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A3 adenosine receptor (A3AR) is a cell membrane protein, which has been found to be overexpressed in a large number of cancer types. This receptor plays an important role in cancer by interacting with adenosine. Specifically, A3AR has a dual nature in different pathophysiological conditions, as it is expressed according to tissue type and stimulated by an adenosine dose-dependent manner. A3AR activation leads to tumor growth, cell proliferation and survival in some cases, while triggering cytostatic and apoptotic pathways in others. This review aims to describe the most relevant aspects of A3AR activation and its ligands whereas it summarizes A3AR activities in cancer. Progress in the field of A3AR modulators, with a potential therapeutic role in cancer treatment are reported, as well.

Summary Objectives Polyomavirus (PyV) infections have been associated with different diseases. BK (BKPyV), JC (JCPyV) and simian virus 40 (SV40) are the three main PyVs whose primary infection occurs early in life. Their vertical transmission was investigated in this study. Methods PyV sequences were analyzed by the digital droplet PCR in blood, serum, placenta, amniotic fluid, vaginal smear from two independent cohorts of pregnant females and umbilical cord blood (UCB) samples. IgG antibodies against the three PyVs were investigated by indirect E.L.I.S.As with viral mimotopes. Results DNAs from blood, vaginal smear and placenta tested BKPyV-, JCPyV- and SV40-positive with a distinct prevalence, while amniotic fluids were all PyVs-negative. A prevalence of 3%, 7%, and 3% for BKPyV, JCPyV and SV40 DNA sequences, respectively, was obtained in UCBs. Serum IgG antibodies from pregnant females reached an overall prevalence of 62%, 42% and 17% for BKPyV, JCPyV and SV40, respectively. Sera from newborns (UCB) tested IgG-positive with a prevalence of 10% for BKPyV/JCPyV and 3% for SV40. Conclusions In this investigation, PyV vertical transmission was revealed by detecting PyV DNA sequences and IgG antibodies in samples from females and their offspring suggesting a potential risk of diseases in newborns.

Published data support the hypothesis that viruses could be trigger agents of multiple sclerosis onset. This link is based on evidence of early exposure to viral agents in patients affected by this neurologic disease. JC (JC polyomavirus [JCPyV]), BK (BKPyV), and simian virus 40 (SV40) neurotropic polyomavirus footprints have been detected in brain tissue specimens and samples from patients affected by different neurological diseases. In this investigation, serum samples from patients affected by multiple sclerosis and other inflammatory and noninflammatory neurologic diseases, as well as healthy subjects representing the control, were investigated for immunoglobulin G (IgG) antibodies against JCPyV. To this end, an immunologic approach was employed, which consists of employing indirect enzyme-linked immunosorbent assay testing with synthetic peptides mimicking viral capsid protein 1 antigens. A significantly lower prevalence of IgG antibodies against JCPyV VP1 epitopes, with a low titer, was detected in serum samples from patients with multiple sclerosis (MS) and other neurologic diseases than in healthy subjects. Our study indicates that the prevalence of JCPyV antibodies from patients with multiple sclerosis is 50% lower than in healthy subjects, suggesting specific immune impairments. These results indicate that patients affected by neurological diseases, including MS, respond poorly to JCPyV VP1 antigens, suggesting specific immunologic dysfunctions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly discovered coronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic. COVID-19 has rapidly become a public health emergency of international concern. Although remarkable scientific achievements have been reached since the beginning of the pandemic, the knowledge behind this novel coronavirus, in terms of molecular and pathogenic characteristics and zoonotic potential, is still relatively limited. Today, there is a vaccine, or rather several vaccines, which, for the first time in the history of highly contagious infectious diseases that have plagued mankind, has been manufactured in just one year. Currently, four vaccines are licensed by regulatory agencies, and they use RNA or viral vector technologies. The positive effects of the vaccination campaign are being felt in many parts of the world, but the disappearance of this new infection is still far from being a reality, as it is also threatened by the presence of novel SARS-CoV-2 variants that could undermine the effectiveness of the vaccine, hampering the immunization control efforts. Indeed, the current findings indicate that SARS-CoV-2 is adapting to transmission in humans more efficiently, while further divergence from the initial archetype should be considered. In this review, we aimed to provide a collection of the current knowledge regarding the molecular, phylogenetic, and pathogenetic insights into SARS-CoV-2. The most recent findings obtained with respect to the impact of novel emerging SARS-CoV-2 variants as well as the development and implementation of vaccines are highlighted.

International audience; 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.

Background Diagnostic biomarkers for detecting chronic obstructive pulmonary disease (COPD) in acute coronary syndrome (ACS) patients are not available. SERPINA1, coding for the most potent circulating anti-inflammatory protein in the lung, has been found to be differentially methylated in blood cells from COPD patients. This study aimed to investigate the methylation profile of SERPINA1 in blood cells from ACS patients, with (COPD+) or without COPD (COPD−). Methods Blood samples were from 115 ACS patients, including 30 COPD+ and 85 COPD− according to lung function phenotype, obtained with spirometry. DNA treated with sodium bisulfite was PCR-amplified at SERPINA1 promoter region. Methylation analysis was carried out by sequencing the PCR products. Lymphocytes count in ACS patients was recorded at hospital admission and discharge. Results SERPINA1 was hypermethylated in 24/30 (80%) COPD+ and 48/85 (56.5%) COPD− (p SERPINA1 hypermethylated (1.98 × 103 ± 0.6 cell/µl) than in COPD− carrying SERPINA1 hypomethylated (1.7 × 103 ± 0.48 cell/µl) (p Conclusions SERPINA1 is hypermethylated in blood cells from COPD+ patients. COPD− carrying SERPINA1 hypermethylated and high lymphocytes count may be at risk of COPD development. Therefore, SERPINA1 hypermethylation may represent a potential biomarker for predicting COPD development in ACS patients.

In recent years, a number of studies focused on the role of epigenetics, including DNA methylation, in spermatogenesis and male infertility. We aimed to provide an overview of the knowledge concerning the gene and genome methylation and its regulation during spermatogenesis, specifically in the context of male infertility etiopathogenesis. Overall, the findings support the hypothesis that sperm DNA methylation is associated with sperm alterations and infertility. Several genes have been found to be differentially methylated in relation to impaired spermatogenesis and/or reproductive dysfunction. Particularly, DNA methylation defects of MEST and H19 within imprinted genes and MTHFR within non-imprinted genes have been repeatedly linked with male infertility. A deep knowledge of sperm DNA methylation status in association with reduced reproductive potential could improve the development of novel diagnostic tools for this disease. Further studies are needed to better elucidate the mechanisms affecting methylation in sperm and their impact on male infertility.

Malignant pleural mesothelioma (MPM) is an aggressive asbestos-related cancer arising from the mesothelial cells lining the pleural cavity. MPM is characterized by a silent clinical progression and a highly resistance to conventional chemo/radio-therapies. MPM patients die in a few months/years from diagnosis. Notch signaling is a well-conserved cell communication system, which regulates many biological processes. In humans, the dysregulation of Notch pathway potentially contributes to cancer onset/progression, including MPM. Metformin is the first-line drug used to treat type 2 diabetes mellitus. Metformin is proven to be an effective antitumor drug in preclinical models of different types of cancer. To date, clinical efficacy is being studied in many clinical trials. In this study, the anti-proliferative effect of metformin on MPM cells and the putative involvement of Notch1 as a mediator of metformin activities, were investigated. MPM cells showed high levels of Notch1 activation compared to normal pleural mesothelial cells. Furthermore, metformin treatment hampered MPM cell proliferation and enhanced the apoptotic process, accompanied by decreased Notch1 activation.

