New inflammatory features of human T helper 17 cells in health and multiple sclerosis

T helper (Th) 17 cells are a subpopulation of CD4 T lymphocytes characterized by the expression of interleukin (IL)-17 and the transcription factor retinoid acid receptor-related orphan receptor (ROR)gt. Pathogenic role of human Th17 cells has been demonstrated in several autoimmune diseases, including multiple sclerosis (MS), where they promote inflammatory processes. However, the mechanisms leading to the pathogenicity of Th17 cells are largely unknown. The main objective of this thesis was to identify new mechanisms and molecules inducing inflammatory functions of human Th17 cells that could be potentially involved in the pathogenic processes of MS. We addressed this aim by using two distinct approaches: 1) analysis of general inflammatory features of Th17 cells acquired during Th17 cell differentiation; 2) analysis of intrinsic features of Th17 cells derived from MS patients. In order to investigate potential mechanisms responsible for pathogenic functions of human Th17 cells, we dissected their differentiation process by performing a transcriptome analysis of cells at 48 hours and 5 days of differentiation. We uncovered three time-regulated modules: early modulation, involving exclusively ‘signalling pathways’ genes; late modulation, characterized by genes involved in response to infections; persistent modulation, involving effector immune functions. To assign them an inflammatory or regulatory potential, we compared Th17 cells differentiated in presence or absence of IL-1b, respectively. We named inflammatory Th17 condition the polarizing milieu containing IL-1b, which is crucially involved in the pathology of Th17-related diseases. In contrast, Th17 regulatory condition refers to the anti-inflammatory IL-10 cytokine produced in Th17 condition lacking IL-1b. We found that most part of the inflammatory genes belong to the persistent or late module, indicating the crucial role of these genes in the late phases of differentiation. Thus, we elucidated the global molecular signature that characterizes the acquisition of the inflammatory profile by human Th17 cells, by analysing all genes differentially expressed in regulatory versus inflammatory Th17 conditions. Among inflammatory genes, we identified those sharing pathogenic functions with murine Th17 cells, including IL17A, IFNG, TBX21, EBI3, IRF8, TNFRSF9, TNFRSF14, CCL5, CD40LG, BATF and TNF. In addition, our analysis allowed the identification of novel effector molecules, including interferon (IFN)K, lymphotoxin (LT)-a, IL1A, platelet derived growth factor (PDGF)-A, and transcriptional regulators, such as transcriptional regulators homeodomain-only protein homeobox (HOP)X, SRY (sex determining region Y)-box 2 (SOX2), expression of which was independently validated. In order to unveil the mechanisms underlying the acquisition of the human Th17 signature, we investigated the potential transcription factors involved in this process. In this context, we analysed the role of RORgt, known master regulator of Th17 cells, and of the two novel transcriptional regulators HOPX and SOX2, by performing RNA-interference experiments. We found that HOPX regulates IL-17A and IFN-g, while SOX2 regulates PDGF-A and IFN-g. As expected, RORgt regulates expression of IL-17A, IL-17F, but also IFN-g, PDGF-A, and IL1A, not previously described. These results, together with the reduced expression of both HOPX and SOX2 in RORC-interfered cells, suggest that HOPX and SOX2 are two transcriptional regulators acting downstream of RORgt signalling in human Th17 cells. In the second approach, we studied the pathogenic features intrinsically associated to Th17 cells using Th17 cells obtained from MS patients. In particular, we compared in-vitro differentiated Th17 cells of MS patients and healthy donors (HD) and we systematically analysed typical features of Th17 cells, including receptors, transcription factors and soluble factors by flow cytometry, ELISA and Luminex assays. We also included in this study the expression analysis of PDGF and LT-a, two novel effector molecules that we found associated to inflammatory Th17 cells in the previous approach. Results from these analyses unveiled the increased expression of pro-inflammatory proteins IL-21, IL-2, and IL-1 receptor1 (IL-1R1) in Th17 cells derived from MS patients compared to those from HD. Moreover, we found that Th17 cells derived from MS patients express higher levels of LT-a compared to those from HD, indicating that the pathogenic signature that we previously identified contains intrinsic inflammatory features of Th17 cells derived from patients affected by Th17-related diseases. In conclusion, the main results of the project were: 1) the identification of the inflammatory signature of human Th17 cells, that includes novel Th17 genes, such as IFNK, IL1A, PDGF-A, and LT-a, and novel transcriptional regulators HOPX and SOX2; 2) the identification of the intrinsic Th17 features specifically overexpressed in Th17 cells of MS patients, that includes IL-21, IL-2, IL-1R1, and LT-a. Importantly, these factors could become new biomarkers or new therapeutic targets in Th17-related autoimmune diseases

