Objective
Despite growing evidence about myocardial injury in hospitalized COronaVIrus Disease 2019 (COVID-19) patients, the mechanism behind this injury is only poorly understood and little is known ...about its association with SARS-CoV-2-mediated myocarditis. Furthermore, definite evidence of the presence and role of SARS-CoV-2 in cardiomyocytes in the clinical scenario is still lacking.
Methods
We histologically characterized myocardial tissue of 40 patients deceased with severe SARS-CoV-2 infection during the first wave of the pandemic. Clinical data were also recorded and analyzed. In case of findings supportive of myocardial inflammation, histological analysis was complemented by RT-PCR and immunohistochemistry for SARS-CoV-2 viral antigens and in situ RNA hybridization for the detection of viral genomes.
Results
Both chronic and acute myocardial damage was invariably present, correlating with the age and comorbidities of our population. Myocarditis of overt entity was found in one case (2.5%). SARS-CoV-2 genome was not found in the cardiomyocytes of the patient with myocarditis, while it was focally and negligibly present in cardiomyocytes of patients with known viral persistence in the lungs and no signs of myocardial inflammation. The presence of myocardial injury was not associated with myocardial inflammatory infiltrates.
Conclusions
In this autopsy cohort of COVID-19 patients, myocarditis is rarely found and not associated with SARS-CoV-2 presence in cardiomyocytes. Chronic and acute forms of myocardial damage are constantly found and correlate with the severity of COVID-19 disease and pre-existing comorbidities.
Graphic abstract
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
In Alzheimer’s disease (AD), the molecular mechanisms involved in the neurodegeneration are still incompletely defined, though this aspect is crucial for a better understanding of the malady and for ...devising effective therapies. Mitochondrial dysfunctions and altered Ca2+ signaling have long been implicated in AD, though it is debated whether these events occur early in the course of the pathology, or whether they develop at late stages of the disease and represent consequences of different alterations. Mitochondria are central to many aspects of cellular metabolism providing energy, lipids, reactive oxygen species, signaling molecules for cellular quality control, and actively shaping intracellular Ca2+ signaling, modulating the intensity and duration of the signal itself. Abnormalities in the ability of mitochondria to take up and subsequently release Ca2+ could lead to changes in the metabolism of the organelle, and of the cell as a whole, that eventually result in cell death. We sought to investigate the role of mitochondria and Ca2+ signaling in a model of Familial Alzheimer’s disease and found early alterations in mitochondria physiology under stressful condition, namely, reduced maximal respiration, decreased ability to sustain membrane potential, and a slower return to basal matrix Ca2+ levels after a mild excitotoxic stimulus. Treatment with an inhibitor of the permeability transition pore attenuated some of these mitochondrial disfunctions and may represent a promising tool to ameliorate mitochondria and cellular functioning in AD and prevent or slow down cell loss in the disease.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The exact quantification of tiny amounts of nucleic acids in biological samples continues to remain a requirement in both the experimental and the diagnostic laboratory. Competitive PCR involves the ...coamplification of a target DNA sample with known amounts of a competitor DNA that shares most of the nucleotide sequence with the target; in this way, any predictable or unpredictable variable affecting PCR amplification has the same effect on both molecular species. Competitive PCR therefore permits the quantification of the absolute number of target molecules in comparison to the amount of competitor DNA. Although requiring intensive post-PCR manipulation, the accuracy of competitive PCR by far exceeds that of any other quantitative PCR procedure, including real-time PCR. This protocol covers all stages in the competitive PCR and RT-PCR methods, from the design and construction of competitor molecules, and the competitive PCR itself, to the analysis of data and quantification of target DNA. Once the correct primers are available, the protocol can be completed in about 24 h.
Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a ...procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy.
Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness and resistance to treatment. We have previously demonstrated that most PDAC ...patients have circulating antibodies against the glycolytic enzyme alpha-enolase (ENO1), which correlates with a better response to therapy and survival. ENO1 is a metabolic enzyme, also expressed on the cell surface where it acts as a plasminogen receptor. ENO1 play a crucial role in cell invasion and metastasis by promoting plasminogen activation into plasmin, a serine-protease involved in extracellular matrix degradation. The aim of this study was to investigate the role of ENO1 in PDAC cell invasion. We observed that ENO1 was expressed on the cell surface of most PDAC cell lines. Mouse anti-human ENO1 monoclonal antibodies inhibited plasminogen-dependent invasion of human PDAC cells, and their metastatic spreading in immunosuppressed mice was inhibited. Notably, a single administration of Adeno-Associated Virus (AAV)-expressing cDNA coding for 72/1 anti-ENO1 mAb reduced the number of lung metastases in immunosuppressed mice injected with PDAC cells. Overall, these data indicate that ENO1 is involved in PDAC cell invasion, and that administration of an anti-ENO1 mAb can be exploited as a novel therapeutic option to increase the survival of metastatic PDAC patients.
Heart failure is preceded by ventricular remodeling, changes in left ventricular mass, and myocardial volume after alterations in loading conditions. Concentric hypertrophy arises after pressure ...overload, involves wall thickening, and forms a substrate for diastolic dysfunction. Eccentric hypertrophy develops in volume overload conditions and leads wall thinning, chamber dilation, and reduced ejection fraction. The molecular events underlying these distinct forms of cardiac remodeling are poorly understood. Here, we demonstrate that miR-148a expression changes dynamically in distinct subtypes of heart failure: while it is elevated in concentric hypertrophy, it decreased in dilated cardiomyopathy. In line, antagomir-mediated silencing of miR-148a caused wall thinning, chamber dilation, increased left ventricle volume, and reduced ejection fraction. Additionally, adeno-associated viral delivery of miR-148a protected the mouse heart from pressure-overload-induced systolic dysfunction by preventing the transition of concentric hypertrophic remodeling toward dilation. Mechanistically, miR-148a targets the cytokine co-receptor glycoprotein 130 (gp130) and connects cardiomyocyte responsiveness to extracellular cytokines by modulating the Stat3 signaling.
