STUDIO DEL DIFFERENZIAMENTO CARDIACO DI CELLULE STAMINALI ISOLATE DA TESSUTI POST-NATALI

In recent years the stem cells are subject of intensive studies because they are able to differentiate in different types of cells and they can be use for regenerative therapies. In particular we are interested to the care of several acquired or genetic cardiac diseases that they are characterized by rhythm disturbances. These diseases include for example severe bradycardia, Sick Sinus Syndrome, atrio-ventricular block and heart block and often the pharmacological treatment is not always applicable. The our major object is to create a biological pacemaker, generally intended as cell substrates with the same features of native sinoatrial node (SAN) myocytes. So far, murine and human embryonic stem cells have been shown to differentiate into pacemaker myocytes, with electrical and molecular characteristics similar to those of SAN cells. Embryonic stem cells are at the moment one of the most promising cell sources for regenerative but they have different ethical and immunological problems. For this purpose we intend to study different type of adult stem cells and, in particular, a population of hematopoietic stem cells CD34+ isolated from umbilical cord blood and a class of vessel-associated clonogenic, self-renewing progenitor cells, called Mesoangioblasts, Mabs. In this work we investigated whether CD34+ endothelial progenitor cells acquire spontaneous beating and pacemaker properties by using a co-culture system onto neonatal cardiac. We found that the CD34+ cells acquire a spontaneously contractile phenotype but it were the result of fusion events between cardiac myocytes and CD34+-derived cells, as assessed by double color labeling experiment and high throughput FACS analysis. Furthermore the electrophysiological properties investigated did not show any difference between EGFP+ cells and neonatal cardiac myocytes, supporting the hypothesis that these autorhythmic cells derive indeed from a fusion events between electrically passive CD34+ cells with cardiac myocytes. Furthermore we have shown that ventricle-derived Mabs when cultured in low-serum medium spontaneously differentiate into cardiomyocytes that express typical cardiac ion channels. A distinct feature of cardiac MABs not described previously in other adult cardiac stem cells is the high spontaneous rate of cardiac differentiation, which allows the prospective use of these cells for systemic delivery and in vivo transplantation. Cardiac MABs express several factors involved in early cardiac differentiation, among these such as Isl-1, Tbx2, Tbx3, GATA-6 are especially relevant to the development of the cardiac conduction system. Upon differentiation, cardiac MABs often displayed spontaneous activity which involved large, syncronous foci, suggesting that at least a fraction of these cells were capable of self-generating action potentials. We have identified a novel type of adult stem cells, the MdPCs, as the subpopulation of cardiac vessel-derived Mabs which differentiate into a pacemaker cell-like phenotype. The cardiac differentiation potency, the expression of HCN channels underlying spontaneous activity and the sensitivity to autonomic modulation of rate are features especially suitable to a potential use of these cells as a substrate for developing implantable biological pacemakers. The major limitation of the use of mesoangioblasti is the low rate of proliferation and cardiac differentiation. One way to overcome the limitations imposed by the use of pluripotent stem cells derived from adult tissues. Two such types of cells are presently available: induced pluripotent stem (iPS) cells and pluripotent stem cells derived from testis. iPS are somatic cells reprogrammed by exogenous expression of a series of genes involved in the maintenance of ES cell pluripotency (Oct3/4, Sox2, c-Myc and Klf4 for murine cells and Oct4, Sox2, nanog and lin28 for human cells). While these cells have the advantage of not being immunogenic, they still require genetic manipulation which could potentially impair their therapeutic application. Recent data have shown that spermatogonial stem cells (SSCs), which are normally located on the basal membrane of the seminiferous tubules and are responsible for spermatogenesis, can in vitro become pluripotent cells which differentiate into derivatives of all three embryonic germ layers and form teratomas when injected in animals. We have developed a protocol of enzymatic and mechanical dissociation from wild type and transgenic mouse. The most promising protocol is the use of transgenic mouse that express the protein reporter GFP under control of trascritional factor Oct4. At the moment we obtained single cells Oct4+/EGFP+ that remain in colture until seven days but we must apply further analysis to find the optimal conditions to enable the survival and the proliferation of this cells

The New Paradigms in Clinical Research: From Early Access Programs to the Novel Therapeutic Approaches for Unmet Medical Needs.

