Retraction Note: Presence of viral spike protein and vaccinal spike protein in the blood serum of patients with long-COVID syndrome.

The article "Presence of viral spike protein and vaccinal spike protein in the blood serum of patients with long-COVID syndrome", by K. Dhuli, M.C. Medori, C. Micheletti, K. Donato, F. Fioretti, A. Calzoni, A. Praderio, M.G. De Angelis, G. Arabia, S. Cristoni, S. Nodari, M. Bertelli, published in Eur Rev Med Pharmacol Sci 2023; 27 (6 Suppl): 13-19-DOI: 10.26355/eurrev_202312_34685-PMID: 38112944 has been retracted by the Editor in Chief for the following reasons. Following some concerns raised on PubPeer, the Editor in Chief has started an investigation to assess the validity of the results. The outcome of the investigation revealed that the manuscript presented major flaws in the following: -       Unclear methodology and patient recruitment -       Discrepancies among data reported in the text and tables -       Unreliable results -       Undeclared conflict of interest Consequently, the Editor in Chief mistrusts the results presented and has decided to withdraw the article. The authors disagree with this retraction. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/34685.

Serum proteomic profiling reveals potential inflammatory biomarkers in long-COVID patients: a comparative analysis with healthy controls.

OBJECTIVE: The highly transmissible severe acute respiratory syndrome-Coronavirus-2 was responsible for the 2020 COVID-19 pandemic. COVID-19 mostly affects the respiratory system; however, this infection also affects several other organs. In addition, the sequelae of this disease affect patients for several months after recovery, resulting in long-COVID syndrome. PATIENTS AND METHODS: In order to characterize the differences between healthy control individuals and long-COVID patients, proteomic profiling of the serum of both groups was performed by mass spectrometry. The obtained data were analyzed with multivariate and univariate statistical analyses. RESULTS: Initially, performing a partial latent square discriminant analysis (PLS-DA) made it possible to identify thirty-three proteins of interest, which were then subjected to a receiver operating characteristic (ROC) analysis. Four proteins were identified as potential stand-alone biomarkers: Sirtuin 1, Natriuretic Peptide B, Hemopexin, and Arachidonate 5-Lipoxygenase. Moreover, a multivariate ROC analysis identified a panel of biomarkers composed of Natriuretic Peptide B, Anterior Gradient 2 Protein, Adiponectin, Endothelin Converting Enzyme 1, Interferon Induced Transmembrane Protein 1, Mannose Binding Lectin 2, Prostaglandin-Endoperoxide Synthase 2, Pirin, Prostaglandin Reductase 1 and Cystatin C. CONCLUSIONS: The identified biomarkers are associated with inflammatory processes, corroborating literature evidence that long-COVID patients develop an inflammatory state that damages many tissues. Nevertheless, these data should be validated in a larger cohort.

Linking pathogenic and likely pathogenic gene variants to long-COVID symptoms.