These findings show the ability of miR-148a to prevent the transition of pressure-overload induced concentric hypertrophic remodeling toward eccentric hypertrophy and dilated cardiomyopathy and provide evidence for the existence of separate molecular programs inducing distinct forms of myocardial remodeling.
Raso et al. show that miR-148a is elevated in concentric hypertrophy and decreased in dilated cardiomyopathy. Adeno-associated viral delivery of miR-148a protects the mouse heart from cardiac dilation. Mechanistically, miR-148a regulates the sensitivity of the heart to extracellular cytokines.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Medulloblastoma (MB), the tumor of the cerebellum, is the most frequent brain cancer in childhood and a major cause of pediatric mortality. Based on gene profiling, four MB subgroups have been ...identified, i.e., Wnt or Sonic Hedgehog (Shh) types, and subgroup 3 or 4. The Shh-type MB has been shown to arise from the cerebellar precursors of granule neurons (GCPs), where a hyperactivation of the Shh pathway leads to their neoplastic transformation. We have previously shown that the gene Tis21 (PC3/Btg2) inhibits the proliferation and promotes the differentiation and migration of GCPs. Moreover, the overexpression or the deletion of Tis21 in Patched1 heterozygous mice, a model of spontaneous Shh-type MB, highly reduces or increases, respectively, the frequency of MB. Here we tested whether Tis21 can inhibit MB allografts. Athymic nude mice were subcutaneously grafted with MB cells explanted from Patched1 heterozygous mice. MB allografts were then injected with adeno-associated viruses either carrying Tis21 (AAV-Tis21) or empty (AAV-CBA). We observed that the treatment with AAV-Tis21 significantly inhibited the growth of tumor nodules, as judged by their volume, and reduced the number of proliferating tumor cells (labeled with Ki67 or BrdU), relative to AAV-CBA-treated control mice. In parallel, AAV-Tis21 increased significantly tumor cells labeled with early and late neural differentiation markers. Overall the results suggest that Tis21-gene therapy slows down MB tumor growth through inhibition of proliferation and enhancement of neural differentiation. These results validate Tis21 as a relevant target for MB therapy.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Adeno-associated virus (AAV) is a small, non-enveloped virus used as vector in gene therapy, mainly produced in human cells and in baculovirus systems. Intense studies on these platforms led to the ...production of vectors with titers between 10
3
and 10
5
viral genomes (vg) per cells. In spite of this, vector yields need to be improved to satisfy the high product demands of clinical trials and future commercialization. Our studies and those of other groups have explored the possibility to exploit the yeast
Saccharomyces cerevisiae
to produce rAAV. We previously demonstrated that yeast supports AAV genome replication and capsid assembly. The purpose of this study was to evaluate the quality of the encapsidated AAV DNA. Here, we report the construction of a yeast strain expressing Rep68/40 from an integrated copy of the
Rep
gene under the control of the yeast constitutive ADH promoter and Capsid proteins from the
Cap
gene under the control of an inducible GAL promoter. Our results indicate that a portion of AAV particles generated by this system contains encapsidated AAV DNA. However, the majority of encapsidated DNA consists of fragmented regions of the transgene cassette, with ITRs being the most represented sequences. Altogether, these data indicate that, in yeast, encapsidation occurs with low efficiency and that rAAVs resemble pseudo-vectors that are present in clinical-grade rAAV preparations.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Muscle regeneration depends on satellite cells (SCs), quiescent precursors that, in consequence of injury or in pathological states such as muscular dystrophies, activate, proliferate, and ...differentiate to repair the damaged tissue. A subset of SCs undergoes self‐renewal, thus preserving the SC pool and its regenerative potential. Unacylated ghrelin (UnAG) is a circulating hormone that protects muscle from atrophy, promotes myoblast differentiation, and enhances ischemia‐induced muscle regeneration. Here we show that UnAG increases SC activity and stimulates Par polarity complex/p38‐mediated asymmetric division, fostering both SC self‐renewal and myoblast differentiation. Because of those activities on different steps of muscle regeneration, we hypothesized a beneficial effect of UnAG in mdx dystrophic mice, in which the absence of dystrophin leads to chronic muscle degeneration, defective muscle regeneration, fibrosis, and, at later stages of the pathology, SC pool exhaustion. Upregulation of UnAG levels in mdx mice reduces muscle degeneration, improves muscle function, and increases dystrophin‐null SC self‐renewal, maintaining the SC pool. Our results suggest that UnAG has significant therapeutic potential for preserving the muscles in dystrophies. Stem Cells 2017;35:1733–1746
(A): Unacylated ghrelin (UnAG) induces the asymmetric co‐segregation of atypical PKCλ/ι and phospho‐p38 within satellite cells (CD34+) on floating myofibers. Scale bar, 50 µm. (B): The segregation of PKCλ/ι and phospho‐p38 MAPK results in the asymmetric division of satellite cells, giving rise to quiescent stem cells (Pax7+/MyoD–) and cells committed to differentiation (Pax7+/MyoD+). Scale bar, 20 µm.
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