Experiencia en el cuadro clínico, diagnóstico y tratamiento de COVID-19 / Experience in the clinical picture, diagnosis and treatment of COVID-19 / Experiência no quadro clínico, diagnóstico e tratamento da COVID-19

Resumen: Introducción: La infección por SARS-CoV-2 en Wuhan, China, ocasionó una pandemia de tal magnitud que ha provocado la muerte por neumonía a causa de enfermedad infecciosa por coronavirus 19 (COVID-19) de millones de personas. Nos dimos a la tarea de recolectar todas las características de los pacientes que estuvieron hospitalizados por esta enfermedad en nuestra UCI adultos. Material y métodos: Se realizó un estudio de tipo analítico, descriptivo, observacional y retrospectivo en pacientes con diagnóstico de COVID-19 ingresados en la Unidad de Cuidados Intensivos (UCI) del Hospital Ángeles Clínica Londres en la Ciudad de México, evaluados en el periodo del 23 de marzo de 2020 al 10 de mayo de 2020. Se revisaron los expedientes y se tomaron los datos de los mismos, se describieron variables de tipo demográfico, factores de riesgo, signos y síntomas, tratamiento médico y atención respiratoria. Se revisaron escalas de mortalidad SAPS III, APACHE II, SOFA y CALL-score. Se formaron dos grupos con y sin mortalidad realizándose análisis bivariado y multivariado de las variables significativas. El análisis estadístico se efectuó con el programa SPSS 25. Resultados: En el periodo considerado, 25 expedientes cumplieron con los criterios de inclusión, de ellos la demografía y factores de riesgo, 18 (72%) correspondieron a hombres y siete (38%) a mujeres con una mortalidad de 10 (40%). Los factores de riesgo más frecuentes fueron diabetes mellitus (DM) en siete (38%) pacientes, hipertensión arterial (HAS) en seis (24%), obesidad en cuatro (16%), enfermedad pulmonar obstructiva crónica (EPOC) en uno (4%), tabaquismo en 11 (44%) y alcoholismo en siete (28%). Se encontraron diferencias estadísticamente significativas en los grupos sin mortalidad y con mortalidad, 15 y 10 pacientes, respectivamente, observando las siguientes significancias: glucosa 105 mg/dL (percentil [PE 88]) versus 171 mg/dL (PE 125) p = 0.012, urea 33 mg/dL (PE 22) versus 95 mg/dL (PE 57) p = 0.03, BUN 15.3 mg/dL (PE 11) versus 44.2 mg/dL (PE 26.28) p = 0.04, TGO 32 U/L (PE 24.4) versus 58 U/L (PE 43.8) p = 0.010, DHL 239 U/L (PE 198) 454 U/L (PE 260) p = 0.003, triglicéridos 148 mg/dL (PE 120) versus 187.5 mg/dL (PE 165) p = 0.002, CPK 70 U/L (PE 35) versus 81 U/L (PE 78.25) p = 0.003, ferritina 446 mg/L (PE 238) versus 1,030 mg/L (PE 665) p = 0.007. Se realizó un análisis bivariado con regresión logística binaria, con la variable mortalidad dicotómica, no resultando significativa con esta prueba. Conclusiones: Se entiende que ninguna variable es predominantemente importante para explicar la mortalidad y que se recurre a muchos factores que se conjugan para explicar este desenlace, uno de éstos es la severidad misma del problema respiratorio en que se encuentre el paciente.

