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.

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.

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

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

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

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

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

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

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.

Colorectal Carcinoma Affected Patients Are Significantly Poor Responders Against the Oncogenic JC Polyomavirus.

Background: Many investigations reported the association between human tumors and JCPyV, a polyomavirus with oncogenic potential. The association has been supported by studies that found JCPyV footprints in CRC and gliomas of different types. Indeed, JCPyV footprints including its nucleic acids and Tag oncoprotein have been revealed in CRC tissues. Methods: Herein, sera from colorectal carcinoma (CRC) affected patients and healthy individuals (HS), employed as control, were analysed for immunoglobulin G (IgG) antibodies against specific JCPyV viral capsid protein 1 (VP1) antigens. The investigation was carried out employing an innovative immunological assay. Indeed, an indirect enzyme-linked immunosorbent assay (ELISA) with JCPyV VP1 mimotopes was used. JCPyV VP1 mimotopes consisted of synthetic peptides mimicking VP1 epitopes. Results: Sera from CRC affected patients, evaluated using indirect ELISAs with synthetic mimotopes, showed a significant lower prevalence of IgG antibodies against JCPyV VP1 mimotopes (26%) compared to HS (51%), p<0.005. These data were confirmed by another method, the hemagglutination inhibition (HAI) assay. Altogether these results, i.e. the prevalence of serum IgG antibodies against JCPyV VP1 mimotopes from patients with CRC is approximately 50% lower than in HS, are of interest. Discussion: Our data suggest that patients with CRC are significantly poor responders against JCPyV VP1 antigens. It is possible that CRC patients are affected by a specific immunological deregulation. This immunological dysfunction, revelled in CRC patients, may account for their predisposition to the colorectal carcinoma onset.

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.

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.

Association between oncogenic human papillomavirus type 16 and Killian polyp.

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< 0.001). None of the antral and nasal KP samples tested positive for HPyV DNA (0/28). 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.

Irrelevance of Panton-Valentine leukocidin in hidradenitis suppurativa: results from a pilot, observational study.

Panton-Valentine leukocidin (PVL) appears to be a virulence factor which, among others, can exacerbate the pathogenicity of Staphylococcus aureus infections, especially inducing severe necrotic, deep-seated skin infections, abscesses, and recurrences. These peculiarities have some overlaps with hidradenitis suppurativa (HS). Our main aim was to assess if S. aureus producing PVL could have some role in influencing clinical features and/or course of HS, specifically in the suppuration and recurrence of lesions. This pilot, mono-centric, observational study included all adult subjects affected with HS consecutively referring to our HS clinic over a 3-month period. Clinically evident suppuration and at least 2 weeks wash out from any antibiotic were the main inclusion criteria. Purulent material from HS skin lesions was collected with swabs in order to isolate micro-organisms, with specific regard to S. aureus. Detection of PVL was performed by real-time quantitative PCR (RT-qPCR). We also analyzed purulent material from suppurative skin lesions other than HS, as a control. Thirty HS patients were included; 29 purulent lesions (96.7%) harbored at least one bacterial species. Five (16.7%) swab samples were positive for S. aureus, none of which was positive for PVL genes. Among the 30 purulent disorders included as controls, 8 (26.3%) were positive for S. aureus; of these, 4 strains (50%) expressed LPV. The study results seem to exclude the pathogenetic involvement of S. aureus producing PVL in HS; as a result, PVL does not seem to represent a potential target in the future development of HS treatments.

Mother-to-child transmission of oncogenic polyomaviruses BKPyV, JCPyV and SV40.

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.

Ticagrelor Increases SIRT1 and HES1 mRNA Levels in Peripheral Blood Cells from Patients with Stable Coronary Artery Disease and Chronic Obstructive Pulmonary Disease.

Ticagrelor is a powerful P2Y12 inhibitor with pleiotropic effects in the cardiovascular system. Consistently, we have reported that in patients with stable coronary artery disease (CAD) and concomitant chronic obstructive pulmonary disease (COPD) who underwent percutaneous coronary intervention (PCI), 1-month treatment with ticagrelor was superior in improving biological markers of endothelial function, compared with clopidogrel. The objective of this study was to investigate the mechanisms underlying these beneficial effects of ticagrelor by conducting molecular analyses of RNA isolated from peripheral blood cells of these patients. We determined mRNAs levels of markers of inflammation and oxidative stress, such as RORγt (T helper 17 cells marker), FoxP3 (regulatory T cells marker), NLRP3, ICAM1, SIRT1, Notch ligands JAG1 and DLL4, and HES1, a Notch target gene. We found that 1-month treatment with ticagrelor, but not clopidogrel, led to increased levels of SIRT1 and HES1 mRNAs. In patients treated with ticagrelor or clopidogrel, we observed a negative correlation among changes in both SIRT1 and HES1 mRNA and serum levels of Epidermal Growth Factor (EGF), a marker of endothelial dysfunction found to be reduced by ticagrelor treatment in our previous study. In conclusion, we report that in stable CAD/COPD patients ticagrelor positively regulates HES1 and SIRT1, two genes playing a protective role in the context of inflammation and oxidative stress. Our observations confirm and expand previous studies showing that the beneficial effects of ticagrelor in stable CAD/COPD patients may be, at least in part, mediated by its capacity to reduce systemic inflammation and oxidative stress.

