Osteosarcoma cell death induced by innovative scaffolds doped with chemotherapeutics.

Osteosarcoma (OS) cancer treatments include systemic chemotherapy and surgical resection. In the last years, novel treatment approaches have been proposed, which employ a drug-delivery system to prevent offside effects and improves treatment efficacy. Locally delivering anticancer compounds improves on high local concentrations with more efficient tumour-killing effect, reduced drugs resistance and confined systemic effects. Here, the synthesis of injectable strontium-doped calcium phosphate (SrCPC) scaffold was proposed as drug delivery system to combine bone tissue regeneration and anticancer treatment by controlled release of methotrexate (MTX) and doxorubicin (DOX), coded as SrCPC-MTX and SrCPC-DOX, respectively. The drug-loaded cements were tested in an in vitro model of human OS cell line SAOS-2, engineered OS cell line (SAOS-2-eGFP) and U2-OS. The ability of doped scaffolds to induce OS cell death and apoptosis was assessed analysing cell proliferation and Caspase-3/7 activities, respectively. To determine if OS cells grown on doped-scaffolds change their migratory ability and invasiveness, a wound-healing assay was performed. In addition, the osteogenic potential of SrCPC material was evaluated using human adipose derived-mesenchymal stem cells. Osteogenic markers such as (i) the mineral matrix deposition was analysed by alizarin red staining; (ii) the osteocalcin (OCN) protein expression was investigated by enzyme-linked immunosorbent assay test, and (iii) the osteogenic process was studied by real-time polymerase chain reaction array. The delivery system induced cell-killing cytotoxic effects and apoptosis in OS cell lines up to Day 7. SrCPC demonstrates a good cytocompatibility and it induced upregulation of osteogenic genes involved in the skeletal development pathway, together with OCN protein expression and mineral matrix deposition. The proposed approach, based on the local, sustained release of anticancer drugs from nanostructured biomimetic drug-loaded cements is promising for future therapies aiming to combine bone regeneration and anticancer local therapy.

Acute Prosthetic Joint Infections with Poor Outcome Caused by Staphylococcus Aureus Strains Producing the Panton-Valentine Leukocidin.

The aim of this study was to investigate whether the presence of Staphylococcus aureus (SA) producing the Panton-Valentine leukocidin (PVL) affects the outcome of Prosthetic Joint Infection (PJI). Patients with acute and chronic PJI sustained by SA were prospectively enrolled at the orthopedic unit of "Casa di Cura Santa Maria Maddalena", from January 2019 to October 2021. PJI diagnosis was reached according to the diagnostic criteria of the International Consensus Meeting on PJI of Philadelphia. Synovial fluid obtained via joint aspirations was collected in order to isolate SA. The detection of PVL was performed via real-time quantitative PCR (RT-qPCR). The outcome assessment was performed using the criteria of the Delphi-based International Multidisciplinary Consensus. Twelve cases of PJI caused by SA were included. Nine (75%) cases were acute PJI treated using debridement, antibiotic and implant retention (DAIR); the remaining three (25%) were chronic PJI treated using two-stage (n = 2) and one-stage revision (n = 1), respectively. The SA strains that tested positive for PVL genes were 5/12 (41.6%,). Treatment failure was documented in three cases of acute PJI treated using DAIR, all supported by SA-PVL strains (p < 0.045). The remaining two cases were chronic PJI treated with a revision arthroplasty (one and two stage, respectively), with a 100% eradication rate in a medium follow-up of 24 months. Although a small case series, our study showed a 100% failure rate in acute PJI, probably caused by SA PVL-producing strains treated conservatively (p < 0.04). In this setting, toxin research should guide radical surgical treatment and targeted antibiotic therapy.

Immortalized erythroid cells as a novel frontier for in vitro blood production: current approaches and potential clinical application.