Abstract: Introduction: The SARS-CoV-2 infection in Wuhan, China caused a pandemic of such magnitude that it has caused the death of millions of people from pneumonia due to infectious disease caused by coronavirus 19 (COVID-19). We took on the task of collecting all the characteristics of the patients who were hospitalized for this disease in our Adult Intensive Care Unit. Material and methods: An analytical, descriptive, observational and retrospective study was carried out in patients with a diagnosis of COVID-19 admitted to the Intensive Care Unit (ICU) of the Hospital Ángeles Clínica Londres in Mexico City, evaluated in the period of March 23 from 2020 to May 10, 2020. The files were reviewed and the data taken from them, demographic variables, risk factors, signs and symptoms, medical treatment and respiratory care were described. SAPS III, APACHE II, SOFA and CALL-score mortality scales were reviewed. Two groups were formed with and without mortality, performing bivariate and multivariate analyzes of the significant variables. Statistical analysis was performed with the SPSS 25 program. Results: In the period considered, 25 files met the inclusion criteria for them: demographics and risk factors were 18 (72%) corresponding to men and seven (38%) to women. With a mortality of 10 (40%). The most frequent risk factors are diabetes mellitus (DM) in seven (38%), arterial hypertension (SAH) six (24%), obesity four (16%), chronic obstructive pulmonary disease (COPD) one (4%), smoking 11 (44%) and alcoholism seven (28%). Statistically significant differences were found in the groups without mortality and with mortality 15 and 10 patients respectively, observing the following significance: glucose 105 mg/dL (percentil [PE] 88) versus 171 mg/dL (PE 125) p = 0.012, urea 33 mg/dL (PE 22) versus 95 mg/dL (PE 57) p = 0.03, BUN 15.3 mg/dL (PE 11) versus 44.2 mg/dL (PE 26.28) p = 0.04, TGO 32 U/L (PE 24.4) versus 58 U/L (PE 43.8) p = 0.010, DHL 239 U/L (PE 198) 454 U/L (PE 260) p = 0.003, triglycerides 148 mg/dL (PE 120) versus 187.5 mg/dL (PE 165) p = 0.002, CPK 70 U/L (PE 35) versus 81 U/L (PE 78.25) p = 0.003, ferritin 446 mg/L (PE 238) versus 1,030 mg/L (PE 665) p = 0.007. A bivariate analysis with binary logistic regression was performed, with the dichotomous mortality variable, not resulting in this significant test. Conclusions: It is understood that no variable is predominantly important to explain mortality and that many factors are involved that are combined to explain this outcome, one of these being the same severity of the respiratory problem in which the patient is.

Resumo: Introdução: A infecção por SARS-CoV-2 em Wuhan China causou uma pandemia de tal magnitude que causou a morte de milhões de pessoas por pneumonia devido a doença infecciosa causada pelo coronavírus 19 (COVID-19). Assumimos a tarefa de coletar todas as características dos pacientes internados por essa doença em nossa unidade de terapia intensiva adulto. Material e métodos: Realizou-se um estudo analítico, descritivo, observacional e retrospectivo em pacientes com diagnóstico de COVID-19 internados na Unidade de Terapia Intensiva (UTI) do Hospital Ángeles Clínica Londres na Cidade do México, validado para o período de 23 de março de 2020 a 10 de maio de 2020. Os prontuários médicos foram revisados e seus dados coletados, as variáveis do tipo demográficas foram descritas, fatores de risco, sinais e sintomas, tratamento médico e cuidados respiratórios. Foram revisadas as escalas de mortalidade SAPS III, APACHE II, SOFA e CALL-score. Dois grupos foram formados com e sem mortalidade, realizando análises bivariadas e multivariadas das variáveis significativas. A análise estatística foi realizada com o programa SPSS 25. Resultados: No período considerado, 25 prontuários atenderam aos critérios de inclusão para os mesmos: dados demográficos e fatores de risco foram 18 (72%) correspondentes a homens e 7 (38%) a mulheres. Com mortalidade de 10 (40%). Os fatores de risco mais frequentes são diabetes mellitus (DM) em 7 (38%), hipertensão arterial (HAS) 6 (24%), obesidade 4 (16%), doença pulmonar obstrutiva crônica (DPOC) 1 (4%), tabagismo 11 (44%) e alcoolismo 7 (28%). Encontrou-se diferenças estatisticamente significativas nos grupos sem mortalidade e com mortalidade de 15 e 10 pacientes respectivamente, observando a seguinte significância: glicose 105 mg/dL (percentil [PE] 88) versus 171 mg/dL (PE 125) p = 0.012, uréia 33 mg/L (PE 22) versus 95 mg/L (PE 57) p = 0.03, BUN 15.3 mg/L (PE 11) versus 44.2 mg/L (PE 26.28) p = 0.04, TGO 32 U/L (PE 24.4) versus 58 U/L (PE 43.8) p = 0.010, DHL 239 U/L (PE 198) 454 (PE 260) p = 0.003, triglicerídeos 148 mg/dL (PE 120) versus 187.5 mg/dL (PE 165) p = 0.002, CPK 70 U/L (PE 35) versus 81 U/L (PE 78.25) p = 0.003, ferritina 446 mg/L (PE 238) versus 1030 mg/L (PE 665) p = 0.007. Realizou-se análise bivariada com regressão logística binária, com a variável mortalidade dicotômica, não resultando em teste significativo. Conclusões: Entende-se que nenhuma variável é predominantemente importante para explicar a mortalidade e que usamos muitos fatores que se conjugam para explicar esse desfecho, sendo um deles a mesma gravidade do problema respiratório em que o paciente se encontra.

