La fosfolipasi C specifica per fosfatidilcolina nelle cellule Natural Killer: studio degli aspetti funzionali e del traffico recettoriale del CD16

Lo scopo di questo lavoro di tesi è quello di ampliare le conoscenze sulla biologia delle cellule Natural Killer umane, una sottopopolazione linfocitaria del sangue periferico protagonista dell’immunità innata grazie alla sua abilità nel lisare cellule bersaglio in assenza di una precedente stimolazione ed in modo non ristretto al complesso maggiore di istocompatibilità. In particolare, in questa tesi l’attenzione è stata rivolta sull’enzima fosfolipasi C, specifico del ciclo della fosfatidilcolina (PC-PLC), responsabile della produzione di diacilglicerolo e fosfocolina. E’ stata evidenziata, sulla membrana plasmatica delle cellule NK, una correlazione diretta tra i livelli di espressione dell’enzima PC-PLC e quelli del recettore CD16. Inoltre, è stato dimostrato come, a seguito di ingaggio con il suo anticorpo specifico, il CD16 vada incontro ad un processo di internalizzazione, indipendente dall’ATP come fonte energetica, ma citoscheletrodipendente, cui segue una degradazione e una ri-espressione sulla membrana plasmatica delle cellule NK. La stimolazione del recettore induce un significativo incremento nell’attività enzimatica della PCPLC, entro 10 minuti dalla stimolazione. Inoltre, analisi più dettagliate della distribuzione dei due antigeni sulla membrana plasmatica hanno evidenziato la presenza sia della PC-PLC che del CD16 a livello dei microdomini di membrana. Dagli esperimenti di citofluorimetria a flusso si evince come la regolazione esercitata dall’enzima PC-PLC sul CD16 sia un fenomeno altamente specifico. Infatti, gli studi effettuati utilizzando l’inibitore dell’enzima PC-PLC, D609, dimostrano che il recettore CD16 è l’unico ad essere fortemente down-modulato dalla superficie cellulare, tra i vari recettori presenti sui linfociti del sangue periferico umano. Ne consegue che l’attività citotossica CD16-mediata risulta essere completamente abolita in presenza dell’inibitore. Questi dati suggeriscono che l’enzima PCPLC potrebbe svolgere un ruolo chiave nella regolazione dell’espressione del recettore CD16 sulla membrana plasmatica delle cellule NK, sui meccanismi litici CD16-mediati e nella cascata di trasduzione del segnale innescata dalla stimolazione del recettore stesso.CD16, the low-affinity FcIgG receptor (FcgammaRIIIA), is predominantly expressed in human NK cells. Our recent findings indicate that CD16 expression on the outer membrane surface of NK cells is correlated with the membrane expression of phosphatidylcholine-specific phospholipase C (PC-PLC), responsible for the production of diacylglycerol and phosphocholine. In the present work we analyse the trafficking of CD16 from the plasma membrane to cytoplasmic regions, after stimulation with specific mAb. The CD16 receptor is internalized, likely degraded and newly synthesized; its endocytosis is independent of ATP, but requires an integral and functional actin cytoskeleton. Antibody-mediated CD16 cross-linking results in an approximately twofold increase in PC-PLC enzymatic activity within 10 minutes. Analysis of PC-PLC and CD16 distribution in NK cell plasma membrane demonstrates that the proteins are physically associated and partially accumulated in lipid rafts. Pre-incubation of NK cells with a PC-PLC inhibitor, D609, determines a dramatic decrease both in CD16 receptor and PC-PLC enzyme expression on the plasma membrane. Interestingly, among phenotype PBL markers, only CD16 is strongly down-modulated by D609 treatment. CD16-mediated cytotoxicity is also reduced after D609 incubation. Taken together, these data suggest that the PC-PLC enzyme could play an important role in regulating CD16 membrane expression, the CD16-mediated cytolytic mechanism and CD16- triggered signal transduction

1,2,4-Triazolo[1,5-a]pyrimidines as a Novel Class of Inhibitors of the HIV-1 Reverse Transcriptase-Associated Ribonuclease H Activity