OBJECTIVE: Long-COVID is a clinical syndrome characterized by the presence of symptoms related to SARS-CoV-2 infection that persist for at least four weeks after recovery from COVID-19. Genetics have been proposed to play an important role in long-COVID syndrome onset. This study aimed to identify genetic pathogenetic and likely pathogenetic causative variants of Mendelian genetic diseases in patients with Long-COVID syndrome. Additionally, we aimed to establish an association between these genetic variants and the clinical symptoms manifested during long-COVID syndrome. PATIENTS AND METHODS: 95 patients affected by long-COVID syndrome were analyzed with a Next-Generation Sequencing (NGS) panel comprising 494 genes. The analyzed genes and the symptoms of the patients collected with an ad-hoc questionnaire were divided into four groups (cardiological, respiratory, immunological, and neurological). Finally, a statistical analysis comprising descriptive statistics, classification based on reported symptoms, and comparative analysis against a control group of healthy individuals was conducted. RESULTS: 12 patients resulted positive for genetic testing with an autosomal dominance (8) or autosomal recessive (4) inheritance, showing a higher prevalence of cardiovascular genetic diseases (9) in the analyzed cohort compared to the normal population. Moreover, the onset of the long-COVID syndrome and its cardiovascular manifestations was compliant with the onset reported in the literature for the identified genetic diseases, suggesting that COVID-19 could manifest late-onset genetic diseases associated with their appearance. Apart from the 12 positive patients, 57 were healthy carriers of genetic diseases. Analyzing the whole cohort, a statistical correlation between prevalent symptomatology and the gene class was established, suggesting an association between the genetic susceptibility of an individual and the possibility of developing specific long-COVID syndrome symptoms, especially cardiovascular symptoms. Furthermore, 17 genetic variants were identified in CFTR. Finally, we identified genetic variants in IFNAR2 and POLG, supporting their respective involvement in inflammation and mitochondria mechanisms, correlated with long-COVID syndrome according to literature data. CONCLUSIONS: This study proposed COVID-19 to act as a manifest of underlying late-onset genetic diseases Mendelian associated with carrier status. Moreover, according to our results, mutations in cardiological genes are more present in patients who show cardiological symptoms during the syndrome. This underscores the necessity for cardiological investigation and genetic screening in long-COVID patients to address existing or potential clinical implications.

Autoantibodies in patients with post-COVID syndrome: a possible link with severity?

OBJECTIVE: Coronavirus disease 2019 is an infectious disease associated with the respiratory system caused by the SARS-CoV-2 virus. Right now, an increasing number of patients with Post-COVID Syndrome show, without clear evidence of organ dysfunction, a plethora of severe symptoms, such as fatigue, pain, shortness of breath, cognitive impairment, and sleep disturbance. It has already been demonstrated that SARS-CoV-2 virus can disrupt the self-tolerance mechanism of the immune system, thus triggering autoimmune conditions. Several studies have recently documented the presence of autoantibodies in the sera of post-COVID patients, but until now, it is unclear whether the persistence of symptoms could be directly correlated with the presence of autoantibodies. PATIENTS AND METHODS: In this study, serum autoantibodies (AAbs) levels against four G protein-coupled receptors in 78 patients with post-COVID syndrome have been analyzed. The AAbs investigated are clustered in two groups: adrenergic receptors (α1 and ß2) and muscarinic acetylcholine receptors (M3 and M4). RESULTS: At least one or more AAbs were detected in 60.3% (47/78) of patients diagnosed with post-COVID syndrome, whereas 37.2% (29/78) of patients were positive for all receptors investigated. Interestingly, a strong correlation has been found between AAbs and pain intensity feeling by the patients measured by Visual Analogic Scale. A significant association was also obtained with insomnia and AABS-positive patients. CONCLUSIONS: The identification of AAbs and their correlation with pathological symptoms seriousness underly the possible role of AAbs as future therapeutic targets.

Presence of viral spike protein and vaccinal spike protein in the blood serum of patients with long-COVID syndrome.

OBJECTIVE: COVID-19 patients experience, in 10-20% of the cases, a prolonged long-COVID syndrome, defined as the persistence of symptoms for at least two months after the infection. The underlying biological mechanisms of this syndrome remain poorly understood. Several hypotheses have been proposed, among which are the potential autoimmunity resulting from molecular mimicry between viral spike protein and human proteins, the reservoir and viral reproduction hypothesis, and the viral integration hypothesis. Although official data state that vaccinal spike protein is harmless and remains at the site of infection, several studies proposed spike protein toxicity and found it in blood circulation several months after the vaccination. To search for the presence of viral and vaccine spike protein in a cohort of long-COVID patients. PATIENTS AND METHODS: In this study, we employed a proteomic-based approach utilizing mass spectrometry to analyze the serum of 81 patients with long-COVID syndrome. Moreover, viral integration in patients' leukocytes was assessed with a preliminary study, without further investigation. RESULTS: We identified the presence of the viral spike protein in one patient after infection clearance and negativity of COVID-19 test and the vaccine spike protein in two patients two months after the vaccination. CONCLUSIONS: This study, in agreement with other published investigations, demonstrates that both natural and vaccine spike protein may still be present in long-COVID patients, thus supporting the existence of a possible mechanism that causes the persistence of spike protein in the human body for much longer than predicted by early studies. According to these results, all patients with long-COVID syndrome should be analyzed for the presence of vaccinal and viral spike protein.

