Dimethyl fumarate-related immune and transcriptional signature is associated with clinical response in multiple sclerosis-treated patients.

Background and objective: Dimethyl fumarate (DMF) is an immunomodulatory drug approved for the therapy of multiple sclerosis (MS). The identification of response biomarkers to DMF is a necessity in the clinical practice. With this aim, we studied the immunophenotypic and transcriptomic changes produced by DMF in peripheral blood mononuclear cells (PBMCs) and its association with clinical response. Material and methods: PBMCs were obtained from 22 RRMS patients at baseline and 12 months of DMF treatment. Lymphocyte and monocyte subsets, and gene expression were assessed by flow cytometry and next-generation RNA sequencing, respectively. Clinical response was evaluated using the composite measure "no evidence of disease activity" NEDA-3 or "evidence of disease activity" EDA-3 at 2 years, classifying patients into responders (n=15) or non-responders (n=7), respectively. Results: In the whole cohort, DMF produced a decrease in effector (TEM) and central (TCM) memory T cells in both the CD4+ and CD8+ compartments, followed by an increase in CD4+ naïve T cells. Responder patients presented a greater decrease in TEM lymphocytes. In addition, responder patients showed an increase in NK cells and were resistant to the decrease in the intermediate monocytes shown by non-responders. Responder patients also presented differences in 3 subpopulations (NK bright, NK dim and CD8 TCM) at baseline and 4 subpopulations (intermediate monocytes, regulatory T cells, CD4 TCM and CD4 TEMRA) at 12 months. DMF induced a mild transcriptional effect, with only 328 differentially expressed genes (DEGs) after 12 months of treatment. The overall effect was a downregulation of pro-inflammatory genes, chemokines, and activators of the NF-kB pathway. At baseline, no DEGs were found between responders and non-responders. During DMF treatment a differential transcriptomic response was observed, with responders presenting a higher number of DEGs (902 genes) compared to non-responders (189 genes). Conclusions: Responder patients to DMF exhibit differences in monocyte and lymphocyte subpopulations and a distinguishable transcriptomic response compared to non-responders that should be further studied for the validation of biomarkers of treatment response to DMF.

Endocannabinoid levels in peripheral blood mononuclear cells of multiple sclerosis patients treated with dimethyl fumarate.

The endocannabinoid system (ECS), a signalling network with immunomodulatory properties, is a potential therapeutic target in multiple sclerosis (MS). Dimethyl fumarate (DMF) is an approved drug for MS whose mechanism of action has not been fully elucidated; the possibility exists that its therapeutic effects could imply the ECS. With the aim of studying if DMF can modulate the ECS, the endocannabinoids 2-arachidonoylglycerol (2-AG), anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) were determined by liquid chromatography-mass spectrometry in peripheral blood mononuclear cells from 21 healthy donors (HD) and 32 MS patients at baseline and after 12 and 24 months of DMF treatment. MS patients presented lower levels of 2-AG and PEA compared to HD. 2-AG increased at 24 months, reaching HD levels. AEA and PEA remained stable at 12 and 24 months. OEA increased at 12 months and returned to initial levels at 24 months. Patients who achieved no evidence of disease activity (NEDA3) presented the same modulation over time as EDA3 patients. PEA was modulated differentially between females and males. Our results show that the ECS is dysregulated in MS patients. The increase in 2-AG and OEA during DMF treatment suggests a possible role of DMF in ECS modulation.

Hyperthermic Intraperitoneal Chemotherapy and Recirculation with CO2: A Safe Technique.

