HUMAN HERPESVIRUS 8 UPREGULATES ACTIVATING TRANSCRIPTION FACTOR 4 (ATF4) and REAL TIME PCR TO ASSESS TOTAL BACTERIAL LOAD IN CHRONIC WOUNDS.

I. Human Herpesvirus 8 Upregulates Activator Transcription Factor 4 BACKGROUND: Human herpesvirus 8 (HHV-8) is the primary etiologic agent of Kaposi’s sarcoma, a highly vascularised neoplasm of endothelial origin characterized by inflammation, neoangiogenesis, and by the presence of characteristic spindle cells. HHV-8 angiogenic activity is due to the activation of NF-kB and the subsequent induction of MCP-1 synthesis. MCP-1 is a chemokine produced by macrophages and endothelial cells in response to different stimuli, and is a direct mediator of angiogenesis. HHV-8-induced angiogenesis is MCP-1 dependent. However, HHV-8 activation of MCP-1 is not completely dependent on NF-kB induction and another cellular factor is involved. A potential candidate is the cellular activating transcription factor 4 (ATF4), a stress responsive gene. ATF4 is upregulated in several condition, including ER-stress, viral infection (e.g. CMV) and in tumours. AIM: The aim of the research is to study the interaction between HHV-8 and ATF4, and verify whether ATF4 is involved in angiogenesis characteristic of HHV-8 infection. METHODS: To demonstrate the effect of HHV-8 on ATF4 expression, Jurkat cells were infected with a cell-free viral inoculum, obtained in our laboratory by stimulating reactivation of latent HHV-8 in chronically infected cells (PEL-derived). HHV-8 reactivation from latency was assessed by transfection of BC-3 and BCBL-1 cells (PEL-derived) with pCG-ATF4 recombinant plasmid. DNA or RNA were analysed by PCR, rtPCR or quantitative real time PCR. Promoters activation was assessed by luciferase assays. RESULTS: HHV-8 upregulates the expression of ATF4 gene, and overexpression of ATF4 is able to increase replication and transcription of HHV-8, but not to reactivate HHV-8 from latency. Preliminary results show that ATF4 activates the MCP-1 promoter in absence of the NF-kB binding sites. CONCLUSION: HHV-8 induces ATF4 expression during the productive infection, probably to obtain advantage for its replication and neoplastic development. ATF4 might be implicated in HHV-8 induced tumorigenesis, being involved in several aspects of viral replication, and could represent a potential therapeutic target for HHV-8 induced transformation. II. Real Time PCR To Assess Total Bacterial Load in Chronic Wounds BACKGROUND: Wounds and wound healing are important issues in chronic patients (i.e. diabetes, pressure). Critical colonization and infection are strictly linked to a delay in wound healing. Analysis of pathogens present in chronic wound is an essential aspect for the wound care. Classic microbiologic methods have several limits to ensure the correct analysis of ulcer environment. AIM: To analyse total bacterial load by a single quantitative real time PCR reaction in swabs and biopsies obtained from infected chronic wounds treated with an innovative hydrophobic dressing. METHODS: Biopsies were collected at the beginning and after 4 weeks of treatment, and swabs were collected once a week for 4 weeks. Real time PCR was carried out on DNA extracted from biopsies and swabs amplifying a region of the 16S rRNA gene, highly conserved among bacteria. Moreover, DNA extracted from biopsies was also analysed for the detection of 2 anaerobic bacteria (B. Fragilis, F. necrophorum, frequently associated with delayed healing) by real time PCR amplifying specific unique regions of their genome. In parallel, classical culturing methods were performed on biopsies searching for Staphylococcus and Pseudomonas species. RESULTS: We evaluated the correlation between the molecular data obtained by real time PCR and the clinical data, in particular considering the area of the wound. We observed a mean 253-fold decrease of the total bacterial load in 10/20 wounds those also showed an average 58% decrease of their area. This 10 wounds showed a positive correlation between clinical and molecular data. In 5/20 wound, we found a non significant 5,2-fold decrease of the total bacterial load, correlate with a 27% increase of the wound’s area. Thus, 75% of molecular results (15/20 wounds) were correlate to the clinical data. In contrast, classical culturing method did not correlate with the clinical data, confirming that classical methods have several limits and disadvantages. B. Fragilis was present in 10/20 wounds, and F. necrophorum in 2/20 wounds. CONCLUSION: The molecular approach can be considered a reliable and rapid test to assess infection levels in chronic wounds, being more sensitive than the classic cultural techniques. The research of specific pathogens is not sufficient to assess the outcome of the wounds, whereas total bacterial load can give a prognostic value to the wound care

HHV-6A infection induces amyloid-beta expression and activation of microglial cells.

