No detectable truncating mutations in large T antigen (LT-Ag) sequence of Merkel cell polyomavirus (MCPyV) DNA obtained from porocarcinomas.

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma (MCC). In tumor cells the MCPyV large T antigen (LT-Ag) is frequently found truncated and this is considered a major tumor-specific signature. The role of MCPyV in other, non-MCC tumours, is little known. Viral DNA and/or tumour-specific mutations have been sometimes detected in different tumours, but such data are not unequivocal and the involvement of the virus in the tumorigenesis is not clear. In a previous study, we demonstrated a significantly higher prevalence of MCPyV DNA in formalin fixed paraffin embedded (FFPE) porocarcinoma tissues compared to the normal skin. In the present study, we investigated the presence of truncating mutations in MCPyV LT-Ag coding region in porocarcinoma specimens. Using several overlapped PCR primer pairs, the complete LT-Ag sequence from two biopsies were obtained. No truncating mutations were detected. The lack of truncating mutations in LT-Ag sequence does not seem to support the role of MCPyV in porocarcinoma oncogenesis. However, an oncogenetic mechanism, different from that proposed for MCC and not associated with the LT-Ag mutations/deletions, cannot be excluded. Further studies of more sequences coding for LT-Ag would be needed to verify this hypothesis.

Molecular investigation of some DNA viruses in mucosal melanoma: Case-control study.

The association between viral infections and both cutaneous and mucosal melanoma (MM) has not been fully investigated. Here, we assessed the prevalence of the DNA of a broad range of viruses in 31 MMs and 15 biopsies of healthy mucosa (HM) using molecular methods. The parvoviruses CuV and B19V, herpesviruses HSV1, HSV2, EBV, HHV6, and HHV8, polyomavirus MCPyV, and α-HPVs were not detected, or rarely found, in MMs, and in HM, of the digestive, respiratory, and female genital tract. The overall prevalence of ß-HPV in MMs was not significantly higher compared to that in HM (70.9% and 53.3% respectively; p = 0.514). However, the number of MMs positive for ß-HPV types belonging to Species 3 and 5 and for some viral types belonging to Species 1, 2, 3, and 5 were significantly higher compared with HM (p < 0.05). Moreover, compared to HM, the MM samples contained a significantly higher number of ß-HPV types, mainly belonging to Species 1, 3, and 5 (p < 0.05). Our data, although suggesting a role for certain ß-HPV types in MM oncogenesis, require additional investigation in larger populations to support this hypothesis.

Insights into the knowledge of complex diseases: Environmental infectious/toxic agents as potential etiopathogenetic factors of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease secondary to three cardinal pathological features: immune-system alterations, diffuse microangiopathy, and fibrosis involving the skin and internal organs. The etiology of SSc remains quite obscure; it may encompass multiple host genetic and environmental -infectious/chemical-factors. The present review focused on the potential role of environmental agents in the etiopathogenesis of SSc based on epidemiological, clinical, and laboratory investigations previously published in the world literature. Among infectious agents, some viruses that may persist and reactivate in infected individuals, namely human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), and parvovirus B19 (B19V), and retroviruses have been proposed as potential causative agents of SSc. These viruses share a number of biological activities and consequent pathological alterations, such as endothelial dysfunction and/or fibroblast activation. Moreover, the acute worsening of pre-existing interstitial lung involvement observed in SSc patients with symptomatic SARS-CoV-2 infection might suggest a potential role of this virus in the overall disease outcome. A variety of chemical/occupational agents might be regarded as putative etiological factors of SSc. In this setting, the SSc complicating silica dust exposure represents one of the most promising models of study. Considering the complexity of SSc pathogenesis, none of suggested causative factors may explain the appearance of the whole SSc; it is likely that the disease is the result of a multifactorial and multistep pathogenetic process. A variable combination of potential etiological factors may modulate the appearance of different clinical phenotypes detectable in individual scleroderma patients. The in-deep investigations on the SSc etiopathogenesis may provide useful insights in the broad field of human diseases characterized by diffuse microangiopathy or altered fibrogenesis.