INTRODUCTION: Hyperthermic IntraPEritoneal Chemotherapy (HIPEC) has evolved as a treatment for peritoneal carcinomatosis in various tumors after a careful and complete cytoreductive surgery, and it demonstrated much better and longer survival than more traditional therapeutic schemas. Our objective has been to examine the safety, efficacy and survival achieved with closed technique with CO2-agitation system Combat PRS® (Peritoneal Recirculation System: PRS). To achieve this, we compared the appearance of adverse events, mortality and survival with the described using classic techniques (open, closed without CO2-agitation) for the treatment of selected patients with peritoneal carcinomatosis; Materials and methods: We studied overall survival, disease-free survival and safety (morbidity and mortality) of the administration of HIPEC through a closed method technique with CO2 recirculation (Combat PRS®) in 482 patients from 11 Spanish hospitals; Results: The mortality of our technique (1.66%) was similar to other published techniques (open, closed). Morbidity exhibited a 9.96% rate of Clavien-Dindo (CD) III/IV complications in 482 patients, which was lower than in other series. Survival (overall survival (OS) and disease-free survival (DFS)) was similar to previously published results: 86% 1y-OS, 54% 3y-OS, 77% 1y-DFS and 31% 3y-DFS; Conclusion: The procedure with closed PRS with CO2 agitation is as safe as standard open and closed procedures for the administration of HIPEC after complete cytoreductive surgery, with similar and very low mortality (1.66%) and lower morbidity (9.96% CD III and IV in our series vs range of 20-40% in the majority of different series); only Kusamura had similar results, with 12% in 205 patients, using the closed technique without CO2 agitation).

Functional and Phylogenetic Characterization of Bacteria in Bovine Rumen Using Fractionation of Ruminal Fluid.

Cattle productivity depends on our ability to fully understand and manipulate the fermentation process of plant material that occurs in the bovine rumen, which ultimately leads to the improvement of animal health and increased productivity with a reduction in environmental impact. An essential step in this direction is the phylogenetic and functional characterization of the microbial species composing the ruminal microbiota. To address this challenge, we separated a ruminal fluid sample by size and density using a sucrose density gradient. We used the full sample and the smallest fraction (5%), allowing the enrichment of bacteria, to assemble metagenome-assembled genomes (MAGs). We obtained a total of 16 bacterial genomes, 15 of these enriched in the smallest fraction of the gradient. According to the recently proposed Genome Taxonomy Database (GTDB) taxonomy, these MAGs belong to Bacteroidota, Firmicutes_A, Firmicutes, Proteobacteria, and Spirochaetota phyla. Fifteen MAGs were novel at the species level and four at the genus level. The functional characterization of these MAGs suggests differences from what is currently known from the genomic potential of well-characterized members from this complex environment. Species of the phyla Bacteroidota and Spirochaetota show the potential for hydrolysis of complex polysaccharides in the plant cell wall and toward the production of B-complex vitamins and protein degradation in the rumen. Conversely, the MAGs belonging to Firmicutes and Alphaproteobacteria showed a reduction in several metabolic pathways; however, they have genes for lactate fermentation and the presence of hydrolases and esterases related to chitin degradation. Our results demonstrate that the separation of the rumen microbial community by size and density reduced the complexity of the ruminal fluid sample and enriched some poorly characterized ruminal bacteria allowing exploration of their genomic potential and their functional role in the rumen ecosystem.

Enfoque oncoplástico en el tratamiento quirúrgico conservador del cáncer de mama. Una revisión bibliográfica / Oncoplastic approach in the conservative surgical treatment of breast cancer. A bibliographic review

Introducción: El cáncer de mama es el más común en el mundo y constituye la principal causa de muerte en mujeres, no obstante, los avances terapéuticos han permitido aumentar sus tasas de supervivencia global, libre de enfermedad. Procurando una mejor calidad de vida surge la cirugía oncoplástica, que combina la cirugía plástica y reconstructiva en el ma-nejo quirúrgico del cáncer de mama localmente avanzado, de mayor tamaño o con ubicación desfavorable, buscando mejorar el resultado estético sin perjuicio del resultado oncológico. Objetivo: Realizar una revisión bibliográfica actualizada del enfoque oncoplástico en el tra-tamiento quirúrgico conservador del cáncer de mama, con el propósito de conocer sus re-sultados oncológicos y estéticos en comparación con los tratamientos quirúrgicos conven-cionales.Material y Métodos: La búsqueda bibliográfica se llevó a cabo en las bases de datos Pub-Med, Elsevier, Hinari, Cochrane y ScienceDirect. Seleccionando los artículos publicados du-rante los últimos 10 años, en inglés o español, que tuvieron relación con información relevan-te en el tratamiento quirúrgico oncoplástico conservador del cáncer de mama.Resultados: Los criterios de búsqueda incluyeron artículos sobre el tratamiento quirúrgico oncoplástico conservador en mujeres con cáncer de mama primario, 99 artículos cumplie-ron los criterios, excluyéndose aquellos publicados hace más de 10 años, con metodología poco clara o no reproducible. Fueron elegibles 35 por ser relevantes al tema, publicados en revistas indexadas entre los años 2011 y 2021, permitiendo determinar los resultados clíni-cos, oncológicos y estéticos de la cirugía oncoplástica conservadora y contrastarlos con las técnicas convencionales. Conclusión: La cirugía oncoplástica conservadora de mama es oncológicamente segura y equiparable a las cirugías convencionales (mastectomía total o parcial), ofreciendo resulta-dos estéticos más satisfactorios y mejorando la calidad de vida de las pacientes.