Despite great efforts have been made in the prevention and therapy of human immunodeficiency virus (HIV-1) infection, however the difficulty to eradicate latent viral reservoirs together with the emergence of multi-drug-resistant strains require the search for innovative agents, possibly exploiting novel mechanisms of action. In this context, the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H), which is one of the few HIV-1 encoded enzymatic function still not targeted by any current drug, can be considered as an appealing target. In this work, we repurposed in-house anti-influenza derivatives based on the 1,2,4-triazolo[1,5-a]-pyrimidine (TZP) scaffold for their ability to inhibit HIV-1 RNase H function. Based on the results, a successive multi-step structural exploration around the TZP core was performed leading to identify catechol derivatives that inhibited RNase H in the low micromolar range without showing RT-associated polymerase inhibitory activity. The antiviral evaluation of the compounds in the MT4 cells showed any activity against HIV-1 (IIIB strain). Molecular modelling and mutagenesis analysis suggested key interactions with an unexplored allosteric site providing insights for the future optimization of this class of RNase H inhibitors

Structural Modifications of the Quinolin-4-yloxy Core to Obtain New Staphylococcus aureus NorA Inhibitors

: Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones

Effects of Mentha suaveolens

Candidosis is the most important cause of fungal infections in humans. The yeast Candida albicans can form biofilms, and it is known that microbial biofilms play an important role in human diseases and are very difficult to treat. The prolonged treatment with drugs has often resulted in failure and resistance. Due to the emergence of multidrug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to analyse the effects induced by essential oil of Mentha suaveolens Ehrh (EOMS) on Candida albicans and its potential synergism when used in combination with conventional drugs. Morphological differences between control and EOMS treated yeast cells or biofilms were observed by scanning electron microscopy and transmission electron microscopy (SEM and TEM resp.,). In order to reveal the presence of cell cycle alterations, flow cytometry analysis was carried out as well. The synergic action of EOMS was studied with the checkerboard method, and the cellular damage induced by different treatments was analysed by TEM. The results obtained have demonstrated both the effects of EOMS on C. albicans yeast cells and biofilms and the synergism of EOMS when used in combination with conventional antifungal drugs as fluconazole (FLC) and micafungin (MCFG), and therefore we can hypothesize on its potential use in therapy. Further studies are necessary to know its mechanism of action

Mutations impairing GSK3-mediated MAF phosphorylation cause cataract, deafness, intellectual disability, seizures, and a down syndrome-like facies

Transcription factors operate in developmental processes to mediate inductive events and cell competence, and perturbation of their function or regulation can dramatically affect morphogenesis, organogenesis, and growth. We report that a narrow spectrum of amino-acid substitutions within the transactivation domain of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a leucine zipper-containing transcription factor of the AP1 superfamily, profoundly affect development. Seven different de novo missense mutations involving conserved residues of the four GSK3 phosphorylation motifs were identified in eight unrelated individuals. The distinctive clinical phenotype, for which we propose the eponym Aymé-Gripp syndrome, is not limited to lens and eye defects as previously reported for MAF/Maf loss of function but includes sensorineural deafness, intellectual disability, seizures, brachycephaly, distinctive flat facial appearance, skeletal anomalies, mammary gland hypoplasia, and reduced growth. Disease-causing mutations were demonstrated to impair proper MAF phosphorylation, ubiquitination and proteasomal degradation, perturbed gene expression in primary skin fibroblasts, and induced neurodevelopmental defects in an in vivo model. Our findings nosologically and clinically delineate a previously poorly understood recognizable multisystem disorder, provide evidence for MAF governing a wider range of developmental programs than previously appreciated, and describe a novel instance of protein dosage effect severely perturbing developmen

TBCE Mutations Cause Early-Onset Progressive Encephalopathy with Distal Spinal Muscular Atrophy

Tubulinopathies constitute a family of neurodevelopmental/neurodegenerative disorders caused by mutations in several genes encoding tubulin isoforms. Loss-of-function mutations in TBCE, encoding one of the five tubulin-specific chaperones involved in tubulin folding and polymerization, cause two rare neurodevelopmental syndromes, hypoparathyroidism-retardation-dysmorphism and Kenny-Caffey syndrome. Although a missense mutation in Tbce has been associated with progressive distal motor neuronopathy in the pmn/pmn mice, no similar degenerative phenotype has been recognized in humans. We report on the identification of an early-onset and progressive neurodegenerative encephalopathy with distal spinal muscular atrophy resembling the phenotype of pmn/pmn mice and caused by biallelic TBCE mutations, with the c.464T>A (p.Ile155Asn) change occurring at the heterozygous/homozygous state in six affected subjects from four unrelated families originated from the same geographical area in Southern Italy. Western blot analysis of patient fibroblasts documented a reduced amount of TBCE, suggestive of rapid degradation of the mutant protein, similarly to what was observed in pmn/pmn fibroblasts. The impact of TBCE mutations on microtubule polymerization was determined using biochemical fractionation and analyzing the nucleation and growth of microtubules at the centrosome and extracentrosomal sites after treatment with nocodazole. Primary fibroblasts obtained from affected subjects displayed a reduced level of polymerized α-tubulin, similarly to tail fibroblasts of pmn/pmn mice. Moreover, markedly delayed microtubule re-polymerization and abnormal mitotic spindles with disorganized microtubule arrangement were also documented. Although loss of function of TBCE has been documented to impact multiple developmental processes, the present findings provide evidence that hypomorphic TBCE mutations primarily drive neurodegeneration

De Novo VPS4A Mutations Cause Multisystem Disease with Abnormal Neurodevelopment.