Despite several innovative medicines gaining worldwide approval in recent years, there are still therapeutic areas for which unsatisfied therapeutic needs persist. For example, high unmet clinical need was observed in patients diagnosed with type 2 diabetes mellitus and hemophilia, as well as in specific age groups, such as the pediatric population. Given the urgent need to improve the therapy of clinical conditions for which unmet clinical need is established, clinical testing, and approval of new medicines are increasingly being carried out through accelerated authorization procedures. Starting from 1992, the Food and Drug Administration and the European Medicines Agency have supported the so-called Early Access Programs (EAPs). Such procedures, which can be based on incomplete clinical data, allow an accelerated marketing authorization for innovative medicines. The growth in pharmaceutical research has also resulted in the development of novel therapeutic approaches, such as biotech drugs and advanced therapy medicinal products, including new monoclonal antibodies for the treatment of asthma, antisense oligonucleotides for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy, and new anticancer drugs that act on genetic biomarkers rather than any specific type of cancer. Even though EAPs and novel therapeutic approaches have brought huge benefits for public health, their implementation is limited by several challenges, including the high risk of safety-related label changes for medicines authorized through the accelerated procedure, the high costs, and the reimbursement and access concerns. In this context, regulatory agencies should provide the best conditions for the implementation of the described new tools

A caveolin-binding domain in the HCN4 channels mediates functional interaction with caveolin proteins

Pacemaker (HCN) channels have a key role in the generation and modulation of spontaneous activity of sinoatrial node myocytes. Previous work has shown that compartmentation of HCN4 pacemaker channels within caveolae regulates important functions, but the molecular mechanism responsible is still unknown. HCN channels have a conserved caveolin-binding domain (CBD) composed of three aromatic amino acids at the N-terminus; we sought to evaluate the role of this CBD in channel-protein interaction by mutational analysis. We generated two HCN4 mutants with a disrupted CBD (Y259S, F262V) and two with conservative mutations (Y259F, F262Y). In CHO cells expressing endogenous caveolin-1 (cav-1), alteration of the CBD shifted channels activation to more positive potentials, slowed deactivation and made Y259S and F262V mutants insensitive to cholesterol depletion-induced caveolar disorganization. CBD alteration also caused a significant decrease of current density, due to a weaker HCN4-cav-1 interaction and accumulation of cytoplasmic channels. These effects were absent in mutants with a preserved CBD. In caveolin-1-free fibroblasts, HCN4 trafficking was impaired and current density reduced with all constructs; the activation curve of F262V was not altered relative to wt, and that of Y259S displayed only half the shift than in CHO cells. The conserved CBD present in all HCN isoforms mediates their functional interaction with caveolins. The elucidation of the molecular details of HCN4-cav-1 interaction can provide novel information to understand the basis of cardiac phenotypes associated with some forms of caveolinopathies

Controversies in the treatment of mild asthma. What novelties and practical implications?

Mild asthma is prevalent in childhood and causes as many as 30%–40% asthma exacerbations requiring emergency visits. The management of "intermittent" and "mild persistent" asthma phenotypes is still a matter of debate, even if the role of inhaled corticosteroids, both continuous and intermittent, is a cornerstone in this field. Recent updates of the guidelines on the strategies to manage these patients are coming, since the role of inflammation in these asthma phenotypes is crucial, as well as the potential side effect and risks of short-acting beta 2 agonists overuse, prescribed as the only "as-needed" treatments. In this paper, we overview the new (r)evolution regarding intermittent and mild persistent asthma management

Management of infectious lymphadenitis in children

Lymphadenopathy is an irregularity in the size and texture of the lymph nodes, which is quite common in childhood. When the enlargement of lymph nodes is caused by inflammatory and infectious processes, it is called lymphadenitis. The main objective of this manuscript is to summarize the common infectious etiologies and presentations of lymphadenitis in children providing a management guide for clinical practice. PubMed was used to search for all of the studies published up to April 2021 using keywords such as “lymphadenitis” and “children”. Literature analysis showed that the differential diagnosis for lymphadenitis in pediatrics is broad. Although lymph node enlargement in children is usually benign and self-limited, it is important to exclude malignant etiology. In most cases, history and physical examination allow to identify the correct diagnosis and start a proper treatment with a prompt resolution of the lymphadenopathy. However, particularly in the case of persistent lymphadenitis, determining the cause of lymph node enlargement may be difficult, and the exact etiology may not be identified despite extensive investigations. Further studies should develop and validate an algorithm to assist pediatricians in the diagnosis and timely treatment of lymphadenitis, suggesting situations in which a watchful waiting may be considered a safe approach, those in which empiric antibiotic therapy should be administered, and those requiring a timely diagnostic work-up