Background: Breast cancer is the most common cancer in the world and represents the main cause of death in women; nevertheless, therapeutic advances have made it possible to increase overall and disease-free survival rates. Looking for a better quality of life, on-coplastic surgery arises, which combines plastic and reconstructive surgery in the surgical management of locally advanced breast cancer, larger or with unfavorable location, seeking to improve the appearance without affecting the oncologic result.Objective: To perform an updated bibliographic review of the oncoplastic approach in the conservative surgical treatment of breast cancer, with the purpose of knowing its oncologic and esthetic results compared to conventional surgical treatments.Material and Methods: The bibliographic search was carried out in the databases PubMed, Elsevier, Hinari, Cochrane and ScienceDirect. Selecting the articles published during the last 10 years, in either English or Spanish, that were related to relevant information in the conser-vative oncoplastic surgical treatment of breast cancer.Results: The search criteria included articles on conservative oncoplastic surgical treatment in women with primary breast cancer, 99 articles met the criteria, excluding those published more than 10 years ago, with unclear or non-reproducible methodology. 35 were eligible, because they were relevant to the subject, published in indexed journals between 2011 and 2021, allowing to determine the clinical, oncological and aesthetic results of conservative oncoplastic surgery and contrast them with conventional techniques. Conclusion: Oncoplastic breast-conserving surgery is oncologically safe and comparable to conventional surgeries (total or partial mastectomy), offering more satisfactory esthetic results and improving the quality of life in patients.

Rotura espontánea de quiste hepático simple: éxito del tratamiento conservador / Spontaneous rupture of a simple liver cyst: success of a conservative approach

RESUMEN Los quistes hepáticos simples (QHS) son las lesiones hepáticas más frecuentes. Cursan habitualmente asintomáticas, aunque cuando alcanzan gran tamaño pueden causar síntomas. El objetivo del presente artículo es presentar una complicación excepcional de los QHS. Presentamos a una mujer de 61 años con historia de QHS múltiples que acude a Urgencias por dolor abdominal brusco sin antecedente traumático. Ante la sospecha de rotura quística espontánea, se realiza tomografía computarizada (TC) abdominal que confirma el diagnóstico. Se decide tratamiento conservador con buena evolución. Tras el episodio agudo es intervenida quirúrgicamente realizándose destechamiento de los quistes. a rotura de los QHS es una complicación excepcional que habitualmente cursa con dolor abdominal. Debido a su baja frecuencia no existe un tratamiento estándar. Se acepta que el tratamiento conservador es una buena opción en pacientes sin signos de peritonitis, mientras que la cirugía urgente está indicada en pacientes con abdomen agudo.

ABSTRACT Simple liver cysts (SLC) are the most common liver tumors. They are usually asymptomatic but large cysts may produce symptoms. The aim of this article is to report a rare complication of SLC. We report the case of a 61-year-old woman with a history of multiple SLCs who sought medical care due to sudden abdominal pain not associated with trauma. A probable diagnosis of spontaneous rupture was made, and the patient underwent computed tomography (CT) scan of the abdomen which confirmed the suspicion. Conservative treatment was decided, with favorable outcome. After the acute episode the patient underwent surgery and the cysts were unroofed. Rupture of SLC is a rare complication that usually presents with abdominal pain. There is no standard of care due to the low incidence of this complication. The conservative approach is a good option in patients without signs of peritonitis, while emergency surgery is indicated in patients with acute abdomen.