BACKGROUND: Blood transfusions represent common medical procedures, which provide essential supportive therapy. However, these procedures are notoriously expensive for healthcare services and not without risk. The potential threat of transfusion-related complications, such as the development of pathogenic infections and the occurring of alloimmunization events, alongside the donor's dependence, strongly limits the availability of transfusion units and represents significant concerns in transfusion medicine. Moreover, a further increase in the demand for donated blood and blood transfusion, combined with a reduction in blood donors, is expected as a consequence of the decrease in birth rates and increase in life expectancy in industrialized countries. MAIN BODY: An emerging and alternative strategy preferred over blood transfusion is the in vitro production of blood cells from immortalized erythroid cells. The high survival capacity alongside the stable and longest proliferation time of immortalized erythroid cells could allow the generation of a large number of cells over time, which are able to differentiate into blood cells. However, a large-scale, cost-effective production of blood cells is not yet a routine clinical procedure, as being dependent on the optimization of culture conditions of immortalized erythroid cells. CONCLUSION: In our review, we provide an overview of the most recent erythroid cell immortalization approaches, while also describing and discussing related advancements of establishing immortalized erythroid cell lines.

Human Mesenchymal Stem Cells and Innovative Scaffolds for Bone Tissue Engineering Applications.

Stem cell-based therapy is a significant topic in regenerative medicine, with a predominant role being played by human mesenchymal stem cells (hMSCs). The hMSCs have been shown to be suitable in regenerative medicine for the treatment of bone tissue. In the last few years, the average lifespan of our population has gradually increased. The need of biocompatible materials, which exhibit high performances, such as efficiency in bone regeneration, has been highlighted by aging. Current studies emphasize the benefit of using biomimetic biomaterials, also known as scaffolds, for bone grafts to speed up bone repair at the fracture site. For the healing of injured bone and bone regeneration, regenerative medicine techniques utilizing a combination of these biomaterials, together with cells and bioactive substances, have drawn a great interest. Cell therapy, based on the use of hMSCs, alongside materials for the healing of damaged bone, has obtained promising results. In this work, several aspects of cell biology, tissue engineering, and biomaterials applied to bone healing/regrowth will be considered. In addition, the role of hMSCs in these fields and recent progress in clinical applications are discussed. Impact Statement The restoration of large bone defects is both a challenging clinical issue and a socioeconomic problem on a global scale. Different therapeutic approaches have been proposed for human mesenchymal stem cells (hMSCs), considering their paracrine effect and potential differentiation into osteoblasts. However, different limitations are still to be overcome in using hMSCs as a therapeutic opportunity in bone fracture repair, including hMSC administration methods. To identify a suitable hMSC delivery system, new strategies have been proposed using innovative biomaterials. This review provides an update of the literature on hMSC/scaffold clinical applications for the management of bone fractures.

Epigenetic investigation into circulating microRNA 197-3p in sera from patients affected by malignant pleural mesothelioma and workers ex-exposed to asbestos.

The epigenetic role of microRNAs is established at both physiological and pathological levels. Dysregulated miRNAs and their targets appear to be a promising approach for innovative anticancer therapies. In our previous study, circulating miR-197-3p tested dysregulated in workers ex-exposed to asbestos (WEA). Herein, an epigenetic investigation on this circulating miRNA was carried out in sera from malignant pleural mesothelioma (MPM) patients. MiR-197-3p was quantified in MPM (n = 75) sera and comparatively analyzed to WEA (n = 75) and healthy subject (n = 75) sera, using ddPCR and RT-qPCR techniques. Clinicopathological characteristics, occupational, non-occupational information and overall survival (OS) were evaluated in correlation studies. MiR-197-3p levels, analyzed by ddPCR, were significantly higher in MPM than in WEA cohort, with a mean copies/µl of 981.7 and 525.01, respectively. Consistently, RT-qPCR showed higher miR-197-3p levels in sera from MPM with a mean copies/µl of 603.7, compared to WEA with 336.1 copies/µl. OS data were significantly associated with histologic subtype and pleurectomy. Circulating miR-197-3p is proposed as a new potential biomarker for an early diagnosis of the MPM onset. Indeed, miR-197-3p epigenetic investigations along with chest X-ray, computed tomography scan and spirometry could provide relevant information useful to reach an early and effective diagnosis for MPM.

