Spatial and seasonal variability of human and fish viruses in mussels inside and offshore of Ravenna's harbour (Adriatic Sea, Italy).

AIMS: This study aims to investigate the presence and spatial-seasonal variability of human and fish viruses in coastal marine systems using Ravenna's harbour area (Adriatic Sea, Italy) as a model. METHODS AND RESULTS: Human viruses (noroviruses and hepatitis A virus) and one of the most threatening finfish pathogens, the nervous necrosis virus (NNV), were investigated in mussels living inside and offshore Ravenna's harbour. Thirty-three and 36·7% of tested mussel samples resulted contaminated by human and fish viruses respectively. A different spatial-seasonal distribution was observed. Human viruses were detected mainly in inner port sites during colder months, while NNV was detected in both inside and offshore of Ravenna's harbour, mainly during warmer months. CONCLUSIONS: The presence of human viruses in the inner port close to the city centre could be attributed to wastewaters carrying pathogens in the port environment and this arises public health concerns, however, the presence of these viruses limited to the canal port during the winter can greatly reduce the risk to human health. Regarding NNV, the accumulation and release of viable virus by mussels, could represent a viral source for susceptible finfish. These findings reflect the different epidemiological features of these infections and indicate the importance to choose the correct indicator to monitor viral contaminations. SIGNIFICANCE AND IMPACT OF THE STUDY: The high frequency of viral contamination pointed out in the study stresses the imperative to monitor the viral presence in all coastal habitats where the high natural value meets several recreational and commercial activities such as the Ravenna's harbour area. Particularly, this study could represent a novel starting point for the development of a more structured bio-monitoring program, in order to ensure improved environmental management and safety of coastal areas.

The Workplace Health Promotion (WHP) programme in an Italian University Hospital.

INTRODUCTION: Description of the lifestyles of employees of the Siena University Hospital (Azienda Ospedaliera Universitaria Senese: AOUS), as assessed through a Workplace Health Promotion (WHP) project in the two-year period 2017-18; assessment of possible short-term effects of integrated health promotion interventions in the workplace, within the framework of the Tuscany WHP network, as applied in the AOUS and involving about 1,000 workers. METHODS: A cross-sectional study and a pre-post evaluation of data collected by means of anonymous questionnaires in two phases: before the beginning of the programme and after 12 months. RESULTS: Twelve months after the start of the programme regarding diet (consumption of fruit and vegetables) and physical activity, the positive effects that emerged were not statistically significant. No differences were observed between gender or professional categories. The employees' perception of the programme was satisfactory. CONCLUSIONS: Albeit within the methodological limits of the assessment, the results showed that the diffusion of some major risk factors for chronic diseases had not decreased after 12 months' exposure to the programme. However, monitoring of these risk factors needs to be continued over a longer period, in order to detect the appearance of the expected changes in the long term. Moreover, it is essential to continue monitoring by sex and professional category, in order to pick out any differences and, if possible, take remedial actions. Further studies, in collaboration with the pertinent physicians, are desirable, since integrating data collected during health surveillance with a limited set of indicators of general risk factors may help to promptly identify possible health needs among employees.

Detection and molecular characterization of betanodaviruses retrieved from bivalve molluscs.

Betanodaviruses are small ssRNA viruses responsible for viral encephalopathy and retinopathy, otherwise known as viral nervous necrosis, in marine fish worldwide. These viruses can be either horizontally or vertically transmitted and have been sporadically detected in invertebrates, which seem to be one of the possible viral sources. Twenty-eight new betanodavirus strains were retrieved in three molluscs species collected from different European countries between 2008 and 2015. The phylogenetic analyses revealed that strains retrieved from bivalve molluscs are closely related to viruses detected in finfish in Southern Europe in the period 2000-2009. Nevertheless, a new betanodavirus strain, markedly different from the other members of the RGNNV genotype, was detected. Such a massive and varied presence of betanodaviruses in bivalve molluscs greatly stresses the risks of transmission previously feared for other invertebrates. Bivalve molluscs reared in the same area as farmed and wild finfish could act as a reservoir of the virus. Furthermore, current European regulations allow relaying activities and the sale of live bivalve molluscs, which could pose a real risk of spreading betanodaviruses across different geographic regions. To our knowledge, this is the first study, which focuses on the detection and genetic characterization of betanodaviruses in bivalve molluscs.

Fate of redspotted grouper nervous necrosis virus (RGNNV) in experimentally challenged Manila clam Ruditapes philippinarum.