Sedentarismo laboral en distintos contextos ocupacionales en México: prevalencia y factores asociados / Sedentary work in different occupational contexts in Mexico: prevalence and associated factors

Resumen: Objetivo: Analizar la prevalencia de sedentarismo laboral en diferentes contextos ocupacionales y estimar los factores asociados de acuerdo con el sector de actividad económica. Material y métodos: Análisis secundario de datos de la Encuesta Nacional de Ocupación y Empleo (ENOE); se identificó la ocupación y se clasificó en cuatro categorías. La variable dependiente fue el sedentarismo laboral; las variables independientes fueron sexo, edad, escolaridad, ingreso, zona de residencia urbana del trabajador, formalidad laboral y región socioeconómica. Los factores asociados se estimaron mediante regresión logística múltiple por sector de actividad económica. Resultados: La mayor prevalencia del sedentarismo laboral se encontró en el sector de servicios (43%, IC95%: 42.3-43.6). Los factores asociados fueron nivel de estudios superior, mayores ingresos, ser mujer y trabajar en el sector formal. Conclusiones: La prevalencia de sedentarismo laboral es alta en sectores productivos estratégicos, por lo que es necesario considerar los factores de riesgo identificados en este trabajo para establecer estrategias de mitigación.

Abstract: Objective: To analyze the prevalence of sedentary work in different occupational contexts and to estimate the associated factors to economic activity sector. Materials and methods: Secondary analysis of the Occupation and Employment National Survey (ENOE, in Spanish) was conducted. Occupation was classified into four categories. Sedentary work was the dependent variable; the independent variables were sex, age, education, income, inhabitant of urban area, work formality and socioeconomic region. The associated factors were estimated using multiple logistic regression by economic activity sector. Results: The highest prevalence of sedentary work was found in services sector (43%, 95%CI= 42.3-43.6). The associated factors were college degree, higher income, being a woman and working in the formal sector. Conclusions: Sedentary work has a high prevalence in strategic productive sectors. It is necessary to consider the risk factors identified here to establish mitigation strategies.

Disentangling the Complexity of the Rumen Microbial Diversity Through Fractionation Using a Sucrose Density Gradient.

The ruminal microbial community is an important element in health, nutrition, livestock productivity, and climate impact. Despite the historic and current efforts to characterize this microbial diversity, many of its members remain unidentified, making it challenging to associate microbial groups with functions. Here we present a low-cost methodology for rumen sample treatment that separates the microbial community based on cell size, allowing for the identification of subtle compositional changes. In brief, the sample is centrifuged through a series of sucrose density gradients, and cells migrate to their corresponding density fraction. From each fraction, DNA is extracted and 16S rRNA gene amplicons are sequenced. We tested our methodology on four animals under two different conditions, fasting, and post-feeding. Each fraction was examined by confocal microscopy showing that the same sucrose fraction consistently separated similar cell-sized microorganisms independent of the animal or treatment. Microbial composition analysis using metabarcoding showed that our methodology detected low abundance bacterial families and population changes between fasting and post-feeding treatments that could not be observed by bulk DNA analysis. In conclusion, the sucrose-based method is a powerful low-cost approximation to untwine, enrich, and potentially isolate uncharacterized members of the ruminal microbiome.

Extracellular Vesicles in Cervical Cancer and HPV Infection.

Since their description, extracellular vesicles (EVs) have shown growing relevance in cancer progression. These cell structures contain and transfer molecules such as nucleic acids (including DNA and RNA), proteins, and lipids. Despite the rising information about EVs' relationship with cancer, there is still scarce evidence about their content and function in cervical cancer. Interestingly, the composition and purposes of some cellular molecules and the expression of oncogenic proteins packaged in EVs seem modified in HPV-infected cells; and, although only the E6 oncogenic protein has been detected in exosomes from HPV-positive cells, both E6/E7 oncogenes mRNA has been identified in EVs; however, their role still needs to be clarified. Given that EVs internalizing into adjacent or distant cells could modify their cellular behavior or promote cancer-associated events like apoptosis, proliferation, migration, or angiogenesis in receptor cells, their comprehensive study will reveal EV-associated mechanisms in cervical cancer. This review summarizes the current knowledge in composition and functions of cervical cancer and HPV Infection-derived EVs.

Unbiased Analysis of Temporal Changes in Immune Serum Markers in Acute COVID-19 Infection With Emphasis on Organ Failure, Anti-Viral Treatment, and Demographic Characteristics.