Specific antibodies reacting to JC polyomavirus capsid protein mimotopes in sera from multiple sclerosis and other neurological diseases-affected patients.

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.

Droplet-digital PCR assay to detect Merkel cell polyomavirus sequences in chorionic villi from spontaneous abortion affected females.

Droplet-digital polymerase chain reaction (ddPCR) technique was set up to detect/quantify Merkel cell polyomavirus (MCPyV) DNA in clinical specimens, including chorionic villi and peripheral blood mononuclear cells (PBMCs) from spontaneous abortion (SA)-affected females. This ddPCR assay showed high accuracy, sensitivity, and specificity in detecting MCPyV DNA cloned in a recombinant plasmid vector, the control. ddPCR was extended to MCPyV DNA to investigate/quantify its sequences in clinical samples. Overall, 400 samples were analyzed, that is, 100 chorionic villi and 100 PBMCs, from SA females (n = 100), the cases, and 100 chorionic villi and 100 PBMCs from females who underwent voluntary pregnancy interruption (VI, n = 100), the control. MCPyV DNA was detected in 4/100 (4%) and 5/100 (5%) of SA and VI chorionic villi, respectively. The mean viral DNA load was 1.99 ( ± 0.94 standard mean deviation [SD]) copy/104 cells in SA and 3.02 ( ± 1.86 [SD]) copy/104 cells in VI. In PBMCs, MCPyV DNA was revealed in 9/100 (9%) and 14/100 (14%) of SA and VI, with a mean of 2.09 ( ± 1.17 [SD]) copy/104 cells and 4.09 ( ± 4.26 [SD]) copy/104 cells in SA and VI, respectively. MCPyV gene expression analysis by quantitative PCR for the large T antigen (LT) and viral capsid protein 1 (VP1) showed their mRNAs in 2/4 (50%) SA- and 2/5 (40%) VI-MCPyV-positive samples. MCPyV DNA was detected/quantified using the ddPCR technique, in chorionic villi and PBMCs from SA and VI. In our experimental conditions, ddPCR provided a powerful tool to detect/quantify MCPyV DNA sequences in clinical samples.

Hydroxylapatite-collagen hybrid scaffold induces human adipose-derived mesenchymal stem cells to osteogenic differentiation in vitro and bone regrowth in patients.

Tissue engineering-based bone graft is an emerging viable treatment modality to repair and regenerate tissues damaged as a result of diseases or injuries. The structure and composition of scaffolds should modulate the classical osteogenic pathways in human stem cells. The osteoinductivity properties of the hydroxylapatite-collagen hybrid scaffold named Coll/Pro Osteon 200 were investigated in an in vitro model of human adipose mesenchymal stem cells (hASCs), whereas the clinical evaluation was carried out in maxillofacial patients. Differentially expressed genes (DEGs) induced by the scaffold were analyzed using the Osteogenesis RT2 PCR Array. The osteoinductivity potential of the scaffold was also investigated by studying the alkaline phosphatase (ALP) activity, matrix mineralization, osteocalcin (OCN), and CLEC3B expression protein. Fifty patients who underwent zygomatic augmentation and bimaxillary osteotomy were evaluated clinically, radiologically, and histologically during a 3-year follow-up. Among DEGs, osteogenesis-related genes, including BMP1/2, ALP, BGLAP, SP7, RUNX2, SPP1, and EGFR, which play important roles in osteogenesis, were found to be upregulated. The genes to cartilage condensation SOX9, BMPR1B, and osteoclast cells TNFSF11 were detected upregulated at every time point of the investigation. This scaffold has a high osteoinductivity revealed by the matrix mineralization, ALP activity, OCN, and CLEC3B expression proteins. Clinical evaluation evidences that the biomaterial promotes bone regrowth. Histological results of biopsy specimens from patients showed prominent ossification. Experimental data using the Coll/Pro Osteon 200 indicate that clinical evaluation of bone regrowth in patients, after scaffold implantation, was supported by DEGs implicated in skeletal development as shown in "in vitro" experiments with hASCs.

Adult Stem Cells for Bone Regeneration and Repair.

The regeneration of bone fractures, resulting from trauma, osteoporosis or tumors, is a major problem in our super-aging society. Bone regeneration is one of the main topics of concern in regenerative medicine. In recent years, stem cells have been employed in regenerative medicine with interesting results due to their self-renewal and differentiation capacity. Moreover, stem cells are able to secrete bioactive molecules and regulate the behavior of other cells in different host tissues. Bone regeneration process may improve effectively and rapidly when stem cells are used. To this purpose, stem cells are often employed with biomaterials/scaffolds and growth factors to accelerate bone healing at the fracture site. Briefly, this review will describe bone structure and the osteogenic differentiation of stem cells. In addition, the role of mesenchymal stem cells for bone repair/regrowth in the tissue engineering field and their recent progress in clinical applications will be discussed.

Association Between Simian Virus 40 and Human Tumors.

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.

New Insights into Molecular Oncogenesis and Therapy of Uveal Melanoma.

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.

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.