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.

Advances in Bone Biology.

Bone is a unique type of mineralised connective tissue that can support and protect soft tissues, contain bone marrow, and allow movement [...].

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.

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.

MicroRNAs Modulate Signaling Pathways in Osteogenic Differentiation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been identified in many adult tissues and they have been closely studied in recent years, especially in view of their potential use for treating diseases and damaged tissues and organs. MSCs are capable of self-replication and differentiation into osteoblasts and are considered an important source of cells in tissue engineering for bone regeneration. Several epigenetic factors are believed to play a role in the osteogenic differentiation of MSCs, including microRNAs (miRNAs). MiRNAs are small, single-stranded, non-coding RNAs of approximately 22 nucleotides that are able to regulate cell proliferation, differentiation and apoptosis by binding the 3' untranslated region (3'-UTR) of target mRNAs, which can be subsequently degraded or translationally silenced. MiRNAs control gene expression in osteogenic differentiation by regulating two crucial signaling cascades in osteogenesis: the transforming growth factor-beta (TGF-ß)/bone morphogenic protein (BMP) and the Wingless/Int-1(Wnt)/ß-catenin signaling pathways. This review provides an overview of the miRNAs involved in osteogenic differentiation and how these miRNAs could regulate the expression of target genes.

Protocol for the long-term culture of human primary keratinocytes from the normal colorectal mucosa.

Procedures for in vitro culturing of human primary keratinocytes from normal colon mucosa specimens have not been fully feasible, thus far. The protocol described herein allows primary keratinocytes from small tissue fragments of colorectal mucosa biopsies to grow in vitro. The procedure develops in three steps: (a) the enzymatic digestion of the tissue biopsy; (b) the use of cloning rings to purify primary keratinocyte colonies, (c) a defined keratinocyte medium to grow these cells in long-term culture. Our cultural method enables normal primary keratinocytes to be obtained by simple and rapid techniques. In our culture condition, primary keratinocytes express specific epithelial markers. Colorectal mucosa keratinocyte colonies require approximately 2 weeks to grow. Compared with previous approaches, our protocol provides a valuable model of study for human primary keratinocytes from normal colorectal (NCR) mucosa both at the cellular and molecular levels. It is well known, that different mutations occurring during the multistep process of carcinogenesis in the NCR mucosa, are strictly associated to the onset/progression of the colorectal carcinoma. On this ground, normal keratinocytes grown with our protocol, may represent an innovative tool to investigate the mechanisms that lead to colorectal carcinoma and other diseases. Our innovative procedure may allow to perform comparative investigations between normal and pathological colorectal cells.

Innovative Biomaterials for Bone Regrowth.

The regenerative medicine, a new discipline that merges biological sciences and the fundamental of engineering to develop biological substitutes, has greatly benefited from recent advances in the material engineering and the role of stem cells in tissue regeneration. Regenerative medicine strategies, involving the combination of biomaterials/scaffolds, cells, and bioactive agents, have been of great interest especially for the repair of damaged bone and bone regrowth. In the last few years, the life expectancy of our population has progressively increased. Aging has highlighted the need for intervention on human bone with biocompatible materials that show high performance for the regeneration of the bone, efficiently and in a short time. In this review, the different aspects of tissue engineering applied to bone engineering were taken into consideration. The first part of this review introduces the bone cellular biology/molecular genetics. Data on biomaterials, stem cells, and specific growth factors for the bone regrowth are reported in this review.

Porous titanium scaffolds modified with Zeolitic Imidazolate Framework (ZIF-8) with enhanced osteogenic activity for the prevention of implant-associated infections.

In this study, zeolitic imidazolate framework 8 (ZIF-8) was coated on porous Ti6Al4V scaffolds, either bare or previously modified using hydroxyapatite (HA) or HA and gelatin (HAgel), via a growing single-step method in aqueous media using two contact times at 6 h and 24 h. The coated scaffolds termed ZIF-8@Ti, ZIF-8@HA/Ti, and ZIF-8@HAgel/Ti were characterized via scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), and molecular plasma-atomic emission spectroscopy (MP-AES). In order to assess the cell proliferation rate, the cytocompatibility of the scaffolds was evaluated in primary osteoblasts (hOBs) using alamarBlue assay, while the osteoconductivity was analyzed in hOBs using a real-time approach, evaluating the expression of secreted phosphoprotein 1 (SPP1). Osteopontin, which is the protein encoded by this gene, represents the major non-collagenous bone protein that binds tightly to HA. The scaffolds were shown to be non-cytotoxic based on hOB proliferation at all time points of analysis (24 h and 72 h). In hOB cultures, the scaffolds induced the upregulation of SPP1 with different fold changes. Some selected scaffolds were assayed in vitro for their antibacterial potential against Staphylococcus epidermidis; the scaffolds coated with ZIF-8 crystals, regardless of the presence of HA and gelatin, strongly inhibited bacterial adhesion to the materials and reduced bacterial proliferation in the culture medium, demonstrating the suitable release of ZIF-8 in a bioactive form. These experiments suggest that the innovative scaffolds, tested herein, provide a good microenvironment for hOB adhesion, viability, and osteoconduction with effective prevention of S. epidermidis adhesion.