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.

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.

Background Killian polyp (KP) is a benign lesion that arises from the maxillary sinus. The etiology of KP is unknown. The aim of this study was to investigate the potential involvement of human papilloma- (HPV) and polyoma-viruses (HPyV) infections in the onset of KP. Methods DNA from antral (n = 14) and nasal (n = 14) KP fractions were analyzed for HPV and HPyV sequences, genotypes, viral DNA load and physical status along with expression of viral proteins and p16 cellular protein. Results The oncogenic HPV16 was detected in 3/14 (21.4%) antral KPs, whilst nasal KPs tested HPV-negative (0/14). The mean HPV16 DNA load was 4.65 ± 2.64 copy/104 cell. The whole HPV16 episomal genome was detected in one KP sample, whereas HPV16 DNA integration in two KPs. P16 mRNA level was lower in the KP sample carrying HPV16 episome than in KPs carrying integrated HPV16 and HPV- negative KPs (p Conclusions A fraction of KP tested positive for the oncogenic HPV16. HPV16 detection in the KP antral portion may be consistent with HPV16 infection derived from the maxillary sinus. HPV16 DNA integration represents a novel finding. Altogether, these data improve our knowledge on the association between KP and HPV infection, whereas it indicates that the KP onset is heterogeneous.

Viral infections are considered to be risk factors for spontaneous abortion (SA). Conflicting results have been reported on the association between Human Papillomavirus (HPV) and SA. HPV DNA was investigated in matched chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from women who experienced SA (n = 80, cases) and women who underwent a voluntary interruption of pregnancy (VI, n = 80, controls) by qualitative PCR and quantitative droplet digital PCR (ddPCR). Viral genotyping was performed using real-time PCR in HPV-positive samples. Specific IgG antibodies against HPV16 were investigated in sera from SA (n = 80) and VI (n = 80) females using indirect ELISA assays. None of the DNA samples from SA subjects was HPV-positive (0/80), whilst HPV DNA was detected in 2.5% of VI women (p &gt, 0.05), with a mean viral DNA load of 7.12 copy/cell. VI samples (n = 2) were found to be positive for the HPV45 genotype. The ddPCR assay revealed a higher number of HPV-positive samples. HPV DNA was detected in 3.7% and 5% of SA and VI chorionic tissues, respectively, with mean viral DNA loads of 0.13 copy/cell in SA and 1.79 copy/cell in VI (p &gt, 0.05) samples. All DNA samples from the PBMCs of SA and VI females tested HPV-negative by both PCR and ddPCR. The overall prevalence of serum anti-HPV16 IgG antibodies was 37.5% in SA and 30% in VI (p &gt, 0.05) women. For the first time, HPV DNA was detected and quantitatively analyzed using ddPCR in chorionic villi tissues and PBMCs from SA and VI women. Circulating IgG antibodies against HPV16 were detected in sera from SA and VI females. Our results suggest that HPV infection in chorionic villi may be a rare event. Accordingly, it is likely that HPV has no significant role in SA.

Background. Previous studies reported human papillomaviruses (HPVs) in middle ear tumors, whereas these viruses have been poorly investigated in chronic inflammatory middle ear diseases. We investigated HPVs in non-tumor middle ear diseases, including chronic otitis media (COM). Methods. COM specimens (n = 52), including chronic suppurative otitis media (CSOM) (n =38) and cholesteatoma (COMC) (n = 14), as well as normal middle ear (NME) specimens (n = 56) were analyzed. HPV sequences and DNA loads were analyzed by quantitative-PCR. HPV genotyping was performed by direct sequencing. Results. HPV DNA was detected in 23% (12/52) of COM and in 30.4% (17/56) of NME (p &gt, 0.05). Specifically, HPV DNA sequences were found in 26.3% (10/38) of CSOM and in 14.3% (2/14) of COMC (p &gt, 0.05). Interestingly, the HPV DNA load was higher in COMC (mean 7.47 copy/cell) than in CSOM (mean 1.02 copy/cell) and NME (mean 1.18 copy/cell) (P = 0.03 and P = 0.017 versus CSOM and NME, respectively). HPV16 and HPV18 were the main genotypes detected in COMC, CSOM and NME. Conclusions. These data suggest that HPV may infect the middle ear mucosa, whereas HPV-positive COMCs are associated with higher viral DNA loads as compared to NME.

18 years ago, in 2002, the world was astonished by the appearance of Severe Acute Respiratory Syndrome (SARS), supported by a zoonotic coronavirus, called SARS-CoV, from the Guangdong Province of southern China. After about 10 years, in 2012, another similar coronavirus triggered the Middle East Respiratory Syndrome (MERS-CoV) in Saudi Arabia. Both caused severe pneumonia killing 774 and 858 people with 8700 cases of confirmed infection for the former, and 2494 for the latter, causing significant economic losses. 8 years later, despite the MERS outbreak remaining in certain parts of the world, at the end of 2019, a new zoonotic coronavirus (SARS-CoV-2) and responsible of coronavirus Disease (COVID-19), arose from Wuhan, Hubei Province, China. It spread rapidly and to date has killed 3,242 persons with more than 81,000 cases of infection in China and causing over 126,000 global cases and 5,414 deaths in 166 other countries around the world, especially Italy. SARS-CoV-2 would seem to have come from a bat, but the intermediate reservoir continues to be unknown. Nonetheless, as for SARS-CoV and MERS CoV, the Spillover effect linked to animal-human promiscuity, human activities including deforestation, illegal bush-trafficking and bushmeat, cannot be excluded. Recently, however, evidence of inter-human only transmission of SARS-CoV-2 has been accumulated and thus, the outbreak seems to be spreading by human-to-human transmission throughout a large part of the world. Herein we will provide with an update on the main features of COVID-19 and suggest possible solutions how to halt the expansion of this novel pandemic.

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 &lt, 0.001). Classical markers correlated with improved OS (p &lt, 0.05). Serological studies showed similar proportions of HPV16 L1 antibodies in all HNSCCs (p &gt, 0.05). Serum E7 oncoprotein was present in 30% HPV-positive patients at diagnosis (p &gt, 0.05) and correlated to HNSCC HPV16 E7 mRNA (p &lt, 0.01), whereas it was associated to worse RFS and OS, especially for oropharyngeal squamous cell carcinoma (OPSCC) (p &lt, 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.

