Tiny percutaneous needle biopsy: An efficient methodfor studying cellular and molecular aspectsof skeletal muscle in humans

Needle biopsy is widely used to obtain specimens for physiological, anatomical and biochemical studies of skeletal muscle (SM). We optimized a procedure which we termed tiny percutaneous needle biopsy (TPNB), to efficiently gather good numbers of human satellite cells and single dissociated fibers for the functional study of skeletal muscle; these samples permit isolation of high-quality RNA and sufficient amounts of proteins to allow molecular analysis. Moreover, TPNB showed a clear advantage in that the technique was easier than other procedures used on healthy volunteers in human trials. TPNB is a very safe minor surgical procedure. It is less traumatic than needle aspiration biopsy, and significant complications are improbable. TPNB should become established as an important tool in the investigation of SM and may be employed to study various physiological aspects of SM in human subjects. We suggest that TPNB should also be used in the study of muscle diseases and disorders including muscular dystrophy, congenital myopathy, and metabolic defects

Signal transduction events induced by extracellular guanosine 5′triphosphate in excitable cells

A better understanding of the physiological effects of guanosine-based purines should help clarify the complex subject of purinergic signalling. We studied the effect of extracellular guanosine 5′triphosphate (GTP) on the differentiation of two excitable cell lines that both have specific binding sites for GTP: PC12 rat pheochromocytoma cells and C2C12 mouse skeletal muscle cells. PC12 cells can be differentiated into fully functional sympathetic-like neurons with 50′00 ng ml−1 of nerve growth factor, whereas serum starvation causes C2C12 cells to differentiate into myotubes showing functional excitation–contraction coupling, with the expression of myosin heavy chain proteins. Our results show that GTP enhances the differentiation of both of these excitable cell lines. The early events in guanosine-based purine signal transduction appear to involve an increase in intracellular Ca2+ levels and membrane hyperpolarization. We further investigated the early activation of extracellular-regulated kinases and phosphoinositide 3-kinase in GTP-stimulated PC12 and C2C12 cells, respectively. We found that GTP promotes the activation of both kinases. Together, our results suggest that, even if there are some differences in the signalling pathways, GTP-induced differentiation in both cell lines is dependent on an increase in intracellular Ca2+

Randomised controlled trial comparing efficacy and safety of high versus low Low-Molecular Weight Heparin dosages in hospitalized patients with severe COVID-19 pneumonia and coagulopathy not requiring invasive mechanical ventilation (COVID-19 HD): a structured summary of a study protocol.

To assess whether high doses of Low Molecular Weight Heparin (LMWH) (i.e. Enoxaparin 70 IU/kg twice daily) compared to standard prophylactic dose (i.e., Enoxaparin 4000 IU once day), in hospitalized patients with COVID19 not requiring Invasive Mechanical Ventilation [IMV], are: a)more effective in preventing clinical worsening, defined as the occurrence of at least one of the following events, whichever comes first: 1.Death2.Acute Myocardial Infarction [AMI]3.Objectively confirmed, symptomatic arterial or venous thromboembolism [TE]4.Need of either: a.Continuous Positive Airway Pressure (Cpap) or Non-Invasive Ventilation (NIV) orb.IMV in patients who at randomisation were receiving standard oxygen therapy5.IMV in patients who at randomisation were receiving non-invasive mechanical ventilationb)Similar in terms of major bleeding risk TRIAL DESIGN: Multicentre, randomised controlled, superiority, open label, parallel group, two arms (1:1 ratio), in-hospital study

Current challenges in the management of patients with sickle cell disease - A report of the Italian experience

Sickle cell disease (SCD) is an inherited red blood cell disorder caused by a structural abnormality of hemoglobin called sickle hemoglobin (HbS). Clinical manifestations of SCD are mainly characterized by chronic hemolysis and acute vaso-occlusive crisis, which are responsible for severe acute and chronic organ damage. SCD is widespread in sub-Saharan Africa, in the Middle East, Indian subcontinent, and some Mediterranean regions. With voluntary population migrations, people harboring the HbS gene have spread globally. In 2006, the World Health Organization recognized hemoglobinopathies, including SCD, as a global public health problem and urged national health systems worldwide to design and establish programs for the prevention and management of SCD. Herein we describe the historical experience of the network of hemoglobinopathy centers and their approach to SCD in Italy, a country where hemoglobinopathies have a high prevalence and where SCD, associated with different genotypes including f-thalassemia, is present in the native population

Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with Covid-19 pneumonia.