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.

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.

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.

Hsa-microRNA-1249-3p/Homeobox A13 axis modulates the expression of ß-catenin gene in human epithelial cells.

Intercellular adhesion is a key function for epithelial cells. The fundamental mechanisms relying on epithelial cell adhesion have been partially uncovered. Hsa-microRNA-1249-3p (hsa-miR-1249-3p) plays a role in the epithelial mesenchymal transition in carcinoma cells, but its physiological function in epithelial cells is unknown. We aimed to investigate the role and molecular mechanisms of hsa-miR-1249-3p on epithelial cell functions. Hsa-miR-1249-3p was overexpressed in human epithelial cells and uterine cervical tissues, compared to cervical carcinoma cells and precancerous tissues, respectively. Hsa-miR-1249-3p was analyzed to verify its regulatory function on Homeobox A13 (HOXA13) target gene and its downstream cell adhesion gene ß-catenin. Functional experiments indicated that hsa-miR-1249-3p inhibition prompted the mRNA and protein overexpression of HOXA13 which, in turn, led to the ß-catenin protein expression. Moreover, hsa-miR-1249-3p inhibition induced a strong colony forming ability in epithelial cells, suggesting the miR involvement in cell adhesion machinery. These data indicate that hsa-miR-1249-3p regulates the expression of HOXA13 and its downstream cell adhesion gene ß-catenin, possible resulting in cell adhesion modification in epithelial cells. This study will allow the set-up of further investigations aimed at exploring the relationship between the hsa-miR-1249-3p/HOXA13 axis and downstream cell adhesion genes.

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.

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.

Bioactive Materials for Soft Tissue Repair.

Over the past decades, age-related pathologies have increased abreast the aging population worldwide. The increased age of the population indicates that new tools, such as biomaterials/scaffolds for damaged tissues, which display high efficiency, effectively and in a limited period of time, for the regeneration of the body's tissue are needed. Indeed, scaffolds can be used as templates for three-dimensional tissue growth in order to promote the tissue healing stimulating the body's own regenerative mechanisms. In tissue engineering, several types of biomaterials are employed, such as bioceramics including calcium phosphates, bioactive glasses, and glass-ceramics. These scaffolds seem to have a high potential as biomaterials in regenerative medicine. In addition, in conjunction with other materials, such as polymers, ceramic scaffolds may be used to manufacture composite scaffolds characterized by high biocompatibility, mechanical efficiency and load-bearing capabilities that render these biomaterials suitable for regenerative medicine applications. Usually, bioceramics have been used to repair hard tissues, such as bone and dental defects. More recently, in the field of soft tissue engineering, this form of scaffold has also shown promising applications. Indeed, soft tissues are continuously exposed to damages, such as burns or mechanical traumas, tumors and degenerative pathology, and, thereby, thousands of people need remedial interventions such as biomaterials-based therapies. It is known that scaffolds can affect the ability to bind, proliferate and differentiate cells similar to those of autologous tissues. Therefore, it is important to investigate the interaction between bioceramics and somatic/stem cells derived from soft tissues in order to promote tissue healing. Biomimetic scaffolds are frequently employed as drug-delivery system using several therapeutic molecules to increase their biological performance, leading to ultimate products with innovative functionalities. This review provides an overview of essential requirements for soft tissue engineering biomaterials. Data on recent progresses of porous bioceramics and composites for tissue repair are also presented.

Serum Antibodies Against the Oncogenic Merkel Cell Polyomavirus Detected by an Innovative Immunological Assay With Mimotopes in Healthy Subjects.

