Autophagy Is Involved in Mesenchymal Stem Cell Death in Coculture with Chondrocytes.

OBJECTIVE: Cartilage formation is stimulated in mixtures of chondrocytes and human adipose-derived mesenchymal stromal cells (MSCs) both in vitro and in vivo. During coculture, human MSCs perish. The goal of this study is to elucidate the mechanism by which adipose tissue-derived MSC cell death occurs in the presence of chondrocytes. METHODS: Human primary chondrocytes were cocultured with human MSCs derived from 3 donors. The cells were cultured in monoculture or coculture (20% chondrocytes and 80% MSCs) in pellets (200,000 cells/pellet) for 7 days in chondrocyte proliferation media in hypoxia (2% O2). RNA sequencing was performed to assess for differences in gene expression between monocultures or coculture. Immune fluorescence assays were performed to determine the presence of caspase-3, LC3B, and P62. RESULTS: RNA sequencing revealed significant upregulation of >90 genes in the 3 cocultures when compared with monocultures. STRING analysis showed interconnections between >50 of these genes. Remarkably, 75% of these genes play a role in cell death pathways such as apoptosis and autophagy. Immunofluorescence shows a clear upregulation of the autophagic machinery with no substantial activation of the apoptotic pathway. CONCLUSION: In cocultures of human MSCs with primary chondrocytes, autophagy is involved in the disappearance of MSCs. We propose that this sacrificial cell death may contribute to the trophic effects of MSCs on cartilage formation.

Inhibition of the epigenetic suppressor EZH2 primes osteogenic differentiation mediated by BMP2.

Bone-stimulatory therapeutics include bone morphogenetic proteins (e.g. BMP2), parathyroid hormone, and antibody-based suppression of WNT antagonists. Inhibition of the epigenetic enzyme enhancer of zeste homolog 2 (EZH2) is both bone anabolic and osteoprotective. EZH2 inhibition stimulates key components of bone-stimulatory signaling pathways, including the BMP2 signaling cascade. Because of high costs and adverse effects associated with BMP2 use, here we investigated whether BMP2 dosing can be reduced by co-treatment with EZH2 inhibitors. Co-administration of BMP2 with the EZH2 inhibitor GSK126 enhanced differentiation of murine (MC3T3) osteoblasts, reflected by increased alkaline phosphatase activity, Alizarin Red staining, and expression of bone-related marker genes (e.g. Bglap and Phospho1). Strikingly, co-treatment with BMP2 (10 ng/ml) and GSK126 (5 µm) was synergistic and was as effective as 50 ng/ml BMP2 at inducing MC3T3 osteoblastogenesis. Similarly, the BMP2-GSK126 co-treatment stimulated osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells, reflected by induction of key osteogenic markers (e.g. Osterix/SP7 and IBSP). A combination of BMP2 (300 ng local) and GSK126 (5 µg local and 5 days of 50 mg/kg systemic) yielded more consistent bone healing than single treatments with either compound in a mouse calvarial critical-sized defect model according to results from µCT, histomorphometry, and surgical grading of qualitative X-rays. We conclude that EZH2 inhibition facilitates BMP2-mediated induction of osteogenic differentiation of progenitor cells and maturation of committed osteoblasts. We propose that epigenetic priming, coupled with bone anabolic agents, enhances osteogenesis and could be leveraged in therapeutic strategies to improve bone mass.

Effect of Saccharomyces boulardii CNCM I-745 as complementary treatment of Helicobacter pylori infection on gut microbiome.

Conventional therapy for H. pylori infection includes the combination of antibiotics and a proton-pump inhibitor. Addition of probiotics as adjuvants for H. pylori antibiotic treatment can increase eradication rate and decrease treatment side effects. Although many studies show the benefits of S. boulardii CNCM I-745 in the treatment of H. pylori infection, the mechanism by which those benefits are achieved is unknown. Here, we report clinical characteristics and fecal microbiota changes comparing conventional anti-H. pylori therapy versus conventional therapy supplemented with S. boulardii CNCM I-745. A total of 74 patients were included in the current study; patients positive for H. pylori (n = 63) were randomly assigned to 2 groups: 34 patients received conventional therapy and 29 antibiotic therapy plus 750 mg of S. boulardii CNCM I-745 daily, for 2 weeks. Eleven patients negative for H. pylori infection were also studied. Patients provided 3 fecal samples: before initiating the antibiotic treatment, upon its completion, and 1 month after treatment. Patients were contacted every 72 h to inquire about side effects and compliance. DNA was extracted, and 16S rRNA was amplified and sequenced on Illumina MiSeq. Bioinformatic analysis was performed using QIIME2. Patients who received the probiotic had a significantly lower frequency of associated gastrointestinal symptoms (P = 0.028); higher number of bacterial diversity evenness (P = 0.0156); higher abundance of Enterobacteria; and lower abundance of Bacteroides and Clostridia upon treatment completion. Addition of S. boulardii CNCM I-745 induced a lower frequency of gastrointestinal symptoms that could be related to changes in gut microbiota.

Molecular characterization of physis tissue by RNA sequencing.