Retinitis pigmentosa (RP) is an inherited retinopathy. Nevertheless, non-genetic biological factors play a central role in its pathogenesis and progression, including inflammation, autophagy and oxidative stress. The retina is particularly affected by oxidative stress due to its high metabolic rate and oxygen consumption as well as photosensitizer molecules inside the photoreceptors being constantly subjected to light/oxidative stress, which induces accumulation of ROS in RPE, caused by damaged photoreceptor’s daily recycling. Oxidative DNA damage is a key regulator of microglial activation and photoreceptor degeneration in RP, as well as mutations in endogenous antioxidant pathways involved in DNA repair, oxidative stress protection and activation of antioxidant enzymes (MUTYH, CERKL and GLO1 genes, respectively). Moreover, exposure to oxidative stress alters the expression of micro-RNA (miRNAs) and of long non-codingRNA (lncRNAs), which might be implicated in RP etiopathogenesis and progression, modifying gene expression and cellular response to oxidative stress. The upregulation of the P2X7 receptor (P2X7R) also seems to be involved, causing pro-inflammatory cytokines and ROS release by macrophages and microglia, contributing to neuroinflammatory and neurodegenerative progression in RP. The multiple pathways analysed demonstrate that oxidative microglial activation may trigger the vicious cycle of non-resolved neuroinflammation and degeneration, suggesting that microglia may be a key therapy target of oxidative stress in RP.

Uveal melanoma (UM), which is the most common cancer of the eye, was investigated in recent years by many teams in the field of biomedical sciences and eye clinicians. New knowledge was acquired on molecular pathways found to be dysregulated during the multistep process of oncogenesis, whereas novel therapeutic approaches gave significant results in the clinical applications. Uveal melanoma-affected patients greatly benefited from recent advances of the research in this eye cancer. Tumour biology, genetics, epigenetics and immunology contributed significantly in elucidating the role of different genes and related pathways during uveal melanoma onset/progression and UM treatments. Indeed, these investigations allowed identification of new target genes and to develop new therapeutic strategies/compounds to cure this aggressive melanoma of the eye. Unfortunately, the advances reported in the treatment of cutaneous melanoma have not produced analogous benefits in metastatic uveal melanoma. Nowadays, no systemic adjuvant therapy has been shown to improve overall survival or reduce the risk of metastasis. However, the increasing knowledge of this disease, and the encouraging results seen in clinical trials, offer promise for future effective therapies. Herein, different pathways/genes involved in uveal melanoma onset/progression were taken into consideration, together with novel therapeutic approaches.

Simian virus 40 (SV40) is a small DNA tumor virus of monkey origin. This polyomavirus was administered to human populations mainly through contaminated polio vaccines, which were produced in naturally infected SV40 monkey cells. Previous molecular biology and recent immunological assays have indicated that SV40 is spreading in human populations, independently from earlier SV40-contaminated vaccines. SV40 DNA sequences have been detected at a higher prevalence in specific human cancer specimens, such as the brain and bone tumors, malignant pleural mesotheliomas, and lymphoproliferative disorders, compared to the corresponding normal tissues/specimens. However, other investigations, which reported negative data, did not confirm an association between SV40 and human tumors. To circumvent the controversies, which have arisen because of these molecular biology studies, immunological researches with newly developed indirect ELISA tests were carried out in serum samples from patients affected by the same kind of tumors as mentioned above. These innovative indirect ELISAs employ synthetic peptides as mimotopes/specific SV40 antigens. SV40 mimotopes do not cross-react with the homologous human polyomaviruses, BKPyV, and JCPyV. Immunological data obtained from indirect ELISAs, using SV40 mimotopes, employed to analyze serum samples from oncological patients, have indicated that these sera had a higher prevalence of antibodies against SV40 compared to healthy subjects. The main data on (i) the biology and genetics of SV40; (ii) the epidemiology of SV40 in the general population, (iii) the mechanisms of SV40 transformation; (iv) the putative role of SV40 in the onset/progression of specific human tumors, and (v) its association with other human diseases are reported in this review.

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.

// Ilaria Bononi 1, * , Manola Comar 2, * , Andrea Puozzo 1, * , Mariarita Stendardo 3 , Piera Boschetto 3 , Sara Orecchia 4 , Roberta Libener 4 , Roberto Guaschino 5 , Silvia Pietrobon 1 , Manuela Ferracin 6 , Massimo Negrini 1, 7 , Fernanda Martini 1 , Massimo Bovenzi 8 , Mauro Tognon 1 1 Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, School of Medicine, University of Ferrara, Ferrara, Italy 2 Institute for Maternal and Child Health - IRCCS “Burlo-Garofolo”– Trieste, University of Trieste, Trieste, Italy 3 Department of Medical Sciences, School of Medicine, University of Ferrara, Ferrara, Italy 4 Mesothelioma BioBank, Pathology Unit and City Hospital, Alessandria, Italy 5 Transfusion Medicine, City Hospital, Alessandria, Italy 6 Department of Experimental, Diagnostic and Specialty Medicine – DIMES, University of Bologna, Bologna, Italy 7 Laboratory for Technologies of Advances Therapies (LTTA), University of Ferrara, Ferrara, Italy 8 Clinical Unit of Occupational Medicine, Department of Medical Sciences, School of Medicine, University of Trieste, Trieste, Italy * These authors have contributed equally to this work Correspondence to: Mauro Tognon, email: tgm@unife.it Fernanda Martini, email: mrf@unife.it Keywords: microRNA, mesothelioma, biomarker, asbestos, worker Received: June 07, 2016 Accepted: September 16, 2016 Published: October 03, 2016 ABSTRACT Malignant pleural mesothelioma (MPM), a fatal cancer, is an occupational disease mostly affecting workers ex-exposed to asbestos fibers. The asbestos, a cancerogenic mineral of different chemical composition, was widely employed in western Countries in industrial manufactures of different types. MPM may arise after a long latency period, up to five decades. MPM is resistant to conventional chemo- and radio-therapies. Altogether, these data indicate that the identification of new and specific markers are of a paramount importance for an early diagnosis and treatment of MPM. In recent years, microRNAs expression was found dysregulated in patients, both in cancer cells and sera, affected by tumors of different histotypes, including MPM. Cell and circulanting microRNAs, found to be dysregulated in this neoplasia, were proposed as new biomarkers. It has been reported that circulating microRNAs are stable in biological fluids and could be employed as potential MPM biomarkers. In this investigation, circulating microRNAs (miR) from serum samples of MPM patients and workers ex-exposed to asbestos fibers (WEA) and healthy subjects (HS) were comparatively analyzed by microarray and RT-qPCR technologies. Our results allowed (i) to select MiR-3665, an endogenous stable microRNA, as the internal control to quantify in our analyses circulating miRNAs; to detect (ii) miR-197-3p, miR-1281 and miR 32-3p up-regulated in MPM compared to HS; (iii) miR-197-3p and miR-32-3p up-regulated in MPM compared to WEA; (iv) miR-1281 up-regulated in both MPM and WEA compared to HS. In conclusion, three circulating up-regulated microRNAs, i.e. miR-197-3p, miR-1281 and miR-32-3p are proposed as potential new MPM biomarkers.