We provide an in-depth investigation of the T cell compartment and functionality, cytokine production and plasma levels in a total of 39 patients affected by Covid-19 pneumonia. At admission, patients were lymphopenic; for all, SARS-CoV-2 was detected in a nasopharyngeal swab specimen by real-time RT-PCR, and pneumonia was subsequently confirmed by X-rays. Detailed 18-parameter flow cytometry coupled with unsupervised data analysis revealed that patients showed similar percentages of CD4+ and CD8+ T cells, but a decreased absolute number in both populations. For CD4+ T lymphocytes, we found a significant decrease in the number of na\uefve, central and effector memory cells and an increased percentage of terminally differentiated cells, regulatory T cells, and of those that were activated or that were expressing PD1 and CD57 markers. Studies on chemokine receptors and lineage-specifying transcription factors revealed that, among CD4+ T cells, patients displayed a lower percentage of cells expressing CCR6 or CXCR3, and of those co-expressing CCR6 and CD161, but higher percentages of 62 CXCR4+ or CCR4+ cells. No differences were noted in the expression of T-bet or GATA-3. Analyses of patients' CD8+ T cells showed decreased numbers of na\uefve and central memory and increased amounts of activated cells, accompanied by increased percentages of activated cells and of lymphocytes expressing CD57, PD1, or both. CD8+ T cells expressed lower percentages of CCR6+, CXCR3+ or T-bet+ cells and of CXCR3+,T-bet+ or CCR6+,CD161+ lymphocytes. We also found higher percentages of cells expressing CCR4+, CXCR4 or GATA-3. Analyses of lymphocyte proliferation revealed that terminally differentiated CD4+ and CD8+ T cell from patients had a lower proliferative index than controls, whereas cellular bioenergetics, measured by the quantification of mitochondrial oxygen consumption and extracellular acidification rate, was similar in CD4+ T cells from both groups. We measured plasma level of 31 cytokines linked to inflammation, including T helper (TH)type-1 and TH2 cytokines, chemokines, galectins, pro- and anti-inflammatory mediators, finding that most were dramatically increased in Covid-19 patients, confirming the presence of a massive cytokine storm. Analysis of the production of different cytokines after stimulation by anti-CD3/CD28 monoclonal antibodies revealed that patients not only had a high capacity to produce tumour necrosis factor (TNF)-\u3b1, interferon (IFN)-\u3b3 and interleukin (IL)-2, but also showed a significant skewing of CD4+ T cells towards the TH17 phenotype. A therapeutic approach now exists based on the administration of drugs that block IL-6pathway, and seems to improve the disease. IL-17 is crucial in recruiting and activating neutrophils, cells that can migrate to the lung and are heavily involved in the pathogenesis of Covid-19. We show here that a skewing of activated T cells towards the TH17 functional phenotype exists in Covid-19 patients. We therefore suggest that blocking the IL-17 pathway by biological drugs that are already used to treat different pathologies could provide a novel, additional strategy to improve the health of patients infected by SARS-CoV-2

Gokyo Khumbu/Ama Dablam Trek 2012

In the expedition Gokyo Khumbu/Ama Dablam Trek 2012, we studied the effects of two 12-day training periods performed both at sea level and at high altitude. The main results on adult women have been published in six original articles. In women, high altitude trekking induced CD69 T cell activation and promoted anti-stress effects of the immune responses and the oxidative balance (1). Low-to-moderate exercise training at s.l. improves the regenerative capacity of skeletal muscle and depicted the epigenetic signature of satellite cells. The cell differentiation was favored by increased [Ca2+]i and fusion index (2). On the contrary, the training in hypobaric-hypoxia induced oxidative stress and impaired the regenerative capacity of satellite cells (6). Although training did not significantly modify muscle phenotype , it induced beneficial adaptations of the oxygen transport-utilization systems witnessed by faster VO2 kinetics at exercise onset (3). The two training periods did not influence the postural stability (4). In young adult women, micturition physiological parameters were affected during adaptation to hypoxia; the correlation with SpO2 strongly suggests a role of hypoxia in these changes (5

Guida alla redazione degli atti amministrativi

La "Guida alla redazione degli atti amministrativi" intende fornire indicazioni per la redazione degli atti per tutti i funzionari della pubblica amministrazione. Si articola in tre parti: (a) la lingua degli atti, (b) la struttura del provvedimento amministrativo, (c) il rinvio ad altri atti. Ne è autore un gruppo di linguisti e giuristi facenti capo all'ITTIG-CNR (Istituto per le Tecniche e Tecnologie dell'Informazione Giuridica) e dell'Accademia della Crusca