Merkel cell polyomavirus (MCPyV), a small DNA tumor virus, has been detected in Merkel cell carcinoma (MCC) and in normal tissues. Since MCPyV infection occurs in both MCC-affected patients and healthy subjects (HS), innovative immunoassays for detecting antibodies (abs) against MCPyV are required. Herein, sera from HS were analyzed with a novel indirect ELISA using two synthetic peptides mimicking MCPyV capsid protein epitopes of VP1 and VP2. Synthetic peptides were designed to recognize IgGs against MCPyV VP mimotopes using a computer-assisted approach. The assay was set up evaluating its performance in detecting IgGs anti-MCPyV on MCPyV-positive (n=65) and -negative (n=67) control sera. Then, the ELISA was extended to sera (n=548) from HS aged 18-65 yrs old. Age-specific MCPyV-seroprevalence was investigated. Performance evaluation indicated that the assay showed 80% sensitivity, 91% specificity and 83.9% accuracy, with positive and negative predictive values of 94.3% and 71%, respectively. The ratio expected/obtained data agreement was 86%, with a Cohen's kappa of 0.72. Receiver-operating characteristic (ROC) curves analysis indicated that the areas under the curves (AUCs) for the two peptides were 0.82 and 0.74, respectively. Intra-/inter-run variations were below 9%. The overall prevalence of serum IgGs anti-MCPyV in HS was 62.9% (345/548). Age-specific MCPyV-seroprevalence was 63.1% (82/130), 56.7% (68/120), 64.5% (91/141), and 66.2% (104/157) in HS aged 18-30, 31-40, 41-50 and 51-65 yrs old, respectively (p>0.05). Performance evaluation suggests that our indirect ELISA is reliable in detecting IgGs anti-MCPyV. Our immunological data indicate that MCPyV infection occurs asymptomatically, at a relatively high prevalence, in humans.

Serum HPV16 E7 Oncoprotein Is a Recurrence Marker of Oropharyngeal Squamous Cell Carcinomas.

Despite improved prognosis for many HPV-positive head and neck squamous cell carcinomas (HNSCCs), some cases are still marked by recurrence and metastasis. Our study aimed to identify novel biomarkers for patient stratification. Classical HPV markers: HPV-DNA, p16 and HPV mRNA expression were studied in HNSCC (n = 67) and controls (n = 58) by qPCR. Subsequently, ELISA tests were used for HPV16 L1 antibody and HPV16 E7 oncoprotein detection in serum at diagnosis and follow-up. All markers were correlated to relapse-free survival (RFS) and overall survival (OS). HPV-DNA was found in HNSCCs (29.85%), HPV16-DNA in 95% of cases, HPV16 E7 mRNA was revealed in 93.75%. p16 was overexpressed in 75% of HPV-positive HNSCC compared to negative samples and controls (p < 0.001). Classical markers correlated with improved OS (p < 0.05). Serological studies showed similar proportions of HPV16 L1 antibodies in all HNSCCs (p > 0.05). Serum E7 oncoprotein was present in 30% HPV-positive patients at diagnosis (p > 0.05) and correlated to HNSCC HPV16 E7 mRNA (p < 0.01), whereas it was associated to worse RFS and OS, especially for oropharyngeal squamous cell carcinoma (OPSCC) (p < 0.01). Detection of circulating HPV16 E7 oncoprotein at diagnosis may be useful for stratifying and monitoring HPV-positive HNSCC patients for worse prognosis, providing clinicians a tool for selecting patients for treatment de-escalation.

The role of microRNAs in the osteogenic and chondrogenic differentiation of mesenchymal stem cells and bone pathologies.

Mesenchymal stem cells (MSCs) have been identified in many adult tissues. MSCs can regenerate through cell division or differentiate into adipocytes, osteoblasts and chondrocytes. As a result, MSCs have become an important source of cells in tissue engineering and regenerative medicine for bone tissue and cartilage. Several epigenetic factors are believed to play a role in MSCs differentiation. Among these, microRNA (miRNA) regulation is involved in the fine modulation of gene expression during osteogenic/chondrogenic differentiation. It has been reported that miRNAs are involved in bone homeostasis by modulating osteoblast gene expression. In addition, countless evidence has demonstrated that miRNAs dysregulation is involved in the development of osteoporosis and bone fractures. The deregulation of miRNAs expression has also been associated with several malignancies including bone cancer. In this context, bone-associated circulating miRNAs may be useful biomarkers for determining the predisposition, onset and development of osteoporosis, as well as in clinical applications to improve the diagnosis, follow-up and treatment of cancer and metastases. Overall, this review will provide an overview of how miRNAs activities participate in osteogenic/chondrogenic differentiation, while addressing the role of miRNA regulatory effects on target genes. Finally, the role of miRNAs in pathologies and therapies will be presented.