriaga, Wilfredo Alejandro. Universidad de Valparaíso; ChileFil: Montes, Héctor Federico. Universidad Nacional de Cuyo. Facultad de Odontologia; ArgentinaFil: Parra, Judith Maria Roxana. Universidad Nacional de Cuyo. Facultad de Odontologia; ArgentinaFil: Guida, Valeria Andrea. Universidad Nacional de Cuyo. Facultad de Odontologia; ArgentinaFil: Gomez, Silvina Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Guerrero Gimenez, Martin Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Fernandez Muñoz, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Zoppino, Felipe Carlos Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Caron, Ruben Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Ezquer, Marcelo Eduardo. Universidad del Desarrollo; ChileFil: Ramires Fernández, Ricardo. Universidad Mayor; ChileFil: Bruna, Flavia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Odontologia; Argentin

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