Parvovirus B19 activates in vitro normal human dermal fibroblasts: a possible implication in skin fibrosis and systemic sclerosis.

OBJECTIVE: Fibrosis is the most characteristic pathological hallmark of SSc, a connective tissue disease characterized by vascular and immunological abnormalities, inflammation and enhanced extracellular matrix production, leading to progressive fibrosis of skin and internal organs. We previously demonstrated that parvovirus B19 (B19V) can infect normal human dermal fibroblasts (NHDFs) and that B19V persists in SSc fibroblasts. In this study, we investigated whether parvovirus B19V is able to activate in vitro NHDFs and to induce in these cells some phenotypic features similar to that observed in the SSc fibroblasts. METHODS: We preliminarily analysed the time course of B19V infection in cultured NHDFs, then we investigated the ability of B19V to induce cell migration, invasive phenotype and mRNA expression of some profibrotic and/or proinflammatory genes. RESULTS: We confirmed our previous findings that B19V infects NHDFs, but the infection is not productive. After incubation with B19V, NHDFs showed a significant increase of both migration and invasiveness, along with mRNA expression of different profibrotic genes (α-SMA, EDN-1, IL-6, TGF-ß1 receptors 1 and 2, Col1α2), some genes associated with inflammasome platform (AIM2, IFI16, IL-1ß, CASP-1) and genes for metalloprotease (MMP 2, 9 and 12). CONCLUSION: These data suggest that B19V can activate dermal fibroblasts and may have a role in the pathogenesis of fibrosis. B19V-induced fibroblast migration and invasiveness could be due to the B19V-associated MMP9 overexpression and activation. Moreover, the up-regulation of MMP12, typical of SSc, could link the B19V infection of fibroblasts to the anti-angiogenic process.

Effects of Parvovirus B19 In Vitro Infection on Monocytes from Patients with Systemic Sclerosis: Enhanced Inflammatory Pathways by Caspase-1 Activation and Cytokine Production.

Parvovirus B19 (B19V) has been proposed as a triggering agent for some autoimmune diseases including systemic sclerosis (SSc). In this study, we investigated whether B19V infection in vitro differently activates inflammatory pathways, including those dependent on caspase-1 activation, in monocytes from patients with SSc and healthy controls. We showed that B19V can infect both THP-1 cells and primary monocytes but is not able to replicate in these cells. B19V infection increases the production of tumor necrosis factor-α and induces NLRP3-mediated caspase-1 activation in both THP-1 cells differentiated with phorbol 12-myristate 13-acetate and in monocytes from patients with SSc but not from healthy controls. B19V infection was sufficient for THP-1 to produce mature IL-1ß. Monocytes from patients with SSc required an additional stimulus, here represented by lipopolysaccharides, to activate cytokine genes. Following B19V infection, however, lipopolysaccharide-activated monocytes from patients with SSc strongly increased the production of IL-1ß and tumor necrosis factor-α. Altogether, these data suggest that viral components might potentiate the response to endogenous and/or exogenous toll-like receptor 4 ligands in monocytes from patients with SSc. The B19V-mediated activation of inflammatory pathways in monocytes might contribute to the disease progression and/or development of specific clinical phenotypes.

Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. OBJECTIVE: To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. METHODS: Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. RESULTS: The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥105 copies per reaction, while the upper limit of ddPCR was 104 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). CONCLUSIONS: The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome.

Detection of Merkel cell polyomavirus and human papillomavirus DNA in porocarcinoma.