Redspotted grouper nervous necrosis virus (RGNNV), genus Betanodavirus, family Nodaviridae, is the causative agent of viral encephalopathy and retinopathy (otherwise known as viral nervous necrosis) and can infect several fish species worldwide. Betanodaviruses, including RGNNV, are very resilient in the aquatic environment, and their presence has already been reported in several wild marine species including invertebrates. In order to investigate the interaction between a bivalve mollusc (Manila clam Ruditapes philippinarum) and RGNNV, we optimised a culture-based method. The bioaccumulation of the pathogenic RGNNV by R. philippinarum and the potential shedding of viable RGNNV from RGNNV-exposed clams were evaluated through a culture-based method. R. philippinarum clearly accumulated viable RGNNV in their hepatopancreatic tissue and were able to release viable RGNNV via faecal matter and filtered water into the surrounding environment. The role of clams as bioaccumulators and shedders of viable RGGNV could put susceptible cohabiting cultured fish at risk. RGNNV-contaminated molluscs could behave as reservoirs for this virus and may modify the virus epidemiology.

Evidence for Tunisian-Like Pestiviruses Presence in Small Ruminants in Italy Since 2007.

The genus Pestivirus, which belongs to the Flaviviridae family, includes ssRNA+ viruses responsible for infectious diseases in pigs, cattle, sheep, goats and other domestic and wild ruminants. Like most of the RNA viruses, pestivirus has high genome variability with practical consequences on disease epidemiology, diagnosis and control. In addition to the officially recognized species in the genus Pestivirus, such as BVDV-1, BVDV-2, BDV and CSFV, other pestiviruses have been detected. Furthermore, most of the ruminant pestiviruses show low or absent species specificity observed in serological tests and are able to infect multiple species. Particularly, small ruminants are receptive hosts of the most heterogeneous group of pestiviruses. The aim of this study was to carry out the molecular characterization of pestiviruses isolated from sheep and goats in Sicily, Italy. Phylogenetic analysis of two viral genomic regions (a fragment of 5'-UTR and the whole Npro regions) revealed the presence of different pestivirus genotypes in the analysed goat and sheep herds. Two of five viral isolates were clustered with BVDV-1d viruses, a strain widespread in Italy, but never reported in Sicily. The other three isolates formed a distinct cluster with high similarity to Tunisian isolates, recently proposed as a new pestivirus species. This represents the first evidence for Tunisian-like pestivirus presence in small ruminants in Italy. Furthermore, one of the isolates was collected from a goat, representing the first isolation of Tunisian-like pestivirus from this species.

Age-related spontaneous lacrimal keratoconjunctivitis is accompanied by dysfunctional T regulatory cells.

In both humans and animal models, the development of Sjögren syndrome (SS) and non-SS keratoconjunctivitis sicca (KCS) increases with age. Here, we investigated the ocular surface and lacrimal gland (LG) phenotype of NOD.B10.H2b mice at 7-14, 45-50, and 96-100 weeks. Aged mice develop increased corneal permeability, CD4+ T-cell infiltration, and conjunctival goblet cell loss. Aged mice have LG atrophy with increased lymphocyte infiltration and inflammatory cytokine levels. An increase in the frequency of CD4+Foxp3+ T regulatory cells (Tregs) was observed with age in the cervical lymph node (CLN), spleen, and LG. These CD4+CD25+ cells lose suppressive ability, while maintaining expression of Foxp3 (forkhead box P3) and producing interleukin-17 (IL-17) and interferon-γ (IFN-γ). An increase of Foxp3+IL-17+ or Foxp3+IFN-γ+ cells was observed in the LG and LG-draining CLN. In adoptive transfer experiments, recipients of either purified Tregs or purified T effector cells from aged donors developed lacrimal keratoconjunctivitis, whereas recipients of young Tregs or young T effector cells failed to develop disease. Overall, these results suggest inflammatory cytokine-producing CD4+Foxp3+ cells participate in the pathogenesis of age-related ocular surface disease.

Erectile dysfunction in young men with type 1 diabetes.