Until less than two decades ago, all known human coronaviruses (CoV) caused diseases so mild that they did not stimulate further advanced CoV research. In 2002 and following years, the scenario changed dramatically with the advent of the new more pathogenic CoVs, including Severe Acute Respiratory Syndome (SARS-CoV-1), Middle Eastern respiratory syndrome (MERS)-CoV, and the new zoonotic SARS-CoV-2, likely originated from bat species and responsible for the present coronavirus disease (COVID-19), which to date has caused 15,581,007 confirmed cases and 635,173 deaths in 208 countries, including Italy. SARS-CoV-2 transmission is mainly airborne via droplets generated by symptomatic patients, and possibly asymptomatic individuals during incubation of the disease, although for the latter, there are no certain data yet. However, research on asymptomatic viral infection is currently ongoing worldwide to elucidate the real prevalence and mortality of the disease. From a clinical point of view, COVID-19 would be defined as “COVID Planet “ because it presents as a multifaceted disease, due to the large number of organs and tissues infected by the virus. Overall, based on the available published data, 80.9% of patients infected by SARS-CoV-2 develop a mild disease/infection, 13.8% severe pneumonia, 4.7% respiratory failure, septic shock, or multi-organ failure, and 3% of these cases are fatal, but mortality parameter is highly variable in different countries. Clinically, SARS-CoV-2 causes severe primary interstitial viral pneumonia and a “cytokine storm syndrome”, characterized by a severe and fatal uncontrolled systemic inflammatory response triggered by the activation of interleukin 6 (IL-6) with development of endothelitis and generalized thrombosis that can lead to organ failure and death. Risk factors include advanced age and comorbidities including hypertension, diabetes, and cardiovascular disease. Virus entry occurs via binding the angiotensin-converting enzyme 2 (ACE2) receptor present in almost all tissues and organs through the Spike (S) protein. Currently, SARS-CoV-2 infection is prevented by the use of masks, social distancing, and improved hand hygiene measures. This review summarizes the current knowledge on the main biological and clinical features of the SARS-CoV-2 pandemic, also focusing on the principal measures taken in some Italian regions to face the emergency and on the most important treatments used to manage the COVID-19 pandemic.

Human osteosarcoma (OS) is a rare human cancer, mostly occurring in children and adolescents. Simian virus 40 (SV40 = Macaca mulatta polyomavirus 1) sequences have been detected in different human cancers, including osteosarcoma. SV40 is an oncogenic virus in vivo, whereas it transforms different kinds of mammalian cells, as well as distinct human cell types. SV40 injected in rodents induces tumors of different histotypes, such as bone and brain tumors. Herein, the association between OS and SV40 large T antigen (Tag) was studied by employing indirect ELISAs using synthetic peptides that mimic different epitopes of the SV40 Tag, the viral oncoprotein. Indirect ELISAs were used to detect serum IgG antibodies against this oncogenic virus in samples from OS patients. Controls were sera from healthy subjects (HS) and oncological patients affect by breast cancer (BC), which is not associated with SV40. It turned out that sera of OS patients had a higher prevalence of SV40 Tag antibodies, 35%, compared to HS, 20% and BC, 19%, respectively. The different prevalence of SV40 Tag antibodies revealed in OS vs. HS and vs. BC is statistically significant with P < 0.05 and P < 0.01, respectively. Our immunological data indicate a significantly higher prevalence of antibodies against SV40 Tag epitopes in serum samples from OS patients compared to HS and BC, the controls. These results suggest an association between OS and SV40 Tag, indicating that this oncogenic virus may be a cofactor in OS development.

Several studies associating BK polyomavirus (BKPyV) and prostate cancer (PCa) suggested that this virus may exert its oncogenic activity at early stages of cancer development. The BKPyV oncogene, the large T antigen (LTag), has frequently been detected in areas of proliferative inflammatory atrophy, which is considered a precursor lesion leading to prostatic intraepithelial neoplasia and overt PCa. In a recently updated systematic review, the presence of BKPyV was significantly higher in PCa tissues than in healthy control tissues, providing an indication for a link between BKPyV infection and cancer risk. In addition, recent original investigations highlighted an association between expression of the virus and the clinical course of PCa. For example, by studying immune responses elicited against BKPyV LTag, a significant association between LTag positive cancer lesions and a peculiar regulatory profiling has been observed in PCa patients with evidence of disease recurrence after surgical radical prostatectomy. Lastly, a study carried out in a larger cohort of patients undergoing radical prostatectomy revealed the IgG response against LTag as an independent predictor of disease recurrence. Although a full picture of the mechanisms potentially responsible for the involvement of BKPyV in PCa is not available yet, continuing work on this topic should help to refine the potential role of BKPyV in PCa patients, perhaps revealing unsuspected associations with the clinical course of this disease.

The association between undifferentiated nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV) is well established. Nevertheless, available evidence suggests that other cofactors are required for the development of undifferentiated NPC. Several investigations reported simian virus 40 (SV40) footprints in human tumors of different histotypes. Serum samples from patients with undifferentiated NPC (n = 64) and healthy subjects (n = 130) were analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) with SV40 synthetic peptides to detect antibodies against viral peptide (VP) capsid proteins VP1, 2, and 3. Immunologic data indicate that in sera from patients with undifferentiated NPC, the prevalence of SV40 antibodies was 25%, whereas in controls it was 16%. This difference is not statistically significant (p > .05). A similar prevalence of SV40 antibodies was detected in undifferentiated NPC and healthy subjects. Our serologic data suggest no association between undifferentiated NPC and SV40 infection. This investigation may stimulate further studies aimed at determining the possible contribution of other risk factors in the pathogenesis of undifferentiated NPC.

BACKGROUND Non-Hodgkin lymphoma (NHL), the most common cancer of the lymphatic system, is of unknown etiology. The identification of etiologic factors in the onset of NHL is a key event that could facilitate the prevention and cure of this malignancy. Simian virus 40 (SV40) has been considered an oncogenic agent in the onset/progression of NHL. METHODS In this study, an indirect enzyme-linked immunosorbent assay with 2 synthetic peptides that mimic SV40 antigens of viral capsid proteins 1 to 3 was employed to detect specific antibodies against SV40. Serum samples were taken from 2 distinct cohorts of NHL-affected patients (NHL1 [n = 89] and NHL2 [n = 61]) along with controls represented by oncologic patients affected by breast cancer (BC; n = 78) and undifferentiated nasopharyngeal carcinoma (UNPC; n = 64) and 3 different cohorts of healthy subjects (HSs; HS1 [n = 130], HS2 [n = 83], and HS3 [n = 87]). RESULTS Immunologic data indicated that in serum samples from NHL patients, antibodies against SV40 mimotopes were detectable with a prevalence of 40% in NHL1 patients and with a prevalence of 43% in NHL2 patients. In HSs of the same median age as NHL patients, the prevalence was 16% for the HS1 group (57 years) and 14% for the HS2 group (65 years). The difference was statistically significant (P

To evaluate the gene expression changes involved in neoplastic progression of cervical intraepithelial neoplasia. Using microarray analysis, large-scale gene expression profile was carried out on HPV16-CIN2, HPV16-CIN3, and normal cervical keratinocytes derived from two HPV16-CIN2, two HPV-CIN3 lesions, and two corresponding normal cervical tissues, respectively. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16-CIN2 keratinocytes and in HPV16-CIN2 keratinocytes compared with HPV16-CIN3 keratinocytes; 37 candidate genes with continuously increasing or decreasing expression during CIN progression were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of CIN toward cancer. Gene expression changes occurring during CIN progression were investigated using microarray analysis, for the first time, in CIN2 and CIN3 keratinocytes naturally infected with HPV16. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression.

