VI Seminario di studio. Salute globale e scenari attuali. Nuovi contributi di ricerca. Istituto Superiore di Sanità. Roma, 16 aprile 2015. Riassunti

Esiste un bisogno crescente di chiare evidenze scientifiche che possano supportare le politiche per la salute. Questo vale per una serie di aspetti che vanno dai protocolli diagnostici applicati alle malattie trasmissibili, agli approcci terapeutici, agli studi epidemiologici, alla definizione e identificazione dei determinanti della salute con partico lare riguardo alle situazioni di grave diseguaglianza, alle contaminazioni ambientali, alla relazione tra malattia e diritti così come alla relazione tra malattia e diritto. L’obiettivo del Dottorato di Ricerca in Malattie Infettive, Microbiologia e Scienze della Salute è proprio quello di fornire momenti di crescita, finalizzati alla formazione di nuovi ricercatori, nel campo delle malattie infettive, della microbiologia, della parassitologia, della sanità pubblica e medicina sociale, nella medicina legale e nelle scienze forensi. L’obiettivo di questo sesto appuntamento, nell’ambito della collaborazione tra Istituto Superiore di Sanità e l’Università̀ Sapienza di Roma, con i dottorandi è proprio quello di fornire una cornice il più possibile unitaria a un quadro complesso di ricerche di base e applicate nell’ambito degli obiettivi del dottorato.There is a growing need for clear scientific evidence that can support health policies. This applies to a number of issues including diagnostic protocols applied to communicable diseases, therapeutic approaches, epidemiological studies, the definition and identification of the health determinants (with particular reference to situations of severe inequality), environmental contamination, the relationship between disease and rights as well as the relationship between disease and law. The objective of the PhD Program in Infectious Diseases, Microbiology and Health Sciences is precisely to provide moments of intellectual growth, aimed at the trai ning of new researchers in the field of infectious diseases, microbiology, parasitology, public health and social medicine, in forensic medicine and forensic sciences. The objective of the sixth meeting with graduate students, part of the collaboration between the Italian National Institute of Health, and the Sapienza University of Rome, is precisely to provide a frame as unitary as possible to a complex picture of basic and applied research within the objectives of the doctorat

Whole Exome Sequencing of HIV-1 long-term non-progressors identifies rare variants in genes encoding innate immune sensors and signaling molecules

Abstract Common CCR5-∆32 and HLA alleles only explain a minority of the HIV long-term non-progressor (LTNP) and elite controller (EC) phenotypes. To identify rare genetic variants contributing to the slow disease progression phenotypes, we performed whole exome sequencing (WES) on seven LTNPs and four ECs. HLA and CCR5 allele status, total HIV DNA reservoir size, as well as variant-related functional differences between the ECs, LTNPs, and eleven age- and gender-matched HIV-infected non-controllers on antiretroviral therapy (NCARTs) were investigated. Several rare variants were identified in genes involved in innate immune sensing, CD4-dependent infectivity, HIV trafficking, and HIV transcription mainly within the LTNP group. ECs and LTNPs had a significantly lower HIV reservoir compared to NCARTs. Furthermore, three LTNPs with variants affecting HIV nuclear import showed integrated HIV DNA levels below detection limit after in vitro infection. HIV slow progressors with variants in the TLR and NOD2 pathways showed reduced pro-inflammatory responses compared to matched controls. Low-range plasma levels of fibronectin was observed in a LTNP harboring two FN1 variants. Taken together, this study identified rare variants in LTNPs as well as in one EC, which may contribute to understanding of HIV pathogenesis and these slow progressor phenotypes, especially in individuals without protecting CCR5-∆32 and HLA alleles

Alternate NF-\u3baB-Independent Signaling Reactivation of Latent HIV-1 Provirus

Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 from infected individuals because of the establishment of proviral latency in long-lived cellular reservoirs. The shock-and-kill approach aims to reactivate viral replication from the latent state (shock) using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells (kill) by specific therapeutics. The NF-\u3baB RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat (LTR). Nevertheless, prolonged NF-\u3baB activation contributes to the development of various autoimmune, inflammatory, and malignant disorders. In the present study, we established a cellular model of HIV-1 latency in J-Lat CD4+ T cells that stably expressed the NF-\u3baB superrepressor I\u3baB-\u3b1 2N\u3944 and demonstrate that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-\u3baB activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors or specific short hairpin RNAs, c-Jun was identified to be an essential factor binding to the LTR enhancer \u3baB sites and mediating the combined synergistic reactivation effect. Furthermore, acetylsalicylic acid (ASA), a potent inhibitor of the NF-\u3baB activator kinase I\u3baB kinase \u3b2 (IKK-\u3b2), did not significantly diminish reactivation in a primary CD4+ T central memory (TCM) cell latency model. The present work demonstrates that the shock phase of the shock-and-kill approach to reverse HIV-1 latency may be achieved in the absence of NF-\u3baB, with the potential to avoid unwanted autoimmune- and or inflammation-related side effects associated with latency-reversing strategies.IMPORTANCE The shock-and-kill approach consists of the reactivation of HIV-1 replication from latency using latency-reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-\u3baB is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-\u3baB in HIV-1-infected patients resulting from the combined administration of different LRAs could represent a potential risk, especially in the case of a prolonged treatment. We demonstrate here that conventional treatments with bryostatin-1 and hexamethylenebisacetamide (HMBA) or ionomycin synergistically reactivate HIV-1 from latency, even under conditions where NF-\u3baB activation is repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-\u3baB in patients under combination antiretroviral therapy during the shock-and-kill approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs

Determinanti di salute: nuovi percorsi per la ricerca scientifica

Nel presente volume sono riportati i lavori di ricerca che saranno presentati nel corso del seminario come comunicazioni orali e poster oltre alle linee di indirizzo dei dottorandi del primo anno. Il seminario, giunto alla quinta edizione, nell’ambito della collaborazione tra Istituto Superiore di Sanità e l’Università della Sapienza di Roma, testimonia la volontà di spingersi sempre più in prossimità delle origini dei problemi di salute affrontandone i determinanti. Il bisogno di evidenze scientifiche chiare per informare e sostenere le scelte politiche non sono mai state così grandi quanto nel momento di attuale cambiamento. La ricerca nel campo dei determinanti di salute, occupandosi degli aspetti fondamentali della vita delle persone, del loro lavoro e degli stili di vita è stata individuata come complessa e stimolante chiave di lettura multidisciplinare della giornata. Il seminario articolato in tre tempi, presenta due sessioni di relazioni orali sulle principali ricerche di microbiologia, chimica ambientale, epidemiologia e scienze sociali orientate alla visione della “Qualità ambientale e alla percezione del rischio”. La sessione intermedia è dedicata alla presentazione dei poster in cui le diversificate anime del dottorato, incardinate nei tre curricula di “Malattie Infettive”, “Scienze della Salute e Medicina Sociale” e “Microbiologia e Parassitologia”, verranno a confronto.This volume collects the research projects that will be presented during the workshop as oral communications and posters in addition to the research lines of PhD students attending the first year of course. The workshop, now in its fifth edition, in the context of an important collaboration between “Istituto Superiore di Sanità” and “Università La Sapienza di Roma”, demonstrates the will to understand health problems and their determinants. The need for clear scientific evidences to inform and support policy choices have never been so great as in this moment of change. Research in the field of health determinants, dealing with the fundamental aspects of people's lives, their jobs and lifestyles, were identified as a complex and challenging key to understanding the multi-disciplinary motive of this day. The workshop, divided into three parts, presents two sessions of oral reports on main researches in microbiology, environmental chemistry, epidemiology and social sciences, all oriente

HIV-1 Tat Recruits HDM2 E3 Ligase To Target IRF-1 for Ubiquitination and Proteasomal Degradation

In addition to its ability to regulate HIV-1 promoter activation, the viral transactivator Tat also functions as a determinant of pathogenesis and disease progression by directly and indirectly modulating the host anti-HIV response, largely through the capacity of Tat to interact with and modulate the activities of multiple host proteins. We previously demonstrated that Tat modulated both viral and host transcriptional machinery by interacting with the cellular transcription factor interferon regulatory factor 1 (IRF-1). In the present study, we investigated the mechanistic basis and functional significance of Tat−IRF-1 interaction and demonstrate that Tat dramatically decreased IRF-1 protein stability. To accomplish this, Tat exploited the cellular HDM2 (human double minute 2 protein) ubiquitin ligase to accelerate IRF-1 proteasome-mediated degradation, resulting in a quenching of IRF-1 transcriptional activity during HIV-1 infection. These data identify IRF-1 as a new target of Tat-induced modulation of the cellular protein machinery and reveal a new strategy developed by HIV-1 to evade host immune responses

Identification of anti-influenza A compounds inhibiting the NS-1 viral protein using a type I IFN-driven screening strategy

There is an urgent need to identify efficient antiviral compounds to combat existing and emerging RNA virus infections, particularly those related to seasonal and pandemic influenza outbreaks. While inhibitors of the influenza viral integral membrane proton channel protein (M2), neuraminidase (NA), and cap-dependent endonuclease are available, circulating influenza viruses acquire resistance over time. Thus, the need for the development of additional anti-influenza drugs with novel mechanisms of action exists. In the present study, a cell-based screening assay and a small molecule library were used to screen for activities that antagonized influenza A non-structural protein 1 (NS1), a highly conserved, multifunctional accessory protein that inhibits the type I interferon response against influenza. Two potential anti-influenza agents, compounds 157 and 164, were identified with anti-NS1 activity, resulting in the reduction of A/PR/8/34(H1N1) influenza A virus replication and the restoration of IFN-β expression in human lung epithelial A549 cells. A 3D pharmacophore modeling study of the active compounds provided a glimpse of the structural motifs that may contribute to anti-influenza virus activity. This screening approach is amenable to a broader analysis of small molecule compounds to inhibit other viral targets