Erectile dysfunction (ED) is a common comorbidity of diabetes mellitus, but few studies investigated its prevalence in type 1 diabetes. The objective of this study was to evaluate the prevalence and correlates of ED in young men with type 1 diabetes treated with different intensive insulin regimens. The study population included 151 type 1 diabetic men, aged 18-35 years, and 60 healthy age-matched controls. Ninety-four men were treated with multiple daily injections of insulin (MDI), and the remaining 71 with continuous subcutaneous insulin infusion (CSII). All participants in the study completed the International Index of Erectile function (IIEF-5), and other validated multiple-choice questionnaires assessing quality of life, physical activity, depressive symptoms and diabetes-related problems. The overall prevalence of ED was higher in diabetic men (37%), as compared with controls (6%, P<0.001). ED prevalence rates were similar in both MDI (36%) and CSII (39%) groups (P=0.326); both were higher compared with controls (P<0.001 for both). More than half of diabetic men (58%) had mild ED. Compared with men without ED, diabetic men with ED showed lower weight, body mass index, fasting glucose, insulin dose and high-density lipoprotein cholesterol levels, and higher self-rating depression score (SRDS). In the multiple regression analysis only the SRDS (P=0.032) were independent predictors of IIEF-5 score in the overall diabetic men. Young men with type 1 diabetes treated with MDI or CSII show a higher prevalence of ED, as compared with healthy age-matched men. Depression was associated with ED in diabetic population.

Outbreak of mortality in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeons associated with sturgeon nucleo-cytoplasmatic large DNA virus.

Diseased outbreaks with high mortality in farmed sturgeon are a limiting factor to the success of this emerging aquaculture sector in Europe. Thorough investigations of outbreaks can determine the aetiological agents, identify important pathological and epidemiological pathways of infections and pave the way for effective control strategies. A thorough investigation of a mortality outbreak in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeons in Italy, demonstrated the primary involvement of a sturgeon nucleo-cytoplasmic large DNA virus (NCLDV). While, the taxonomy classification of this new virus is still uncertain, its involvement in sturgeon mortality outbreaks in Europe is, for the first time, fully investigated and described. Furthermore, the coinfection of bacteria such as motile Aeromonas spp. and Acinetobacter spp. was reported. Genetic characterisation showed the close relationship between the European sturgeon NCLDV with North American sturgeon NCLDVs. Similarly to the latter, the European sturgeon NCLDV persists in survivors. Furthermore, a systemic distribution of the European sturgeon NCLDV was evident in diseased A. baerii and A. gueldenstaedtii and in recovered A. gueldenstaedtii. These epidemiological and pathological findings will help in the identification of effective control strategies for sturgeon NCLDV infection, which afflicts an important and emerging European aquaculture sector.

FAS-ligand regulates differential activation-induced cell death of human T-helper 1 and 17 cells in healthy donors and multiple sclerosis patients.

Functionally distinct T-helper (Th) subsets orchestrate immune responses. Maintenance of homeostasis through the tight control of inflammatory Th cells is crucial to avoid autoimmune inflammation. Activation-Induced Cell Death (AICD) regulates homeostasis of T cells, and it has never been investigated in human Th cells. We generated stable clones of inflammatory Th subsets involved in autoimmune diseases, such as Th1, Th17 and Th1/17 cells, from healthy donors (HD) and multiple sclerosis (MS) patients and we measured AICD. We find that human Th1 cells are sensitive, whereas Th17 and Th1/17 are resistant, to AICD. In particular, Th1 cells express high level of FAS-ligand (FASL), which interacts with FAS and leads to caspases' cleavage and ultimately to cell death. In contrast, low FASL expression in Th17 and Th1/17 cells blunts caspase 8 activation and thus reduces cell death. Interestingly, Th cells obtained from healthy individuals and MS patients behave similarly, suggesting that this mechanism could explain the persistence of inflammatory IL-17-producing cells in autoimmune diseases, such as MS, where their generation is particularly substantial.

Effect of mass media on influenza vaccine coverage in the season 2014/2015: a regional survey in Lazio, Italy.

INTRODUCTION: Adherence to vaccination program for Influenza virus is an important issue of Public Health in presence of many no-vaccine tendencies. The media event about some deaths, occurring after MF59 adjuvanted vaccine administration, has characterized the season 2014/15 vaccination program in Italy. Aim of the study is vaccination adherence assessment of the current season with regards to local health units (LHU) coordinators's perceptions in Lazio Region (IT). METHODS: LHU coordinators's perceptions were collected from a questionnaire that was send via email to the all 12 LHU coordinators. The questionnaire was built with 4 questions concerning the impression about the vaccination adherence of elderly people in the current season. Data from questionnaire was compared with the official coverage rate obtained by the Regional Authority. Severe adverse events were collected by 1 LHU. RESULTS: All the 12 LHU coordinators answered to our questionnaire: 7/12 (50%) predicted a coverage rate of at least 50%; 3/12 (25%) referred a coverage rate around 40-45%; 2/12 (17%) predicted a reduction of 5-10% less than the previous season. Indeed, a mean 49.1% vaccination coverage in the elderly has been reported by the Regional Authority highlighting a reduction of 10% less than the 2013/14 season coverage. No severe adverse events were observed. DISCUSSION: In our survey an important effect of media event on anti-flu vaccination program adherence has been evidenced, with a failure in communication and joint management of Public Health Institutions in Italy about efficacy and safety information of flu vaccine.