BACKGROUND: The control of viral infections in the brain involves the activation of microglial cells, the macrophages of the brain that are constantly surveying the central nervous system, and the production of amyloid-beta (Aβ) as an anti-microbial molecule. Recent findings suggest a possible implication of HHV-6A in AD. We evaluated the effect of HHV-6A infection on microglial cell expression Aβ and the activation status, determined by TREM2, ApoE, cytokines, and tau expression. METHODS: We have infected microglial cells (HMC3, ATCC®CRL-3304), in monolayer and human peripheral blood monocyte-derived microglia (PBM-microglia) spheroid 3D model, with HHV-6A (strain U1102) cell-free virus inocula with 100 genome equivalents per 1 cell. We collected the cells 1, 3, 7, and 14 days post-infection (d.p.i.) and analyzed them for viral DNA and RNA, ApoE, Aβ (1-40, 1-42), tau, and phospho-tau (Threonine 181) by real-time immunofluorescence and cytokines by immunoenzymatic assay. RESULTS: We observed a productive infection by HHV-6A. The expression of Aβ 1-42 increased from 3 d.p.i., while no significant induction was observed for Aβ 1-40. The HHV-6A infection induced the activation (TREM2, IL-1beta, ApoE) and migration of microglial cells. The secretion of tau started from 7 d.p.i., with an increasing percentage of the phosphorylated form. CONCLUSIONS: In conclusion, microglial cells are permissive to HHV-6A infection that induces the expression of Aβ and an activation status. Meanwhile, we hypothesize a paracrine effect of HHV-6A infection that activates and induces microglia migration to the site of infection

Honeybees' physiological and behavioural immunity deficit induced by DW Viruses

Item does not contain fulltextThe purpose of the present study was to determine the effect of a spinal cord injury (SCI) on resting vascular resistance in paralyzed legs in humans. To accomplish this goal, we measured blood pressure and resting flow above and below the lesion (by using venous occlusion plethysmography) in 11 patients with SCI and in 10 healthy controls (C). Relative vascular resistance was calculated as mean arterial pressure in millimeters of mercury divided by the arterial blood flow in milliliters per minute per 100 milliliters of tissue. Arterial blood flow in the sympathetically deprived and paralyzed legs of SCI was significantly lower than leg blood flow in C. Because mean arterial pressure showed no differences between both groups, leg vascular resistance in SCI was significantly higher than in C. Within the SCI group, arterial blood flow was significantly higher and vascular resistance significantly lower in the arms than in the legs. To distinguish between the effect of loss of central neural control vs. deconditioning, a group of nine SCI patients was trained for 6 wk and showed a 30% increase in leg blood flow with unchanged blood pressure levels, indicating a marked reduction in vascular resistance. In conclusion, vascular resistance is increased in the paralyzed legs of individuals with SCI and is reversible by training

Real-world treatment patterns for metastatic non-small cell lung cancer at IRST Italy

n/

Investigating Serum sHLA-G Cooperation With MRI Activity and Disease-Modifying Treatment Outcome in Relapsing-Remitting Multiple Sclerosis

Relapsing-remitting multiple sclerosis (RRMS) is a demyelinating disease in which pathogenesis T cells have a major role. Despite the unknown etiology, several risk factors have been described, including a strong association with human leukocyte antigen (HLA) genes. Recent findings showed that HLA class I-G (HLA-G) may be tolerogenic in MS, but further insights are required. To deepen the HLA-G role in MS inflammation, we measured soluble HLA-G (sHLA-G) and cytokines serum level in 27 patients with RRMS at baseline and after 12 and 24 months of natalizumab (NTZ) treatment. Patients were divided into high (sHLA-G>20 ng/ml), medium (sHLA-G between 10 and 20 ng/ml), and low (sHLA-G <10 ng/ml) producers. Results showed a heterogeneous distribution of genotypes among producers, with no significant differences between groups. A significant decrease of sHLA-G was found after 24 months of NTZ in low producers carrying the +3142 C/G genotype. Finally, 83.3% of high and 100% of medium producers were MRI-activity free after 24 months of treatment, compared to 63.5% of low producers. Of note, we did not find any correlation of sHLA-G with peripheral cell counts or cytokines level. These findings suggest that serum sHLA-G level may partly depend on genotype rather than peripheral inflammation, and that may have impacted on MRI activity of patients over treatment