BACKGROUND Simian virus 40 (SV40) has been considered to be an oncogenic viral agent in the development of osteosarcoma (OS), which to the authors' knowledge continues to be of unknown etiology. METHODS In the current study, serum samples from patients with OS were investigated with an indirect enzyme-linked immunoadsorbent assay (ELISA) to test for the presence of immunoglobulin G antibodies, which react with SV40 antigens. In ELISA, SV40 antigens were represented by 2 synthetic polypeptides that mimic epitopes of the viral capsid proteins 1 to 3. Additional sera from patients with breast cancer and undifferentiated nasopharyngeal carcinoma as well as healthy subjects were the controls. RESULTS Immunologic results suggested that antibodies that react with SV40 mimotopes were more prevalent (44%) in serum samples from patients with OS compared with healthy subjects (17%). The difference in prevalence between these cohorts was statistically significant (P

Ocular or choroidal nevus (CN) is a rare benign neoplastic lesion of the eye. The cause of CN onset/progression, which arises from the transformation of ocular melanocytes, is not known. A fraction of CN patients may develop uveal melanoma. The objective of this study was to investigate the association between CN and BK polyomavirus (BKPyV), a small DNA tumour virus. Serum IgG antibodies which react with BKPyV antigens were analysed. A indirect E.L.I.S.A. using synthetic peptides that mimic BKPyV antigens was employed. Serum antibodies against BKPyV were also investigated by haemagglutination inhibition (HAI) assay. Sera were from CN patients and healthy subject (HS) were the control. A statistically significant higher prevalence of antibodies against BKPyV capsid protein antigens in serum samples from CN patients was detected, compared to HS, using two independent techniques, indirect E.L.I.S.A. and HAI (87.3% CN vs. 62.1% HS and 91.5% CN vs. 64.4% HS, respectively; p

JC polyomavirus (JCPyV) was identified in 1971 in the brain tissue of a patient (J.C.) affected by the progressive multifocal leukoencephalopathy (PML). JCPyV encodes for the oncoproteins large T antigen (Tag) and small t-antigen (tag). These oncoproteins are responsible of the cell transformation and tumorigenesis in experimental animals. JCPyV is ubiquitous in human populations. After the primary infection, which is usually asymptomatic, JCPyV remains lifelong in the host in a latent phase. Its reactivation may occur in heathy subjects and immunocompromised patients. Upon reactivation, JCPyV could reach (i) the CNS inducing the PML, (ii) the kidney of transplant patients causing the organ rejection. Association between JCPyV, which is a small DNA tumor virus, and gliomas and colorectal carcinomas has been published. In the present investigation, we report on a new indirect ELISA with two specific synthetic peptides mimicking JCPyV VP1 immunogenic epitopes to detect specific serum IgG antibodies against JCPyV. Serum samples of healthy subjects (n = 355) ranging 2-100 years old, were analyzed by this new indirect ELISA. The linear peptides VP1 K and VP1 N resemble the natural JCPyV VP1 capsidic epitopes constituting a docking site for serum antibodies. Data from this innovative immunologic assay indicate that the overall prevalence of JCPyV-VP1 antibodies in healthy subjects is at 39%. The innovative indirect ELISA with JCPyV VP1 mimotopes seems to be a useful method to detect specific IgG antibodies against this virus, without cross-reactivity with the closely related SV40 and BKPyV polyomaviruses.

Purpose: The purpose of this investigation was to characterize Merkel cell carcinomas (MCC) arisen in patients affected by autoimmune diseases and treated with biologic drugs. Experimental Design: Serum samples from patients with MCC were analyzed for the presence and titer of antibodies against antigens of the oncogenic Merkel cell polyomavirus (MCPyV). IgG antibodies against the viral oncoproteins large T (LT) and small T (ST) antigens and the viral capsid protein-1 were analyzed by indirect ELISA. Viral antigens were recombinant LT/ST and virus-like particles (VLP), respectively. MCPyV DNA sequences were studied using PCR methods in MCC tissues and in peripheral blood mononuclear cells (PBMC). Immunohistochemical (IHC) analyses were carried out in MCC tissues to reveal MCPyV LT oncoprotein. Results: MCPyV DNA sequences identified in MCC tissues showed 100% homology with the European MKL-1 strain. PBMCs from patients tested MCPyV-negative. Viral DNA loads in the three MCC tissues were in the 0.1 to 30 copy/cell range. IgG antibodies against LT/ST were detected in patients 1 and 3, whereas patient 2 did not react to the MCPyV LT/ST antigen. Sera from the three patients with MCC contained IgG antibodies against MCPyV VP1. MCC tissues tested MCPyV LT-antigen–positive in IHC assays, with strong LT expression with diffuse nuclear localization. Normal tissues tested MCPyV LT–negative when employed as control. Conclusions: We investigated three new MCCs in patients affected by rheumatologic diseases treated with biologic drugs, including TNF. A possible cause–effect relationship between pharmacologic immunosuppressive treatment and MCC onset is suggested. Indeed, MCC is associated with MCPyV LT oncoprotein activity. Clin Cancer Res; 23(14); 3929–34. ©2017 AACR.

A new immunological investigation was carried out to study the association between non-Hodgkin lymphoma and Simian virus 40 (SV40). To this end, a new indirect ELISA was employed with two mimotopes from SV40 large T antigen (Tag), the viral oncoprotein, to analyse for specific reactions to antibodies in sera from non-Hodgkin lymphoma patients and controls, represented by healthy subjects (HS) and breast carcinoma (BC) patients. This study allowed us to assay a new sera collection from non-Hodgkin lymphoma patients (NHL, n = 254). To verify the association between NHL and SV40 Tag, two totally independent cohorts were analysed: NHL1 n = 150 and NHL2 n = 104. The epidemiological survey included sera from HS1, n = 150; HS2, n = 104 and BC, n = 78. This new indirect ELISA revealed that antibodies against SV40 Tag mimotopes are detectable in NHL1 and NHL2 sera with a prevalence of 37 and 36%, respectively. The prevalence of SV40-antibodies detected in both NHL1 and NHL2 cohorts differs statistically from controls, at 19% for HS1 (p

Background: The relevance of human leukocyte antigen (HLA)-G in dimeric form in multiple sclerosis (MS) is still unknown. Objective: To investigate the contribution of cerebrospinal fluid (CSF) HLA-G dimers in MS pathogenesis. Methods: CSF amounts of 78-kDa HLA-G dimers were measured by western blot analysis in 80 MS relapsing–remitting MS (RRMS) patients and in 81 inflammatory and 70 non-inflammatory controls. Results: CSF amounts of 78kDa HLA-G dimers were more frequent in RRMS than in inflammatory ( pConclusion: Our findings suggest that HLA-G dimers may be implicated in termination of inflammatory response occurring in MS.