CD8⁺ cells regulate the T helper-17 response in an experimental murine model of Sjögren syndrome.

This study investigated the regulatory function of CD8⁺ cells in T helper-17 (Th17) cell-mediated corneal epithelial barrier disruption that develops in a murine desiccating stress (DS) model that resembles Sjögren syndrome. CD8⁺ cell depletion promoted generation of interleukin-17A (IL-17A)-producing CD4⁺ T cells via activation of dendritic cells in both the ocular surface and draining cervical lymph nodes in C57BL/6 mice subjected to DS. T-cell-deficient nude recipient mice receiving adoptively transferred CD4⁺ T cells from CD8⁺ cell-depleted donors exposed to DS displayed increased CD4⁺ T-cell infiltration and elevated IL-17A and CC-chemokine attractant ligand 20 levels in the ocular surface, which was associated with greater corneal barrier disruption. Enhanced DS-specific corneal barrier disruption in CD8-depleted donor mice correlated with a Th17-mediated expression of matrix metalloproteinases (MMP-3 and MMP-9) in the recipient corneal epithelium. Co-transfer of CD8⁺CD103⁺ regulatory T cells did not affect the ability of DS-specific pathogenic CD4⁺ T cells to infiltrate and cause ocular surface disease in the nude recipients, showing that CD8⁺ cells regulate the efferent arm of DS-induced immune response. In summary, CD8⁺ regulatory cells suppress generation of a pathogenic Th17 response that has a pivotal role in DS-induced disruption of corneal barrier function.

Immunization coverage and timeliness of vaccination in Italian children with chronic diseases.

Since children with chronic diseases represent a primary target for immunization strategies, it is important that their immunization coverage and timeliness of vaccines is optimal. We performed a study to measure immunization coverage and timeliness of vaccines in children with type 1 diabetes, HIV infection, Down syndrome, cystic fibrosis, and neurological diseases. A total of 275 children aged 6 months-18 years were included in the study. Coverage for diphtheria-tetanus-pertussis (DTP), polio (Pol), and hepatitis B (HBV) vaccines approximated 85% at 24 months, while measles-mumps-rubella (MMR) coverage was 62%. Immunization coverage for seasonal influenza was 59%. The analysis of timeliness revealed that there was heterogeneity among children with different chronic diseases. A proportional hazard model showed that children with HIV infection had the longest time to complete three doses of DTP, Pol, and HBV, and those with neurological diseases received the first dose of MMR later than the other categories. Causes of missing or delayed vaccination mostly included a concurrent acute disease. Children with chronic diseases should be strictly monitored for routine and recommended vaccinations, and health care providers and families should be properly informed to avoid false contraindications.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13.

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK- populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

Disruption of transforming growth factor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation.

Transforming growth factor-beta (TGF-beta) signaling members, TGF-beta receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.

Influenza like Illness (ILI): prescribing behaviour of 83 sentry doctors in Lazio region in the period 2001-2002. Is it adherent to Italian ILI guidelines?

To describe different doctors' attitudes in drug prescribing in case of influenza-Like-Illness during 2001-2002 influenza season in Lazio region, with regard to consumption and expenditure and its adherence to Italian Guidelines on ILI Management. Prospective study aimed to assess doctors' behaviour in prescribing in respect with the following events: (a) ILI and subsequent controls after diagnosis, (b) complications, (c) adverse events to influenza vaccine. 7,629 subjects, have been identified and only 17% presents one or more comorbidities. There are totally 7,766 cases of ILI: 23% are complicated and out of the remaining 77%, only 14% presents comorbidities. Almost all elderly people have been vaccinated. Antibiotics were prescribed to complicated cases (82%) with comorbidities (55%); 1,075 patients (12%) had second or third contact with doctors in a period longer than 7 days and about 65% of them received antibiotics in case of acute bronchitis; children received mainly ibuprofen and paracetamol for fever control; acetylsalicylic acid in children group has been delivered only in 1% of cases. Overall, doctors' attitude in prescribing is generally coherent with Italian Guidelines on ILI even though a high variability still persists.

Sphingosine 1-phosphate interferes on the differentiation of human monocytes into competent dendritic cells.