Dendritic cell vaccination in metastatic melanoma turns \u201cnon-T cell inflamed\u201d into \u201cT-cell inflamed\u201d tumors

Dendritic cell (DC)-based vaccination effectively induces anti-tumor immunity, although in the majority of cases this does not translate into a durable clinical response. However, DC vaccination is characterized by a robust safety profile, making this treatment a potential candidate for effective combination cancer immunotherapy. To explore this possibility, understanding changes occurring in the tumor microenvironment (TME) upon DC vaccination is required. In this line, quantitative and qualitative changes in tumor-infiltrating T lymphocytes (TILs) induced by vaccination with autologous tumor lysate/homogenate loaded DCs were investigated in a series of 16 patients with metastatic melanoma. Immunohistochemistry for CD4, CD8, Foxp3, Granzyme B (GZMB), PDL1, and HLA class I was performed in tumor biopsies collected before and after DC vaccination. The density of each marker was quantified by automated digital pathology analysis on whole slide images. Co-expression of markers defining functional phenotypes, i.e., Foxp3+ regulatory CD4+ T cells (Treg) and GZMB+ cytotoxic CD8+ T cells, was assessed with sequential immunohistochemistry. A significant increase of CD8+ TILs was found in post-vaccine biopsies of patients who were not previously treated with immune-modulating cytokines or Ipilimumab. Interestingly, along with a maintained tumoral HLA class I expression, after DC vaccination we observed a significant increase of PDL1+ tumor cells, which significantly correlated with intratumoral CD8+ T cell density. This observation might explain the lack of a significant concurrent cytotoxic reactivation of CD8+ T cell, as measured by the numbers of GZMB+ T cells. Altogether these findings indicate that DC vaccination exerts an important role in sustaining or de novo inducing a T cell inflamed TME. However, the strength of the intratumoral T cell activation detected in post-DC therapy lesions is lessened by an occurring phenomenon of adaptive immune resistance, yet the concomitant PDL1 up-regulation. Overall, this study sheds light on DC immunotherapy-induced TME changes, lending the rationale for the design of smarter immune-combination therapies

IFLA Library Reference Model (LRM) : Un modello concettuale per le informazioni bibliografiche

IFLA Library Reference Model (LRM) is a high-level conceptual reference model developed within an entity-relationship modelling framework. It is the consolidation of the separately developed IFLA conceptual models: FRBR, FRAD, FRSAD. IFLA LRM was developed to resolve inconsistencies between the three separate models. Every user task, entity, attribute and relationship from the original three models was examined, definitions had to be revised, but also some remodelling was required in order to develop a meaningful consolidation. The result is a single, streamlined, and logically consistent model that covers all aspects of bibliographic data and that at the same time brings the modelling up-to-date with current conceptual modelling practices. IFLA LRM was designed to be used in linked data environments and to support and promote the use of bibliographic data in linked data environments

Cell cycle block by p53 activation reduces SARS-CoV-2 release in infected alveolar basal epithelial A549-hACE2 cells

SARS-CoV viruses have been shown to downregulate cellular events that control antiviral defenses. They adopt several strategies to silence p53, key molecule for cell homeostasis and immune control, indicating that p53 has a central role in controlling their proliferation in the host. Specific actions are the stabilization of its inhibitor, MDM2, and the interference with its transcriptional activity. The aim of our work was to evaluate a new approach against SARS-CoV-2 by using MDM2 inhibitors to raise p53 levels and activate p53-dependent pathways, therefore leading to cell cycle inhibition. Experimental setting was performed in the alveolar basal epithelial cell line A549-hACE2, expressing high level of ACE2 receptor, to allow virus entry, as well as p53 wild-type. Cells were treated with several concentrations of Nutlin-3 or RG-7112, two known MDM2 inhibitors, for the instauration of a cell cycle block steady-state condition before and during SARS-CoV-2 infection, and for the evaluation of p53 activation and impact on virus release and related innate immune events. The results indicated an efficient cell cycle block with inhibition of the virion release and a significant inhibition of IL-6, NF-kB and IFN-λ expression. These data suggest that p53 is an efficient target for new therapies against the virus and that MDM2 inhibitors deserve to be further investigated in this field