One year of surgical mask testing at the University of Bologna labs: Lessons learned from data analysis.

The outbreak of SARS-CoV-2 pandemic highlighted the worldwide lack of surgical masks and personal protective equipment, which represent the main defense available against respiratory diseases as COVID-19. At the time, masks shortage was dramatic in Italy, the first European country seriously hit by the pandemic: aiming to address the emergency and to support the Italian industrial reconversion to the production of surgical masks, a multidisciplinary team of the University of Bologna organized a laboratory to test surgical masks according to European regulations. The group, driven by the expertise of chemical engineers, microbiologists, and occupational physicians, set-up the test lines to perform all the functional tests required. The laboratory started its activity on late March 2020, and as of the end of December of the same year 435 surgical mask prototypes were tested, with only 42 masks compliant to the European standard. From the analysis of the materials used, as well as of the production methods, it was found that a compliant surgical mask is most likely composed of three layers, a central meltblown filtration layer and two external spunbond comfort layers. An increase in the material thickness (grammage), or in the number of layers, does not improve the filtration efficiency, but leads to poor breathability, indicating that filtration depends not only on pure size exclusion, but other mechanisms are taking place (driven by electrostatic charge). The study critically reviewed the European standard procedures, identifying the weak aspects; among the others, the control of aerosol droplet size during the bacterial filtration test results to be crucial, since it can change the classification of a mask when its performance lies near to the limiting values of 95 or 98%.

High-Frequency Vibration Treatment of Human Bone Marrow Stromal Cells Increases Differentiation toward Bone Tissue.

In order to verify whether differentiation of adult stem cells toward bone tissue is promoted by high-frequency vibration (HFV), bone marrow stromal cells (BMSCs) were mechanically stimulated with HFV (30 Hz) for 45 minutes a day for 21 or 40 days. Cells were seeded in osteogenic medium, which enhances differentiation towards bone tissue. The effects of the mechanical treatment on differentiation were measured by Alizarin Red test, (q) real-time PCR, and protein content of the extracellular matrix. In addition, we analyzed the proliferation rate and apoptosis of BMSC subjected to mechanical stimulation. A strong increase in all parameters characterizing differentiation was observed. Deposition of calcium was almost double in the treated samples; the expression of genes involved in later differentiation was significantly increased and protein content was higher for all osteogenic proteins. Lastly, proliferation results indicated that stimulated BMSCs have a decreased growth rate in comparison with controls, but both treated and untreated cells do not enter the apoptosis process. These findings could reduce the gap between research and clinical application for bone substitutes derived from patient cells by improving the differentiation protocol for autologous cells and a further implant of the bone graft into the patient.

The role of PKCε-dependent signaling for cardiac differentiation.

Protein kinase Cepsilon (PKCε) exerts a well-known cardio-protective activity in ischemia-reperfusion injury and plays a pivotal role in stem cell proliferation and differentiation. Although many studies have been performed on physiological and morphological effects of PKCε mis-expression in cardiomyocytes, molecular information on the role of PKCε on early cardiac gene expression are still lacking. We addressed the molecular role of PKCε in cardiac cells using mouse cardiomyocytes and rat bone marrow mesenchymal stem cells. We show that PKCε is modulated in cardiac differentiation producing an opposite regulation of the cardiac genes NK2 transcription factor related, locus 5 (nkx2.5) and GATA binding protein 4 (gata4) both in vivo and in vitro. Phospho-extracellular regulated mitogen-activated protein kinase 1/2 (p-ERK1/2) levels increase in PKCε over-expressing cells, while pkcε siRNAs produce a decrease in p-ERK1/2. Indeed, pharmacological inhibition of ERK1/2 rescues the expression levels of both nkx2.5 and gata4, suggesting that a reinforced (mitogen-activated protein kinase) MAPK signaling is at the basis of the observed inhibition of cardiac gene expression in the PKCε over-expressing hearts. We demonstrate that PKCε is critical for cardiac cell early gene expression evidencing that this protein is a regulator that has to be fine tuned in precursor cardiac cells.