Mg-based materials diminish tumor spreading and cancer metastases.

Cancer metastases are the most common causes of cancer-related deaths. The formation of secondary tumors at different sites in the human body can impair multiple organ function and dramatically decrease the survival of the patients. In this stage, it is difficulty to treat tumor growth and spreading due to arising therapy resistances. Therefore, it is important to prevent cancer metastases and to increase subsequent cancer therapy success. Cancer metastases are conventionally treated with radiation or chemotherapy. However, these treatments elicit lots of side effects, wherefore novel local treatment approaches are currently discussed. Recent studies already showed anticancer activity of specially designed degradable magnesium (Mg) alloys by reducing the cancer cell proliferation. In this work, we investigated the impact of these Mg-based materials on different steps of the metastatic cascade including cancer cell migration, invasion, and cancer-induced angiogenesis. Both, Mg and Mg-6Ag reduced cell migration and invasion of osteosarcoma cells in coculture with fibroblasts. Furthermore, the Mg-based materials used in this study diminished the cancer-induced angiogenesis. Endothelial cells incubated with conditioned media obtained from these Mg and Mg-6Ag showed a reduced cell layer permeability, a reduced proliferation and inhibited cell migration. The tube formation as a last step of angiogenesis was stimulated with the presence of Mg under normoxia and diminished under hypoxia.

Immunological evidence of an early seroconversion to oncogenic Merkel cell polyomavirus in healthy children and young adults.

Oncogenic Merkel cell polyomavirus (MCPyV) provokes a widespread and asymptomatic infection in humans. Herein, sera from healthy children and young adults (HC, n = 344) aged 0-20 years old were evaluated for anti-MCPyV immunoglobulin G (IgG) and IgM antibodies employing a recently developed immunoassay. Serum MCPyV IgG data from healthy subjects (HS, n = 510) and elderlies (ES, n = 226), aged 21-65/66-100 years old, from our previous studies, were included. The anti-MCPyV IgG and IgM rates in HC sera were 40.7% and 29.7%, respectively. A lower prevalence of anti-MCPyV IgGs was found in HC aged 0-5 years old (13%) compared to 6-10 (52.3%), 11-15 (60.5%) and 16-20 years old (61.6%) cohorts. Age-stratified HCs exhibited similar anti-MCPyV IgM rates (27.9%-32.9%). Serological profiles indicated that anti-MCPyV IgGs and IgMs had low optical densities (ODs) during the first years of life, while IgM ODs appeared to decrease throughout young adulthood. A lower anti-MCPyV IgGs rate was found in HC (40.7%) than HS (61.8%) and ES (63.7%). Upon the 5-years range age-stratification, a lower anti-MCPyV IgGs rate was found in the younger HC cohort aged 0-5 years old compared to the remaining older HC/HS/ES cohorts (52.3%-72%). The younger HC cohort exhibited the lowest anti-MCPyV IgG ODs than the older cohorts. Low anti-MCPyV IgMs rates and ODs were found in the 21-25 (17.5%) and 26-30 (7.7%) years old cohorts. Our data indicate that, upon an early-in-life seroconversion, the seropositivity for oncogenic MCPyV peaks in late childhood/young adulthood and remains at high prevalence and relatively stable throughout life.

Prevalence of IgG antibodies against Malawi polyomavirus in patients with autoimmune diseases and lymphoproliferative disorders subjected to bone marrow transplantation.