Activation of latent HIV-1 T cell reservoirs with a combination of innate immune and epigenetic regulators

The presence of T cell reservoirs in which human immunodeficiency virus (HIV) establishes latency by integrating into the host genome represents a major obstacle to an HIV cure and has prompted the development of strategies aimed at the eradication of HIV from latently infected cells. The "shock-and-kill" strategy is one of the most pursued approaches to the elimination of viral reservoirs. Although several latency-reversing agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. In this study, we evaluated a novel immune system-mediated approach to clearing the HIV reservoir, based on a combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP (cyclic GMP-AMP) and the FDA-approved histone deacetylase inhibitor resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro. Reductions in the proportion of HIV-harboring cells and the total amount of HIV DNA were also observed in CD4(+) central memory T (T-CM) cells, a primary cell model of latency, where resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cell-associated HIV RNA were detected ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4(+) T cells from individuals on suppressive antiretroviral therapy (ART). Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4(+) T cells. Collectively, these results represent a promising step toward HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation for reducing the viral reservoir and inducing specific death of HIV-infected cells.IMPORTANCE One of the challenges associated with HIV-1 infection is that despite antiretroviral therapies that reduce HIV-1 loads to undetectable levels, proviral DNA remains dormant in a subpopulation of T lymphocytes. Numerous strategies to clear residual virus by reactivating latent virus and eliminating the reservoir of HIV-1 (socalled "shock-and-kill" strategies) have been proposed. In the present study, we use a combination of small molecules that activate the cGAS-STING antiviral innate immune response (the di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV-infected T cell killing in cell lines, primary T lymphocytes, and patient samples. These studies represent a novel strategy for HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells

I kappa B kinase (IKK)-epsilon targets interferon regulatory factor 1 in activated T lymphocytes.

IB kinase (IKK-) has an essential role as a regulator of innate immunity, functioning downstream of pattern recognition receptors to modulate NF-B and interferon (IFN) signaling. In the present study, we investigated IKK- activation following T cell receptor (TCR)/CD28 stimulation of primary CD4 T cells and its role in the stimulation of a type I IFN response. IKK- was activated following TCR/CD28 stimulation of primary CD4 T cells; however, in T cells treated with poly(I·C), TCR/CD28 costimulation blocked induction of IFN- transcription. We demonstrated that IKK- phosphorylated the transcription factor IFN regulatory factor 1 (IRF-1) at amino acid (aa) 215/219/221 in primary CD4 T cells and blocked its transcriptional activity. At the mechanistic level, IRF-1 phosphorylation impaired the physical interaction between IRF-1 and the NF-B RelA subunit and interfered with PCAF-mediated acetylation of NF-B RelA. These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKK- activation, which in turn contributes to suppression of IFN- production

New Monoclonal Antibodies Specific for Different Epitopes of the Spike Protein of SARS-CoV-2 and Its Major Variants: Additional Tools for a More Specific COVID-19 Diagnosis

The emergence of the new pathogen SARS-CoV-2 determined a rapid need for monoclonal antibodies (mAbs) to detect the virus in biological fluids as a rapid tool to identify infected individuals to be treated or quarantined. The majority of commercially available antigenic tests for SARS-CoV-2 rely on the detection of N antigen in biologic fluid using anti-N antibodies, and their capacity to specifically identify subjects infected by SARS-CoV-2 is questionable due to several structural analogies among the N proteins of different coronaviruses. In order to produce new specific antibodies, BALB/c mice were immunized three times at 20-day intervals with a recombinant spike (S) protein. The procedure used was highly efficient, and 40 different specific mAbs were isolated, purified and characterized, with 13 ultimately being selected for their specificity and lack of cross reactivity with other human coronaviruses. The specific epitopes recognized by the selected mAbs were identified through a peptide library and/or by recombinant fragments of the S protein. In particular, the selected mAbs recognized different linear epitopes along the S1, excluding the receptor binding domain, and along the S2 subunits of the S protein of SARS-CoV-2 and its major variants of concern. We identified combinations of anti-S mAbs suitable for use in ELISA or rapid diagnostic tests, with the highest sensitivity and specificity coming from proof-of-concept tests using recombinant antigens, SARS-CoV-2 or biological fluids from infected individuals, that represent important additional tools for the diagnosis of COVID-19