// Francesca Amoroso 1 , Erica Salaro 1 , Simonetta Falzoni 1 , Paola Chiozzi 1 , Anna Lisa Giuliani 1 , Giorgio Cavallesco 1 , Pio Maniscalco 1 , Andrea Puozzo 1 , Ilaria Bononi 1 , Fernanda Martini 1 , Mauro Tognon 1 , Francesco Di Virgilio 1 1 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy Correspondence to: Francesco Di Virgilio, email: fdv@unife.it Mauro Tognon, email: mauro.tognon@unife.it Keywords: cancer, mesothelioma, purinergic signalling, extracellular ATP, P2X7 Received: December 16, 2015 Accepted: June 26, 2016 Published: July 06, 2016 ABSTRACT Malignant pleural mesothelioma (MPM) is an aggressive tumor refractory to anti-blastic therapy. MPM cells show several genetic and biochemical defects, e.g. overexpression of oncogenes, downregulation of onco-suppressor genes, dysregulation of microRNA, or alteration of intracellular Ca 2+ homeostasis and of apoptosis. No information is as yet available on purinergic signalling in this tumor. Signalling via the P2X7 (P2RX7 or P2X7R) purinergic receptor is attracting increasing attention as a pathway involved in cancer cell death or proliferation. In this report we show that the P2X7R is expressed by three MPM cell lines established from MPM patients but not by mesothelial cells from healthy subjects (healthy mesothelial cells, HMCs). MPM cell proliferation was inhibited by in vitro incubation in the presence of selective P2X7R antagonists, as well as by stimulation with the P2X7R agonist BzATP. Systemic administration of the selective P2X7R blocker AZ10606120 inhibited in vivo growth of MPM tumors whether implanted subcutaneously (s.c.) or intraperitoneally (i.p.). Our findings suggest that the P2X7R might be a novel target for the therapy of mesothelioma.

STUDY QUESTION Is the methylation status of the methylenetetrahydrofolate reductase (MTHFR) promoter region in semen samples associated with 'recurrent spontaneous abortion' (RSA)? SUMMARY ANSWER MTHFR promoter hypermethylation is more frequent in semen samples from RSA couples than in semen samples from infertile couples with no history of RSA (NRSA) and affects the whole sperm population significantly more often. WHAT IS KNOWN ALREADY Modifications to the MTHFR gene such as polymorphisms and promoter methylations are associated with male infertility. STUDY DESIGN, SIZE AND DURATION Retrospective cohort study of semen samples from 20 RSA couples, 147 NRSA couples and 20 fertile men between 2011 and 2012. MATERIALS, SETTING AND METHODS DNA from the semen samples of RSA, NRSA and fertile men were analyzed by methylation-specific PCR amplification using primers which anneal to the methylated or unmethylated cytosine-phosphodiester bond guanine (CpG) islands within the promoter region of MTHFR. The specificity of the PCR products was assessed by DNA sequencing. MAIN RESULTS AND THE ROLE OF CHANCE The methylated MTHFR epigenotype (including samples where it co-existed with unmethylated MTHFR epigenotypes) was detected in 75% of RSA men, 54% of NRSA men and 15% of fertile men. MTHFR methylation was observed in the whole sperm population in semen samples from 55% of RSA men compared with 8% in NRSA men (P < 0.05) and 0% in fertile men (P < 0.05). DNA sequencing analysis was fully concordant with the PCR results and revealed that when MTHFR methylation occurred, CpG islands within the promoter region were 100% methylated (hypermethylation of MTHFR promoter). LIMITATIONS, REASONS FOR CAUTION The relatively small sample size of RSA infertile couples. WIDER IMPLICATIONS OF THE FINDINGS The hypermethylation of the MTHFR gene promoter should be taken into consideration as a novel putative risk factor in RSA etiology. STUDY FUNDING/COMPETING INTEREST(S) Our institution has received an FAR research grant from the University of Ferrara, Ferrara, Italy. No competing interests declared.

Hepatitis E virus (HEV) is a fecally and orally transmitted human pathogen of worldwide distribution. In industrial countries, HEV is observed in an increasing number of autochthonous cases and is considered to be an emerging pathogen. A growing body of evidence suggests that HEV is a zoonotic disease, and pig handlers and pig veterinarians have been reported to be high-risk groups for HEV infection. The aims of the present study were to establish the prevalence of anti-HEV in wild boars in France and to identify whether forestry workers are at a higher risk of HEV infection. Three different anti-HEV tests were used to compare their effectiveness in detecting anti-HEV in the general population. The most sensitive test was then used to investigate HEV seroprevalence in 593 forestry workers and 421 wild boars. Anti-HEV was detected in 31% of the forestry workers and 14% of the wild boars. Detection of anti-HEV in humans was correlated with age, geographical location, and occupational activity and in wild boars was correlated with geographical location. HEV infection is frequent in woodcutters in France, and it varies geographically. Further studies are needed to confirm these findings and to elucidate the transmission route and the exact virus reservoirs. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

The size of human cervical intraepithelial neoplasia (CIN) biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small CIN a tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue-derived fibroblasts and keratinocytes were co-cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17, and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate.

Background Malignant pleural mesothelioma (MPM) is a rare but aggressive asbestos-related cancer that develops by mesothelial cell transformation. At present, there are no effective therapies for MPM. Great efforts have been made in finding specific markers/mechanisms for MPM onset, including studies into microRNAs (miRNAs). Recent studies have shown the differential expression of mature miRNAs in several human cancers, suggesting their potential role as oncogenes or tumor suppressor genes. Methods In this study, we investigated miRNAs profile in five human normal pleural mesothelial short-term cell cultures (HMCs) and five MPMs, with microarray approach. These results were confirmed by real-time quantitative reverse-transcriptase polymerase chain reaction and Western blotting. Results A comparative analysis of miRNA expression in MPM and HMCs was carried out. Microarray profiling showed different miRNA expression between MPM and HMCs. Specifically, members of the oncomiRNA miR 17-92 cluster and its paralogs, namely miR 17-5p, 18a, 19b, 20a, 20b, 25, 92, 106a, 106b , were markedly upregulated. Besides, in our investigation, additional miRNAs, such as miR-7, miR-182, miR-214, and miR-497 were found to be dysregulated in MPM. Conclusions These data are in agreement with results that have previously been reported on dysregulated miRNAs for other solid human tumors. Moreover, in our investigation, additional miRNAs were found to be dysregulated in MPM. Interestingly, gene products that regulate the cell cycle are targets and predicted targets for these miRNAs. Our data suggest that specific miRNAs could be key players in MPM development/progression. In addition, some of these miRNAs may represent MPM markers and potential targets for new therapeutic approaches.

Rationale: A strong link has been established between exposure to asbestos and increased risk for pleural malignant mesothelioma (MM). Adenosine plays a key role in inflammatory processes and cancer, where it is involved in the regulation of cell death and proliferation.Objectives: The primary aim of this study was to investigate the presence of adenosine receptors (ARs) in human MM pleura (MMP) and healthy mesothelial pleura (HMP). To shed some light on the interaction between adenosine and MM, the presence and functionality of ARs were explored in human healthy mesothelial cells (HMC) and in malignant mesothelioma cells (MMC).Methods: ARs were analyzed by using reverse transcriptase–polymerase chain reaction, Western blotting, and saturation binding assays. HMC were treated with crocidolite asbestos, which is the principal risk factor for MM. The role of A3ARs on these cellular models, evaluating cAMP production, Akt phosphorylation, and nuclear factor (NF)-κB activation, was investigated. The dual effe...