The physis is a well-established and anatomically distinct cartilaginous structure that is crucial for normal long-bone development and growth. Abnormalities in physis function are linked to growth plate disorders and other pediatric musculoskeletal diseases. Understanding the molecular pathways operative in the physis may permit development of regenerative therapies to complement surgically-based procedures that are the current standard of care for growth plate disorders. Here, we performed next generation RNA sequencing on mRNA isolated from human physis and other skeletal tissues (e.g., articular cartilage and bone; n = 7 for each tissue). We observed statistically significant enrichment of gene sets in the physis when compared to the other musculoskeletal tissues. Further analysis of these upregulated genes identified physis-specific networks of extracellular matrix proteins including collagens (COL2A1, COL6A1, COL9A1, COL14A1, COL16A1) and matrilins (MATN1, MATN2, MATN3), and signaling proteins in the WNT pathway (WNT10B, FZD1, FZD10, DKK2) or the FGF pathway (FGF10, FGFR4). Our results provide further insight into the gene expression networks that contribute to the physis' unique structural composition and regulatory signaling networks. Physis-specific expression profiles may guide ongoing initiatives in tissue engineering and cell-based therapies for treatment of growth plate disorders and growth modulation therapies. Furthermore, our findings provide new leads for therapeutic drug discovery that would permit future intervention through pharmacological rather than surgical strategies.

Asociación de variables clínicas de hipoperfusión con el lactato y la mortalidad / Association of clinical variables of hypoperfusion with lactate and mortality

Resumen Introducción: entre los pacientes con infección la hiperlactatemia identifica una población de mayor gravedad. Este estudio pretende determinar en pacientes de urgencias la correlación y asociación entre los parámetros clínicos de perfusión y los valores de lactato en el momento de admisión; así como el cambio en los parámetros clínicos con la depuración del lactato. Además, determinar la asociación entre estas variables y la mortalidad intrahospitalaria. Métodos: cohorte prospectiva de pacientes que ingresaron con sospecha de infección a un hospital de tercer nivel. Se midió el lactato en la admisión a las 6 y 24 horas, concomitantemente con las variables llenado capilar, índice de choque y presión de pulso, entre otras. Se realizó correlación de Spearman entre las variables clínicas, los niveles de lactato y su depuración; así como curvas ROC para determinar la capacidad discriminativa de las variables clínicas para detectar hiperlactatemia. Se realizó un modelo de regresión logística multivariable para mortalidad. Resultados: se evaluaron 2257 pacientes, 651 correspondían a infección confirmada. No se encontró ninguna correlación de utilidad entre las variables clínicas y el lactato (r<0.25); y tampoco se detectó adecuada capacidad discriminativa para la detección de hiperlactatemia con ninguna variable clínica (AUC<0.61). En el modelo de regresión logística multivariada el valor del lactato al ingreso fue la única variable que se asoció de manera independiente con mortalidad (OR=1.4, IC95%=1.3-1.6). Conclusiones: entre los pacientes que ingresan a urgencias con infección no se encontró correlación entre las variables clínicas y el lactato; sin embargo, el lactato al ingreso es un marcador pronóstico independiente de mortalidad. (Acta Med Colomb 2017: 42: 97-105).

Abstract Introduction: among patients with infection, hyperlactatemia identifies a population of greater severity. This study aims to determine the correlation and association between clinical perfusion parameters and lactate values in emergency patients at the time of admission, as well as the change in clinical parameters with lactate clearance. In addition, to determine the association between these variables and in-hospital mortality. Methods: Prospective cohort of patients admitted with suspected infection to a third level hospital. Lactate was measured at admission, at 6 and 24 hours, concomitantly with the variables capillary filling, shock index and pulse pressure, among others. Among the clinical variables, Spearman correlation, lactate levels and their clearance, as well as ROC curves to determine the discriminative ability of clinical variables to detect hyperlactatemia were performed. A multivariate logistic regression model for mortality was carried out. Results: 2257 patients were evaluated. 651 were confirmed with infections. No utility correlation was found between clinical variables and lactate (r <0.25), and no discriminative capacity was detected for the detection of hyperlactatemia with any clinical variable (AUC <0.61). In the multivariate logistic regression model the lactate value at admission was the only variable that was independently associated with mortality (OR = 1.4, 95% CI = 1.3-1.6). Conclusions: no correlation was found between clinical variables and lactate among patients admitted to the emergency department with infection; however lactate at admission is an independent prognostic marker of mortality. (Acta Med Colomb 2017: 42: 97-105).

Ronda clínica y epidemiológica. Introducción al análisis multivariable (parte II) / Clinical and epidemiological round. Introduction to multivariable analysis (part II) / Ronda clínica e epidemiológica. Introdução à análise multi-variáveis (parte II

En la primera parte de este tema presentamos su definición, principales usos y los tres métodos de análisis multivariable más utilizados en la literatura científica. En esta segunda parte profundizaremos en los criterios para la incorporación de variables independientes al análisis, las herramientas para evaluar qué tan adecuado es el modelo seleccionado y la interpretación de los resultados y de los coeficientes en cada tipo de regresión.

In the first part of this topic, we explained the definition of multivariable analysis, its main uses, and the three most commonly used methods in the scientific literature. In this second part, we will delve into the inclusion criteria of independent variables to the analysis, the tools to assess how the model fits the data and the interpretation of the results and coefficients in each regression model.

Na primeira parte deste tema apresentamos sua definição, principais usos e os três métodos de análises multi-variável mais utilizados na literatura científica. Nesta segunda parte aprofundaremos nos critérios para a incorporação de variáveis independentes à análise, as ferramentas para avaliar que tão adequado é o modelo selecionado e a interpretação dos resultados e dos coeficientes em cada tipo de regressão.