Contractility of Induced Pluripotent Stem Cell-Cardiomyocytes With an MYH6 Head Domain Variant Associated With Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease; however, its etiology remains largely unknown. We previously demonstrated that genetic variants in the MYH6 gene are significantly associated with HLHS. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from an HLHS-affected family trio (affected parent, unaffected parent, affected proband) carrying an MYH6-R443P head domain variant demonstrated dysmorphic sarcomere structure and increased compensatory MYH7 expression. Analysis of iPSC-CMs derived from the HLHS trio revealed that only beta myosin heavy chain expression was observed in CMs carrying the MYH6-R443P variant after differentiation day 15 (D15). Functional assessments performed between D20-D23 revealed that MYH6-R443P variant CMs contracted more slowly (40 ± 2 vs. 47 ± 2 contractions/min, P < 0.05), shortened less (5.6 ± 0.5 vs. 8.1 ± 0.7% of cell length, P < 0.05), and exhibited slower shortening rates (19.9 ± 1.7 vs. 28.1 ± 2.5 µm/s, P < 0.05) and relaxation rates (11.0 ± 0.9 vs. 19.7 ± 2.0 µm/s, P < 0.05). Treatment with isoproterenol had no effect on iPSC-CM mechanics. Using CRISPR/Cas9 gene editing technology, introduction of the R443P variant into the unaffected parent's iPSCs recapitulated the phenotype of the proband's iPSC-CMs, and conversely, correction of the R443P variant in the proband's iPSCs rescued the cardiomyogenic differentiation, sarcomere organization, slower contraction (P < 0.05) and decreased velocity phenotypes (P < 0.0001). This is the first report to identify that cardiac tissues from HLHS patients with MYH6 variants can exhibit sarcomere disorganization in atrial but not ventricular tissues. This new discovery was not unexpected, since MYH6 is expressed predominantly in the postnatal atria in humans. These findings demonstrate the feasibility of employing patient-derived iPSC-CMs, in combination with patient cardiac tissues, to gain mechanistic insight into how genetic variants can lead to HLHS. Results from this study suggest that decreased contractility of CMs due to sarcomere disorganization in the atria may effect hemodynamic changes preventing development of a normal left ventricle.

Investigation of a dilated cardiomyopathy-associated variant in BAG3 using genome-edited iPSC-derived cardiomyocytes.

