ALDH1A3 Is the Key Isoform That Contributes to Aldehyde Dehydrogenase Activity and Affects <i>in Vitro</i> Proliferation in Cardiac Atrial Appendage Progenitor Cells.

High aldehyde dehydrogenase (ALDH &lt;sup&gt;hi&lt;/sup&gt; ) activity has been reported in normal and cancer stem cells. We and others have shown previously that human ALDH &lt;sup&gt;hi&lt;/sup&gt; cardiac atrial appendage cells are enriched with stem/progenitor cells. The role of ALDH in these cells is poorly understood but it may come down to the specific ALDH isoform(s) expressed. This study aimed to compare ALDH &lt;sup&gt;hi&lt;/sup&gt; and ALDH &lt;sup&gt;lo&lt;/sup&gt; atrial cells and to identify the isoform(s) that contribute to ALDH activity, and their functional role. &lt;b&gt;Methods and Results:&lt;/b&gt; Cells were isolated from atrial appendage specimens from patients with ischemic and/or valvular heart disease undergoing heart surgery. ALDH &lt;sup&gt;hi&lt;/sup&gt; activity assessed with the Aldefluor reagent coincided with primitive surface marker expression (CD34 &lt;sup&gt;+&lt;/sup&gt; ). Depending on their ALDH activity, RT-PCR analysis of ALDH &lt;sup&gt;hi&lt;/sup&gt; and ALDH &lt;sup&gt;lo&lt;/sup&gt; cells demonstrated a differential pattern of pluripotency genes (Oct 4, Nanog) and genes for more established cardiac lineages (Nkx2.5, Tbx5, Mef2c, GATA4). ALDH &lt;sup&gt;hi&lt;/sup&gt; cells, but not ALDH &lt;sup&gt;lo&lt;/sup&gt; cells, formed clones and were culture-expanded. When cultured under cardiac differentiation conditions, ALDH &lt;sup&gt;hi&lt;/sup&gt; cells gave rise to a higher number of cardiomyocytes compared with ALDH &lt;sup&gt;lo&lt;/sup&gt; cells. Among 19 ALDH isoforms known in human, ALDH1A3 was most highly expressed in ALDH &lt;sup&gt;hi&lt;/sup&gt; atrial cells. Knocking down ALDH1A3, but not ALDH1A1, ALDH1A2, ALDH2, ALDH4A1, or ALDH8A1 using siRNA decreased ALDH activity and cell proliferation in ALDH &lt;sup&gt;hi&lt;/sup&gt; cells. Conversely, overexpressing ALDH1A3 with a retroviral vector increased proliferation in ALDH &lt;sup&gt;lo&lt;/sup&gt; cells. &lt;b&gt;Conclusions:&lt;/b&gt; ALDH1A3 is the key isoform responsible for ALDH activity in ALDH &lt;sup&gt;hi&lt;/sup&gt; atrial appendage cells, which have a propensity to differentiate into cardiomyocytes. ALDH1A3 affects &lt;i&gt;in vitro&lt;/i&gt; proliferation of these cells

Characterization of compound 584, an Abl kinase inhibitor with lasting effects

Background: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosomepositive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). Design and Methods: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotransplanted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. Results: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC50: 8 nM) and in cell-based assays (IC50: 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC50: 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. Conclusions: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former. ©2008 Ferrata Storti Foundation

COVID-19 in rheumatic diseases in Italy: first results from the Italian registry of the Italian Society for Rheumatology (CONTROL-19)