Mass Spectrometry Analysis of Hepcidin Peptides in Experimental Mouse Models

The mouse is a valuable model for unravelling the role of hepcidin in iron homeostasis, however, such studies still report hepcidin mRNA levels as a surrogate marker for bioactive hepcidin in its pivotal function to block ferroportin-mediated iron transport. Here, we aimed to assess bioactive mouse Hepcidin-1 (Hep-1) and its paralogue Hepcidin-2 (Hep-2) at the peptide level. To this purpose, fourier transform ion cyclotron resonance (FTICR) and tandem-MS was used for hepcidin identification, after which a time-of-flight (TOF) MS-based methodology was exploited to routinely determine Hep-1 and -2 levels in mouse serum and urine. This method was biologically validated by hepcidin assessment in: i) 3 mouse strains (C57Bl/6; DBA/2 and BABL/c) upon stimulation with intravenous iron and LPS, ii) homozygous Hfe knock out, homozygous transferrin receptor 2 (Y245X) mutated mice and double affected mice, and iii) mice treated with a sublethal hepatotoxic dose of paracetamol. The results showed that detection of Hep-1 was restricted to serum, whereas Hep-2 and its presumed isoforms were predominantly present in urine. Elevations in serum Hep-1 and urine Hep-2 upon intravenous iron or LPS were only moderate and varied considerably between mouse strains. Serum Hep-1 was decreased in all three hemochromatosis models, being lowest in the double affected mice. Serum Hep-1 levels correlated with liver hepcidin-1 gene expression, while acute liver damage by paracetamol depleted Hep-1 from serum. Furthermore, serum Hep-1 appeared to be an excellent indicator of splenic iron accumulation. In conclusion, Hep-1 and Hep-2 peptide responses in experimental mouse agree with the known biology of hepcidin mRNA regulators, and their measurement can now be implemented in experimental mouse models to provide novel insights in post-transcriptional regulation, hepcidin function, and kinetics

Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses

<p>Abstract</p> <p>Background</p> <p>Hereditary Hemochromatosis (HH) is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y <it>HFE </it>associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER). In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH.</p> <p>Results</p> <p>Vector constructs of Wild type HFE and Mutant C282Y HFE were made and transfected into HEK293 cell lines. We have shown that expression of C282Y HFE protein triggers both an unfolded protein response (UPR), as revealed by the increased GRP78, ATF6 and CHOP expression, and an ER overload response (EOR), as indicated by NF-κB activation. Furthermore, C282Y HFE protein induced apoptotic responses associated with activation of ER stress. Inhibition studies demonstrated that tauroursodeoxycholic acid, an endogenous bile acid, downregulates these events. Finally, we found that the co-existence of both C282Y HFE and Z alpha 1-antitrypsin protein (the protein associated with the liver disease of Z alpha 1-antitrypsin deficiency) expression on ER stress responses acted as potential disease modifiers with respect to each other.</p> <p>Conclusion</p> <p>Our novel observations suggest that both the ER overload response (EOR) and the unfolded protein response (UPR) are activated by mutant C282Y HFE protein.</p

Hepcidin Expression in Iron Overload Diseases Is Variably Modulated by Circulating Factors

Hepcidin is a regulatory hormone that plays a major role in controlling body iron homeostasis. Circulating factors (holotransferrin, cytokines, erythroid regulators) might variably contribute to hepcidin modulation in different pathological conditions. There are few studies analysing the relationship between hepcidin transcript and related protein expression profiles in humans. Our aims were: a. to measure hepcidin expression at either hepatic, serum and urinary level in three paradigmatic iron overload conditions (hemochromatosis, thalassemia and dysmetabolic iron overload syndrome) and in controls; b. to measure mRNA hepcidin expression in two different hepatic cell lines (HepG2 and Huh-7) exposed to patients and controls sera to assess whether circulating factors could influence hepcidin transcription in different pathological conditions. Our findings suggest that hepcidin assays reflect hepatic hepcidin production, but also indicate that correlation is not ideal, likely due to methodological limits and to several post-trascriptional events. In vitro study showed that THAL sera down-regulated, HFE-HH and C-NAFLD sera up-regulated hepcidin synthesis. HAMP mRNA expression in Huh-7 cells exposed to sera form C-Donors, HFE-HH and THAL reproduced, at lower level, the results observed in HepG2, suggesting the important but not critical role of HFE in hepcidin regulation