Sphingosine 1-phosphate (S1P) is a lipidic messenger known to exert several physiological functions within the cell. We tested here whether the stimulation of human monocytes with different doses of S1P might interfere with their differentiation into competent dendritic cells (DC). Monocytes cultured with granulocyte macrophage colony stimulating factor, interleukin-4 (IL-4) and S1P differentiated into a DC population lacking CD1a molecules on the surface and acquired some aspects of mature DC (mDC), though in the absence of maturation stimuli. When stimulated with lipopolisaccharide (LPS), CD1a(-) DC produce high amounts of tumour necrosis factor-alpha and IL-10, but not IL-12. Accordingly, these CD1a(-) DC were not capable of stimulating allogenic T lymphocytes so well as CD1a(+) DC generated from untreated monocytes and maturated with LPS. S1P monocyte-derived DC lost their polarizing capacity abrogating the production of gamma-interferon/IL-4 by co-cultured naïve CD4(+)CD45RA(+) T cells. Our findings suggest a mechanism through which S1P can favour the development of immune-related pathological states.

RING finger-dependent ubiquitination by PRAJA is dependent on TGF-beta and potentially defines the functional status of the tumor suppressor ELF.

In gastrointestinal cells, biological signals for transforming growth factor-beta (TGF-beta) are transduced through transmembrane serine/threonine kinase receptors that signal to Smad proteins. Smad4, a tumor suppressor, is often mutated in human gastrointestinal cancers. The mechanism of Smad4 inactivation, however, remains uncertain and could be through E3-mediated ubiquitination of Smad4/adaptor protein complexes. Disruption of ELF (embryonic liver fodrin), a Smad4 adaptor protein, modulates TGF-beta signaling. We have found that PRAJA, a RING-H2 protein, interacts with ELF in a TGF-beta-dependent manner, with a fivefold increase of PRAJA expression and a subsequent decrease in ELF and Smad4 expression, in gastrointestinal cancer cell lines (P < 0.05). Strikingly, PRAJA manifests substantial E3-dependent ubiquitination of ELF and Smad3, but not Smad4. Delta-PRAJA, which has a deleted RING finger domain at the C terminus, abolishes ubiquitination of ELF. A stable cell line that overexpresses PRAJA exhibits low levels of ELF in comparison to a Delta-PRAJA stable cell line, where ELF expression is high compared to normal controls. The alteration of ELF and/or Smad4 expression and/or function in the TGF-beta signaling pathway may be induced by enhancement of ELF degradation, which is mediated by a high-level expression of PRAJA in gastrointestinal cancers. In hepatocytes, half-life (t(1/2)) and rate constant for degradation (k(D)) of ELF is 1.91 h and 21.72 min(-1) when coupled with ectopic expression of PRAJA in cells stimulated by TGF-beta, compared to PRAJA-transfected unstimulated cells (t(1/2) = 4.33 h and k(D) = 9.6 min(-1)). These studies reveal a mechanism for tumorigenesis whereby defects in adaptor proteins for Smads, such as ELF, can undergo degradation by PRAJA, through the ubiquitin-mediated pathway.

Impact of a new dosing regimen of epoetin alfa on quality of life and anemia in patients with low-risk myelodysplastic syndrome.

This study evaluated the impact of a new epoetin alfa dosing regimen on quality of life (QOL), transfusion requirements, and hemoglobin (Hb) levels in 133 patients with low-risk myelodysplastic syndrome (MDS) and Hb < or =10 g/dl. Epoetin alfa 40,000 IU was given subcutaneously twice weekly; after 4 weeks, the dose could be reduced to 40,000 IU weekly in patients achieving erythroid response. QOL was assessed using the functional assessment of cancer therapy-anemia (FACT-An) questionnaire. FACT-An scores increased on average by 7.5 after 4 weeks and by 8.8 after 8 weeks compared with baseline. FACT-An scores were positively associated with Hb values (r=0.53, P<0.01). The mean FACT-An score increase at week 8 was 10.2 in responders and 5.6 in nonresponders. The overall erythroid response rate at week 8 was 68%: 74% in transfusion-independent patients and 59% in transfusion-dependent patients. Of all responders at week 8, response was maintained in 86% at week 12, 71% at week 16, 65% at week 20, and 54% at week 24. Treatment was generally well tolerated. Our data provide new and encouraging results regarding the benefits of 40,000 IU biweekly induction doses followed by 40,000 IU weekly in improving QOL, correcting anemia, and reducing transfusion requirements in low-risk MDS patients.