Introduction: Human polyomaviruses (HPyVs) cause persistent/latent infections in a large fraction of the population. HPyV infections may cause severe diseases in immunocompromised patients. Malawi polyomavirus (MWPyV) is the 10th discovered human polyomavirus (HPyV 10). MWPyV was found in stool samples of healthy children. So far, the few investigations carried out on HPyV 10 did not find an association with human disease. Methods: In this study, to verify the putative association between MWPyV and human diseases, MWPyV seroprevalence was investigated in patients affected by i) lymphoproliferative disorders (LPDs) and ii) immune system disorders, i.e., autoimmune diseases (ADs), and in iii) healthy subjects. An indirect ELISA, employing virus-like particles (VLPs) to detect serum IgG antibodies against MWPyV/HPyV 10, was carried out. The study also revealed the prevalence of another polyomavirus, Merkel cell polyomavirus (MCPyV). Results: Sera from patients with distinct autoimmune diseases (n = 44; mean age 20 years) had a prevalence of MWPyV antibodies of 68%, while in patients with lymphoproliferative disorders (n = 15; mean age 14 years), subjected to bone marrow transplantation, the prevalence was 47%. In healthy subjects (n = 66; mean age 13 years), the prevalence of MWPyV antibodies was 67%. Our immunological investigation indicates that MWPyV/HPyV 10 seroconversion occurs early in life and MWPyV/HPyV 10 appears to be another polyomavirus ubiquitous in the human population. A significantly lower MWPyV antibody reactivity together with a lower immunological profile was detected in the sera of LPD patients compared with HS2 (*p < 0.05) (Fisher's exact test). LPD and AD patients have a similar MCPyV seroprevalence compared with healthy subjects. Discussion: MWPyV seroprevalence indicates that this HPyV is not associated with lymphoproliferative and autoimmune diseases. However, the ability to produce high levels of antibodies against MWPyV appears to be impaired in patients with lymphoproliferative disorders. Immunological investigations indicate that MWPyV seroconversion occurs early in life. MCPyV appears to be a ubiquitous polyomavirus, like other HPyVs, in the human population.

Corrigendum: Serum IgG antibodies from pregnant women reacting to mimotopes of Simian virus 40 large T antigen, the viral oncoprotein.

[This corrects the article DOI: 10.3389/fimmu.2017.00411.].

Predictive Factors of Surgical Site Infection in Prosthetic Joint Surgery: A Prospective Study on 760 Arthroplasties.

Purpose: The success of total joint arthroplasty (TJA) has led to consistent growth in the use of arthroplasty in progressively younger patients. However, more than 10 percent of patients require revision surgery due to implant failure caused by aseptic or septic inflammation. Among the latter, surgical site infection (SSI) represents one of the worst complications of TJA, potentially resulting in the removal of the prosthesis. The aim of our study was to identify potential risk factors for SSIs in a population of patients undergoing TJA. Methods: TJA were prospectively recruited at Casa di Cura Santa Maria Maddalena from February 2019 to April 2020. Age, sex, major comorbidities, American Society of Anesthesiologists (ASA) class, length of surgery, type of surgical suture, total hospital length of stay, and clinical laboratory data were collected. The study population was then divided into two groups: Group A, normal postoperative course, and Group B, patients who developed SSI at follow-up (17-25 days). Results: 25/760 (3.3%) patients developed SSIs at follow-up. Clinical and demographic parameters were not different between the two groups. Total leucocyte and neutrophil values at discharge resulted to be significatively higher in Group B compared to Group A (p = 0.025 and p = 0.016, respectively). Values of 7860/µL for total leucocyte and 5185/µL for neutrophil count at discharge significantly predicted the future development of SSI (AUC 0.623 and AUC 0.641, respectively; p < 0.05) independently from confounding factors (total leukocytes: O.R. = 3, 69 [95% C.I. 1,63-8,32]; neutrophils: O.R. = 3, 98 [95% C.I. 1,76-8,97]). Deep SSIs has been diagnosed significantly before superficial SSIs (p = 0,008), with a median advance of 9 days. Conclusion: Total leukocytes and neutrophils at discharge seem useful to identify a population at risk for the development of septic inflammation at the surgical site following TJA. Further studies with larger populations are needed to develop a predictive SSIs risk score that should include those variables.