Genetic diversity in the env V1-V2 region of proviral quasispecies from long-term controller MHC-typed cynomolgus macaques infected with SHIVSF162P4cy

Intra-host evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) has been shown by viral RNA analysis in subjects who naturally suppress plasma viremia to low levels, known as controllers. However, little is known about the variability of proviral DNA and the inter-relationships among contained systemic viremia, rate of reservoir reseeding and specific major histocompatibility complex (MHC) genotypes, in controllers. Here, we analysed the proviral DNA quasispecies of the env V1-V2 region, in PBMCs and in anatomical compartments of 13 long-term controller monkeys after 3.2 years of infection with simian/human immunodeficiency virus (SHIV)SF162P4cy. A considerable variation in the genetic diversity of proviral quasispecies was present among animals. Seven monkeys exhibited env V1-V2 proviral populations composed of both clusters of identical ancestral sequences and new variants, whereas the other six monkeys displayed relatively high env V1-V2 genetic diversity with a large proportion of diverse novel sequences. Our results demonstrate that in SHIVSF162P4cy-infected monkeys there exists a disparate pattern of intra-host viral diversity and that reseeding of the proviral reservoir occurs in some animals. Moreover, even though no particular association has been observed between MHC haplotypes and the long-term control of infection, a remarkably similar pattern of intra-host viral diversity and divergence was found within animals carrying the M3 haplotype. This suggests that in animals bearing the same MHC haplotype and infected with the same virus, viral diversity follows a similar pattern with similar outcomes and control of infection

La valutazione del paziente sintomatico affetto da COVID-19: la scala CovidScore e la stratificazione del rischio clinico nelle unità sub-intensive

INTRODUCTION: During the pandemic peaks of COVID-19, it was necessary to promptly assess and recognize critical patients early through effective clinical framing supported by specific instruments; therefore, the CovidScore scale was designed, including among its items the specific characteristics of the Sars-CoV-2 patient. OBJECTIVES: To test whether the CovidScore scale results in better risk framing and stratification than the NEWS2 scale and instrument-free nursing assessment. MATERIALS AND METHODS: A longitudinal, single-center, prospective study was performed by enrolling 182 patients. Aggregate scores and risk levels defined by CovidScore and NEWS2 were determined through data collected from nurses, who also provided their assessment at intake. RESULTS: A moderate but statistically significant concordance was found between risk estimated by CovidScore and nurse score (K=0.239.; p<0.00022); between CovidScore and NEWS2, the concordance found was null (K=0.089, p<0.019); low concordance values were found between nurse assessment and NEWS2 (K=0.033, p<0.05). CONCLUSIONS: The NEWS2 scale seems to underestimate the clinical status of the COVID-19 patient compared with the assessments by CovidScore and nurses. Specific standardized clinical assessment and response systems for the COVID-19 patient, such as the CovidScore scale, could improve the management of large inpatient numbers and positively impact patient outcomes. Further studies with robust methodology are needed to test these hypotheses and strengthen the evidence found.INTRODUZIONE: durante i picchi epidemici da COVID-19 è stato necessario valutare e riconoscere precocemente i pazienti critici attraverso un efficace inquadramento clinico, supportato da strumenti specifici; per questo motivo è stata ideata la scala CovidScore, includendo fra i propri items le caratteristiche specifiche del paziente affetto da Sars-CoV-2. OBIETTIVI: verificare se la scala CovidScore determini un migliore inquadramento e stratificazione del rischio rispetto alla scala NEWS2 ed alla valutazione infermieristica senza strumenti. MATERIALI E METODI: E’ stato condotto uno  studio prospettico longitudinale monocentrico arruolando un campione di 182 pazienti. I punteggi aggregati ed i livelli di rischio definiti da CovidScore e NEWS2 sono stati determinati attraverso i dati raccolti dagli infermieri, che hanno fornito anche una loro valutazione alla presa in carico. RISULTATI: Tra rischio stimato dal punteggio CovidScore e quello dell’infermiere è stata riscontrata una discreta ma statisticamente significativa concordanza (K=0.239.; p<0.00022); tra CovidScore e NEWS2 la concordanza riscontrata è stata nulla (K=0.089, p<0.019); bassi valori di concordanza sono stati rinvenuti tra valutazione infermieristica e NEWS2 (K=0.033, p<0.05). CONCLUSIONI: La scala NEWS2 sembrerebbe sottostimare lo stato clinico del paziente affetto da COVID-19 rispetto le valutazioni effettuate da CovidScore ed infermieri. Sistemi specifici di valutazione e risposta clinica standardizzati per il paziente COVID-19, come la scala CovidScore, potrebbero migliorare la gestione di grandi numeri di ricoverati ed avere ripercussioni positive sull’outcome dei pazienti. Sono necessari ulteriori studi con metodologia robusta per la verifica di tali ipotesi e per rafforzare le evidenze riscontrate