Mutations in B cell lymphoma 2-associated athanogene 3 (BAG3) are recurrently associated with dilated cardiomyopathy (DCM) and muscular dystrophy. Using isogenic genome-edited human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we examined how a DCM-causing BAG3 mutation (R477H), as well as complete loss of BAG3 (KO), impacts myofibrillar organization and chaperone networks. Although unchanged at baseline, fiber length and alignment declined markedly in R477H and KO iPSC-CMs following proteasome inhibition. RNA sequencing revealed extensive baseline changes in chaperone- and stress response protein-encoding genes, and protein levels of key BAG3 binding partners were perturbed. Molecular dynamics simulations of the BAG3-HSC70 complex predicted a partial disengagement by the R477H mutation. In line with this, BAG3-R477H bound less HSC70 than BAG3-WT in coimmunoprecipitation assays. Finally, myofibrillar disarray triggered by proteasome inhibition in R477H cells was mitigated by overexpression of the stress response protein heat shock factor 1 (HSF1). These studies reveal the importance of BAG3 in coordinating protein quality control subsystem usage within the cardiomyocyte and suggest that augmenting HSF1 activity might be beneficial as a means to mitigate proteostatic stress in the context of BAG3-associated DCM.

Mitochondrial Respiratory Capacity is Restored in Hibernating Cardiomyocytes Following Co-Culture with Mesenchymal Stem Cells.

Hibernating myocardium is a subset of ischemic cardiac disease characterized by viable but dysfunctional tissue. Standard treatment for hibernating myocardium is coronary artery bypass graft, which reduces arrhythmias and improves survival but does not fully restore function, presenting a gap in currently available treatments. Large animal studies of hibernating myocardium have identified impaired mitochondrial dynamics as a root cause of persistent cardiac dysfunction despite surgical revascularization. This study presents a novel in vitro model of hibernating myocardium cardiomyocytes to study active mitochondrial respiration in hibernating myocardium cells, and to test the paracrine effect of mesenchymal stem cells on impaired mitochondrial function. Exposure of cardiomyocytes to hypoxic conditions of 1% oxygen for 24 hours resulted in a phenotype consistent with hibernating myocardium cardiac tissue, including decreased respiratory capacity under high work states, decreased expression of mitochondrial proteins, and preserved cellular viability. Co-culture of hibernating myocardium cardiomyocytes with mesenchymal stem cells restored mitochondrial respiratory function, potentially via an increase in proliferator-activated receptor gamma coactivator 1-alpha-driven mitochondrial biogenesis. Co-culture treatment of hibernating myocardium cardiomyocytes with mesenchymal stem cells shows improvement in both mitochondrial function and ATP production, both of which are critical for effectively functioning cardiac tissue. These results suggest that mesenchymal stem cell therapy as an adjunct treatment to revascularization may address the current gap in treatment for hibernating myocardium patients.

The Role of Cellular Proliferation in Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

Mitotic clonal expansion has been suggested as a prerequisite for adipogenesis in murine preadipocytes, but the precise role of cell proliferation during human adipogenesis is unclear. Using adipose tissue-derived human mesenchymal stem cells as an in vitro cell model for adipogenic study, a group of cell cycle regulators, including Cdk1 and CCND1, were found to be downregulated as early as 24 h after adipogenic initiation and consistently, cell proliferation activity was restricted to the first 48 h of adipogenic induction. Cell proliferation was either further inhibited using siRNAs targeting cell cycle genes or enhanced by supplementing exogenous growth factor, basic fibroblast growth factor (bFGF), at specific time intervals during adipogenesis. Expression knockdown of Cdk1 at the initiation of adipogenic induction resulted in significantly increased adipocytes, even though total number of cells was significantly reduced compared to siControl-treated cells. bFGF stimulated proliferation throughout adipogenic differentiation, but exerted differential effect on adipogenic outcome at different phases, promoting adipogenesis during mitotic phase (first 48 h), but significantly inhibiting adipogenesis during adipogenic commitment phase (days 3-6). Our results demonstrate that cellular proliferation is counteractive to adipogenic commitment in human adipogenesis. However, cellular proliferation stimulation can be beneficial for adipogenesis during the mitotic phase by increasing the population of cells capable of committing to adipocytes before adipogenic commitment.