Neurological Disease-Affected Patients, including Multiple Sclerosis, Are Poor Responders to BKPyV, a Human Polyomavirus.

Multiple sclerosis (MS) is a neurological disease characterized by immune dysregulations. Different viruses may act as MS triggering agents. MS patients respond differently to distinct viruses. The aim of our study is to verify the association between the polyomavirus BKPyV and MS, together with other neurological diseases, through the investigation of serum IgG antibodies against the virus. Sera were from patients affected by MS and other neurologic diseases, both inflammatory (OIND) and noninflammatory (NIND). Control sera were from healthy subjects (HS). Samples were analyzed for IgG antibodies against BKPyV with an indirect ELISA with synthetic peptides mimicking the viral capsid protein 1 (VP1) antigens. As control, ELISAs were carried out to verify the immune response against the Epstein-Barr virus (EBV) of patients and controls. In addition, we assessed values for total IgG in each experimental groups. A significant lower prevalence of IgG antibodies against BKPyV VP 1 epitopes, together with a low titer, was detected in sera from MS patients and other inflammatory neurologic diseases than HS. In MS patients and OIND and NIND groups, the EBV-antibody values and total IgG did not differ from HS. Experimental data indicate that patients affected by neurological diseases, including MS, are poor responders to BKPyV VP 1 antigens, thus suggesting specific immunologic dysfunctions for this polyomavirus. Our findings are relevant in understanding the immune reactions implicated in neurological disorders.

Stem Cell Fate and Immunomodulation Promote Bone Regeneration via Composite Bio-Oss®/AviteneTM Biomaterial.

Bone defects in maxillofacial regions lead to noticeable deformity and dysfunctions. Therefore, the use of biomaterials/scaffolds for maxillofacial bone regrowth has been attracting great interest from many surgical specialties and experts. Many approaches have been devised in order to create an optimal bone scaffold capable of achieving desirable degrees of bone integration and osteogenesis. Osteogenesis represents a complex physiological process involving multiple cooperating systems. A tight relationship between the immune and skeletal systems has lately been established using the concept of "osteoimmunology," since various molecules, particularly those regulating immunological and inflammatory processes, are shared. Inflammatory mediators are now being implicated in bone remodeling, according to new scientific data. In this study, a profiler PCR array was employed to evaluate the expression of cytokines and chemokines in human adipose derived-mesenchymal stem cells (hASCs) cultured on porous hydroxylapatite (HA)/Collagen derived Bio-Oss®/Avitene scaffolds, up to day 21. In hASCs grown on the Bio-Oss®/Avitene biomaterial, 12 differentially expressed genes (DEGs) were found to be up-regulated, together with 12 DEG down-regulated. Chemokine CCL2, which affects bone metabolism, tested down-regulated. Interestingly, the Bio-Oss®/Avitene induced the down-regulation of pro-inflammatory inter-leukin IL-6. In conclusion, our investigation carried out on the Bio-Oss®/Avitene scaffold indicates that it could be successfully employed in maxillofacial surgery. Indeed, this composite material has the advantage of being customized on the basis of the individual patients favoring a novel personalized medicine approach.

Sera from Patients with Malignant Pleural Mesothelioma Tested Positive for IgG Antibodies against SV40 Large T Antigen: The Viral Oncoprotein.