Bone production by human maxillary sinus mucosa cells.

The Schneider membrane is the mucosa that covers the inner part of the maxillary sinus cavities. The free surface is a ciliated pseudostratified epithelium, while the deeper portion is a highly vascularized connective tissue. The stromal fraction, bordering the bony wall of the sinus, after tooth loss can exhibit increased osteoclastic activity resulting in resorption of the bone in the posterior maxilla. Goal of our study was to isolate and characterize mesenchymal progenitors in the Schneider's membrane connective net and to evaluate their self ability to differentiate toward osteoblastic lineage, in absence of osteoinductive factors and osteoconductive biomaterials of support. This should indicate that maxillary sinus membrane represents an useful an approachable source of MSCs for bone tissue engineering and cell therapy and owns the intrinsic capacity to restore maxillary bone after tooth loss without the needing of biomaterials.

The differentiation of human adipose-derived stem cells (hASCs) into osteoblasts is promoted by low amplitude, high frequency vibration treatment.

Several studies have demonstrated that tissue culture conditions influence the differentiation of human adipose-derived stem cells (hASCs). Recently, studies performed on SAOS-2 and bone marrow stromal cells (BMSCs) have shown the effectiveness of high frequency vibration treatment on cell differentiation to osteoblasts. The aim of this study was to evaluate the effects of low amplitude, high frequency vibrations on the differentiation of hASCs toward bone tissue. In view of this goal, hASCs were cultured in proliferative or osteogenic media and stimulated daily at 30Hz for 45min for 28days. The state of calcification of the extracellular matrix was determined using the alizarin assay, while the expression of extracellular matrix and associated mRNA was determined by ELISA assays and quantitative RT-PCR (qRT-PCR). The results showed the osteogenic effect of high frequency vibration treatment in the early stages of hASC differentiation (after 14 and 21days). On the contrary, no additional significant differences were observed after 28days cell culture. Transmission Electron Microscopy (TEM) images performed on 21day samples showed evidence of structured collagen fibers in the treated samples. All together, these results demonstrate the effectiveness of high frequency vibration treatment on hASC differentiation toward osteoblasts.

The homeobox gene Arx is a novel positive regulator of embryonic myogenesis.

Skeletal muscle fibers form in overlapping, but distinct phases that depend on the generation of temporally different lineages of myogenic cells. During primary myogenesis (E10.5-E12.5 in the mouse), embryonic myoblasts fuse homotypically to generate primary fibers, whereas during later development (E14.5-E17.5), fetal myoblasts differentiate into secondary fibers. How these myogenic waves are regulated remains largely unknown. Studies have been hampered by the lack of markers which would distinguish embryonic from fetal myoblast populations. We show here that the homeobox gene Arx is strongly expressed in differentiating embryonic muscle, downstream of myogenic basic helix-loop-helix (bHLH) genes. Its expression progressively decreases during development. When overexpressed in the C2C12 myogenic cell line, Arx enhances differentiation. Accordingly, it stimulates the transcriptional activity from the Myogenin promoter and from multimerized E-boxes when co-expressed with MyoD and Mef2C in CH310T1/2. Furthermore, Arx co-immunoprecipitates with Mef2C, suggesting that it participates in the transcriptional regulatory network acting in embryonic muscle. Finally, embryonic myoblasts isolated from Arx-deficient embryos show a delayed differentiation in vivo together with an enhanced clonogenic capacity in vitro. We propose here that Arx acts as a novel positive regulator of embryonic myogenesis by synergizing with Mef2C and MyoD and by establishing an activating loop with Myogenin.