Efficient Precision Genome Editing in iPSCs via Genetic Co-targeting with Selection.

Genome editing in induced pluripotent stem cells is currently hampered by the laborious and expensive nature of identifying homology-directed repair (HDR)-modified cells. We present an approach where isolation of cells bearing a selectable, HDR-mediated editing event at one locus enriches for HDR-mediated edits at additional loci. This strategy, called co-targeting with selection, improves the probability of isolating cells bearing HDR-mediated variants and accelerates the production of disease models.

Efectividad y seguridad del sorafenib en pacientes con carcinoma hepatocelular avanzado / Effectiveness and safety of sorafenib in patients with advanced hepatocellular carcinoma

Informe rápido regional de evaluación de tecnología sobre la efectividad y seguridad del uso de Sorafenib en el tratamiento de pacientes con carcinoma renal avanzado. Medicamento: Sorafenib. Indicación: Carcinoma renal avanzado. Dado que no hay beneficio con respecto a los comparadores y las guías de manejo lo recomiendan por la escaza disponibilidad de medicamentos de alta efectividad en este subgrupo de pacientes se sugiere dar cubrimiento restringido.

Dosage and cell line dependent inhibitory effect of bFGF supplement in human pluripotent stem cell culture on inactivated human mesenchymal stem cells.

Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.

Resveratrol exerts dosage and duration dependent effect on human mesenchymal stem cell development.

Studies in the past have illuminated the potential benefit of resveratrol as an anticancer (pro-apoptosis) and life-extending (pro-survival) compound. However, these two different effects were observed at different concentration ranges. Studies of resveratrol in a wide range of concentrations on the same cell type are lacking, which is necessary to comprehend its diverse and sometimes contradictory cellular effects. In this study, we examined the effects of resveratrol on cell self-renewal and differentiation of human mesenchymal stem cells (hMSCs), a type of adult stem cells that reside in a number of tissues, at concentrations ranging from 0.1 to 10 µM after both short- and long-term exposure. Our results reveal that at 0.1 µM, resveratrol promotes cell self-renewal by inhibiting cellular senescence, whereas at 5 µM or above, resveratrol inhibits cell self-renewal by increasing senescence rate, cell doubling time and S-phase cell cycle arrest. At 1 µM, its effect on cell self-renewal is minimal but after long-term exposure it exerts an inhibitory effect, accompanied with increased senescence rate. At all concentrations, resveratrol promotes osteogenic differentiation in a dosage dependent manner, which is offset by its inhibitory effect on cell self-renewal at high concentrations. On the contrary, resveratrol suppresses adipogenic differentiation during short-term exposure but promotes this process after long-term exposure. Our study implicates that resveratrol is the most beneficial to stem cell development at 0.1 µM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its dosage dependent effect on hMSCs.

Herpes simplex virus type-1 (HSV-1) entry into human mesenchymal stem cells is heavily dependent on heparan sulfate.

Hematopoietic stem cells recipients remain susceptible to opportunistic viral infections including herpes simplex virus type-1 (HSV-1). The purpose of this investigation was to analyze susceptibility of human mesenchymal stem cells (hMSCs) to HSV-1 infection and identify the major entry receptor. Productive virus infection in hMSCs was confirmed by replication and plaque formation assays using a syncytial HSV-1 KOS (804) virus. To examine the significance of entry receptors, RT-PCR and antibody-blocking assays were performed. RT-PCR data showed the expression of gD receptors: nectin-1, 3-O sulfotransferase-3 (3-OST-3), and HVEM. Antibody-blocking assay together with heparinase treatment suggested an important role for HS and 3-O-sulfated heparan sulfate (3-OS HS), but not nectin-1 or HVEM, in mediating HSV-1 entry and spread in hMSCs. Taken together, our results provide strong evidence demonstrating that HSV-1 is capable of infecting hMSCs and HS and 3-OS HS serve as its entry receptors during this process.