Identification of novel immune biomarkers to gauge the underlying pathology and severity of COVID-19 has been difficult due to the lack of longitudinal studies. Here, we analyzed serum collected upon COVID-19 admission (t1), 48 hours (t2), and seven days later (t3) using Olink proteomics and correlated to clinical, demographics, and therapeutic data. Older age positively correlated with decorin, pleiotrophin, and TNFRS21 but inversely correlated with chemokine (both C-C and C-X-C type) ligands, monocyte attractant proteins (MCP) and TNFRS14. The burden of pre-existing conditions was positively correlated with MCP-4, CAIX, TWEAK, TNFRS12A, and PD-L2 levels. Individuals with COVID-19 demonstrated increased expression of several chemokines, most notably from the C-C and C-X-C family, as well as MCP-1 and MCP-3 early in the course of the disease. Similarly, deceased individuals had elevated MCP-1 and MCP-3 as well as Gal-9 serum levels. LAMP3, GZMB, and LAG3 at admission correlated with mortality. Only CX3CL13 and MCP-4 correlated positively with APACHE score and length of stay, while decorin, MUC-16 and TNFRSF21 with being admitted to the ICU. We also identified several organ-failure-specific immunological markers, including those for respiratory (IL-18, IL-15, Gal-9) or kidney failure (CD28, VEGF). Treatment with hydroxychloroquine, remdesivir, convalescent plasma, and steroids had a very limited effect on the serum variation of biomarkers. Our study identified several potential targets related to COVID-19 heterogeneity (MCP-1, MCP-3, MCP-4, TNFR superfamily members, and programmed death-ligand), suggesting a potential role of these molecules in the pathology of COVID-19.

Sedentarismo laboral en distintos contextos ocupacionales en México: prevalencia y factores asociados.

Objetivo. Analizar la prevalencia de sedentarismo laboral en diferentes contextos ocupacionales y estimar los factores asociados de acuerdo con el sector de actividad económica. Material y métodos. Análisis secundario de datos de la Encuesta Nacional de Ocupación y Empleo (ENOE); se identificó la ocupación y se clasificó en cuatro categorías. La variable dependiente fue el sedentarismo laboral; las variables independientes fueron sexo, edad, escolaridad, ingreso, zona de residencia urbana del trabajador, formalidad laboral y región socioeconómica. Los factores asociados se estimaron mediante regresión logística múltiple por sector de actividad económica. Resultados. La mayor prevalencia del seden-tarismo laboral se encontró en el sector de servicios (43%, IC95%: 42.3-43.6). Los factores asociados fueron nivel de estudios superior, mayores ingresos, ser mujer y trabajar en el sector formal. Conclusiones. La prevalencia de seden-tarismo laboral es alta en sectores productivos estratégicos, por lo que es necesario considerar los factores de riesgo identificados en este trabajo para establecer estrategias de mitigación.

Combination of Tocilizumab and Steroids to Improve Mortality in Patients with Severe COVID-19 Infection: A Spanish, Multicenter, Cohort Study.

BACKGROUND: We aimed to determine the impact of tocilizumab use on severe COVID-19 (coronavirus disease 19) pneumonia mortality. METHODS: We performed a multicentre retrospective cohort study in 18 tertiary hospitals in Spain from March to April 2020. Consecutive patients admitted with severe COVID-19 treated with tocilizumab were compared to patients not treated with tocilizumab, adjusting by inverse probability of the treatment weights (IPTW). Tocilizumab's effect in patients receiving steroids during the 48 h following inclusion was analysed. RESULTS: During the study period, 506 patients with severe COVID-19 fulfilled the inclusion criteria. Among them, 268 were treated with tocilizumab and 238 patients were not. Median time to tocilizumab treatment from onset of symptoms was 11 days [interquartile range (IQR) 8-14]. Global mortality was 23.7%. Mortality was lower in patients treated with tocilizumab than in controls: 16.8% versus 31.5%, hazard ratio (HR) 0.514 [95% confidence interval (95% CI) 0.355-0.744], p < 0.001; weighted HR 0.741 (95% CI 0.619-0.887), p = 0.001. Tocilizumab treatment reduced mortality by 14.7% relative to no tocilizumab treatment [relative risk reduction (RRR) 46.7%]. We calculated a number necessary to treat of 7. Among patients treated with steroids, mortality was lower in those treated with tocilizumab than in those treated with steroids alone [10.9% versus 40.2%, HR 0.511 (95% CI 0.352-0.741), p = 0.036; weighted HR 0.6 (95% CI 0.449-0.804), p < 0.001] (interaction p = 0.094). CONCLUSIONS: These results show that survival of patients with severe COVID-19 is higher in those treated with tocilizumab than in those not treated and that tocilizumab's effect adds to that of steroids administered to non-intubated patients with COVID-19 during the first 48 h of presenting with respiratory failure despite oxygen therapy. Randomised controlled studies are needed to confirm these results. TRIAL REGISTRATION: European Union electronic Register of Post-Authorization Studies (EU PAS Register) identifier, EUPAS34415.

Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness.

The Type VI secretion system (T6SS) is a protein translocation nanomachine widespread among Gram-negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. These effectors have a wide variety of functions within target cells that ultimately help the secreting cell gain a competitive fitness advantage. Here, we discuss the different ways in which these effectors can be delivered by the T6SS and the diverse mechanisms by which they exert their noxious action upon recipient cells. We also highlight the existence of roles for T6SS effectors beyond simply the killing of neighbouring cells.

Presence of HPV DNA in extracellular vesicles from HeLa cells and cervical samples / Presencia de ADN de VPH en vesículas extracelulares de células HeLa y muestras cervicales

INTRODUCTION: The main cause of cervical cancer is an infection of keratinocytes in the basal layer of the stratified epithelium of the cervix by human papillomavirus (HPV). Other than in cervical samples, HPV DNA has been found in serum and other fluids but its origin is unclear. Extracellular vesicles (EV) could be a conveyance of viral DNA given their emerging role in cellular communication. The content of EV derived from cervical cells has not been properly explored and it is not known whether or not they contain HPV DNA. METHODS: We evaluated the DNA content of exosomes purified from cultures of HeLa cells by Next Generation Sequencing (NGS) and confirmed its presence by PCR. The presence of HPV DNA was also evaluated by PCR and NGS in EV from HPV-positive cervical samples without apparent lesion or with LSIL. RESULTS: We detected the integrated form of viral-DNA in exosomes from HeLa cells by NGS and confirmed its presence by PCR. The search for HPV sequences in EV obtained from cervical exudate samples without apparent lesion or with LSIL, where we expected to find the viral genome as an episome, indicated that HPV DNA, including the E6 and E7 oncogenes, is present in these EV. CONCLUSIÓN: HPV DNA, including the viral oncogenes E6/E7, is found in exosomes regardless of the integration status of the virus in the infected cell

INTRODUCCIÓN: La principal causa del cáncer de cérvix es la infección de los queratinocitos de la capa basal del epitelio estratificado del cuello uterino por el virus del papiloma humano (VPH). El ADN del VPH se ha encontrado en muestras cervicales, pero también en suero y otros fluidos, aunque su origen en estos últimos no está claro. Las vesículas extracelulares (VE) podrían ser el medio de transporte del ADN viral considerando su papel emergente en la comunicación celular. El contenido de las VE derivadas de células cervicales ha sido poco explorado y la presencia en ellas de ADN de VPH sigue siendo desconocida. MÉTODOS: Evaluamos el ADN de exosomas purificados a partir de cultivos de células HeLa mediante secuenciación de nueva generación (NGS) y confirmamos su presencia a través de PCR. La presencia de ADN de VPH también se evaluó mediante PCR y NGS en VE de muestras cervicales positivas a VPH, sin lesión aparente o con LSIL. RESULTADOS: Detectamos la forma integrada del ADN viral en exosomas de células HeLa mediante NGS, y confirmamos su presencia a través de PCR. La búsqueda de secuencias de VPH en VE obtenidas a partir de muestras de exudado cervical sin lesión aparente o con LSIL, donde esperamos encontrar el genoma viral en forma episomal, indicó que el DNA de VPH incluyendo los oncogenes E6 y E7, está presente en estas VE. CONCLUSIÓN: El ADN del VPH incluyendo el correspondiente con los oncogenes virales E6/E7 se encuentra en exosomas independientemente del estado de integración del virus en la célula infectada

Presence of HPV DNA in extracellular vesicles from HeLa cells and cervical samples.