Malignant pleural mesothelioma (MPM), a fatal tumor, is mainly linked to the asbestos exposure. It has been reported that together with the inhalation of asbestos fibers, other factors are involved in the MPM onset, including simian virus 40 (SV40). SV40, a polyomavirus with oncogenic potential, induces (i) in vitro the mesenchymal cell transformation, whereas (ii) in vivo the MPM onset in experimental animals. The association between MPM and SV40 in humans remains to be elucidated. Sera (n = 415) from MPM-affected patients (MPM cohort 1; n = 152) and healthy subjects (HSs, n = 263) were investigated for their immunoglobulin G (IgG) against simian virus 40 large tumor antigen (Tag), which is the transforming protein. Sera were investigated with an indirect enzyme-linked immunosorbent assay (ELISA) using two synthetic peptides from SV40 Tag protein. SV40 Tag protein was evaluated by immunohistochemical (IHC) staining on MPM samples (MPM cohort 2; n = 20). Formalin-fixed and paraffin-embedded (FFPE) samples were obtained from MPM patients unrelated to MPM serum donors. The proportion of sera, from MPM patients, showing antibodies against SV40 Tag (34%) was significantly higher compared to HSs (20%) (odds ratio 2.049, CI 95% 1.32-3.224; p=0.0026). Immunohistochemical staining (IHS) assays showed SV40 Tag expression in 8/20, 40% of MPM specimens. These results indicate that SV40 is linked to a large fraction of MPM. It is worth noting that the prevalence of SV40 Tag antibodies detected in sera from cohort 1 of MPM patients is similar to the prevalence of SV40 Tag found to be expressed in FFPE tissues from MPM cohort 2.

Slow degrading Mg-based materials induce tumor cell dormancy on an osteosarcoma-fibroblast coculture model.

Osteosarcoma is one of the most common cancers in young adults and is commonly treated using surgery and chemotherapy. During the past years, these therapy approaches improved but failed to ameliorate the outcomes. Therefore, novel, targeted therapeutic approaches should be established to enhance treatment success while preserving patient's quality of life. Recent studies suggest the application of degradable magnesium (Mg) alloys as orthopedic implants bearing a potential antitumor activity. Here, we examined the influence of Mg-based materials on an osteosarcoma-fibroblast coculture. Both, Mg and Mg-6Ag did not lead to tumor cell apoptosis at low degradation rates. Instead, the Mg-based materials induced cellular dormancy in the cancer cells indicated by a lower number of Ki-67 positive cancer cells and a higher p38 expression. This dormancy-like state could be reversed by reseeding on non-degrading glass slides but could not be provoked by inhibition of the protein kinase R-like endoplasmic reticulum kinase. By investigating the influence of the disjunct surface-near effects of the Mg degradation on cell proliferation, an increased pH was found to be a main initiator of Mg degradation-dependent tumor cell proliferation inhibition.

Genetics and Epigenetics of Bone Remodeling and Metabolic Bone Diseases.

Bone metabolism consists of a balance between bone formation and bone resorption, which is mediated by osteoblast and osteoclast activity, respectively. In order to ensure bone plasticity, the bone remodeling process needs to function properly. Mesenchymal stem cells differentiate into the osteoblast lineage by activating different signaling pathways, including transforming growth factor ß (TGF-ß)/bone morphogenic protein (BMP) and the Wingless/Int-1 (Wnt)/ß-catenin pathways. Recent data indicate that bone remodeling processes are also epigenetically regulated by DNA methylation, histone post-translational modifications, and non-coding RNA expressions, such as micro-RNAs, long non-coding RNAs, and circular RNAs. Mutations and dysfunctions in pathways regulating the osteoblast differentiation might influence the bone remodeling process, ultimately leading to a large variety of metabolic bone diseases. In this review, we aim to summarize and describe the genetics and epigenetics of the bone remodeling process. Moreover, the current findings behind the genetics of metabolic bone diseases are also reported.

Antioxidant Activity of Resveratrol Diastereomeric Forms Assayed in Fluorescent-Engineered Human Keratinocytes.

