A modified animal-free serum technique for efficient isolation and proliferation of mesenchymal stem cells from Hoffa fat pad.

We focus on this study in designing an alternative technique for obtaining mesenchymal stem cells (MSCs) from residual tissue, Hoffa fat, in arthroscopic procedures. Two males and two females were included, and underwent knee arthroscopy; a sample of infrapatellar adipose tissue was obtained with basket forceps. The primary culture was made using the explant method and the culture media: DMEM-high glucose, supplemented with 10% of inactivated human allogeneic serum. All the cellular cultures remained under culture conditions for three weeks, after that by flow cytometry the cells were characterized by MSCs antibody panel: CD105, CD73 and CD90. Subsequently, in the first pass, the MSCs were cultured in commercial human chondrogenic, osteogenic and adipogenic mediums, respectively. After primary culture, we obtained on average 95,600.00 ± 7,233.26 cells/cm2, and the duplication time of MSCs isolate from Hoffa fat pad was established in 39 hours. By flow cytometry, we found that surface markers percentage for expanded MSCs (CD105, CD73, CD90) in primary culture significantly increased and its morphology was fibroblastic-like. After differentiation culture which was made in the first pass, by immunofluorescence, we obtained positive cell markers for three lineages of differentiation, adipocytes: LPL protein, osteocytes: RUNX2, Osteopontin, chondrocytes: SOX9, Aggrecan and COL2A1. We managed to isolate a significant number of MSCs from this source using an easy method to implement and minimal nutrient supplementation, with high potential for differentiation to mature mesenchymal tissues and potential use in basic experimental, preclinical and even clinical research.

New prophylaxis regimen for SARS-CoV-2 infection in health professionals with low doses of hydroxychloroquine and bromhexine: a randomised, double-blind placebo clinical trial (ELEVATE Trial).

INTRODUCTION: SARS-CoV-2 infection in Mexico has caused ~2.7 million confirmed cases; around 20%-25% of health workers will be infected by the virus at their workplace, with approximately 4.4% of mortality. High infectivity of SARS-CoV-2 is related with cell entry mechanism, through the ACE receptor. SARS-CoV-2 requires transmembrane protease serine 2 to cleave its spike glycoprotein and ensure fusion of host cell and virus membrane. We propose studying prophylactic treatment with hydroxychloroquine (HCQ) and bromhexine (BHH), which have been shown to be effective in preventing SARS-CoV-2 infection progression when administered in early stages. The aim of this study is to assess the efficacy of HCQ and BHH as prophylactic treatments for SARS-CoV-2 infection in healthy health workers exposed to the virus. METHODS AND ANALYSIS: Double-blind randomised clinical trial, with parallel allocation at a 1:1 ratio with placebo, of low doses of HCQ plus BHH, for 60 days. Study groups will be defined as follows: (1) HCQ 200 mg/day+BHH 8 mg/8 hours versus (2) HCQ placebo plus BHH placebo. Primary endpoint will be efficacy of both interventions for the prevention of SARS-CoV-2 infection, determined by the risk ratio of infected personnel and the absolute risk. At least a 16% reduction in absolute risk is expected between the intervention and placebo groups; a minimum of 20% infection is expected in the placebo group. The sample size calculation estimated a total of 214 patients assigned: two groups of 107 participants each. ETHICS AND DISSEMINATION: This protocol has been approved by the local Medical Ethics Committee (National Institute of Rehabilitation 'Luis Guillermo Ibarra Ibarra', approval number INRLGII/25/20) and by the Federal Commission for Protection against Sanitary Risks (COFEPRIS, approval number 203 300 410A0058/2020). The results of the study will be submitted for publication in peer-reviewed journals and disseminated through conferences. TRIAL REGISTRATION NUMBER: NCT04340349.

A deletion in the PRKAR1A gene is associated with Carney complex.