INTRODUCTION: The main cause of cervical cancer is an infection of keratinocytes in the basal layer of the stratified epithelium of the cervix by human papillomavirus (HPV). Other than in cervical samples, HPV DNA has been found in serum and other fluids but its origin is unclear. Extracellular vesicles (EV) could be a conveyance of viral DNA given their emerging role in cellular communication. The content of EV derived from cervical cells has not been properly explored and it is not known whether or not they contain HPV DNA. METHODS: We evaluated the DNA content of exosomes purified from cultures of HeLa cells by Next Generation Sequencing (NGS) and confirmed its presence by PCR. The presence of HPV DNA was also evaluated by PCR and NGS in EV from HPV-positive cervical samples without apparent lesion or with LSIL. RESULTS: We detected the integrated form of viral-DNA in exosomes from HeLa cells by NGS and confirmed its presence by PCR. The search for HPV sequences in EV obtained from cervical exudate samples without apparent lesion or with LSIL, where we expected to find the viral genome as an episome, indicated that HPV DNA, including the E6 and E7 oncogenes, is present in these EV. CONCLUSION: HPV DNA, including the viral oncogenes E6/E7, is found in exosomes regardless of the integration status of the virus in the infected cell.

NKG2D Polymorphism in Melanoma Patients from Southeastern Spain.

BACKGROUND: Natural killer (NK) and CD8+ T cells are involved in the immune response against melanoma. C-Type lectin-like NK cell receptors are located in the Natural Killer Complex (NKC) region 12p13.2-p12.3 and play a critical role in regulating the activity of NK and CD8+ T cells. An association between polymorphisms in the NKC region, including the NKG2D gene and NKG2A promoter, and the risk of cancer has been previously described. The aim of this study was to analyze the association of polymorphisms in the NKC region with cutaneous melanoma in patients from southeastern Spain. METHODS: Seven single-nucleotide polymorphisms (SNPs) in the NKG2D gene (NKC3,4,7,9,10,11,12), and one SNP in the NKG2A promoter (NKC17) were genotyped by a TaqMan 5' Nuclease Assay in 233 melanoma patients and 200 matched healthy controls. RESULTS: A linkage disequilibrium analysis of the SNPs performed in the NKC region revealed two blocks of haplotypes (Hb-1 and Hb-2) with 14 and seven different haplotype subtypes, respectively. The third most frequent haplotype from the block Hb-2-NK3 (CAT haplotype)-was significantly more frequent on melanoma patients than on healthy controls (p = 0.00009, Pc = 0.0006). No further associations were found when NKC SNPs were considered independently. CONCLUSIONS: Our results suggest an association between NKG2D polymorphisms and the risk of cutaneous malignant melanoma.

Dens invaginatus: reporte de un caso clínico / Dens invaginatus: case report

RESUMEN Introducción: De las malformaciones dentales una de las más frecuentes es el dens in vaginatus o dens in dente, esta alteración anatómica representa un reto para el clínico, debido a su anatomía compleja; sin embargo, gracias a los auxiliares de diagnóstico como la tomografía cone-beam, podremos planear el tratamiento para ofrecer el mejor pronóstico. El presente artículo relata un caso clínico de dens in dente presente en diente veintiuno, con necrosis pulpar y periodontitis apical asintomática. Se solicitó TAC cone-beam, se diagnosticó como tipo II según Oehlers; es instrumentado, irrigado con ultrasonido y obturado con técnica «onda continua de calor¼, con un éxito radiográfi co a seis meses de evolución. Actualmente, gracias a los métodos de diagnóstico y auxiliares de tratamiento tales como el ultrasonido, los sistemas de inyección de gutapercha, se pueden ofrecer tratamientos más efi cientes en la terapéutica endodóncica.

ABSTRACT Introduction: The Dens Invaginatus or Dens in dent it is the most common dental abnormal structure, this anatomic variation means a true clinic challenge because of its complex anatomy, however , thanks to the auxiliary tools for diagnosis just like Cone Beam Computerized Tomography, we can plan a good treatment to offer the best diagnosis. This article describes a clinic case about Dens in Dent in twenty one tooth with pulp necrosis and asymptomatic apical periodontitis. Cone Beam Computerized Tomography was requested, the image helped to classify the kind of Dens in Dent which is type II according to Oehlers´s classifi cation. It is instrumented, irrigated with ultrasound and sealed with "continuous wave of heat "technique. This technique ensures a radiographic success with six months of evolution. Currently, thanks to diagnostic methods and treatment aids such as ultrasound, gutta-percha injection system more effi cient treatments can be offered in endodontic therapeutics.