Resveratrol is a powerful antioxidant molecule. In the human diet, its most important source is in Vitis vinifera grape peel and leaves. Resveratrol exists in two isoforms, cis- and trans. The diastereomeric forms of many drugs have been reported as affecting their activity. The aim of this study was to set up a cellular model to investigate how far resveratrol could counteract cytotoxicity in an oxidant agent. For this purpose, a keratinocyte cell line, which was genetically engineered with jelly fish green fluorescent protein, was treated with the free radical promoter Cumene hydroperoxide. The antioxidant activity of the trans-resveratrol and its diastereomeric mixture was evaluated indirectly in these treated fluorescent-engineered keratinocytes by analyzing the cell number and cell proliferation index. Our results demonstrate that cells, which were pre-incubated with resveratrol, reverted the oxidative damage progression induced by this free radical agent. In conclusion, fluorescent-engineered human keratinocytes represent a rapid and low-cost cellular model to determine cell numbers by studying emitted fluorescence. Comparative studies carried out with fluorescent keratinocytes indicate that trans-resveratrol is more efficient than diastereomeric mixtures in protecting cells from the oxidative stress.

Circulating microRNA-197-3p as a potential biomarker for asbestos exposure.

Asbestos is considered the main cause of diseases in workers exposed to this mineral in the workplace as well as an environmental pollutant. The association between asbestos and the onset of different diseases has been reported, but asbestos exposure specific biomarkers are not known. MicroRNAs (miRNAs) are small, single-strand, non-coding RNAs, with potential value as diagnostic, prognostic, and predictive markers in liquid biopsies. Sera collected from workers ex-exposed to asbestos (WEA) fibers were compared with sera from healthy subjects (HS) of similar age, as liquid biopsies. The expression of the circulating miRNA 197-3p was investigated employing two different highly analytical PCR methods, i.e. RT-qPCR and ddPCR. MiR-197-3p levels were tested in sera from WEA compared to HS. MiR-197-3p tested dysregulated in sera from WEA (n = 75) compared to HS (n = 62). Indeed, miR-197-3p was found to be 2.6 times down-regulated in WEA vs. HS (p = 0.0001***). In addition, an inverse correlation was detected between miR-197-3p expression level and cumulative asbestos exposure, being this miRNA down-regulated 2.1 times in WEA, with high cumulative asbestos exposure, compared to WEA with low exposure (p = 0.0303*). Circulating miR-197-3p, found to be down regulated in sera from WEA, is proposed as a new potential biomarker of asbestos exposure.

Decreased IgG Antibody Response to Viral Protein Mimotopes of Oncogenic Merkel Cell Polyomavirus in Sera From Healthy Elderly Subjects.

Merkel cell polyomavirus (MCPyV) is the main causative agent of Merkel cell carcinoma (MCC), a rare but aggressive skin tumor with a typical presentation age >60 years. MCPyV is ubiquitous in humans. After an early-age primary infection, MCPyV establishes a clinically asymptomatic lifelong infection. In immunocompromised patients/individuals, including elders, MCC can arise following an increase in MCPyV replication events. Elders are prone to develop immunesenescence and therefore represent an important group to investigate. In addition, detailed information on MCPyV serology in elders has been debated. These findings cumulatively indicate the need for new research verifying the impact of MCPyV infection in elderly subjects (ES). Herein, sera from 226 ES, aged 66-100 years, were analyzed for anti-MCPyV IgGs with an indirect ELISA using peptides mimicking epitopes from the MCPyV capsid proteins VP1-2. Immunological data from sera belonging to a cohort of healthy subjects (HS) (n = 548) aged 18-65 years, reported in our previous study, were also included for comparisons. Age-/gender-specific seroprevalence and serological profiles were investigated. MCPyV seroprevalence in ES was 63.7% (144/226). Age-specific MCPyV seroprevalence resulted as 62.5% (25/40), 71.7% (33/46), 64.9% (37/57), 63.8% (30/47), and 52.8% (19/36) in ES aged 66-70, 71-75, 76-80, 81-85, and 86-100 years, respectively (p > 0.05). MCPyV seroprevalence was 67% (71/106) and 61% (73/120) in ES males and females, respectively (p > 0.05). Lack of age-/gender-related variations in terms of MCPyV serological profiles was found in ES (p > 0.05). Notably, serological profile analyses indicated lower optical densities (ODs) in ES compared with HS (p < 0.05), while lower ODs were also determined in ES males compared with HS males (p < 0.05). Our data cumulatively suggest that oncogenic MCPyV circulates in elders asymptomatically at a relatively high prevalence, while immunesenescence might be responsible for a decreased IgG antibody response to MCPyV, thereby potentially leading to an increase in MCPyV replication levels. In the worse scenario, alongside other factors, MCPyV might drive MCC carcinogenesis, as described in elders with over 60 years of age.

