Alessandro Sinigaglia

Isolation of infectious Zika virus from saliva and prolonged viral RNA shedding in a traveller returning from the Dominican Republic to Italy, January 2016

We report the isolation of infectious Zika virus (ZIKV) in cell culture from the saliva of a patient who developed a febrile illness after returning from the Dominican Republic to Italy, in January 2016. The patient had prolonged shedding of viral RNA in saliva and urine, at higher load than in blood, for up to 29 days after symptom onset. Sequencing of ZIKV genome showed relatedness with strains from Latin America.

Isolation of West Nile Virus from Urine Samples of Patients with Acute Infection

ABSTRACT This study demonstrated that West Nile virus (WNV) excreted in the urine of patients with acute infection can be isolated in cell cultures. In addition, the protocols for WNV isolation from urine samples were standardized, and factors that may affect the efficiency of WNV isolation were identified.

MGMT expression and promoter methylation status may depend on the site of surgical sample collection within glioblastoma: a possible pitfall in stratification of patients?

We recently described a three-layer concentric model of a glioblastoma (GBM) related to a specific distribution of molecular and phenotypic characteristics driven by the intratumoral hypoxic gradient in which the cancer stem cells niche is located in the hypoxic necrotic core of the tumour. The purpose of this study was to investigate the relationship between O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation status and MGMT expression in GBM samples collected according to the three-layer concentric model. Multiple tissue samples were obtained, by means of image-guided surgery, from the three concentric layers of newly diagnosed GBM. Two samples from each layer were collected from 12 patients (total 72 samples). Immunohistochemical analysis was performed on formalin-fixed paraffin-embedded tissue samples. The methylation status of the MGMT promoter was determined by methylation-specific polymerase-chain-reaction analysis. In all tumours, MGMT protein expression decreased progressively from the inner to the outer layer, and methylation of the MGMT promoter was unrelated to tumour layer. In particular, the MGMT promoter was unmethylated in all layers in 41.7% of tumours, methylated in all layers in 25%, and variably methylated in the three layers in 33.3%. We recorded concordance between MGMT expression and MGMT promoter methylation status within the GBM in only 58.8% of the samples collected. Our data suggest that both MGMT expression and promoter methylation data may be variable throughout GBM and that they may, consequently, depend on the site of surgical sample collection, even in the same patient. However, whereas MGMT expression is pre-operatively predictable when sampling is performed according to the three-layer concentric model, MGMT promoter methylation is not. These results must be considered when sample collection is performed for assessment of MGMT data.

Profiling of Expression of Human Papillomavirus–Related Cancer miRNAs in Penile Squamous Cell Carcinomas

Penile squamous cell carcinoma (PSCC) is a rare tumor associated with high-risk human papillomavirus (HR-HPV) infection in 30% to 60% of cases. Altered expression of miRNAs has been reported in HPV-related cervical and head and neck cancers, but such data have not been available for PSCC. We analyzed a series of 59 PSCCs and 8 condylomata for presence of HPV infection, for p16(INK4a), Ki-67, and p53 immunohistochemical expression, and for expression of a panel of cellular miRNAs (let-7c, miR-23b, miR-34a, miR-145, miR-146a, miR-196a, and miR-218) involved in HPV-related cancer. HR-HPV DNA (HPV16 in most cases) was detected in 17/59 (29%) PSCCs; all penile condylomata (8/8) were positive for low-risk HPV6 or HPV11. HR-HPV(+) PSCCs overexpressed p16(INK4a) in 88% cases and p53 in 35% of cases, whereas HR-HPV(-) PSCCs were positive for p16(INK4a) and p53 immunostaining in 9% and 44% of cases, respectively. Among the miRNAs investigated, expression of miR-218 was lower in PSCCs with HR-HPV infection and in p53(-) cancers. Hypermethylation of the promoter of the SLIT2 gene, which contains miR-218-1 in its intronic region, was frequently observed in PSCCs, mainly in those with low miR-218 expression. Epigenetic silencing of miR-218 is a common feature in HR-HPV(+) PSCCs and in HR-HPV(-) PSCCs without immunohistochemical detection of p53.

Vaccination of mice using the West Nile virus E-protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects mice against a lethal challenge.

West Nile virus (WNV) is a mosquito-borne flavivirus that is endemic in Africa, the Middle East, Europe and the United States. There is currently no antiviral treatment or human vaccine available to treat or prevent WNV infection. DNA plasmid-based vaccines represent a new approach for controlling infectious diseases. In rodents, DNA vaccines have been shown to induce B cell and cytotoxic T cell responses and protect against a wide range of infections. In this study, we formulated a plasmid DNA vector expressing the ectodomain of the E-protein of WNV into nanoparticles by using linear polyethyleneimine (lPEI) covalently bound to mannose and examined the potential of this vaccine to protect against lethal WNV infection in mice. Mice were immunized twice (prime – boost regime) with the WNV DNA vaccine formulated with lPEI-mannose using different administration routes (intramuscular, intradermal and topical). In parallel a heterologous boost with purified recombinant WNV envelope (E) protein was evaluated. While no significant E-protein specific humoral response was generated after DNA immunization, protein boosting of DNA-primed mice resulted in a marked increase in total neutralizing antibody titer. In addition, E-specific IL-4 T-cell immune responses were detected by ELISPOT after protein boost and CD8+ specific IFN-γ expression was observed by flow cytometry. Challenge experiments using the heterologous immunization regime revealed protective immunity to homologous and virulent WNV infection.