EV20-mediated delivery of cytotoxic auristatin MMAF exhibits potent therapeutic efficacy in cutaneous melanoma.

Abstract Cutaneous melanoma is one of the cancers with the fastest rising incidence and in its advanced metastatic form is a highly lethal disease. Despite the recent approval of several new drugs, the 5-year overall survival rate for advanced cutaneous melanoma is still below 20% and therefore, the development of novel treatments remains a primary need. Antibody-Drug Conjugates are an emerging novel class of anticancer agents, whose preclinical and clinical development has recently seen a remarkable increase in different tumors, including melanoma. Here, we have coupled the anti-HER-3 internalizing antibody EV20 to the cytotoxic drug monomethyl auristatin F (MMAF) to form a novel antibody-drug conjugate (EV20/MMAF). In a panel of human melanoma cell lines, this novel ADC shows a powerful, specific and target-dependent cell killing activity, independently of BRAF status. Efficacy studies demonstrated that a single administration of EV20/MMAF leads to a long-lasting tumor growth inhibition. Remarkably, the effect of this novel ADC was superior to the BRAF inhibitor vemurafenib in preventing kidney, liver and lung melanoma metastases. Overall, these results highlight EV20/MMAF as a novel ADC with promising therapeutic efficacy, warranting extensive pre-clinical evaluation in melanoma with high levels of HER-3 expression

Distinct Expression of Inflammatory Features in T Helper 17 Cells from Multiple Sclerosis Patients

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). T helper (Th) 17 lymphocytes play a role in the pathogenesis of MS. Indeed, Th17 cells are abundant in the cerebrospinal fluid and peripheral blood of MS patients and promote pathogenesis in the mouse model of MS. To gain insight into the function of Th17 cells in MS, we tested whether Th17 cells polarized from naïve CD4 T cells of healthy donors and MS patients display different features. To this end, we analysed several parameters that typify the Th17 profile during the differentiation process of naïve CD4 T cells obtained from relapsing-remitting (RR)-MS patients (n = 31) and healthy donors (HD) (n = 28). Analysis of an array of cytokines produced by Th17 cells revealed that expression of interleukin (IL)-21, tumour necrosis factor (TNF)-β, IL-2 and IL-1R1 is significantly increased in Th17 cells derived from MS patients compared to healthy donor-derived cells. Interestingly, IL-1R1 expression is also increased in Th17 cells circulating in the blood of MS patients compared to healthy donors. Since IL-2, IL-21, TNF-β, and IL-1R1 play a crucial role in the activation of immune cells, our data indicate that high expression of these molecules in Th17 cells from MS patients could be related to their high inflammatory status