BACKGROUND: Increasing evidences support the role of Merkel cell polyomavirus (MCPyV) and human papillomavirus (HPV) in non-cutaneous and cutaneous tumours. Porocarcinoma is a rare malignant neoplasm that arises from the intraepidermal ductal portion of the eccrine sweat glands. The aetiology of porocarcinoma is largely unknown and no systematic studies have been done to investigate the implication of infectious agents in the pathogenesis of this tumour. OBJECTIVES: To investigate the possible association between MCPyV and/or HPV infection and porocarcinoma. STUDY DESIGN: Forty-four formalin-fixed paraffin-embedded (FFPE) porocarcinomas (40 primary and 4 metastatic) and 10 healthy skin specimens (controls), were analysed for the presence of MCPyV and HPV DNA using molecular detection methods. RESULTS: MCPyV DNA was found in 27/40 (68%) primary porocarcinomas and in 3/10 (30%) controls (Fisher exact test: p<0.04). No significant difference in viral load was observed between tumours and healthy skin. Moreover, 2/40 primary porocarcinomas tested positive for high-risk HPV16. Cutaneous beta-HPV infection was detected in 16/40 (40%) porocarcinomas and in 6/10 (60%) controls. No particular beta-HPV types were significantly associated with tumour or with healthy skin. Two out of 4 metastatic biopsies were MCPyV DNA positive. All metastatic samples had mixed infections with cutaneous HPV types. CONCLUSIONS: This study demonstrated a significantly high prevalence of MCPyV and the presence of a broad spectrum of HPV types in porocarcinoma and provided the first available data about viral infections in this tumour. To understand the role, if any, of viral infections in the pathogenesis of porocarcinoma further studies are needed.

Pattern of HPV infection in basal cell carcinoma and in perilesional skin biopsies from immunocompetent patients.

BACKGROUND: The association between human papillomavirus (HPV) infection and non-melanoma skin cancers (NMSCs) such as squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) is not yet fully understood. We analysed the prevalence and spectrum of cutaneous beta-HPV types and mucosal/genital HPV types in paired biopsies (tumour and corresponding perilesional skin) obtained from 50 BCC immunocompetent patients. A small group of SCC patients (n=9) was also included. We also evaluated some previously postulated risk factors for HPV infection in NMSC patients. RESULTS: All biopsies were negative for mucosal/genital HPV types. Overall, beta-HPV DNA was detected more often in SCC compared to BCC patients (78% vs 55% of total samples). The frequency of infection increased with the patient's age [OR=4.88 (95% CI 1.29-18.39)]. There was no significant correlation between beta-HPV positivity and sex, skin type and UV exposure. The prevalence of beta-HPV species 1 types was significantly higher than those belonging to other beta-HPV species in biopsies from BCC (p=0.022) but not from SCC subjects (p=0.091). There was no significant difference in the overall prevalence of beta-HPV infection and the number of viral types between tumour lesions and perilesional skin. BCC samples were significantly more likely to be infected with beta-HPV species 1 types compared to perilesional skin (p=0.036) and showed a higher frequency of mixed infections (p=0.028). CONCLUSIONS: These findings demonstrate that beta-HPV types belonging to species 1 are the most common HPV types detected in the skin of BCC patients. Moreover beta-1-HPV types and mixed infections are significantly more frequent in tumour samples than in healthy perilesional skin. Our results suggest that beta-1-HPVs as well as co-infection with more than one viral type could be important in NMSC and in particular in BCC.Further studies aimed to compare the biological activity of viral types in tumours and in healthy skin (viral replication and expression, interference of infection with cellular functions) are necessary to understand the role of HPV infection in skin cancer.

Multistep drug intercalation: molecular dynamics and free energy studies of the binding of daunomycin to DNA.

Atomic-scale molecular dynamics and free energy calculations in explicit aqueous solvent are used to study the complex mechanism by which a molecule can intercalate between successive base pairs of the DNA double helix. We have analyzed the intercalation pathway for the anticancer drug daunomycin using two different methods: metadynamics and umbrella sampling. The resulting free energy pathways are found to be consistent with one another and point, within an equilibrium free energy context, to a three-step process. Daunomycin initially binds in the minor groove of DNA. An activated step then leads to rotation of the drug, coupled with DNA deformation that opens a wedge between the base pairs, bends DNA toward the major groove, and forms a metastable intermediate that resembles structures seen within the interfaces between DNA and minor-groove-binding proteins. Finally, crossing a small free energy barrier leads to further rotation of daunomycin and full intercalation of the drug, reestablishing stacking with the flanking base pairs and straightening the double helix.