Surface modification of a porous polyurethane through a culture of human osteoblasts and an electromagnetic bioreactor.

There is increasing interest in new biomaterials and new culture methods for bone tissue engineering, in order to produce, in vitro, living constructs able to integrate in the surrounding tissue. Using an electromagnetic bioreactor (magnetic field intensity, 2 mT; frequency, 75 Hz), we investigated the effects of electromagnetic stimulation on SAOS-2 human osteoblasts seeded onto a porous polyurethane. In comparison with control conditions, the electromagnetic stimulation caused higher cell proliferation, increased surface coating with decorin and type-I collagen, and higher calcium deposition. The immunolocalization of decorin and type-I collagen showed their colocalization in the cell-rich areas. The use of an electromagnetic bioreactor aimed at obtaining the surface modification of the porous polyurethane in terms of cell colonization and coating with calcified matrix. The superficially modified biomaterial could be used, in clinical applications, as an implant for bone repair.

Cell therapy of primary myopathies.

Mesoangioblasts are multipotent progenitors of mesodermal tissues. In vitro mesoangioblasts differentiate into many mesoderm cell types, such as smooth, cardiac and striated muscle, bone and endothelium. After transplantation mesoangioblasts colonize mostly mesoderm tissues and differentiate into many cell types of the mesoderm. When delivered through the arterial circulation, mesoangioblasts significantly restore skeletal muscle structure and function in a mouse model of muscular dystrophy. Their ability to extensively self-renew in vitro, while retaining multipotency, qualifies mesoangioblasts as a novel class of stem cells. Phenotype, properties and possible origin of mesoangioblasts are addressed in the first part of this paper. In the second part we will focus on the cell therapy approach for the treatment of Muscular Dystrophy and we will describe why mesangioblasts appear to be promising candidates for this strategy.

A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay-Sachs disease.

Therapy for neurodegenerative lysosomal Tay-Sachs (TS) disease requires active hexosaminidase (Hex) A production in the central nervous system and an efficient therapeutic approach that can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, we re-established the Hex A activity and totally removed the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month's treatment. In our studies, no adverse effects were observed due to the viral vector, injection site or gene expression and on the basis of these results, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect.

Restoration of the GM2 ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model.

The therapeutic potential of bone marrow-derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow-derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the alpha-subunit of the lysosomal enzyme beta-hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow-derived stromal cells have beta-hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, beta-hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.

Cardiomyocytes induce endothelial cells to trans-differentiate into cardiac muscle: implications for myocardium regeneration.

The concept of tissue-restricted differentiation of postnatal stem cells has been challenged by recent evidence showing pluripotency for hematopoietic, mesenchymal, and neural stem cells. Furthermore, rare but well documented examples exist of already differentiated cells in developing mammals that change fate and trans-differentiate into another cell type. Here, we report that endothelial cells, either freshly isolated from embryonic vessels or established as homogeneous cells in culture, differentiate into beating cardiomyocytes and express cardiac markers when cocultured with neonatal rat cardiomyocytes or when injected into postischemic adult mouse heart. Human umbilical vein endothelial cells also differentiate into cardiomyocytes under similar experimental conditions and transiently coexpress von Willebrand factor and sarcomeric myosin. In contrast, neural stem cells, which efficiently differentiate into skeletal muscle, differentiate into cardiomyocytes at a low rate. Fibroblast growth factor 2 and bone morphogenetic protein 4, which activate cardiac differentiation in embryonic cells, do not activate cardiogenesis in endothelial cells or stimulate trans-differentiation in coculture, suggesting that different signaling molecules are responsible for cardiac induction during embryogenesis and in successive periods of development. The fact that endothelial cells can generate cardiomyocytes sheds additional light on the plasticity of endothelial cells during development and opens perspectives for cell autologous replacement therapies.