The endosomal sorting complexes required for transport (ESCRTs) are essential for multiple membrane modeling and membrane-independent cellular processes. Here we describe six unrelated individuals with de novo missense variants affecting the ATPase domain of VPS4A, a critical enzyme regulating ESCRT function. Probands had structural brain abnormalities, severe neurodevelopmental delay, cataracts, growth impairment, and anemia. In cultured cells, overexpression of VPS4A mutants caused enlarged endosomal vacuoles resembling those induced by expression of known dominant-negative ATPase-defective forms of VPS4A. Proband-derived fibroblasts had enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane. VPS4A function was also required for normal endosomal morphology and IST1 localization in iPSC-derived human neurons. Mutations affected other ESCRT-dependent cellular processes, including regulation of centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, mitotic spindle formation, and cell cycle progression. We thus characterize a distinct multisystem disorder caused by mutations affecting VPS4A and demonstrate that its normal function is required for multiple human developmental and cellular processes.This work was supported by: UK Medical Research Council Project Grants [MR/M00046X/1], [MR/R026440/1] and Project grant from National Institute of Health Research Biomedical Research Centre at Addenbrooke's Hospital (to E.R.), Fondazione Bambino Gesù (Vite Coraggiose) and Italian Ministry of Health (CCR-2017-23669081) (to M.T.), National Institute for Health Research (NIHR) for the Cambridge Biomedical Research Centre and NIHR BioResource (Grant Number RG65966) (to F.L.R.), and a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (Grant Number 216370/Z/19/Z) (to J.E.). CIMR was supported by a Wellcome Trust Strategic Award [100140] and Equipment Grant [093026]. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The 100,000 Genomes Project is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support

Integrase defective lentiviral vector as a vaccine platform for delivering Influenza antigens

Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV) as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA) and nucleoprotein (NP) were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp) with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively). Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp+MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA)-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by Enzyme-Linked Lectine Assay (ELLA). IFN?-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate that IDLV can be harnessed for producing a vaccine able to induce a comprehensive immune response, including functional antibodies directed towards HA and NA proteins present on the vector particles in addition to a functional T cell response directed to the protein transcribed from the vector

Imported arboviral infections in Italy, July 2014-October 2015: A National Reference Laboratory report

BACKGROUND: Imported cases of infections due to Dengue (DENV) and Chikungunya (CHIKV) viruses and, more recently, Zika virus (ZIKV) are commonly reported among travelers returning from endemic regions. In areas where potentially competent vectors are present, the risk of autochthonous transmission of these vector-borne pathogens is relatively high. Laboratory surveillance is crucial to rapidly detect imported cases in order to reduce the risk of transmission. This study describes the laboratory activity performed by the National Reference Laboratory for Arboviruses (NRLA) at the Italian National Institute of Health in the period from July 2014 to October 2015. METHODS: Samples from 180 patients visited/hospitalized with a suspected DENV/CHIKV/ZIKV infection were sent to the NRLA from several Italian Hospitals and from Regional Reference Laboratories for Arboviruses, in agreement with the National Plan on human surveillance of vector-borne diseases. Both serological (ELISA IgM test and Plaque Reduction Neutralization Test-PRNT) and molecular assays (Real Time PCR tests, RT-PCR plus nested PCR and sequencing of positive samples) were performed. RESULTS: DENV infection was the most frequently diagnosed (80 confirmed/probable cases), and all four genotypes were detected. However, an increase in imported CHIKV cases (41 confirmed/probable cases) was observed, along with the detection of the first ZIKV cases (4 confirmed cases), as a consequence of the recent spread of both CHIKV and ZIKV in the Americas. CONCLUSIONS: Main diagnostic issues highlighted in our study are sensitivity limitations of molecular tests, and the importance of PRNT to confirm serological results for differential diagnosis of Arboviruses. The continuous evaluation of diagnostic strategy, and the implementation of laboratories networks involved in surveillance activities is essential to ensure correct diagnosis, and to improve the preparedness for a rapid and proper identification of viral threats