Adding a "Notch" to Cardiovascular Disease Therapeutics: A MicroRNA-Based Approach.

Dysregulation of the Notch pathway is implicated in the pathophysiology of cardiovascular diseases (CVDs), but, as of today, therapies based on the re-establishing the physiological levels of Notch in the heart and vessels are not available. A possible reason is the context-dependent role of Notch in the cardiovascular system, which would require a finely tuned, cell-specific approach. MicroRNAs (miRNAs) are short functional endogenous, non-coding RNA sequences able to regulate gene expression at post-transcriptional levels influencing most, if not all, biological processes. Dysregulation of miRNAs expression is implicated in the molecular mechanisms underlying many CVDs. Notch is regulated and regulates a large number of miRNAs expressed in the cardiovascular system and, thus, targeting these miRNAs could represent an avenue to be explored to target Notch for CVDs. In this Review, we provide an overview of both established and potential, based on evidence in other pathologies, crosstalks between miRNAs and Notch in cellular processes underlying atherosclerosis, myocardial ischemia, heart failure, calcification of aortic valve, and arrhythmias. We also discuss the potential advantages, as well as the challenges, of using miRNAs for a Notch-based approach for the diagnosis and treatment of the most common CVDs.

In Vitro Osteoinductivity Assay of Hydroxylapatite Scaffolds, Obtained with Biomorphic Transformation Processes, Assessed Using Human Adipose Stem Cell Cultures.

In this study, the in vitro biocompatibility and osteoinductive ability of a recently developed biomorphic hydroxylapatite ceramic scaffold (B-HA) derived from transformation of wood structures were analyzed using human adipose stem cells (hASCs). Cell viability and metabolic activity were evaluated in hASCs, parental cells and in recombinant genetically engineered hASC-eGFP cells expressing the green fluorescence protein. B-HA osteoinductivity properties, such as differentially expressed genes (DEG) involved in the skeletal development pathway, osteocalcin (OCN) protein expression and mineral matrix deposition in hASCs, were evaluated. In vitro induction of osteoblastic genes, such as Alkaline phosphatase (ALPL), Bone gamma-carboxyglutamate (gla) protein (BGLAP), SMAD family member 3 (SMAD3), Sp7 transcription factor (SP7) and Transforming growth factor, beta 3 (TGFB3) and Tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11)/Receptor activator of NF-κB (RANK) ligand (RANKL), involved in osteoclast differentiation, was undertaken in cells grown on B-HA. Chondrogenic transcription factor SRY (sex determining region Y)-box 9 (SOX9), tested up-regulated in hASCs grown on the B-HA scaffold. Gene expression enhancement in the skeletal development pathway was detected in hASCs using B-HA compared to sintered hydroxylapatite (S-HA). OCN protein expression and calcium deposition were increased in hASCs grown on B-HA in comparison with the control. This study demonstrates the biocompatibility of the novel biomorphic B-HA scaffold and its potential use in osteogenic differentiation for hASCs. Our data highlight the relevance of B-HA for bone regeneration purposes.