Determination, diversification and multipotency of mammalian myogenic cells.

In amniotes, myogenic commitment appears to be dependent upon signaling from neural tube and dorsal ectoderm, that can be replaced by members of the Wnt family and by Sonic hedgehog. Once committed, myoblasts undergo different fates, in that they can differentiate immediately to form the myotome, or later to give rise to primary and secondary muscle fibers. With fiber maturation, satellite cells are first detected; these cells contribute to fiber growth and regeneration during post-natal life. We will describe recent data, mainly from our laboratory, that suggest a different origin for some of the cells that are incorporated into the muscle fibers during late development. We propose the possibility that these myogenic cells are derived from the vasculature, are multi-potent and become committed to myogenesis by local signaling, when ingressing a differentiating muscle tissue. The implications for fetal and perinatal development of the whole mesoderm will also be discussed.

Skeletal myogenic potential of human and mouse neural stem cells.

Distinct cell lineages established early in development are usually maintained throughout adulthood. Thus, adult stem cells have been thought to generate differentiated cells specific to the tissue in which they reside. This view has been challenged; for example, neural stem cells can generate cells that normally originate from a different germ layer. Here we show that acutely isolated and clonally derived neural stem cells from mice and humans could produce skeletal myotubes in vitro and in vivo, the latter following transplantation into adult animals. Myogenic conversion in vitro required direct exposure to myoblasts, and was blocked if neural cells were clustered. Thus, a community effect between neural cells may override such myogenic induction. We conclude that neural stem cells, which generate neurons, glia and blood cells, can also produce skeletal muscle cells, and can undergo various patterns of differentiation depending on exposure to appropriate epigenetic signals in mature tissues.

Ultrastructural changes in human gingival fibroblasts in vitro after exposure to vapour phase smoke components.

Tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including gingival fibroblasts. Because normal gingival fibroblast functioning is fundamental to the maintenance of the oral connective tissue as well as to wound healing, we examined the effect of two vapour phase smoke components (acrolein and acetaldehyde) on proliferation and ultrastructure of human gingival fibroblasts (HGFs) in culture. A human gingival fibroblast strain derived from healthy individuals was used in this study. The cells were incubated in the presence of different concentrations of acrolein and acetaldehyde and cell proliferation and fine morphology were evaluated. The results show that acrolein and acetaldehyde produced dose dependent inhibition of HGF viability and alteration of cytoplasmic organelles. The main ultrastructural finding for the HGF cytoplasm was the presence of vacuoles and lysosomal structures which became prominent with increasing concentration of acrolein and acetaldehyde. Our results suggest that the ultrastructural alterations we observed in HGFs may be due to the uptake and storage of acrolein and acetaldehyde by the cells.

Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration.

Skeletal muscle in vertebrates is derived from somites, epithelial structures of the paraxial mesoderm, yet many unrelated reports describe the occasional appearance of myogenic cells from tissues of nonsomite origin, suggesting either transdifferentiation or the persistence of a multipotent progenitor. Here, we show that clonable skeletal myogenic cells are present in the embryonic dorsal aorta of mouse embryos. This finding is based on a detailed clonal analysis of different tissue anlagen at various developmental stages. In vitro, these myogenic cells show the same morphology as satellite cells derived from adult skeletal muscle, and express a number of myogenic and endothelial markers. Surprisingly, the latter are also expressed by adult satellite cells. Furthermore, it is possible to clone myogenic cells from limbs of mutant c-Met-/- embryos, which lack appendicular muscles, but have a normal vascular system. Upon transplantation, aorta-derived myogenic cells participate in postnatal muscle growth and regeneration, and fuse with resident satellite cells.The potential of the vascular system to generate skeletal muscle cells may explain observations of nonsomite skeletal myogenesis and raises the possibility that a subset of satellite cells may derive from the vascular system.