Merkel cell carcinoma (MCC) is an uncommon neuroendocrine tumour of the skin; rare cases have been reported within the lymph nodes without a primary site. The detection of Merkel cell polyomavirus (MCPyV) DNA, integrated within the genome of MCC, suggests its role for the onset of this tumour. We report a case of MCC in an inguinal lymph node of a patient with Von Willebrand disease (VWD), who underwent multiple blood transfusions following haemorrhoidectomy. The diagnosis was performed on the bases of morphology and immunohistochemistry; genomic sequences of LT and VP1 regions of MCPyV were amplified from MCC using a quantitative polymerase chain reaction (qPCR) assay. High levels of MCPyV antibodies were detected in the patient's serum by ELISA method. We discuss the role of MCPyV in the development of this tumour, the use of viral DNA detection for confirming the diagnosis of MCC in unusual sites and the possibility of MCPyV transmission from blood donors.

IMPORTANCE: Molecular alterations in lichen sclerosus–associated vulvar squamous cell carcinoma (LS-VSCC) are largely unknown. OBJECTIVE: To determine whether the retinoic acid receptor β (RARβ) tumor-suppressor gene is involved in the onset and/or progression of LS-VSCC. DESIGN, SETTING, AND PARTICIPANTS: The case-control study, conducted at University-Hospital of Ferrara, Italy, included 20 LS-VSCC (mean [SD] age, 75 [3] years) and 20 cancer-associated vulvar LS (caVLS; mean [SD] age, 62 [11] years) formalin-fixed embedded tissue specimens, 20 cancer-free vulvar LS (cfVLS), and 20 normal skin fresh specimens from diagnostic biopsies and women surgically treated for nonmalignant skin lesions, respectively. RARβ gene expression and promoter methylation were investigated in LS-VSCC and caVLS adjacent to VSCC specimens, and in cfVLS and normal skin specimens, as controls, by RT-Q real-time polymerase chain reaction (PCR) analysis, and sequencing of PCR-amplified bisulfite-treated DNA. c-Jun expression, an RARβ pathway–related gene, was also investigated. MAIN OUTCOMES AND MEASURES: RARβ expression, correlation with its promoter methylation and c-Jun expression, and association with onset or progression of LS-VSCC. RESULTS: In LS-VSCC, RARβ messenger RNA was 3.4-, 3.6-, and 4.8-fold lower than in caVLS (P = .001), cfVLS (P = .005), and normal skin (P

International audience; Viral agents seem to be linked to multiple sclerosis (MS). This association is based on evidence of (1) early exposure to viruses and MS onset; (2) increased prevalence of MS disease in specific geographic regions; (3) likelihood of developing MS being more prevalent in high-risk areas; (4) altered immune responses to different viruses. In this study, sera from patients affected by MS and controls, represented by sera from patients with other neurologic diseases, both inflammatory and non-inflammatory, and from healthy donors, were investigated for the presence of antibodies against neurotropic polyomaviruses BKV, JCV and SV40 in their sera. Our study has indicated that the prevalence of BKV antibodies in sera from MS patients is higher than that detected in normal individuals, while levels of antibodies against BKV and JCV are lower in MS patients compared to those of normal subjects.

The failure of apoptosis may contribute to the formation of cancer and to its resistance to therapy. Malignant pleural mesothelioma (MPM) is an aggressive tumor that responds poorly to standard chemo- and radio-therapies. Several studies have demonstrated that a plethora of oncogenes and tumor suppressors contribute to MPM onset/progression. Importantly, most of these genes are involved in the regulation of calcium (Ca2+)-handling. Cellular Ca2+ signaling is an important regulator of many physiological processes, and it has been widely reported to participate in the regulation of apoptotic cell death in cancer cells and tissues. However, in MPM the role of cellular Ca2+ has been poorly investigated. Therefore, we examined whether Ca2+ is involved in MPM. We found that mesothelioma cell lines and short-term cultures obtained from MPM-affected patients exhibited a critical dysregulation in Ca2+ signaling. We determined that this characteristic was associated with resistance to apoptotic stimuli and that correction of intracellular Ca2+ signaling resulted in the rescue of efficient apoptotic responses. In addition, we discovered that mitochondrial Ca2+-uptake plays a pivotal role as an inducer of apoptosis in MPM. Altogether, these findings suggest the identification of new MPM markers, which in turn could be potential targets for new therapeutic approaches.

Pleural malignant mesothelioma is an uncommon neoplasm usually associated with asbestos exposure. The increasing incidence of malignant mesothelioma cases involving individuals with low levels of asbestos exposure suggests a complex carcinogenetic process with the involvement of other cofactors. Cytogenetic studies revealed the complexity of the genetic changes involved in this neoplasm reflecting the accumulation of genomic damage. One of the most used methodologies for assessing genomic damage is the cytokinesis-blocked micronucleus test applied in peripheral blood lymphocytes (PBL). This approach allows the detection of chromosomal alterations expressed in binucleated cells after nuclear division in vitro. This marker could provide a tool for assessing genetically determined constitutional differences in chromosomal instability. A biomonitoring study was carried out to evaluate the micronuclei frequency in PBLs of patients with pleural malignant mesothelioma with respect to lung cancer, healthy, and risk controls as a marker of cancer susceptibility in correlation with the presence of SV40. A significant increased micronuclei frequency was observed in patients with malignant mesothelioma in comparison with all the other groups, the mean micronuclei frequency was double in patients with malignant mesothelioma compared with healthy controls, risk controls, and patients with lung adenocarcinoma (median 11.4 binucleated cells with micronuclei/1,000 binucleated cells versus 6.2, 6.1, and 5.1, respectively). Our data indicate that human T lymphocyte samples carry DNA sequences coding for SV40 large T antigen at low prevalence, both in cancer cases and controls. Evidence of cytogenetic damage revealed as micronuclei frequency in mesothelioma cancer patients could be related to exogenous and endogenous cofactors besides asbestos exposure.

Background. Late-onset hemorrhagic cystitis (HC) is a well-known severe complication of bone marrow transplantation (BMT), both in adults and in children. Protracted postengraftment HC is associated with graft-versus-host disease and viral infections, mainly caused by BK virus (BKV) or adenovirus (AV). This study investigated whether simian virus 40 (SV40) DNA sequences can be detected in specimens from pediatric patients affected by severe postengraftment HC. Methods. The clinical diagnosis of HC was made in 7 of 28 BMT children. DNA from peripheral blood mononuclear cells (PBMC) and urine sediment cells and supernatants was analyzed by polymerase chain reaction (PCR) for human cytomegalovirus (HCMV), AV, BKV, JC virus (JCV), and SV40. DNA filter hybridization and sequencing was carried out in SV40-positive samples. Results. SV40 footprints were detected in two of seven cases of HC. Specific SV40 DNA sequences were detected by PCR and by filter hybridization both in urine and in PBMC samples at the HC onset and during the follow-up. The DNA sequencing proved that the amplicons belonged to the SV40 wild-type. Urine samples of the two HC cases tested negative by cell cultures, PCR, or both for HCMV, BKV, JCV, and AV. Conclusions. The detection of SV40 DNA sequences suggest that this simian polyomavirus could be involved, at least in some cases, in the HC occurring in children after BMT.