Chapter Facing Malaria Parasite with Mosquito Symbionts

Optical physic

Facing Malaria Parasite with Mosquito Symbionts

Optical physic

The RNA-binding protein MEX3A is a prognostic factor and regulator of resistance to gemcitabine in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer. Most patients present with advanced disease at diagnosis, which only permits palliative chemotherapeutic treatments. RNA dysregulation is a hallmark of most human cancers, including PDAC. To test the impact of RNA processing dysregulation on PDAC pathology, we performed a bioinformatics analysis to identify RNA-binding proteins (RBPs) associated with prognosis. Among the 12 RBPs associated with progression-free survival, we focused on MEX3A because it was recently shown to mark an intestinal stem cell population that is refractory to chemotherapeutic treatments, a typical feature of PDAC. Increased expression of MEX3A was correlated with higher disease stage in PDAC patients and with tumor development in a mouse model of PDAC. Depletion of MEX3A in PDAC cells enhanced sensitivity to chemotherapeutic treatment with gemcitabine, whereas its expression was increased in PDAC cells selected upon chronic exposure to the drug. RNA-sequencing analyses highlighted hundreds of genes whose expression is sensitive to MEX3A expression, with significant enrichment in cell cycle genes. MEX3A binds to its target mRNAs, like cyclin-dependent kinase 6 (CDK6), and promotes their stability. Accordingly, knockdown of MEX3A caused a significant reduction in PDAC cell proliferation and in progression to the S phase of the cell cycle. These findings uncover a novel role for MEX3A in the acquisition and maintenance of chemoresistance by PDAC cells, suggesting that it may represent a novel therapeutic target for PDAC

Inhibition of Asaia in adult mosquitoes causes male-specific mortality and diverse transcriptome changes

Mosquitoes can transmit many infectious diseases, such as malaria, dengue, Zika, yellow fever, and lymphatic filariasis. Current mosquito control strategies are failing to reduce the severity of outbreaks that still cause high human morbidity and mortality worldwide. Great expectations have been placed on genetic control methods. Among other methods, genetic modification of the bacteria colonizing different mosquito species and expressing anti-pathogen molecules may represent an innovative tool to combat mosquito-borne diseases. Nevertheless, this emerging approach, known as paratransgenesis, requires a detailed understanding of the mosquito microbiota and an accurate characterization of selected bacteria candidates. The acetic acid bacteria Asaia is a promising candidate for paratransgenic approaches. We have previously reported that Asaia symbionts play a beneficial role in the normal development of Anopheles mosquito larvae, but no study has yet investigated the role(s) of Asaia in adult mosquito biology. Here we report evidence on how treatment with a highly specific anti-Asaia monoclonal antibody impacts the survival and physiology of adult Anopheles stephensi mosquitoes. Our findings offer useful insight on the role of Asaia in several physiological systems of adult mosquitoes, where the influence differs between males and females

The European Research Infrastructures of the ESFRI Roadmap in Biological and Medical Sciences: status and perspectives

Introduction. Since 2002, the European Strategy Forum on Research Infrastructures  identified  the  needs  for  Research  Infrastructures  (RIs)  in  Europe  in  priority  fields  of  scientific research and drafted a strategic document, the ESFRI Roadmap, defining the  specific RIs essential to foster European research and economy. The Biological and Medical Sciences RIs (BMS RIs) were developed thanks to the active participation of many  institutions  in  different  European  member  states  associated  to  address  the  emerging  needs in biomedicine and, among these, the Italian National Institute of Health (ISS),  in virtue of its role in public health and research, has been specifically involved in the  national development and implementation of three RIs: the Biobanking and Biomolecu-lar Resources Research Infrastructure (BBMRI), the European Advanced Translational  Research Infrastructure in Medicine (EATRIS) and the European Clinical Research Infrastructures Network (ECRIN). Aim.  This  article  outlines  the  design  and  development  of  these  RIs  up  to  the  recent  achievement of the ERIC status, their importance in the Horizon 2020 programme and  their societal and economic potential impact, with special attention to their development  and significance in Italy. Conclusions.  The  ISS  plays  a  unique  role  in  fostering  a  coordinated  participation  of  excellence Italian institutes/facilities to different European biomedical RIs, thus contributing to health innovation, healthcare optimization, and healthcare cost containment.