Mutations of the PRKAR1A gene are an important cause of Carney complex (CC). The PRKAR1A gene encodes the type 1A regulatory subunit of cAMP-dependent protein kinase A. We have identified one mutation of PRKAR1A (553delG) in three members of the same family affected by CC. This mutation was not identified in six unaffected family members, 12 patients with sporadic cardiac myxoma and 100 non-related healthy individuals. The novel mutation (553delG) is predicted to produce a frameshift leading to a premature stop codon. RNA analysis in the index patient showed normal size transcripts in RT-PCR amplicons of several exons, but an overall tendency to lower amounts of transcripts in relation to GAPDH controls. In Western blot analyses only full-length protein was present without any evidence of truncated product. These data suggest that the mutant allele might be a null allele due to degradation of the mutant mRNA via nonsense-mediated decay.

Oxidative stress impairs endocytosis of the scavenger receptor class A.

We report the characterization of a cell system employing Chinese hamster ovary (CHO) cells and CHO cells transfected with the scavenger receptor class A (CHO-SRA) using extracellularly produced reactive oxygen species (ROS) in order to study the endocytic function of the scavenger receptor. The oxidative environment was produced using tert-butyl hydroperoxide (TBH) and characterized by flow cytometry and cell viability. Once an adequate oxidative environment was established, binding and internalization studies of radiolabeled acetylated LDL particles (125I-labeled Ac-LDL) with CHO-SRA cells were carried out. RT-PCR analysis using total RNAs from CHO-SRA cells revealed that oxidative stress does not alter the expression of the scavenger receptor. However, internalization of 125I-labeled Ac-LDL through this receptor carried out by these cells was completely abolished under extracellularly oxidative conditions. Together, these results support the idea that an oxidative stress produced extracellularly, inhibiting the endocytosis of the scavenger receptor, could help to understand and explain the mechanisms by which several physiologically important ligands are accumulated in the extracellular space with its consequent cell damage.

[The scavenger receptor and its importance in amyloid processes]. / El receptor "pepenador" y su importancia en procesos amiloidóticos.

The scavenger receptor (SRA or RPA) belongs to a wide family of receptor proteins. The classification is based on sequence homologies and structural similarities; nevertheless, it has been useful to group them on the basis of ligand specificity. The SRA was first identified as a receptor for modified low-density lipoproteins, where such modification permits to regulate the uptake of modified LDL by macrophages leading to a massive cholesterol accumulation. Moreover, SRA facilitates the clearance by phagocytic cells of microbial pathogens and senescent cells. SRA is a transmembrane glycoprotein that exists as a trimer comprised of a cystein-linker dimer and a non-covalently bound monomer. SRA has an a-helical coiled coil domain, which is essential for both trimer formation and acid-dependent ligand dissociation. It also contains a collagenous domain, essential for ligand binding. The majority of these ligands are polyanionic molecules, such as the A beta-peptide, important in the development of Alzheimer's disease. Present findings including our own consider that binding of these peptides to SRA activates an inflammatory response with the production of oxidative stress.

El receptor "pepenador" y su importancia en procesos amiloidáticos / The scavenger receptor and its importance in amyloid processes

The scavenger receptor (SRA or RPA) belongs to a wide family of receptor proteins. The classification is based on sequence homologies and structural similarities; nevertheless, it has been useful to group them on the basis of ligand specificity. The SRA was first identified as a receptor for modified low-density lipoproteins, where such modification permits to regulate the uptake of modified LDL by macrophages leading to a massive cholesterol accumulation. Moreover, SRA facilitates the clearance by phagocytic cells of microbial pathogens and senescent cells. SRA is a transmembrane glycoprotein that exists as a trimer comprised of a cystein-linker dimer and a non-covalently bound monomer. SRA has an a-helical coiled coil domain, which is essential for both trimer formation and acid-dependent ligand dissociation. It also contains a collagenous domain, essential for ligand binding. The majority of these ligands are polyanionic molecules, such as the A beta-peptide, important in the development of Alzheimer's disease. Present findings including our own consider that binding of these peptides to SRA activates an inflammatory response with the production of oxidative stress.