Characterization of Intra-Type Variants of Oncogenic Human Papillomaviruses by Next-Generation Deep Sequencing of the E6/E7 Region.

Different human papillomavirus (HPV) types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV) types are characterized by increased oncogenic activity and probability to induce cancer. In this study, we designed and validated a new method based on multiplex PCR-deep sequencing of the E6/E7 region of HR-HPV types to characterize HPV intra-type variants in clinical specimens. Validation experiments demonstrated that this method allowed reliable identification of the different lineages of oncogenic HPV types. Advantages of this method over other published methods were represented by its ability to detect variants of all HR-HPV types in a single reaction, to detect variants of HR-HPV types in clinical specimens with multiple infections, and, being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity.

Modeling viral infectious diseases and development of antiviral therapies using human induced pluripotent stem cell-derived systems

The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host–pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance.

Mycobacterium tuberculosis-induced miR-155 subverts autophagy by targeting ATG3 in human dendritic cells

Autophagy is a primordial eukaryotic pathway, which provides the immune system with multiple mechanisms for the elimination of invading pathogens including Mycobacterium tuberculosis (Mtb). As a consequence, Mtb has evolved different strategies to hijack the autophagy process. Given the crucial role of human primary dendritic cells (DC) in host immunity control, we characterized Mtb-DC interplay by studying the contribution of cellular microRNAs (miRNAs) in the post-transcriptional regulation of autophagy related genes. From the expression profile of de-regulated miRNAs obtained in Mtb-infected human DC, we identified 7 miRNAs whose expression was previously found to be altered in specimens of TB patients. Among them, gene ontology analysis showed that miR-155, miR-155* and miR-146a target mRNAs with a significant enrichment in biological processes linked to autophagy. Interestingly, miR-155 was significantly stimulated by live and virulent Mtb and enriched in polysome-associated RNA fraction, where actively translated mRNAs reside. The putative pair interaction among the E2 conjugating enzyme involved in LC3-lipidation and autophagosome formation-ATG3-and miR-155 arose by target prediction analysis, was confirmed by both luciferase reporter assay and Atg3 immunoblotting analysis of miR-155-transfected DC, which showed also a consistent Atg3 protein and LC3 lipidated form reduction. Late in infection, when miR-155 expression peaked, both the level of Atg3 and the number of LC3 puncta per cell (autophagosomes) decreased dramatically. In accordance, miR-155 silencing rescued autophagosome number in Mtb infected DC and enhanced autolysosome fusion, thereby supporting a previously unidentified role of the miR-155 as inhibitor of ATG3 expression. Taken together, our findings suggest how Mtb can manipulate cellular miRNA expression to regulate Atg3 for its own survival, and highlight the importance to develop novel therapeutic strategies against tuberculosis that would boost autophagy.

Characterization of a new CDC73 missense mutation that impairs Parafibromin expression and nucleolar localization.

Mutations of the Cell Division Cycle 73 (CDC73) tumor suppressor gene (previously known as HRPT2), encoding for parafibromin, are associated with the Hyperparathyroidism-Jaw Tumor (HPT-JT) syndrome, an autosomal dominant disease whose clinical manifestations are mainly parathyroid tumors and, less frequently, ossifying fibromas of the jaws, uterine and renal tumors. Most mutations of CDC73 are nonsense or frameshift, while missense mutations are rare and generally affect the N-terminal domain of parafibromin, a region that is still poorly characterized. The aim of this study was to characterize a novel somatic CDC73 missense mutation (Ile60Asn) identified in the mandibular tumor of a HPT-JT patient carrying a germline CDC73 inactivating mutation. Immunostaining of the tumor showed reduced nuclear parafibromin immunoreactivity. Western blotting and confocal microscopy of transfected cells demonstrated that the Ile60Asn mutant parafibromin was less expressed than the wild-type protein and exhibited impaired nucleolar localization. Treatment of transfected cells with translation and proteasome inhibitors demonstrated a decreased stability of the Ile60An mutant, partially due to an increase in proteasomal degradation. Overexpression of the Ile60Asn mutant led to increased cell proliferation and to accumulation in the G2/M phase of cell cycle. Moreover, mutant parafibromin lost the ability to down-regulate c-myc expression. In conclusion, our study shows that a missense mutation in the N-terminus of parafibromin, identified in an ossifying fibroma from a HPT-JT patient, stimulated cell proliferation and impaired parafibromin expression and nucleolar localization, suggesting a relevant role of the N-terminal domain for parafibromin function.

Changes in microRNA expression during disease progression in patients with chronic viral hepatitis

MicroRNAs (miRNAs) have been involved in hepatocarcinogenesis, but little is known on their role in the progression of chronic viral hepatitis. Aim of this study was to identify miRNA signatures associated with stages of disease progression in patients with chronic viral hepatitis.