OBJECTIVES: Italy was one of the first countries significantly affected by the coronavirus disease 2019 (COVID-19) epidemic. The Italian Society for Rheumatology promptly launched a retrospective and anonymised data collection to monitor COVID-19 in patients with rheumatic and musculoskeletal diseases (RMDs), the CONTROL-19 surveillance database, which is part of the COVID-19 Global Rheumatology Alliance. METHODS: CONTROL-19 includes patients with RMDs and proven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) updated until May 3rd 2020. In this analysis, only molecular diagnoses were included. The data collection covered demographic data, medical history (general and RMD-related), treatments and COVID-19 related features, treatments, and outcome. In this paper, we report the first descriptive data from the CONTROL-19 registry. RESULTS: The population of the first 232 patients (36% males) consisted mainly of elderly patients (mean age 62.2 years), who used corticosteroids (51.7%), and suffered from multi-morbidity (median comorbidities 2). Rheumatoid arthritis was the most frequent disease (34.1%), followed by spondyloarthritis (26.3%), connective tissue disease (21.1%) and vasculitis (11.2%). Most cases had an active disease (69.4%). Clinical presentation of COVID-19 was typical, with systemic symptoms (fever and asthenia) and respiratory symptoms. The overall outcome was severe, with high frequencies of hospitalisation (69.8%), respiratory support oxygen (55.7%), non-invasive ventilation (20.9%) or mechanical ventilation (7.5%), and 19% of deaths. Male patients typically manifested a worse prognosis. Immunomodulatory treatments were not significantly associated with an increased risk of intensive care unit admission/mechanical ventilation/death. CONCLUSIONS: Although the report mainly includes the most severe cases, its temporal and spatial trend supports the validity of the national surveillance system. More complete data are being acquired in order to both test the hypothesis that RMD patients may have a different outcome from that of the general population and determine the safety of immunomodulatory treatments

MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation.

BACKGROUND: The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models. RESULTS: In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression. CONCLUSIONS: This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo

A PiggyBac-mediated approach for muscle gene transfer or cell therapy

An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable gene expression when transferred to cultured mesoangioblasts and into murine muscles. Without selection, up to 8% of the mesoangioblasts expressed the transgene from 1 to 2 genomic copies of the piggyBac vector. Integration occurred mostly in intergenic genomic DNA and transgene expression was stable in vitro. Intramuscular transplantation of mouse Tibialis anterior muscles with mesoangioblasts containing the transposon led to sustained myofiber GFP expression in vivo. In contrast, the direct electroporation of the transposon-donor plasmids in the mouse Tibialis muscles in vivo did not lead to sustained transgene expression despite molecular evidence of piggyBac transposition in vivo. Together these findings provide a proof-of-principle that piggyBac transposon may be considered for mesoangioblast cell-based therapies of muscular dystrophies

Table_2_ALDH1A3 Is the Key Isoform That Contributes to Aldehyde Dehydrogenase Activity and Affects in Vitro Proliferation in Cardiac Atrial Appendage Progenitor Cells.DOCX

<p>High aldehyde dehydrogenase (ALDH^hi) activity has been reported in normal and cancer stem cells. We and others have shown previously that human ALDH^hi cardiac atrial appendage cells are enriched with stem/progenitor cells. The role of ALDH in these cells is poorly understood but it may come down to the specific ALDH isoform(s) expressed. This study aimed to compare ALDH^hi and ALDH^lo atrial cells and to identify the isoform(s) that contribute to ALDH activity, and their functional role.</p><p>Methods and Results: Cells were isolated from atrial appendage specimens from patients with ischemic and/or valvular heart disease undergoing heart surgery. ALDH^hi activity assessed with the Aldefluor reagent coincided with primitive surface marker expression (CD34⁺). Depending on their ALDH activity, RT-PCR analysis of ALDH^hi and ALDH^lo cells demonstrated a differential pattern of pluripotency genes (Oct 4, Nanog) and genes for more established cardiac lineages (Nkx2.5, Tbx5, Mef2c, GATA4). ALDH^hi cells, but not ALDH^lo cells, formed clones and were culture-expanded. When cultured under cardiac differentiation conditions, ALDH^hi cells gave rise to a higher number of cardiomyocytes compared with ALDH^lo cells. Among 19 ALDH isoforms known in human, ALDH1A3 was most highly expressed in ALDH^hi atrial cells. Knocking down ALDH1A3, but not ALDH1A1, ALDH1A2, ALDH2, ALDH4A1, or ALDH8A1 using siRNA decreased ALDH activity and cell proliferation in ALDH^hi cells. Conversely, overexpressing ALDH1A3 with a retroviral vector increased proliferation in ALDH^lo cells.</p><p>Conclusions: ALDH1A3 is the key isoform responsible for ALDH activity in ALDH^hi atrial appendage cells, which have a propensity to differentiate into cardiomyocytes. ALDH1A3 affects in vitro proliferation of these cells.</p

Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis

International audienceFunctional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells

Misdiagnosis in fibromyalgia: a multicentre study

Background. Fibromyalgia (FM) is the second most common cause of visits to rheumatologists after osteoarthritis, and may be difficult to diagnose in many patients. It is associated with various rheumatic disorders such as rheumatoid arthritis, spondyloarthropathies (SpA) and connective tissue disease (CTD), and a late diagnosis or misdiagnosis is a common and underestimated problem. Objectives. The aim of this study was to investigate the "underdiagnosis" of FM, and which rheumatic diseases tend to be confused with it. Methods. The following data were collected at baseline: symptoms, disease duration, physical examination findings, previous and current investigations and management, laboratory tests, tender point count, tender and swollen joint counts, and spinal pain. The clinimetric evaluation included the Fibromyalgia Impact Questionnaire (FIQ) and Fibromyalgia Assessment Status (FAS). Results. The study population consisted of 427 outpatients (418 females and 9 males; mean age 49.3 years; mean disease duration 8.5 years). Fifty-seven patients (13.3%) had been previously misdiagnosed as having other musculoskeletal disorders (MSDs); 370 patients had been. previous correctly diagnosed as having FM, or were diagnosed as having it during the course of the study. The FM and MSD groups were comparable in terms of demographic data and referral patterns. Disease duration was longer and the erythrocyte sedimentation rate was higher in the MSD patients, who also had less severe FIQ and lower pain visual analogue scale scores. Moreover, the FIQ and FAS scores correlated in the MS group. Conclusions. The findings of this study suggest that, although FM is a well-known clinical entity, differential diagnosis with SpA, CTD and inflammatory arthritis can still be a challenge for rheumatologists and general practitioners

Table_1_ALDH1A3 Is the Key Isoform That Contributes to Aldehyde Dehydrogenase Activity and Affects in Vitro Proliferation in Cardiac Atrial Appendage Progenitor Cells.DOCX

<p>High aldehyde dehydrogenase (ALDH^hi) activity has been reported in normal and cancer stem cells. We and others have shown previously that human ALDH^hi cardiac atrial appendage cells are enriched with stem/progenitor cells. The role of ALDH in these cells is poorly understood but it may come down to the specific ALDH isoform(s) expressed. This study aimed to compare ALDH^hi and ALDH^lo atrial cells and to identify the isoform(s) that contribute to ALDH activity, and their functional role.</p><p>Methods and Results: Cells were isolated from atrial appendage specimens from patients with ischemic and/or valvular heart disease undergoing heart surgery. ALDH^hi activity assessed with the Aldefluor reagent coincided with primitive surface marker expression (CD34⁺). Depending on their ALDH activity, RT-PCR analysis of ALDH^hi and ALDH^lo cells demonstrated a differential pattern of pluripotency genes (Oct 4, Nanog) and genes for more established cardiac lineages (Nkx2.5, Tbx5, Mef2c, GATA4). ALDH^hi cells, but not ALDH^lo cells, formed clones and were culture-expanded. When cultured under cardiac differentiation conditions, ALDH^hi cells gave rise to a higher number of cardiomyocytes compared with ALDH^lo cells. Among 19 ALDH isoforms known in human, ALDH1A3 was most highly expressed in ALDH^hi atrial cells. Knocking down ALDH1A3, but not ALDH1A1, ALDH1A2, ALDH2, ALDH4A1, or ALDH8A1 using siRNA decreased ALDH activity and cell proliferation in ALDH^hi cells. Conversely, overexpressing ALDH1A3 with a retroviral vector increased proliferation in ALDH^lo cells.</p><p>Conclusions: ALDH1A3 is the key isoform responsible for ALDH activity in ALDH^hi atrial appendage cells, which have a propensity to differentiate into cardiomyocytes. ALDH1A3 affects in vitro proliferation of these cells.</p