Immunophenotype and Transcriptome Profile of Patients With Multiple Sclerosis Treated With Fingolimod: Setting Up a Model for Prediction of Response in a 2-Year Translational Study.

BACKGROUND: Fingolimod is a functional sphingosine-1-phosphate antagonist approved for the treatment of multiple sclerosis (MS). Fingolimod affects lymphocyte subpopulations and regulates gene expression in the lymphocyte transcriptome. Translational studies are necessary to identify cellular and molecular biomarkers that might be used to predict the clinical response to the drug. In MS patients, we aimed to clarify the differential effects of fingolimod on T, B, and natural killer (NK) cell subsets and to identify differentially expressed genes in responders and non-responders (NRs) to treatment. MATERIALS AND METHODS: Samples were obtained from relapsing-remitting multiple sclerosis patients before and 6 months after starting fingolimod. Forty-eight lymphocyte subpopulations were measured by flow cytometry based on surface and intracellular marker analysis. Transcriptome sequencing by next-generation technologies was used to define the gene expression profiling in lymphocytes at the same time points. NEDA-3 (no evidence of disease activity) and NEDA-4 scores were measured for all patients at 1 and 2 years after beginning fingolimod treatment to investigate an association with cellular and molecular characteristics. RESULTS: Fingolimod affects practically all lymphocyte subpopulations and exerts a strong effect on genetic transcription switching toward an anti-inflammatory and antioxidant response. Fingolimod induces a differential effect in lymphocyte subpopulations after 6 months of treatment in responder and NR patients. Patients who achieved a good response to the drug compared to NR patients exhibited higher percentages of NK bright cells and plasmablasts, higher levels of FOXP3, glucose phosphate isomerase, lower levels of FCRL1, and lower Expanded Disability Status Scale at baseline. The combination of these possible markers enabled us to build a probabilistic linear model to predict the clinical response to fingolimod. CONCLUSION: MS patients responsive to fingolimod exhibit a recognizable distribution of lymphocyte subpopulations and a different pretreatment gene expression signature that might be useful as a biomarker.

Description of the new HLA-A*11:288 allele found in a healthy individual from the Canary Islands.

A new HLA-A allele, A*11:288, was characterized in a Spanish individual from the Canary Islands.

Mechanisms of action of cannabidiol in adoptively transferred experimental autoimmune encephalomyelitis.

Cannabidiol (CBD) is one of the most important compounds in Cannabis sativa, lacks psychotropic effects, and possesses a high number of therapeutic properties including the amelioration of experimental autoimmune encephalomyelitis (EAE). The aim of this study was to analyse the relative efficacy of CBD in adoptively transferred EAE (at-EAE), a model that allows better delineation of the effector phase of EAE. Splenocytes and lymph nodes from mice with actively induced EAE were cultured in the presence of MOG35-55 and IL-12 and inoculated intraperitoneally in recipient female C57BL/6J mice. The effects of CBD were evaluated using clinical scores and magnetic resonance imaging (MRI). In the central nervous system, the extent of cell infiltration, axonal damage, demyelination, microglial activation and cannabinoid receptors expression was assessed by immunohistochemistry. Lymph cell viability, apoptosis, oxidative stress and IL-6 production were measured in vitro. Preventive intraperitoneal treatment with CBD ameliorated the clinical signs of at-EAE, and this improvement was accompanied by a reduction of the apparent diffusion coefficient in the subiculum area of the brain. Inflammatory infiltration, axonal damage, and demyelination were reduced, and cannabinoid receptor expression was modulated. Incubation with CBD decreased encephalitogenic cell viability, increasing early apoptosis and reactive oxygen species (ROS) and decreasing IL-6 production. The reduction in viability was not mediated by CB1, CB2 or GPR55 receptors. CBD markedly improved the clinical signs of at-EAE and reduced infiltration, demyelination and axonal damage. The CBD-mediated decrease in the viability of encephalitogenic cells involves ROS generation, apoptosis and a decrease in IL-6 production and may contribute to the therapeutic effect of this compound.