Evidence of abnormal scalar timing property in alexithymia

: Evidence suggests that incidental modulation of affective states affects the ability to keep track of time. Alexithymia represents an ideal condition to further address the emotion-time processing link, as it refers to a trait characterized by a deficit of affective processing. 31 healthy participants completed an online version of the TAS-20 scale, which measures alexithymia, and a time reproduction task of visual stimuli related to positive (i.e., happiness) and negative (i.e., anger) facial expressions. Results documented a positive correlation between TAS-20 score and the variability in reproducing sub-second durations of the anger expression stimuli We also found an overestimation of sub-second durations of non-affective expressions in borderline/alexithymic participants. Finally, in line with the literature, we confirmed the overall tendency to overestimate the duration of anger expression stimuli. These findings, which can be interpreted in terms of abnormal scalar timing property in alexithymia, expand previous investigations linking this personality trait with abnormal processing of negative emotions. The evidence that alexithymia predicts the reproduction variability of sub-second durations of negative affective stimuli corroborates previous neuroimaging studies documenting cerebellar deficits in these individuals

The Role of Renin-Angiotensin-Aldosterone System in the Heart and Lung: Focus on COVID-19

The renin-angiotensin-aldosterone system (RAAS) firstly considered as a cardiovascular circulating hormonal system, it is now accepted as a local tissue system that works synergistically or independently with the circulating one. Evidence states that tissue RAAS locally generates mediators with regulatory homeostatic functions, thus contributing, at some extent, to organ dysfunction or disease. Specifically, RAAS can be divided into the traditional RAAS pathway (or classic RAAS) mediated by angiotensin II (AII), and the non-classic RAAS pathway mediated by angiotensin 1–7. Both pathways operate in the heart and lung. In the heart, the classic RAAS plays a role in both hemodynamics and tissue remodeling associated with cardiomyocyte and endothelial dysfunction, leading to progressive functional impairment. Moreover, the local classic RAAS may predispose the onset of atrial fibrillation through different biological mechanisms involving inflammation, accumulation of epicardial adipose tissue, and electrical cardiac remodeling. In the lung, the classic RAAS regulates cell proliferation, immune-inflammatory response, hypoxia, and angiogenesis, contributing to lung injury and different pulmonary diseases (including COVID-19). Instead, the local non-classic RAAS counteracts the classic RAAS effects exerting a protective action on both heart and lung. Moreover, the non-classic RAAS, through the angiotensin-converting enzyme 2 (ACE2), mediates the entry of the etiological agent of COVID-19 (SARS-CoV-2) into cells. This may cause a reduction in ACE2 and an imbalance between angiotensins in favor of AII that may be responsible for the lung and heart damage. Drugs blocking the classic RAAS (angiotensin-converting enzyme inhibitors and angiotensin receptor blockers) are well known to exert a cardiovascular benefit. They are recently under evaluation for COVID-19 for their ability to block AII-induced lung injury altogether with drugs stimulating the non-classic RAAS. Herein, we discuss the available evidence on the role of RAAS in the heart and lung, summarizing all clinical data related to the use of drugs acting either by blocking the classic RAAS or stimulating the non-classic RAAS

Severe bleeding and absent ADP-induced platelet aggregation associated with inherited combined CalDAG-GEFI and P2Y12 deficiencies

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Neuroinflammation and Neurotransmission Mechanisms Involved in Neuropsychiatric Disorders

Some classical psychiatric disorders, such as schizophrenia, autism, major depression, bipolar and obsessive‐compulsive disorders, have been related to neuroinflammatory process, immunological abnormalities, and neurotransmission impairment beyond genetic mutations. Neuroinflammation is mostly regulated by glial cells, which respond to physiological and pathological stimuli by anti‐ and pro‐inflammatory cytokine and chemokine signaling; moreover, recent studies have indicated that glial cells also respond to the neurotransmitters. Neurotransmitters regulate many biological processes, such as cell proliferation and synaptogenesis, which contribute to the formation of functional circuits. Alterations in the neurotransmission can lead to many pathological changes that occur in brain disorders. For example, studies have shown that neuroinflammation can alter the metabolism of glutamate as well as the function of its transporters, resulting in cognitive, behavioral, and psychiatric impairments. Cytokines as IL‐1β and IL‐6 appear to have an important influence in the dopaminergic and serotoninergic neurons. These data together suggest that glial cells via cytokines and abnormal regulation of neurotransmitters can influence psychiatric disorders. The present knowledge about this issue does not allow answering whether neuroinflammation is the cause or the consequence of neurotransmission imbalance and emphasizes the importance to improve in vivo imaging methods and models to elucidate this enigma