Human parvovirus PARV4 DNA in tissues from adult individuals: a comparison with human parvovirus B19 (B19V).

BACKGROUND: PARV4 is a new member of the Parvoviridae family not closely related to any of the known human parvoviruses. Viremia seems to be a hallmark of PARV4 infection and viral DNA persistence has been demonstrated in a few tissues. Till now, PARV4 has not been associated with any disease and its prevalence in human population has not been clearly established. This study was aimed to assess the tissue distribution and the ability to persist of PARV4 in comparison to parvovirus B19 (B19V). RESULTS: PARV4 and B19V DNA detection was carried out in various tissues of individuals without suspect of acute viral infection, by a real time PCR and a nested PCR, targeting the ORF2 and the ORF1 respectively. Low amount of PARV4 DNA was found frequently (>40%) in heart and liver of adults individuals, less frequently in lungs and kidneys (23,5 and 18% respectively) and was rare in bone marrow, skin and synovium samples (5,5%, 4% and 5%, respectively). By comparison, B19V DNA sequences were present in the same tissues with a higher frequency (significantly higher in myocardium, skin and bone marrow) except than in liver where the frequency was the same of PARV4 DNA and in plasma samples where B19V frequency was significantly lower than that of PARV4 CONCLUSIONS: The particular tropism of PARV4 for liver and heart, here emerged, suggests to focus further studies on these tissues as possible target for viral replication and on the possible role of PARV4 infection in liver and heart diseases. Neither bone marrow nor kidney seem to be a common target of viral replication.

Human parvovirus B19 (B19V) infection in systemic sclerosis patients.

BACKGROUND: Our previous reports suggested a possible association between parvovirus B19 (B19V) infection and systemic sclerosis (SSc), based on higher prevalence of B19V DNA in SSc patients in respect to controls. METHODS: In the present study, to further evaluate the differences in the pattern of B19 infection in SSc, skin biopsies and bone marrow samples from patients and controls were analysed for B19V DNA detection, genotyping and viral expression. RESULTS: B19V DNA was detected in skin biopsies from 39/49 SSc patients and from 20/28 controls. Bone marrow showed positive in 17/29 SSc patients, 5/21 haematological patients and 0/10 healthy controls. Genotype 1 was more frequent in skin and bone marrow from patients than from controls. Simultaneous persistence of 2 genotypes was detected in SSc skin and bone marrow samples, never in controls. Viral mRNA for capsid protein was detected in the skin of genotype 1-positive patients and not in control skins. CONCLUSION: The results outline some differences in the rate of persistence of B19V DNA, in the simultaneous persistence of 2 genotypes and in the pattern of viral expression among SSc patients and controls.

Tissue persistence of parvovirus B19 genotypes in asymptomatic persons.

Parvovirus B19 (B19V) can persist in immunocompetent symptomatic and non-symptomatic individuals, as demonstrated by the finding of viral DNA in different tissues, in absence of viremia and of anti-B19V IgM. The spread and the nature of this phenomenon have not been clearly determined. In order to investigate the frequency of persistence and the tissue distribution of the three genotypes of B19V, the viral load of the persistent virus and its expression in the affected tissues, 139 tissue samples and 102 sera from 139 asymptomatic individuals have been analyzed by consensus PCRs and genotype specific PCRs for B19V detection and genotyping. Viral load was measured by real time PCR and viral mRNAs were detected by RT-PCR. Altogether, 51% individuals carried B19V DNA, more frequently in solid tissues (65%) than in bone marrow (20%). Genotype 1 was found in 28% tissue samples, genotype 2 in 68% and genotype 3 in 3% only. Viral load ranged from less then 10 copies to 7 x 10(4) copies per 10(6) cells, with the exception of two samples of myocardium with about 10(6) copies per 10(6) cells. mRNA of capsid proteins was present in two bone marrow samples only. In conclusion, in asymptomatic individuals B19V persistence is more common in solid tissues than in bone marrow, and genotype 2 persists more frequently than genotype 1. The results suggest that the virus persists without replicating, at sub-immunogenic levels.