Simian virus 40 (SV40) sequences of the early region coding for the large T antigen (Tag) oncoprotein were investigated in DNA samples from human pleomorphic adenoma (PA) of parotid glands. Specific SV40 sequences were detected, by PCR and filter hybridization with an internal oligoprobe, in 28 of 45 (62%) human PA specimens. None of the DNA samples from 11 normal salivary gland tissues was SV40-positive. DNA sequence analysis, carried out in all PCR amplified products from SV40-positive PA specimens, confirmed the SV40 specificity and indicated that PCR products had a sequence not distinguishable from SV40 DNA wild-type strain 776. SV40 Tag expression was revealed by immunohistochemistry with the specific monoclonal antibody Pab 101 in PA thin sections with a highly sensitive technical approach which retrieved the nuclear viral oncoprotein in 26 out of 28 (93%) samples previously found SV40-positive by PCR. Detection of SV40 sequences and Tag expression in human PA suggests that this oncogenic virus may play a role as a cofactor in the onset and/or progression of this benign neoplasm, or that SV40 DNA could replicate and express the Tag in PA cells.

Merkel cell polyomavirus (MCPyV), a DNA tumor virus, has been found to be associated with Merkel cell carcinoma and chronic lymphocytic leukemia. MCPyV sequences have also been detected in various normal tissues in tumor-affected patients. Immunologic studies have detected MCPyV antibodies in as many as 80% of healthy blood donors. This high seroprevalence suggests that MCPyV infection is widespread in humans. In our study, buffy coats, which were examined for MCPyV DNA Tag sequences, showed a prevalence of 22%. Viral DNA load was revealed in blood samples from 10 to 100 molecules/100 000 cells. DNA sequencing confirmed that polymerase chain reaction amplicons belong to the MCPyV strain, MKL-1. To interpret the putative role of MCPyV in chronic lymphocytic leukemia, we may infer that, during a long period of viral persistence in blood cells, this DNA tumor virus may generate mutants, which are able to participate as cofactors in the multistep process of cell transformation.

Glioblastoma multiforme (GBM) is the most severe form of cancer among gliomas. Indeed, GBM is a World Health Organization grade IV tumor of the CNS, with a mean survival time of 0.5 years.1 Its incidence is ∼1/100 000, which represents 30% of all CNS malignancies.2 According to the recent Eurocare 3 report,3 the mean age of patients affected by glioblastoma is 66 years. The extremely grim prognosis of GBM patients probably explains why such a rare tumor accounts for 2% of all cancer-related deaths in both Europe and the United States.4 GBM is characterized by high genotypic and phenotypic heterogeneities, but it is invariably and homogeneously resistant to conventional chemo/radiotherapy.1 The vast majority (95%) of GBM is represented by primary de novo glioblastomas.5 GBM is considered, like other tumors, a genetic disease of the somatic cell due to an impressive series of alterations of the cellular genome, such as point mutations, gene amplifications, translocations, and deletions, as well as epigenetic modifications, including methylation of specific genes. A limited percentage (5%) of GBM is linked to genetic predispositions, which are mainly associated with rare hereditary syndromes, such as Li–Fraumeni syndrome, Turcot syndrome, Peutz–Jegher syndrome, and neurofibromatosis 1 and 2.2 Exposure to chemical and physical carcinogenic agents, the genetic predisposition, and/or infections by oncogenic viruses, including simian virus 40 (SV40), have been repeatedly advocated in the literature.6,7 SV40 is a nonenveloped small DNA virus of ∼5.2 kb in size. SV40 was recognized in the 1960s as a contaminant of both inactivated (Salk) and live attenuated (Sabin) antipoliomyelitis vaccines. As a consequence, SV40 was inadvertently administered to human populations worldwide mainly by antipoliomyelitis vaccines, produced in SV40 naturally infected monkey kidney cells. Soon after its isolation, SV40 was characterized as a transforming and tumorigenic viral agent.6,7 Cell transformation and tumorigenesis by SV40 is induced by 2 oncoproteins, the large T antigen (Tag) and the small t antigen (tag), which target key cellular proteins, such as the tumor suppressor p53 and retinoblastoma family proteins, inactivating their functions.6,8 This small DNA tumor virus was found to be associated with some human cancers, including GBM. SV40 footprints were detected at high prevalence in GBM specimens, but SV40 sequences have also been revealed, although at lower prevalence, in blood specimens from healthy donors.9–11 In this context it is worth recalling that other investigations did not find SV40 footprints in human brain tumor specimens12,13 or higher SV40 antibodies in serum samples from brain tumor patients than in normal individuals.14 It has been reported that SV40-negative data on human tumor samples obtained by some groups were affected by technical flaws that invalidate the conclusions.15–17 A considerable debate, because of these contrasting reports, has developed in the scientific community.6,7 SV40 antibody detection had been performed in most cases using serologic methods with SV40 virus-like particles, but due to the high protein homology among the 3 main polyomaviruses—SV40, BKV, and JCV—final results were always influenced by some cross-reactivity.18–24 Altogether these reports prompted us to use an indirect ELISA with synthetic peptides employed as specific mimotopes, mimicking SV40 viral capsid antigens. Indeed, recent investigations have reported on the development of a specific and sensitive serologic test for SV40. The test is based on an indirect ELISA employing synthetic peptides as mimotopes/antigens of SV40 viral capsid proteins (VPs), without cross-reactivity with other closely related human polyomaviruses, such as BKV and JCV.25–28 This immunologic assay was used to detect specific serum antibodies against SV40 VPs in normal individuals of different ages and oncologic patients affected by malignant pleural mesothelioma and breast cancer (BC).25–28 In this investigation, serum samples from GBM patients were comparatively analyzed for the presence and titer of antibodies against SV40 infection, with serum samples from healthy individuals and from other reliable controls. No other slow virus infections were investigated.

Many investigations were carried out on the association between viruses and multiple sclerosis (MS). Indeed, early studies reported the detections of neurotropic virus footprints in the CNS of patients with MS. In this study, sera from patients affected by MS, other inflammatory (OIND) and non-inflammatory neurologic diseases (NIND) were analyzed for antibodies against the polyomavirus, Simian Virus 40 (SV40). An indirect enzyme-linked immunosorbent assay (ELISA), with two synthetic peptides, which mimic SV40 antigens, was employed to detect specific antibodies in sera from patients affected by MS, OIND, NIND and healthy subjects (HS). Immunologic data indicate that in sera from MS patients antibodies against SV40 mimotopes are detectable with a low prevalence, 6%, whereas in HS of the same mean age, 40 yrs, the prevalence was 22%. The difference is statistically significant (P = 0.001). Significant is also the difference between MS vs. NIND patients (6% vs. 17%; P = 0.0254), whereas no significant difference was detected between MS vs OIND (6% vs 10%; P>0.05). The prevalence of SV40 antibodies in MS patients is 70% lower than that revealed in HS.